The method of cultivation, a fibroblast, a way of separating cells

 

(57) Abstract:

The invention relates to biotechnology and can be used for growing cultures of hematopoietic cells. Culturing human stem cells carried out ex vivo in a liquid culture medium with the replacement of the medium with a speed of 50 to 100% daily replacement at a density of cells 1104-11071 ml of medium. The cultivation is carried out in the presence of growth factors and/or transformed stromal cells, secreting growth factor. Additionally on Wednesday cultivation type vector gene transfer, providing a stable genetic transformation. For the Department of neoplastic cells from normal hematopoietic cells, combined with stromal cells influence the fluid flow, creating a shear stress equal to or more than 1.0 Dyne/cm2. The invention allows to optimize the cultivation of stem cells and precursor cells. 3 S. and 18 C.p. f-crystals, 8 tab., 5 Il.

The present invention relates to methods, bioreactors and compositions for culturing and transformation of mammalian cells in culture medium, in particular for the cultivation and transformation of Kul the following erythrocytes, leukocytes, platelets and lymphocytes, are produced in the bone marrow as precursor cells during differentiation. These cells in turn are produced from very Mature cells called undifferentiated cells predecessors who, as the analysis shows, produce colonies of Mature hemocytes 1-3-week cultures in semi-solid medium, such as methylcellulose or agar. These precursor cells essentially originate from the class of progenitor cells, called stem cells. Stem cells have the ability dividing as for self-maintenance and semidifferential in undifferentiated cells predecessors. Thus, stem cell division produce as the embryoblast cells, and a few more differentiated precursor cells. In addition to producing Gemalto stem cells can also increase the output of osteoblasts and osteoclasts and possibly cells of other tissues. In this description of the disclosed methods and compositions, which for the first time allow for successful cultivation in vitro of hemocytoblasts, leading to their proliferation and differentiation in cells-the precursors and Mature hemocyte.

And although it became possible to migrate retroviral genes in cultured mouse osteocytes, however, this transfer was still possible in the bone marrow cells of a person, because to date culture dexterous type (long-lived culture of bone marrow cells) is limited by their longevity, and their ability to ensure the survival of stem cells and to produce precursor cells throughout life.

Suspension cultures of bone marrow cells h is its reduced output precursor cells and Mature hemocytes, moreover, the synthesis of the cells cease to 6-8 weeks. Subsequent modifications originally derived suspension cultures have led to improved methods of cultivation. The solution of this problem is of such great importance that it is impossible to estimate, as it would allow, for example, to replicate human stem cells and precursor cells for bone marrow transplantation and protection from the effects of chemotherapy, to choose and to carry out various manipulations with such cells, that is, to use for gene transfer and synthesize Mature hemocyte person for transfusion therapy.

Studies of hematopoiesis and cultivation of bone marrow cells installed inside the adhesive layer, as the Central elements of the cellular stroma are fibroblasts and endocyte. These cells how to create an active binding sites for the growth of hematopoietic cells, and can induce the secretion of hematopoietic growth factors that stimulate proliferation and differentiation of progenitor cells. To these the hematopoietic growth factors include granulocyte-colony stimulating factor (G-CSF), granulocyte-micropar-colon is x adhesive layers in vitro, however, did not give promising results. Unlike similar cultures related species such as shrews and tupai, suspension cultures of osteocytes person is not capable of producing significant quantities as not related to substrate hematopoietic precursor cells and the count of clonogenic progenitor cells within 6-8 weeks. Although it is known from literature about continuously producing cultures within 3-5 months, there's no information about cultural environments, which provide stable continuous synthesis of precursor cells from stem cells for more than 4-6 weeks.

Moreover, extracellular synthesis and the production of progenitor cells for even such a short life of these cultures is usually decreased enough to understand that these cultures do not provide a survival or proliferation of stem cells. Besides nonactivated cells bone marrow stromal studied in the isolated state, secrete a small amount, if it is possible to determine, hematopoietic growth factors (HGF).

The absence of a stable synthesis of precursor cells and seligeria for continuous regeneration and increase the production of stem cells. It was therefore hypothesized that the specified culture or do not contain critical stimulator (stimulants) stem cells and/or contain new inhibitor (inhibitors). But at the same time explaining the failure to identify cultures of hematopoietic growth factors and pendulumed stromal cells was nominated for the null hypothesis that United failed to identify HGF and relative disadvantages of suspension cultures of human osteocytes, suggesting that such systems are continuous cultivation used in vitro, do not provide full recovery of hemopoiesis source of bone marrow stroma in vivo.

The increase in stem cells and precursor cells for bone marrow transplantation is a potential use of long-lived cultures of human osteocytes. Currently, the use of autologous and allogeneic bone marrow transplantation as treatment methods for diseases such as leukemia, lymphoma, and other life-threatening systemic diseases. However, to conduct this therapy, you must have a large number of donor bone marrow, which would provide the required number and stem cells and cells progenitors, reduces the need of taking a large number of donor bone marrow and allows you to take a small amount of bone marrow cells, and then increase the number of stem cells and cells progenitors in vitro before introducing them to the recipient. It is also known that a small number of stem cells and progenitor cells circulate in the blood circulatory system. If such cells could be collected electrophoretic methods and to increase their volume, then it would be possible to obtain the required number of stem cells and precursor cells for transplantation of peripheral blood, and there would not be a need to capture donor bone marrow.

When bone marrow transplantation is necessary the introduction of approximately 1x10 - 2x10 mononuclear bone marrow cells per 1 kg of patient's weight for their engraftment. To obtain such a large number of cells need to take a bone marrow donor in an amount of about 70 ml of brain 70 kg of body weight of the donor. And although 70 ml of a small fraction of bone marrow donors, the procedure of its capture requires a considerable investment of time and fill a large blood loss in the process of its implementation. If the production of stem cells and cell-preds is maintained only by the intake of these cells from the peripheral blood with subsequent reproduction of their in vitro.

The increase in the production of progenitor cells is also suitable as a substitution maintenance therapy to chemotherapy and represents another aspect of applying a long-lived cell cultures of human bone marrow. The dilemma faced by oncologists, is that the majority of chemotherapeutic drugs used to destroy cancer cells, is having a devastating effect on all cells passing through the cell division cycle. Bone marrow is one of the most productive tissues in the body and, therefore, often refers to the body, which is primarily subject to destruction when exposed to chemotherapeutic drugs. This leads to a rapid disruption of the blood in the treatment of chemotherapy drugs, and it is therefore necessary to interrupt the course for recovery of hemopoiesis, before proceeding with this treatment. May take a month or even more to resting stem cells were able to raise to the desired level, the number of cells to resume chemotherapy, during which repeatedly occurs a reduction in the number of erythrocytes. Unfortunately, although leukocytes vastasmaja to increase their resistance to chemotherapeutic agents due to their natural ability election.

To reduce the gap between ongoing courses of chemotherapy, a large number of progenitor cells and immature blood cells could normalize the patient's condition. This would have a significant impact on the reduction of the periods of time when the patient is marked low blood corpuscles, which would allow faster to resume chemotherapy. The longer course of chemotherapy and shorter intervals between doses of chemotherapeutic drugs, the greater the likelihood of successful treatment of cancer. Hematopoietic cells are needed to increase production of progenitor cells can be obtained either by taking the donor's bone marrow or collection of cells from the peripheral blood. Harvesters bone cells provide approximately 4x10 granulocyte-macrophage colony forming units (CFU-GM) progenitor cells. During electrophoresis 5 l of peripheral blood is generally collected about 10 CFG-GM, although this number can be increased to 10 by pretreatment of the donor GM-CSF factor. For rapid normalization of the patient's condition requires an infusion of approximately 1x10 - 5x10 CFU-GM, which is approximately 100-1000 times greater than the quantity that obrazom, increases the production of cells from the donor bone marrow or they are collected from the peripheral blood to increase the number of CFU-GM by 2-3 orders of magnitude has a significant impact on the use and treatment of cancer chemotherapy.

Gene therapy refers to the rapidly evolving field of medicine, and therapeutic potential of which is also difficult to assess. Gene therapy methods have many potential applications in the treatment of diseases (see, for example, Boggs, Int. J. All Cloning (1990), 8:80-96, Kohn and others, Cancer Invest. (1989), 7 (2):179-192, Lehn. Bone Marrow Transp. (1990), 5:287-293 and Verma, Scientific Amer., (1990), page 68-34. Genetically transformed human stem cells possess a wide range of potential applications in clinical medicine as a drug for gene therapy.

However, there are several problems that must be overcome for successful gene therapy. The first problem is to provide the possibility of introducing a desired therapeutic gene into the specified cell. Secondly, the specified gene will be properly expressed in the target cell, resulting in the desired concentration of the gene product. And, finally, produced RNA or white the beard, that is, to gene therapy influenced the clinical picture of the disease treatment. Table 1 shows several methods of inserting a gene into human cells in vitro.

Many laboratories are under development other such methods such as homologous recombination. Research in the field of gene therapy was conducted over several years in several types of cells in vitro, and then continued on animals, and finally, recently entered the first stage of clinical trials on humans.

The blood system is an ideal material as a delivery system, the gene product of gene therapy. Hematopoietic cells can be accessed easily by drawing the bone marrow or by collecting mononuclear cells from peripheral blood. Immediately after the implementation of the genetic insertions in vitro treated cells can re-enter intravenous infusion, after which genetically transformed cells return to their place and continue to grow in the bone marrow. Because Mature blood cells circulate throughout the body, genetic modified cells can deliver specific gene product to any fabric-MICS, which have great potential (possibly unlimited) to heal itself. By this we mean that if stable transduction of genetic material in these stem cells, when reinfused hematopoietic tissue these modified stem cells capable of proliferation and repopulation in the brain tissue with cells that ensure the expression of the new gene. This leads to long-term, possibly throughout their life cycle, the delivery of the desired gene product. Similarly, successful stable gene transfer into stem cells that are located in other organs, or embryonic stem cells also leads to continuous long-term delivery of a gene product.

Gene therapy using hematopoietic stem cells has a wide range of applications such as for the treatment of diseases that are specific to the blood system and the systemic diseases of other organs. Within the framework of the hematopoietic system, gene therapy, stem cells can be used to treat both inherited and acquired diseases. So, for example, conditions associated with a deficiency of hemoglobin, in particular alpha - and beta-thalassemia, what sledovatelnot, passed erythrocytes high tissue-specific affinity (see Grosvell and other Cell, (1987), 51:975-986). Similarly, you can adjust sickle cell anemia by genetic insertion of a gene of fetal globin in hematopoietic stem cells, as regulated expression of high concentrations of fetal hemoglobin sufficient to prevent generation of sickle cell erythrocytes despite apresentacao hemoglobin sickle cells (see Sunshine and others, J. Molec. Biol. (1979), 133:435).

Genetic disorder of neuropile, due to lack of functional activity of proteins, such as failure lymphoreticulosis determinants (LAD) or chronic Wegener (CGD), can successfully be treated by genetic insertion of the gene encoding the defective or lost gene, in combination with regulatory DNA sequences that convey a high degree of tissue-specific expression in hematopoietic stem cells (see Wilson and others, Science (1990), 248:1413-1416). Hereditary diseases, including thrombopathy, such as angiohemophilia, can be treated by insertion of the gene coding for example, the factor of Villebranda in combination with sequences, maintains the static stem cells for replacement of the congenital defective genes in the treatment of lymphocyte-mediated immunodeficiency complexes, for example, as distinct immunodeficiency, caused by the lack of adenozindezaminazy. Retroviral gene therapy of circulating T-cells in combination with the gene coding for adenoidectomies determined to be effective to reduce the clinical picture in patients with immune deficiency, but these effects are only temporary, because transfection T-lymphocytes have a limited period of life in vivo (see Kasid and others, Proc. Nat. Acad. Sci. (USA) (1990), 37:473-477, or Culver and others, Proc. Nat. Acad. Sci. (USA) (1991), 38:3155-3159). If this gene could be successfully transferred in such hematopoietic stem cells, then any T-cells are produced from these stem cells would contain and expressed gene adenozindezaminazy. Thus, due to the fact that transfection stem cells have the ability to survive and rapid reproduction throughout the life of the patient, T-cell-mediated ADA deficiency is usually treated by the method of maintenance therapy by gene transfer into stem cells (see Wilson and others, Proc. Natl. Acad. Sci., (USA) (1990), 87:439-443).

In addition to congenital disorders of the enzymatic processes of the blood, a method of gene therapy based on stem to the impact of exogenous factors, such as viruses and chemotherapy. So, for example, found that gene transfer DNA sequence that encodes the binding site antigenspecific T-lymphocytes (TAR) transactionbased factor TAT HIV, protects T cells from spreading HIV infection (see Sullenger and other Cell, (1990), 63:601-608). Stable transduction of these sequences in hematopoietic stem cells leads to aggregation of T-cells produced from these stem cells, which are relatively or absolutely resistency to the spread of HIV infection.

Similarly successful transfection of genes encoding resistance gene combination products (MDR), in hematopoietic stem cells of bone marrow, which are relatively resistant to the effects of anticancer chemotherapy treatment. After conducting autologous bone marrow transplantation on the basis of such genetically transformed cells, patients will be able to endure therapy chemotherapy, from the effects of which defended stem cells due to profound suppression of bone marrow function, which is called these anticancer drugs. As a result, patients will be able to take more effective is that gene-based therapy of hematopoietic stem cells is also suitable for the treatment of acquired diseases of the blood, for example, leukemia, lymphoma and aplastic anemia. Immediately after the establishment of the genetic nature of these diseases, the introduction of a gene product, which either resolves the cause of the disease gene in a cell or gene itself (possibly by splicing or replacing the gene), corrects deviations from the norm.

On a broader level, however, gene-based therapy of hematopoietic stem cells can also be used for treatment of diseases not related to the blood system. Gene transfer of DNA sequences that carry drug-soluble proteins, can lead to the production of Mature blood cells, which continuously secrete the required amount of therapeutic molecules. For example, this method can also be used to treat, for example, diabetes by introducing DNA sequences of insulin in combination with regulatory DNA sequences, providing adequate expression transfectional gene insulin, possibly in response to increased sugar content in the blood plasma. Systemic hypertension can be treated by genetic insertion of stem cells with DNA sequences encoding the secretory pepti analyzes vasculature smooth muscle or adrenergic receptors. Alzheimer's disease, in all probability, can be treated by genetic insertion into stem cells DNA sequences encoding enzymes that destroy amyloid plaques in the Central nervous system.

Thus, there are many applications of gene therapy, in particular the introduction of a gene product in stem cells (see above Boggs and others, Kohn and others , and/or Verma and others). And, of course, there are many cases some progress in ensuring the transfer of therapeutic gene in differentiated human stem cells, as described, for example, in the case of T-lymphocytes (see, Kalsd and others, Proc. Nat. Acad. Sci. (USA) (1990), 37:473-477, and Culver and others, Proc. Nar. Acad. Sci. (USA) (1991), 88:3155-3159).

Unfortunately, before the creation of the present invention to provide stable gene transfer into human stem cells was unsuccessful. Although several groups of researchers have demonstrated the possibility of implementing retrovirus-mediated gene transfer into human hematopoietic cells, was an unsuccessful attempt transfection primitive elements of hematopoietic stem cells. The results of these studies differ sharply from experiments conducted on mice, which showed the possibility ad. Sci. (USA) (1990), 87:439-443).

The main difficulty for successful gene therapy on the basis of hematopoietic stem cells was the inability to provide for the insertion of genes into human gamecity in terms of the division and proliferation of stem cells. Successful sustainable insertions of the gene in the target cell requires that such cell-target was at least one cell division cycle. Thus, if the stem cells do not divide in the presence of the target genetic product, that the product shall be unstable in these stem cells. Before the creation of the present invention lacked any system that supports ex vivo division and proliferation of human stem cells and allowing for the successful genetic transformation of such stem cells.

In the literature, relevant to the present description of the invention, also includes U.S. patent N 4721096, which revealed a 3-dimensional system, comprising stromal cells for growth hemocytes. Cm. also contrasted in this description of the sources Glanville and other, Nature (1981), 292: 267-269 describes gene product mouse metallothionein; Wong and others, Science (1985), 228: 810-815 describes the forehead is Kera hematopoietic stem cells and methods for tracking the development of these cells after their transplantation; Yang and others, Cell (1986), 47: 3-10 describes the obtaining of the human interleukin-3; Chen et al, Mol. Cell. Biol. (1987). 7: 2745-2752 describes the transformation of mammalian cells plasmid DNA; Greaves and others, Cell (1989), 58: 979-986 describes the human CD2 gene; Civin, etc., J. Immunol. (1984), 133:1576 reveals the CD34 antigen; Martin and others, Cell (1990), 63: 20-211 describes human S-CSF; Forrester and others, J. Cell Science (1984), 70: 93-110 describes a flow-through chamber for parallel immunoelectrophoresis; Coulembel etc., J. Clin. Invest., (1986), 75: 961 describes the shortage of CML cells in static cultures.

Based on the foregoing, there is a great need for developing methods and compositions for the ex vivo replication and stable genetic transformation of human stem cells and optimization of cultures of human hematopoietic precursor cells, especially in light of the huge potential to increase the yield of stem cells and precursor cells, as well as the empowerment of gene therapy, which provide the specified system. Unfortunately, to date, attempts to solve these problems did not live up to expectations.

Disclosure of the invention

In accordance with the foregoing objective of the present invention is the creation of novaform human stem cells.

Another objective of the present invention is to provide new methods, including culture conditions and reactors to optimize (I) cultures of human hematopoietic precursor cells and (II) stable genetically transformed human hematopoietic precursor cells.

The present invention is based on the results of the author's research, in which new methods are developed, including culture conditions and reactors for the ex vivo replication and stable genetic transformation of human stem cells and/or to optimize cultures of human hematopoietic precursor cells. In the basis of these methods is the cultivation of human stem cells and/or human hematopoietic precursor cells in a liquid culture medium which is replaced, preferably perfusion, either continuously or periodically at a flow rate of 1 ml medium per 1 ml of culture after about 24 to 48 hours with the subsequent removal of metabolites and compensation of the deficit of nutrients while maintaining a physiologically acceptable condition specified culture. In a particularly preferred variant done by InEU environment hematopoietic growth factors.

In the course of developing the present invention, the authors unexpectedly found that increasing the intensity of the exchange in such an environment, which is used in accordance with the present invention, with the optional addition of hematopoietic growth factors in a rapidly changing culture medium (1) ensure the viability of the cultures in which the growth of human stem cells for an extended period of time - about 5 months, (2) provide support to the cultures in which there is generation of human hematopoietic precursor cells by division and differentiation of human stem cells for an extended period of time, about 5 months, and (3) stimulates increased metabolism of human stromal cells and the release of these GM-CSF factors, including the stromal cells of human bone marrow. The present invention allows for the first time to ensure the survival of human stem cells and their proliferation in culture.

The present invention also provides an ex vivo system, which provides continuous proliferation of human stem cells that can successfully enter gereformeerden human stem cells. This variant implementation of the invention can be used for the transfer of any genetic material, which can be introduced by genetic engineering recombinant retrovirus, and any vector gene transfer, which requires cell division. Genetically transformed human stem cells obtained in accordance with the proposed method can be used to treat a wide range of the aforementioned diseases.

The present invention also provides methods for increasing the efficiency of transfer of genetic material into human hematopoietic precursor cells in combination with methods that ensure a stable genetic transformation of human stem cells and/or human hematopoietic precursor cells.

The present invention also provides bioreactors and compositions that provide effective proliferation of human cells in culture, particularly cells at early stages of their maturation, including stem cells. In these methods use stroma cells transformed by standard methods, which provide permanent or induced hematopoietic cells. Providing continuous perfusion and recycling of the respective cells, can significantly increase the density of the distribution of viable hematopoietic cells and their output. In the bioreactor as a source material used protein carrier for stromal cells. This protein membrane or other substrate to provide the Department of stromal and hematopoietic cells.

A brief description of the drawings.

In Fig. 1 shows a schematic view of the perfusion chamber.

In Fig. 2 shows a schematic view and a flow chart of passing the perfusion solution.

In Fig. 3A shows a schematic view of a flow chamber for measuring the shear stress at the division of cells.

In Fig. 3B shows a side projection of the flow cells according to Fig. 3A.

In Fig. 3C shows a graphical profile of the shear stress hematopoietic cells.

In Fig. 4A and 4B shows a horizontal and lateral projection of the flow cells to grow and branch hematopoietic cells.

In Fig. 5A and 5B shows a perspective view of the flow cells consistently remove the substrate for continuous cultivation of stromal cells.

aloudat when it is used with reference to any standard system of liquid cultures of human stem and/or progenitor cells, for example, as ustanovlivena in hematopoietic cell culture (cultures). When using methods with the rapid replacement of the culture medium used according to the invention in combination with the optional addition of growth factors hematopoietic cells, the authors found that this allows you to create a standard system suspension cultures of human hematopoietic cells, which include culture, operating in the presence or absence of serum or blood plasma of the animal, including equine, bovine, fetal calf or human serum.

Suspension culture of human hematopoietic cells, which are suitable for use according to the present invention can be prepared when the distribution density of cells from 104up to 109cells in 1 ml culture using standard well-known components to obtain nutrient environments, such as environment Dulbecco, modified by the method of Claims (IMDM), modified Dulbecco Wednesday Needle (DMEM), minimum essential medium (MEM), RPMI medium 1640, medium alpha or MSO's environment that can be used in combination with serum albumin, cholesterylester corticosteroids, for example hydrocortisone at a concentration of 10-4- 10-7M, or other corticosteroids in the same dosage, for example cortisone, dexamethasone or solumedrol. The cultivation is usually carried out at a pH approaching the pH of the body, i.e. in the range of from 6.9 to 7.4. The culture medium is usually kept in oxygen-containing atmosphere comprising from 4 to 20 vol.%, preferably from 6 to 8% vol. the oxygen.

When using these standard methods of cultivation used cell mass may be filled to the desired size, for example up to 103times the amount or even more or due to the content of stem cells, either by hematopoietic precursor cells. To provide such enrichment can be used well-known methods in accordance either with the methods of election of negative reaction, or the election of a positive reaction. For example, for the electoral backlash Mature cells are removed using immunoassay methods, for example, the introduction of labels not containing stem cells or precursor cells pools when using the microplate with mouse anti-human monoclonal antibodies with subsequent Ishenim Ig (see for example, Amerson and others, J. Clin. Invest. (1985), 76: 1286-1230).

The basis of the invention lies in a fundamental change of culture conditions suspended human bone marrow cells using any of the above conditions for the maintenance of culture, rapid replenishment of the nutrient medium. In accordance with the standard method of cultivation of the invention it is necessary to replace the nutrient medium and serum every week, either conduct a full replacement weekly or half medium and half of the serum two times a week. In accordance with the proposed invention, the nutrient medium of this culture is substituted preferably in the form of a perfusion solution, either continuously or periodically, at a flow rate of about 1 ml of medium per 1 ml of culture for about 24 to 48 h, and the density of cultured cells is from 2x10 to 1x10 cells per 1 ml of medium. To ensure the distribution density of cells from about 1x10 to 2x10 cells per 1 ml at filling you can use the same amount of nutrient medium. To increase the density of cells more than 10 cells per ml, you must increase the consumption of the replaceable environment in proportion to their nutritional environment in accordance with the invention can be performed by any method, which provides its recovery, for example by taking aliquots of the spent culture medium and replacing it with a fresh aliquot. Specified aliquot can be added by gravity or by a pump or other suitable means. The flow medium can flow in any direction or you can use a variety of threads supplied from different directions depending on configuration and media culture. Preferably, fresh culture medium was done so as to ensure its contact with the entire cell mass. In the most preferred embodiment, the nutrient medium was added to the culture like the process of blood circulation occurring in vivo, i.e. provide perfusion at least through a portion of the cell mass, and more preferable that it passed through the whole biomass.

Another optional, but important aspect of the implementation of the present invention is that cultures with a fast metabolic process added hematopoietic growth factors, including synthetic growth factors. In a particularly preferred embodiment of the present invention, the culture is added cytokines IL-3 and GM-CSF together with nutritious with up to 2 ng/ml/day. Erythropoietin (Epo) can be added to the nutrient medium in an amount of from 0.001 to 10 u/ml/day, preferably from 0.5 to 0.15 u/ml/day. The growth factors of the fat cells (MCF, the ligand c-complement factor of Steele) can be added to the nutrient medium in an amount of from 1 to 100 ng/ml/day, preferably from 10 to 50 ng/ml/day. IL-1 (a - or b-) can also be added to the nutrient medium in the amount of 100 u/ml for 3-5 days. You can also add to the nutrient medium IL-6, GM-CSF, fibroblast growth factors, IL-7, IL-8, IL-9, IL-10, IL-11, PDGF or EGF in an amount of from 1 to 100 ng/ml/day.

During the construction of the present invention found that when IL-3, GM-CSF and Epo use in accordance with the conditions described above, it is possible to provide specific differentiation of the growth of red blood cells. Alternatively, if use of IL-3 and GM-CSF in the presence or absence of IL-6 or G-CSF specified culture produces predominantly granulocytes. There is also the fact that the culture of the present invention during the whole time was lost T - and B-lymphocytes.

The level of metabolic products in the environment is usually retained within the specified range. Glucose is usually kept in the range from primernaya in the range of approximately 1-3 mm. The concentration of ammonium is usually maintained at a level less than 35 mm. These concentrations can be adjusted by holding or periodic measurements or continuous measurements of the content in real-time using traditional methods (see. for example, Caldwell and others, J. Cell Physiol. (1991), 147: 344-353).

As cells that can be grown according to the present invention, it is possible to use any human stem cells, or cell mass containing such stem cells, including human mononuclear cells from peripheral blood, human osteocytes, embryonic liver cells human embryonic stem cells and/or human sickle cells. Each of these cellular compositions contains stem cells and/or human hematopoietic precursor cells. In other cell compositions containing human stem cells can also be used in accordance with the invention any human stem cells human bone marrow.

In a preferred embodiment of the present invention, the specified cell culture can be enriched by additional is accordance with the above methods, and this enriched culture, if used in accordance with the present invention is primarily suitable as a tool for gene therapy by transfer of genetic material into human stem cells, including stem cells contained in the bone marrow of humans and human stem cells in the bone marrow. As stem cells from human bone marrow can use cells which can be isolated from the tissues of human bone marrow, peripheral blood, embryonic hepatocytes, heart or blood cells.

Generally speaking, this aspect of the implementation of the present invention to provide a stable genetic transformation of human stem cells these cells, cultivated in accordance with the proposed methods, add the cell line with a defect in the packaging that is infected with a retrovirus, or a supernatant obtained by culturing such a cell line with a defect in the packaging, or any other vector gene transfer material. The present invention improves the maintenance of stem cells and the replication of human hematopoietic precursor cells, but protivopul the Cove while reducing its intensity (i.e. the "dying culture"). The proposed method of culturing cells makes it possible to provide the first increase in cell mass in the culture, requiring the infection of cells by the retrovirus. Known up to the present time of the system, where they spent the infection by the retrovirus on evanescent cultures, did not provide the infected cells at early stages of their growth. The present invention, especially if it is implemented in combination with the enrichment of the pool of stem cells, and even more when added hematopoietic growth factors, including synthesized growth factors, it is possible to obtain a very effective means for the infection of stem cells in vitro.

During the construction of the present invention found that the addition of supernatants containing recombinant retroviruses, to these cultures lead to the introduction of viruses and genes that they carry, in human (gemopoeticheskoi) stem cells. Precursor cells formed by the division and differentiation of these stem cells and Mature blood cells, resulting from further division and differentiation of these precursor cells contain transfection DNA sequences throughout qualem stage of cultivation, you get transfection precursor cells and Mature the formed elements of blood that can go from stem cells, proliferation and stable genetic transformation, which was achieved only at the beginning of the biosynthesis, since no infected by retroviral cell cannot infect the next cell. Thus, precursor cells and more Mature cells bearing the target genetic material, took a gene from a more primitive stem cells that have been genetically transformed at the initial stage of retroviral infection.

In a preferred realisation of the invention in human hematopoietic cells or isolated from bone marrow, peripheral blood, fetal liver cells or blood taken from the umbilical cord, first enriched relative to the increase in the content of stem cells by removing more Mature hemocytes. This enrichment is carried out by incubation of hematopoietic cells with mouse monoclonal antibodies that recognize epitopes on Mature namorita and precursor cells of the bone marrow, but not stem cells. Then remove the labeled cells E negative reaction to the differentiation (Lin-) and then incubated in the presence of a retroviral or other vector gene transfer in accordance with the invention. In a preferred embodiment, the cultivation is performed in the presence of the factor GM-CSF, preferably in quantities of 1 mg/ml/day) and IL-3 (preferably in quantities of 1 mg/ml/day) in the presence or without IL-1 (preferably at a dose of 50 IU/ml over a 4-day period), and in the presence of or without a c-complement-binding ligand (growth factor fat cells) (preferably at a dose of 10 IU/ml/day).

Retroviral infection can be done either by introduction into the specified culture medium of supernatants (for example, from 5 to 20%, vol/vol), derived from a cell line with a defect packaging, infected with recombinant retrovirus for 2-21, preferably 10-14 days from the beginning of the cultivation or growing of Lin-cells directly on the previously specified cell line, or both.

Preferably using retroviral supernatants and that the incubation period in the presence of the virus ranged from 12 to 16 days. Incubation of cell lines with defective packaging preferably carried out under conditions close to the state of confluences, with the replacement of the culture medium, after which the incubation is conducted for 12-15 hours After this cultural gaining you can use any well-known retroviral cell lines with defective packaging, and their cultivation conduct any known methodology (see, for example, Wilson and others , Science (1990), 248: 1413-1416 and/or Sullenger, etc., Cell (1990), 63: 601-608). As examples of such cell lines with defective packaging, you can specify cell line NIH 3T3 and cell line 5337 carcinoma of the kidney.

Any gene that is being introduced in the recombinant retrovirus together with appropriate promoters and enhancers, which ensures its expression, you can type in human stem cells and hematopoietic precursor cells. The present invention first provides conditions that ensure the viability of stem cells and their proliferation

in these cultures, which makes it possible to obtain stable transfection and genetically modified stem cells in these cultures. The terms "stable transfection" and "stably transformed" in this context is used to emphasize that the introduction of exogenous DNA into the chromosomes of human stem cells has been made possible by the present invention because it allows the impact of this DNA fissile human stem cells ex vivo.

In accordance poetic precursor cells by division and differentiation of stem cells throughout the cultivated period of time, and at least for 5 months.

The data obtained indicate that the rate of perfusion medium very significantly varies when determining the behavior of extracellular cultures of human bone marrow. These data showed that when he increased the intensity upgrade of the specified environment from the traditional once a week dexterous type to the replacement frequency environment daily about 7 volumes per week, you receive a significant positive effect of the process of hematopoiesis ex vivo. In experiments conducted in the course of developing the invention, all cultures showed a significant loss of cells during the first 3-4 weeks. After this decline, the culture was stabilized, and the effect of the intensity of perfusion in the environment has become more pronounced. The refresh rate of the nutrient medium 3.5 amount per week resulted in the greatest proliferation of cell cultures, and in addition to the very long continuous cultivation period from the point of view of production of progenitor cells. It should be noted that during the time interval from 4 to 10 weeks the number of non-adhesive cells, produced twice a week, was in fact stable or tended to povyshenie increased almost three times compared to traditional method of cultivation dexterous type. Moreover, stable output precursor cells stored up to 18 weeks.

Human stromal cells, such as that identified in the tissue of human bone marrow may or may not necessarily be included in the culture of the present invention. In standard cultures of these cells are contained in an amount of about 104up to 109(stromal cells of the total cells).

In another aspect of the present invention offer cell culture, as is unexpectedly set to lead to the increase of the intensity of metabolic process and secretion from stromal cells from human bone marrow GM-CSF and IL-6. While not detected the presence of GM-CSF factor in the supernatant stromal cells from human bone marrow, a quick update of the nutrient medium in accordance with the invention leads to stimulation of stromal cells of the human brain to release from 300 ng/ml/day to 200 ng/ml/day factor GM-CSF. The secretion of IL-6 stromal cells from human bone marrow also increases with the rapid update of the nutrient medium in accordance with the invention with 1-2 ng/ml/day to 2-4 ng/ml/day. Such uvelicheniya update a nutrient medium with simultaneous introduction into it of hematopoietic growth factors. On the basis of obtained by the authors of the present invention data the impact of rapid replacement of the culture medium on the production of cytokines from human stromal cells can be observed when using these stromal cells in any complex system tissue cultures.

In the example used in the present invention the culture medium may contain three main components. The first component includes the environment Dulbecco, modified by the method of Claims (IMDM), modified Dulbecco Wednesday Needle (DMEM), minimum essential medium (MEM), RPMI medium 1640, medium alpha or McCoy''s environment, or an equivalent component for the preparation of known cultural environments. The second component is whey component, which includes at least a horse or human serum, and optionally it may also contain fetal calf serum, newborn calf serum and/or bovine serum. As the third component can be used corticosteroids, such as hydrocortisone, cortisone, dexamethasone or solumedrol, or combinations thereof, but preferably using hydrocortisone.

Composition for the preparation of RA whom t may be present in the culture in the amount of at least 1 to 50% (vol/vol). The concentration of serum in the culture can make in a preferred embodiment, from about 15% to 30% (vol/vol). When the concentration of serum component in proportion to increases the rate of metabolism. The third component of culture can be present in an amount of from 10 to 10 M, and preferably from 5x10 up to 4x10 - M This component of culture medium used to balance, resulting in the sum of all three components is 100%. Alternatively, serum component can be replaced by any of several standard serum replacement mixtures, which typically includes insulin, albumin and lecithin or cholesterol (see, for example, Migliaceio etc. , Exp. Hematol. (1990), 18: 1043-1045, Iscove, etc., Exp. Cell Res. (1980), 126: 121-126 and Dainiak, etc., J. Clin. Invest. (1985) 76: 1237-1242).

To illustrate the concentration of human hematopoietic stem cells can be increased in the following way. Erythrocytes separated from the bone marrow punctate method of centrifugation in density gradient ficol-vipaka, after which mononuclear cells are incubated together with the "cocktail" of antibodies, which recognize Mature hemocyte, including erythrocytes, granulocytes, macrophages and Mature lymphocytes (kovanye precursor cells (including anti-CD33). Then the Mature cells are removed by any of many known methods, including penninga, immunovative on magnetic beads or cell sorting on a laser sorter cells according to the intensity of fluorescence (FACS). When the removal of Mature blood elements greatly increases the possibility of infection with the recombinant virus, and therefore, the transfer of the desired gene (genes).

In another embodiment, the present invention provides methods of cultivation of hematopoietic cells in culture using fibroblastoid, usually modified, which provide the possibility of introducing growth factors together with protein components in the compounds of fibroblasts and hematopoietic cells and almost continuous perfusion with carrying out the recycling if desired, and thereby provide a stable and effective state growth environment.

Description of proposed method of the present invention can be divided into multiple parts, which relate to the conditions of the perfusion, the bioreactor and its design transformation of fibroblasts.

The inventive bioreactor includes a reaction vessel, which may be of any Konsta, the removal of waste products of metabolism, optional recycling of hematopoietic cells, replacement of stromal cells and the collection of hematopoietic cells. Such a reactor must provide, almost close to the bone perfusion. In vivo spent approximately 0.08 ml serum 1 ml bone marrow in a minute, or if you say otherwise, 0.3 ml serum 10 cells daily. Such an environment, therefore, must be replaced on average at least 50%, preferably 100% every 24 h for stabilization of the normal products of metabolism, which should not be limited in growth. Consumption when replacing typically varies from 0.5 to 1.0 perfusion solution on 10 cells every day, which is similar to empirical estimates of perfusion in vivo.

The intensity of perfusion in a bioreactor varies depending on the density of cells in the reactor. For cells grown at their expense 2-1010 cells/ml, the average is 1 ml/ml volume reactor (24-48 h, if the medium used contains 20% serum or 10% fetal calf serum and 10% horse serum or 20% fetal calf serum). With the increase of density of cells grows in proportion and rate of perfusion that t with their density 510 cells/ml of medium, the perfusion rate is 0.1 ml/ml of reactor volume per minute. These consumption figures are important for the stimulation of endogenous production of hematopoietic growth factors of healthy stromal cells of human bone marrow culture. As a hematopoietic growth factors induced at these rates of consumption, serum and environment, you can use GM-CSF, as well as S-CSF, g-38 IL-6, G-CSF and other hematopoietic growth factors. Such rules are usually set in the bioreactors so that the shear stress of longitudinally incoming flow, which have stem cells and precursor cells in their sites of attachment to stromal cell was in the range of 1.0-5.0 Dyne/cm2.

Many different mediums can be used for the cultivation of hematopoietic and stromal cells. As an example, you can specify MEM, IMDM and RPMI, which can be supplemented with combinations of 5-20% fetal calf serum, 5-20% calf serum and 0-15% HP, and/or not containing serum medium, supplemented with PDGF, EGF, HGF, PGF and other growth factors to stimulate stromal and stem cells. In addition to the growth factors that provide transformed fibroblasts, can at will Preboot site-specific differentiation. In the amount of growth factors that can be included in such perfusion environment or when they are secreting stem cells, either by adding to it, includes GM-CSF, G-CSF or M-CSF, interleukins 1 to 7, especially 1, 3, 6, and 7, TGF-a or b, erythropoietin and the like, preferably human growth factors. Of particular interest is the combination containing about 0.5 to 2, preferably 1 ng/ml GM-CSF and 0.5-2, preferably 1 ng/ml, and 0.1-2 u/ml/day final cultivation eritropoyetina, from approximately 100-300 u/ml/day G-CSF and about 1-10 u/ml/day (factor known as factor in the growth of the fat cells or kit-ligand). It is clear that one or more, preferably at least two growth factor, will be supported by the secretion of transformed cells, which are contained in a quantity sufficient to maintain the required level of these growth factors in this perfusion medium.

For convenience, the bioreactor support physiological temperature, 37oC, although it is also possible to use lower temperatures, including the 33oC, but usually not below the 25oC. the Humidity is usually 100%, and in air containing about 5% carbon dioxide. Perfusion environment can us the for example, oxygenation inside the bioreactor can provide hollow fibers, porous sintered disks, through the silicone tubing or other membranes with appropriate permeability and hydrophobicity. The level of nutrients and metabolismof is normally maintained in a relatively wide range. The glucose content typically varies in the range of from about 5 to 20 mm, usually from 10 to 20 mm, the concentration of lactate is usually maintained at a level below 35 mm, but its content can reach about 20%. The concentration of glutamine is maintained typically in the range of 1 to 3 mm, usually from 1.5 to 2.5 mm, while ammonium is below approximately 2.5 mm, preferably below 2.0 mm. The flow medium can flow by gravity, a pump or other means, and it can move in any direction or in the form of many different streams coming from different directions depending on the packing of the reactor. Preferably the use of a laminar flow, where the stream passes horizontally through the reactor or vertically up from the bottom of the reactor, or Vice versa.

When there is a suspicion that human hematopoietic cells contain neoplastic cells, Lacrimosa or carcinoma, ski hematopoietic cells. Healthy hematopoietic precursor cells, as installed, are attached to the stroma and matrix proteins with affinity, capable of withstanding the voltage on the offset created by the horizontal flow of about 1.5 to 2.0 Dyne/cm2In contrast, the neoplastic cells and their predecessors have significantly lower affinity for stroma in the range of approximately 0.05 to 1.2 Dyne/cm2. Hold the feed rate of the perfusion solution, which provides a measure of the shear stress between the portable normal and neoplastic cells predecessors, usually more than 1 Dyne/cm2you can ensure separation of neoplastic progenitor cells from healthy progenitor cells by perfusion around for at least two days, preferably for at least four days and more preferably for a period of seven days or more. This way you can increase the amount of healthy hematopoietic cells in a patient simultaneously in the use of appropriate feed rate of flow to separate the neoplastic cells.

To illustrate the use of the shear stress for the Department of hematopoietic tumor cells from healthy g is the shift to CML cells varies in the range from 0.05-1.2 Dyne/cm2. This difference allows you to effectively remove CML cells together with the individual breakdown of the bone marrow. When using the rate of shear of the order of 1.2-1.5, preferably 1.3 Dyne/cm2you can successfully separate the CML cell.

Resistance to shear stress in bone marrow cells inducible patient can be determined using a radial cone-shaped flow chamber. This camera is the shear stress that can be experienced cells, decreases with the distance d from the beginning of the camera as a function of 1/d. The strips can then be analyzed on the size of the cell population, and a measure of the shear stress determines at what level should be defined population of cells.

For the Department of leukemia stem cells, precursor cells and stem cells from the bone marrow punctate patients with leukemia are placed in a radial flow chamber. This camera consists of two parallel plates made of polycarbonate resin or glass.

Based on the results of these measurements establish a cascade of parallel rectangular cells, where the flow rate of the perfusion solution (see Fig. 4A and 4B) on the lower level creates a shear stress, which sets forth the notches. In the case of bone marrow of a patient with chronic myeloid leukemia is the shear stress is usually 1.01-0.05 Dyne/cm2. The actual flow rate of the perfusion solution depends on the size and geometry of these cells. The bone marrow cells of this patient were grown in these rectangular cells at a concentration of 5x10 - 50x10/ml in the medium, Dulbecco modified by the method of Claims with 5-20% (usually 10%) of horse serum with or without hydrocortisone 10 M Above the bone marrow cells were incubated for 12-24 hours without feeding the perfusion solution, and then began to enter the perfusion solution. Cells were cultured for 3-7 days, removing all the non-adhesive cells. Adhesive cells extracted from these rectangular tablets aspiration or mechanical shaking, after which they were collected. These cells then can be directly injected to the patient or to store them in liquid nitrogen by standard methods before use.

In addition to the cells of the blood can also be divided cells of other organs using the difference in tolerance to lateral pressure. Thus, where there are distinct subpopulations of cells within a given range of cells, videopal, for example, skin tissue, liver, muscle, nervous tissue or epithelium. Of particular interest is the division of cells within a population of healthy cells. Partial population of cells is brought into contact with stromal substrate, as will be described later in the description, such as purified protein or cellular component is bound to the investigated cells. The stability of the shear stress of each of the adhesive subpopulations determined by the above method. The feed flow of perfusion solution can then be adjusted to maintain the desired subpopulation of cells on stroma. Target cells are then harvested by the above method.

Different packing can be used in the proposed reactor to ensure adhesion cell growth, while providing a physical separation between stromal and hematopoietic cells, as well as for the possibility of some or interaction between stromal and hematopoietic cells. In this case, factors released from stromal cells, can be easily absorbed hematopoietic cells to facilitate their proliferation and proper differentiation and maturation.

Protein side, and porous collagen beads, and these sponges or microspheres consist of an extracellular protein matrix of the bone marrow or protein-coated membranes, where the protein may be collagen, fibronectin, gameaction, peptide, protein blend with bone marrow matrix, and the like. The pore size of the membrane is typically vary from 1 to 5 MS in order to ensure the interaction between different cell types, although there is still physical separation.

You can use the protein-coated membranes. To obtain membranes can be used a variety of materials such as polypropylene, polyethylene, polycarbonate and polysulfone resin. The membrane must be sufficiently small pores, through which should not be modified cells, but they can grow and form confluently layer on the side surface of such membrane. Typically, the pores of these membranes vary in the range of from about 1 to 5 milliseconds. In this case, hematopoietic stem cells can grow on the opposite side of the membrane and interact with the modified cells, resulting in growth factors can be transferred directly from the transformed cells of the Yu be attached to the side of the cytoplasmic projections, which are specified in the pores. Hematopoietic differentiation of stem cells occurs on one side of the membrane, and differentiated precursor cells are unable to pass back through the membrane, which is already mostly covered with a layer of stromal cells, when almost or already achieved the status of continuity. For example, you can install multiple cells, which can grow stromal cells and hematopoietic cells can be moved in accordance with the cell that have the stromal cells on subconfluent level. Thus, if the movable membrane between cells, when stromal cells are approaching confluentes state, usually in approximately 8-12 weeks, you can open or remove this barrier between cells and provide the ability to move stromal cell to a new cell and hematopoietic cells to come into interaction with subconfluent stromal cells, although these subconfluent stromal cells move the growth factors to the cell containing hematopoietic cells (Fig. 5A and 5B). The migration of hematopoietic cells can be achieved by controlling the rate of flow of perfusion solution or other well-known the existing partitions, and after contamination of one hole, when cells reach confluentes state, then they will move to the next hole, and then after sowing likewise will move to the next hole. Another modification of the system is that after 8-12 weeks in culture of hematopoietic cells affect new proliferating stromal cells. This is achieved in several ways. According to the first method of cell culture process within 3-5 min EDUC (EDTA), which removes from stromal cells of hematopoietic stem cells. Isolated cells are then transferred into a vessel with fresh medium, which may itself contain stromal cells in the bone marrow, planted for 3-7 days before. This process is repeated every 8-12 weeks. And, finally, small organic molecules or proteins, especially hormones, such as growth factors derived from platelets (at a concentration of 100-500 ng/ml), interleukin-1-alpha, alpha-tumor necrosis factor or growth factor basic fibroblast or other molecules, mitogenic relatively fibroblasts, can be added to the indicated cultures within 3-7 days. Such effects on stromal mitogen-stimulating factorya growth factors. Thus, it is possible to provide a continuous phase subconfluent development of stromal cells.

A continuous flow of perfusion solution can also be used to separate healthy from cancerous cells within a population of bone marrow cells. In the said method initially used radial flow cell to determine the specific adhesion of healthy cells to cancerous, then use the rectangular flow cell at the required speeds of the feed streams, which allow the shear stress sufficient to separate cancer cells.

The claimed method and apparatus also provide the opportunity for recycling stem cells, lost in the flow of perfusion solution. The surface marker CD34 membrane provides separation of immature hematopoietic cells from Mature. Thus, the capture and recycling of cells which are CD34+, you can avoid losses in the environment of stem cells.

To capture and return the immature fraction of cells in the reactor can be used a variety of methods. For example, you can type a label antibodies specific for CD34 in these cells, and then use antibodies against antibodies for sbcla, in the result, you can ensure the removal of Mature cells, using antibodies against various markers associated with Mature cells, such as antibodies to glycophorin, CD33, MO1, OKT3, CKT4, OKT8, OKT11, OKT16, OKM1, OKM5, Leu 7, Leu 9, Leu M1, Leu M3, and the like. There are many different antibodies for markers specific for Mature cells of various hematopoietic differentsirovat, lymphoid, myeloid and erythroid, and these antibodies can be used for removal of Mature cells from affluently flows from the reactor and subsequent collection of the remaining cells and returning them to the reactor.

Separation when using or antibody-based test markers can provide different ways, using standard methods individually or in combination, for example as penning, sorting cells with amplification by fluorescence, antibody binding to different media, for example with the surface of polystyrene microspheres, metallotropic and magnetic media, and so on, Antibodies bind to surfaces, which allow the separation of the adhesive from the non-adhesive cells or antibodies enter a label directly or indirectly, resulting in a choice between labeled and unlabeled cells.

In addition, adhering to the proposed in the present invention methods can significantly increase the intensity of the division of stem cells that enables efficient introduction of retroviral-transfectional genetic material. Genes introduced appropriate retroviral vector within the first two weeks after the infection, you can Express up to 10-30% in all cells-the precursors and cells-the precursors produced during further cultivation for four months in culture. The proposed methods, thus, ensure the successful delivery of the gene product in vysokopoligonalnye human hematopoietic stem cells.

In Fig. 1 shows schematic the military at the base of the reactor, connecting bolts 16 with fixing their position wing nut 18. Use three screws to prevent twisting. The camera 20 has three compartments, the middle compartment 22 contains supporting matrix for stromal cells landing stromal cells and bone marrow cells. The Central part of the compartment 22 is separated from the upper section 24 and lower section 23 of the membranes or screens 28 and 30, respectively. For convenience, you can use polysulphonate membrane or a stainless steel sieve, the size of the sieve mesh is sufficient, so that the cells could get into the Central part of the chamber. Dividing the interfacial partition can be set in the camera when using the internal cylinder 27, which is divided into sections to provide mechanical support for a separation membrane. The upper section 24 and lower section 26 may have different profiles and contain a cross section of tubing or membrane, where the replacement of the nutrient medium and gases. Gases pass through the hydrophobic, for example, silicone tube, the size of which (and therefore the contact area of the gas-liquid) may vary to provide an adequate number of incoming flows to meet the needs of cellular populace to flow by gravity into the top and bottom of the camera through the hole 24, or to act upon the pipe 34.

If necessary, the upper and lower sections can be deleted by replacing them on an external oxygenator. In this case, the separation membrane is fixed by means of a glass cylinder 36, which is mounted in a cylindrical grooves of the plates 12 and 14, and the outer cylindrical surface of the groove is the same as its interior that provides excellent distribution of the incoming flow across the membrane. This geometry allows to provide mixed stream from the destination input holes for the stabilization of the radial pressure. This arrangement is suitable for cameras that have relatively few cells, resulting in the oxidation process does not become limiting.

In Fig. 2 shows a schematic view of the circuit that connects the perfusion chamber with a reservoir with a nutrient medium, an oxygenator, measuring (touch) cell and holes for submitting samples and download environment.

External source of fresh medium 50 is pumped by pump 52 to the tank through the pipe 56, and the old medium is removed by pipe 58 from the reservoir 54 through the pump 52 to the tank waste product dloce the oxygenator 66. Wednesday is sent by pipeline 68 into the first chamber of the bioreactor 70. For convenience, the tool is installed to discharge the framework component 82, which pipe 68 transports the medium in the first chamber of the bioreactor 70. As this component can be analyzed materials, additional growth factors and the like. The nutrient medium from the bioreactor 70 is directed through the Central part of the chamber 72 into the second chamber 74 of the bioreactor. Hence, the medium flows through the pipe 76 in sensors operating in real-time to determine changes in the composition environment.

For example, it is desirable that the ratio of glutamine to glucose was in the range of 1:5-8 depending on the cell lines, for example, preferably a ratio of 1:8 for transfection cell line 3T3. Moreover, the concentration of ammonium is preferably less than 2.0 mm, and the concentration of lactate is preferably less than about 40 mm. Regulation of flows delivered from the bioreactor, it is possible to provide the modification introduced into the bioreactor environment, change the value of oxygen partial pressure and change the feed rate of oxygen, the addition of various components or reduction or uvelichival 54.

When using the above circuit feed streams environment in the side tank slowly exchanged. This arrangement allows independent control of frequency of replacement of the environment (external pump) and the rate of passage of flow through the oxygenator and the perfusion chamber. The first is used for long-term control variables in the composition of the medium and conditions of perfusion, while the latter can be used to control the pressure of dissolved oxygen and the flow regimes of streams in the camera. Use a small biocompatible mesh membrane can provide the movement of the piston in the chamber, and this, in turn, to exercise strict control over the supply of growth factors and other special compounds, which are preferably added to the hematopoietic and stromal cells in fixed quantities.

After treatment in the autoclave chamber elements of the feeding path specified bioreactor is harvested under sterile conditions. The medium can circulate through the side path and the camera in a few days, at the same time adjusting the degree of contamination. If provided sterile Assembly, carry out the inoculation of the Central section of the camera or when Espanola matrix, which contains stromal cells. After that stromal cells either (1) maintain in the chamber for several days, at the same time adjusting the parameters of their metabolism and/or sensitivity of the growth factor, and if the results are satisfactory, carry out the inoculation of bone marrow, or (2) immediately inoculant bone marrow. In any case, the suspended cells incubated in the lower part of the Central compartment of the camera. The cells are then put on additional extracellular matrix and immobilized specified layer of cells in the partition wall. At this time, the camera may be removed, and the layer of cells will move to the top of the specified center section. With this configuration, maturing cells settle to the bottom of the Central chamber, because they have lost their adhesive ability relative to stromal cells. This feature is important to prevent damage to the stromal layer and/or less Mature hematopoietic cells when exposed to Mature cells. This feature also provides continuous over easy separation of Mature cells.

Stromal cells are mainly fibroblasts modified by one or more gene of the host, moreover, the same or other cells can be used for many different genes.

You can use a wide variety of healthy cells or stable cell lines. However, not all cell strains, as installed, can be used as the transformation of some cell lines can lead to excessive cell growth. It is desirable that cells used were not neoplasticity cells, and likely it will immobilization on the substrate. For mammalian cells is not necessary that they belonged to a human or were premirovany. Many of the untransformed cells can be used in the adhesive layers of cells, including healthy adhesive cells of the human spleen and intact epithelial cells of the human thymus.

Well-known methods of transformation of mammalian cells, and there is a lot of literature on this issue, although in this description are only a few sources. These designs can be used in natural-occurring regulatory region of transcription initiation, containing the promoter and the corresponding enhancer or you can include another region of transcription initiation, which may be the General, and these include chromosomal promoters such as the promoters of mouse or human metallothionein-I and II, the actin promoter, etc. or viral promoters such as the promoter of the early gene SU 40, the promoter SMU, adenoviral promoters, the promoters associated with LTRS of retroviruses and so on, These promoters are readily available, they can easily be inserted into appropriate vectors that contain

printery for insertion site of transcription initiation, as well as the test gene. In other examples using expression vectors that contain polylinker between the region of transcription initiation and termination region transcription for the transmission of various signals associated with the processing of messenger broadcast, that is, the signal site kupirovaniya and polyadenylation. When designing polygenic expressing the cluster containing the regulatory region and the structural gene, you can use one or more restriction enzymes, adapters, polylinkers, in vitro mutagenesis, primerno reparation, reselection etc.

Polygenic expressing the cluster is usually part of a vector containing the marker and one or more systems for replication. Marker obespecheno to use various markers especially markers that confer resistance to the toxin, especially the antibiotic. It is preferable to use resistance to gentamicin, which provides resistance to G418 of miclette the host mammal. As replication system, you can use system replication prokaryotes, which allow cloning in different stages, assembling the individual components (polygenic expressing) cluster.

With the introduction of polygenic expressing cluster in the cell-host, you can use any of the well-known techniques, including transformation, precipitiously calcium DNA, transfection, infection, electroporation, ballistic particles, and the like. Immediately after modifying host cells can hold their amplification in an appropriate nutrient medium containing a selective agent to select for those cells that contain the marker. The surviving cells can then amplify and use them in the future.

As host cells, you can use the cell line of African green monkey CV-1, murine cells, NIH-3T3, healthy human fibroblasts bone marrow, human fibroblasts spleen, healthy thinking is s depending on the chosen vector and cell line cells become neoplastic condition. Important is the fact that the resulting transformed cells are capable of adhesion, whereby they provide immobilization on a substrate, such as a protein sponge, protein-coated membranes, and the like.

Immediately after construction of vector for expression of the desired growth factor can be used to transform cells by any known method. The obtained transformed cells can be used for inoculation of the substrate, as mentioned previously. These substrates can be introduced into the reactor, or they may be present in the reactor during inoculation.

The reaction mass is then possible to inoculate properly together with hematopoietic cells. Hematopoietic cells can contain almost pure stem cells, a mixture of hematopoietic cells, containing no Mature hematopoietic cells of one or more differentsirovat, or mixture, including any one (or almost all) the differentiation of the haematopoietic system at different stages of their maturation.

Cells pokasivaut almost continuous passage of the perfusion solution through the reactor and the regulation of the levels of PI is yaytsa stromal cells, the result is usually restored normal levels of growth factors. Because air supernatant determined to have efficiency in the cultivation of hematopoietic growth factors, it is possible to provide value to stromal hematopoietic cells, which supports the growth factor at the proper level of concentration in the specified reactor.

Transfectional stroma allows the introduction of genes into human stem cells. Mice with retrovirus-mediated gene transfer into stem cells can be performed pre-treatment of mice with 5-FV with subsequent cultivation of Mature bone marrow cells in air-conditioned environment WEHI containing IL-3 and GM-CSF (Lemischka) Cell (1986), 45: 917). Synthesized stroma, cultured cell line with a defect packaging, secreting studied retroviral vector can be used to ensure the efficient introduction of genes in human stem cells. So, for example, human T-cells can be made resistant to HIV infection by infection stem cell retroviral vector containing antisense HIV-sequence under the control of regulatory posledovich must have factor providing cell line retroviral packaging: this factor is not present in hematopoietic target cells. Once this virus is transferred to the hematopoietic target cells, they immediately lose their ability to replicate.

In Fig. 3A and shows a radial flow chamber 100 having an inlet 102 and outlet 104, a camera 106, where the arrows 108 shows the direction of flow. Hematopoietic cells 110 are seeded in the stromal layer 112 in the camera and cultivated. The flow rate will be determined by the cell, which is capable of adhesion, and the non-adhesive cells 114 is discharged through the outlet 104.

In Fig. 4A and 4B Luggage for cultivation 120 is equipped with inlet 122 and the outlet 124. In Fig. 4B, the inlet port 122 contains a valve system 128, which provides separate chambers 126 containing cells 110 and stroma 112 and intended for cultivation and separation.

Figure 5A and 5B shows the camera for cultivation, in which the partitions 134, 136, 138 are removed in the process of cultivation according to the following scheme: barriers 134 remove approximately 8-10 weeks, barrier 135 remove approximately 18-20 weeks, and the barrier 138 remove priblizitelino above, provide concrete examples that illustrate, but are not considered limiting of the invention unless otherwise stated.

Illustrative division of cells and the staining procedure

Division of bone marrow cells on Ficoll

1. Throw a bone marrow sample in the ratio of 1:4 in I-MDM environment, maintained at room temperature (environment Dulbecco, modified according to the method Claims, GIBCO, cat. N 430-2200).

2. Carefully layer of 5 ml of the diluted sample is transferred into a 15 ml Ficoll-Paque at room temperature (specific gravity of 1.077 g/cm3, Pharmacia; cat. N 17-0840-02) in 50-ml centrifuge tube.

3. Centrifuged at 700g (1800 rpm on Beckmah) for 30 min at room temperature (20oC).

4. After centrifugation remove most of the top layer (removing approximately 5 ml over the interphase), collect interfacial layer (bone marrow cells) and washed three times in ice environment I-MDM as follows:

first wash: 1400 rpm, 15 min, 4oC;

second wash: 1200 rpm, 10 min, 4oC;

the third rinsing: 1200 rpm, 10 min, 4oC.

5. After the third wash, the cells are suspended either in the environment or in a balanced of soletek in acetic acid (10 μl of cell suspension = 90 μl of 2% acetic acid in PBS, that allows to calculate only leukocytes, since erythrocytes are subjected to lysis in acetic acid).

6. The cells are then suspended to the desired final concentration in an appropriate environment (for various applications).

Fluorescent staining MU-10 positive cells in the bone marrow

Reagents

Standard buffer:

the powder Bacto-buffer DIFCO (Baxter, cat. N 2314-15GB) - 200 grams

10% NaN3(sodium azide) - 20 g,

thermoinactivation amniotic calf serum (56oC, 30 min) to 200 ml.

The volume was adjusted to 20 l in dd - H2O, pH 7,15-7,25, stored at 4oC. Suitable for use within 1 month

2% Paraformaldehyde solution:

paraformaldehyde - 10 grams

dd - H2O - 500 ml,

10 N. NaOH (under exhaust box) - 8-20 drops

powder dry Bacto-buffer DIFCO - 5,

Pour dd - H2O in a 500-ml flask and stirred in a hot plate, heated to 60oC in a fume hood.

Add 10 g of paraformaldehyde.

Drop by drop add NaOH until then, until the solution becomes transparent.

Add 5 g DIFCO.

The solution is cooled and the pH adjusted to 7.35-7.45 from using 2 N. HCl.

1. After the treatment tip can spenderat in standard buffer at a concentration of 2105cells/ml.

3. Two 50-µl aliquots of cells is placed in 215-ml centrifuge tubes.

4. In one test tube add 50 ál of the 1:5 dilution of anti-HPCA-1 (antigen against human precursor cells, Becton Dickinson, cat. N 7660, diluted 1:5 in standard buffer). In another test tube and add 50 ál of the 1: 5 dilution MIg (mouse IgG1 control, Becton Dickinson, cat. N 9040, diluted 1:5 in standard buffer).

5. Then both tubes incubated on ice for an hour.

6. After incubation, the cells washed twice in 5-ml standard buffer (1000 rpm, 5 min, 4oC).

7. After a second washing step of cellular debris resuspended in 50 μl of 1: 40 dilution of GAM-FITC (affine selected goat F(ab'), 2 antimurine IgG and IgM adsorbed human Ig, fluorescin-anywhereman, TAGO, cat. N 4353, diluted 1:40 in the standard buffer).

8. Cells incubated for 1/2 and 1 h in the dark on ice.

9. After incubation, the cells washed twice in 5 ml of a standard buffer (100 rpm, 5 min, 4oC), and each debris resuspending 100 ál standard buffer + 100 ál of 2% paraformaldehyde solution.

10. The cells are then examined for fluorescence using flow cytometry MIg.

Fluorescent staining of bone marrow cells for the selection of Mature precursor cells

Goals

The purpose of this painting is the enrichment of hematopoietic stem cells (the most primitive stem cells) by removing Mature cell populations using flow cytometer or magnetic microspheres. Almost you should always keep some cells in the total bone marrow cells (after separation on Ficoll) dye for MY-10-positive cells in order to compare the efficiency of sorting and determining the degree of enrichment.

1. The cells divide on Ficoll-Paque as described above. (Emit 0,5106cells and divided into two parts for painting anti-HPCA-1/GAM-FITG and MIg/CAM-FITC).

2. After the third washing, the cell debris is suspended in a cocktail containing monoclonal antibody (see description of the making of this cocktail), using 1 ml of a cocktail of 107cells, and the cells incubated on ice for 1 h

3. The cells are then washed three times in excess of ice I-MDM-medium (1000 rpm, 5 min, 4oC).

4. After the third wash, the cells are suspended in a 1:40 dilution of GAM-FITC (diluted in I-MDM custom buffer) when the cells washed three times in ice I-MDM environment, and after the last wash, the cells are suspended in 2-4 ml ice I-MDM and kept on ice prior to sorting.

6. The cells are then sorted by fluorescence flow cytometer to exclude the top 85% of the fluorescence histogram. For the best enrichment sorting can be repeated twice.

7. After sorting cells counted, washed and aliquot paint on MY-10-positive cells (as described above) in order to determine the degree of enrichment compared with the painted aliquot full bone marrow cells.

The selection of immature b cells using magnetic antibodies

1. After implementation stages 1-3 in the procedure of fluorescent staining of bone marrow cells sort of Mature cells.

Attention! sodium azide is not included in any of the buffers.

2. While the corresponding number of magnetic goat antimisting Ig (Biomag: Collaborative Research; cat. N 74340-50, 1 mg/ml, 5108particles/ml) are washed three times in ice I-MDM environment at 1500 rpm, 5 min, 4oC (for washing of sodium azide, which is used as a preservative).

3. Resuspending cellular debris, obtained after the third PC, and, therefore, 0.1 ml Biomag).

4. Cells precipitate in the flask for tissue culture T-25 or T-75 (depending on the number of cells) and incubated for half an hour on ice, shaking occasionally.

5. After incubation, the flask is placed on a flat magnet (supplied Biomag) and fix with the help of rubber or sticky tape, and incubated for 10-15 min at 4oC.

6. The magnet and the flask is put in an upright position and collect the supernatant.

7. Stages 4-6 are repeated twice.

8. Count the cells washed once in ice I-MDM, allocate aliquot of dye for MY-10-positive cells and resuspended in appropriate environments for future use.

I. Replacement environment

Materials and methods

Cells: cells of human bone marrow was obtained from heparinised aspiration biopsy specimen taken from the iliac crest of the volunteers who gave this informed consent. Bone marrow was isolated by centrifugation in density gradient Ficoll-Paque (Pharmacia, N 17-0840-02), and cells with a low density (<of 1.077 g/cm3) were collected and three times washed with environment Dulbecco modified by the method of Claims (IMDM). Between the second and third washings, kleinem duplicate at 1, 2 and 5106cells/ml at 322 μl per well.

Conditions for long-lived cultures: cells with low density were incubated in IMDM, supplemented with 10% serum, amniotic calf Hyclone Laboratories, 10% horse serum (Hyclone Laboratories), 1% penicillin/streptomycin (Sigma, 10000 u/ml penicillin G and 10 mg/ml streptomycin, cat. N P3539) and 10-5M hydrocortisone (17-hydroxycorticosterone, Sigma, cat. N H0888) in a humidified atmosphere containing 5% CO2and 95% air. The culture was treated with one of three environments for sharing: 100% environment for daily replacement (7/week), 50% the environment for daily replacement (5,5/week.) or 50% medium replacement twice per week (1/week). Twice a week during the replacement of the medium from each well with the culture was removed 50% of the non-adhesive cells and was calculated using hemocytometer.

When the cells were removed for counting (2/week), all environments that are removed in the process of filing a 3.5/week.- 1/week.-cultures were maintained for counting cells in the wells were served a fresh environment. 7/weeks.-cultures half was retained for counting cells and the non-adhesive cells were centrifuged and returned to the remaining half of a removable medium. Then to each well was added a fresh environment to replace the medium removed for counting cells. If cells kletki centrifuged and returned in the original holes with added fresh environment.

The methylcellulose and morphological analyses: once in two weeks the non-adhesive cells are removed for counting, were sown in methylcellulose in the presence of erythropoietin, GM-CSF and IL-3 and counted granulocyte-macrophage colony forming unit macrophage (CFV-GM). Aliquots of remote cells were cytocentrifuged, were stained by Wright-Giemsa and carried out the calculation of differentiated cells.

Statistical analyses: the results of the production of cells for two weeks was expressed as the average value of the average square of Osh. from duplicate cultures. The likelihood of significant differences between groups of cultures was determined by comparing the normalized values of the cumulative products of the cells derived from the quickly replaceable cultures (7/week. 3.5/week.) in comparison to the control cultures (1/week) using bilateral T-test. Statistical significance was considered at 5% level.

Results

The kinetics of production of the non-adhesive cells: production of non-adhesive cells were evaluated as a function of cell density of the inoculum (within 1-5106cells/ml) and exchange rate environment. The exchange rate of the medium was varied from sharing the same volume of medium per week (traditional kultuurkapital by dividing by the number of cells, inoculated into the culture.

For each exchange rate environment curves normalized values of the number of collected cells do not show significant changes depending on the density of the inoculum. The production of cell cultures, supported by the three modes of exchange: 7/weeks., 3,5/week. 1/week, it was the same after normalization to the number of inoculated cells in culture. Comparison of ultimate cumulative production cells at different inoculum densities showed no significant differences in any exchange mode (p > 0.2 at a bilateral T-criteria for all pairs of samples).

In contrast, the exchange rate environment is very important for the speed and duration of the producing cells in these cultures. Producing cells in cultures that are replaced when the modes 1/week. (control), 3.5 weeks. and 7/week. declined in the first few weeks. However, this difference in productivity of crops became noticeable after 3 weeks. After 3-10 weeks producing cells remained constant at 7/week.-cultures, constant at a lower level in 1/week.-cultures, but exponentially increased 3.5/week.-the cultures. After 10-12 weeks producing cells was decreased, is equivalently those that are usually seen in traditional cultures dexterous type in some systems, whereas bathroomamenities culture 3,5 and 7/week. find high cellular productivity compared to previously used methods of optimal cultivation. Culture, in which half of the daily environment exchanged (3,5/week), maintained high cell productivity for a longer time than the control (1/week) or completely replaced daily culture (7/week). Through 3-9 weeks the number of non-adhesive cells collected from 3.5/week.-exchanging cultures, exponentially doubled every 2,1 weeks.

Producing cells in the modes of 3.5/week. 1/week. can be directly estimated by constructing a curve producing cells in the exchange rate of 3.5/week. as a percentage of the production cultures compared to cultures with a regime of exchange of 1/week. These comparisons showed that the duration of the initial phase of reducing the production of cells for the two modes are the same. However, after 3.5-18 weeks of cellular productivity of crops with exchange mode 3,5/week. remains at a consistently higher level.

Proliferative Potentate. The normalized cumulative number of production cells obtained after 3 weeks (ni= 7' Ci/C0) does not depend on the density of the inoculum for exchange environments 7/week. 3.5/week. Data producing cells obtained for cultures with similar modes of exchange environment, are quantitatively and statistically similar, and therefore, they were averaged and combined (bottom panel) for a much larger statistical sample. The average density of the cumulative products of the cells after 3.5-20 weeks was: 0,22 7/week., 0,40 for 3.5/week. and 0.15 to 1/week.-cultures.

Thus, the increase in the rate of exchange environment from 1/week. to 7/week. contributed to the increased production of cells by approximately 60% compared with the productivity of traditional crops dexterous type. As a result, the rate of exchange of medium of 3.5 weeks. gives almost 3-fold increase in the production of cells compared with the productivity of crops dexterously exchange mode (1/week). Statistical analysis of these data using bilateral T-test showed significant difference between cultures with 7/week. 1/week. -modes, but also between cultures with 3.5/week.- 1/week.-modes at a significance level of 5%. The same is m dexterously mode 1/week.

Production of precursor cells of granulocytes and macrophages

The analysis on the production of progenitor cells was carried out by playback experiments with this mode of perfusion medium and the inoculum density (table 2). The perfusion rate environment had a significant impact on the number of produced precursor cells of granulocytes and macrophages. Longest duration of production of progenitor cells was detected in the culture exchange mode 3,5/week. These cultures have produced predecessors at a stable level through 4-18 weeks from the beginning of cultivation.

Optimal conditions for the production of progenitor cells gave culture with a regime of exchange of medium of 3.5/week. and density of inoculation 5106cells /ml of These cultures have produced a significant number of progenitor cells up to 20 weeks after the start of cultivation. Statistical analysis using bilateral T-test showed that culture with the specified optimal exchange mode (i.e., 3,5/week.) produce a significantly larger number of precursor cells of granulocytes and macrophages after 8 weeks than 7/week.- 1/week.-culture in all three dps factor because it is an indirect indicator of the recovery of stem cells. Precursor cells may be present in the culture only a few weeks later when the differentiation from earlier cells, preferably stem cells, which are still in culture. Thus, the obtained data suggest that a more physiological quick change environment/serum and higher density of cells can provide conditions under which will be maintained a certain level of regeneration of stem cells for five months.

The morphology of the non-adhesive cells: to determine whether there are qualitative differences between prolongirovannogo haematopoiesis, supported 3.5/week.-cultures from hemopoiesis in other cultures, the non-adhesive cells were harvested 10-19 weeks, were stained and were identified by morphological. When exchange regimes 1/week. and 7/week. produced cells were predominantly macrophages to 15 weeks later (table 3), which was identical with the results obtained in experiments carried out in other laboratories. In contrast, culture exchange mode 3.5 volumes of medium per week at a density of inoculum 5106cells/ml Productionist inoculum are more effective for recovery granulopoiesis in vitro.

II. Replacement environment in combination with the addition of environment hematopoietic growth factors

Materials and methods

Cells: cells of human bone marrow was obtained from represiroannyh aspiration biopsies taken from the bone marrow of the iliac crest volunteers who gave this informed consent, in accordance with the procedure validated by the University of Michigan Committee for human research. Bone marrow was isolated by centrifugation in density gradient Ficoll-Paque (Pharmacia), and cells with a low density (<of 1.077 g/cm3) were collected and washed 3 times with IMDM. Between the second and third washes the cells were counted. Then cells were sown in 6-hole tablets for tissue cultures (Costar N 3406) or 6-hole tablets coated with collagen (rat tail collagen type I, Biocoat. Collaborative Research Inc., cat. N 40400) in duplicate 5106cells/ml at 1.5 ml per well.

Cultural environment: used IMDM medium (Gibo Laboratories, cat. N 430-2200) containing 10% amniotic calf serum (Hyclone Laboratories), 10% horse serum (Hyclone Laboratories), 1% penicillin/streptomycin (Sigma, 10000 u/ml penicillin G and 10 mg/ml streptomycin, cat. N P3539) and 10-5hydrocortisone (17-hydroxycorticosterone, Sigma, cat. N H0888).

ultor dexterous type, are suboptimal, and that can be obtained in vitro culture, which is the best approximation to the conditions of hemopoiesis in vivo and is more effective for the regeneration of bone marrow ex vivo.

Physical characteristics: the rate of exchange environment significantly affects the appearance of the crops. On the 10th week 7/week.-cultures there is a large number of lipocytes in the stroma, whereas 3,5/week.-cultures have very little fat cells, and 1/week.-culture do not have fat cells. With the depletion of the culture at the 26th week stroma 7/week.-cultures consists of approximately 20-30% of fat cells, while the 3.5/week. cultures have by this time only a few fat cells. The distribution of adhesive colonies also varies depending on the speed of perfusion medium. Adhesive colonies 3.5/week.-cultures are more persistent than colonies in 7/week.- 1/week.-the cultures.

Hematopoietic growth factors (HGH): it was found that due to frequent replacement of the culture through a rapid change environment added to the environment of hematopoietic growth factors at a concentration of approximately 1/20 contribute maximum to the formation of colonies in clonal analyses 4. This used the following concentrate dotirovanie hematopoietic precursor cells: the non-adhesive hematopoietic cells, isolated from the culture were counted and were sown in methylcellulose at a concentration of cells 1105cells/ml or less. Then in methylcellulose was added GM-CSF and Epo at 20 ng/ml and 2 u/ml, respectively. These cells were sown in 24-hole tablets at 0.25 ml/well and incubated for 14 days at 37oC. then counted the number of colonies using an inverted microscope, and colonies with more than 50 cells were counted as colony-forming units of granulocytes and macrophages (CFU-GM), erythrocyte burebasaga unit (BFU-E), or colony-forming unit granulocyte-erythrocyte-macrophage-megakaryocytes (CFU-GEMM).

TBMC-conditions: the culture incubated at 37oC in humidified atmosphere containing 5% CO2and 95% air, and perfesional with daily change of 50% environment. In the first week of cultivation 50% of all the non-adhesive cells were removed from the cultures twice a week during the exchange environment, mononuclear cells were counted, and the wells were returned to fresh medium. In the remaining five days a week, when the cells were counted from each well with the culture was removed 50% of the medium was replaced with fresh medium, and the remote environment centrifuged, the environment decantation with cellular precipitate, and the cells were returned to pami cultures was determined by comparing the normalized values of the cumulative products of the cells from rapidly perfoirmance cultures, supplemented with hematopoietic growth factors with untreated control cultures, using bilateral T-test. Statistical significance was considered at the 5% level. This was not observed statistical differences between fast perfoirmance LTBMC, cultured on plastic and collagen type I, rat tail, at a significance level of 5%. Therefore, data for plastic and collagen matrix combined for presentation on the charts, and for conducting statistical analyses.

Results

The kinetics of production of cells in quickly replaced and supplemented with growth factor LTBMAc crops

Since the first test was performed to test the hypothesis that the duration and productivity of long-lived cultures of bone marrow (LTBMC) are limited by insufficient production of the people's Assembly, were used quickly replaceable ex vivo culture of bone marrow, which were added to IL-3 or Epo. In these cultures, 50% of the medium was removed daily and replaced in an equal volume of fresh medium supplemented with IL-3 or Epo. Then the removed cells are centrifuged, the environment decantation and discarded, the cells resuspendable and returned in the original culture. IL-3 and Epo separately useouu degree producing cells due to considerable end erythroid differentiation. However, on the fourth week erythropoiesis was stopped, and the rate of production of cells was decreased to the level of the control cultures. IL-3 and Epo induced on average increased production of non-adhesive cells compared with control within 18 weeks of culture on 175 and 173%, respectively.

The Association of growth factors gave more effective in increasing the speed of production of the non-adhesive cells. The greatest degree of cell productivity was observed with the combination of IL-3+GM-CSF+Epo. These cultures have produced about 25% of the number of cells inoculated twice a week during the first six weeks, and had on average a 4.8-fold increase in the production of non-adhesive cells compared with control within 2-8 weeks. The combination of IL-3+GM-CSF was given in approximately 3.5-fold increase in the production of non-adhesive cells compared with control for eight weeks. In separate experiments, the addition of either IL-6 or G-CSF in combination with IL-3+GM-CSF+Epo instead of the expected increase in production of non-adhesive cells did not give noticeable differences from cultures containing IL-3+GM-CSF. In all cases a stimulating effect on cell productivity induced by the addition of HGF, b the entire period of cultivation.

The combination of HGF give high absolute values of the non-adhesive cells produced in quickly replaced LTBMC-cultures. The productivity of crops can be demonstrated by comparing the cumulative number of cells produced during the period of time (ni= 1, Ci; Cirepresents the number of non-adhesive cells collected at the time (i) in relation to the number of inoculated cells (C0) by constructing a curve of the relationship (ni= 1, CiC0from time. If this ratio is greater than one, the culture produces more cells than it was inoculated, and this culture leads to expansion in the number of cells.

The combination of IL-3+GM-CSF+Epo promotes the induction of the cumulative products of the cells, which is three times the number of inoculated cells. The degree producing cells was highest during the first six weeks of cultivation, during which the culture has produced about as many cells as was inoculated every two weeks. This maximum degree producing cells was 15% of the estimated in vivo extent producing cells in the bone marrow, where the daily genericvaltrex, comparable to those given by the combination of IL-3+GM-CSF+Epo within 3-7 weeks. Raw quickly replaceable (daily exchange of 50% protection) and slowly replaced (sharing 50% of the medium twice a week) of the control culture, which was not supplemented with HGF, was produced cells, the number of which amounted to approximately 1 and 0.37-fold significance in relation to the number of inoculated cells after 18 weeks, respectively. It is important to note that more than half of all cells that have been removed from these nezapolnennim crops come from the first two samples, which indicates that many of these cells originate from the original inoculum and add crops with HGF necessary to induce a noticeable alternation precursors and stem cells.

Morphological analysis of the non-adhesive cells: add multiple quantities of HGF also leads to an increase in the diversity of myeloid cells produced in cultures. The control culture was produced non-adhesive cells that three weeks were predominantly macrophages. The production of erythroid cells was falling fast, and very little erythroid cells was detected after 5 weeks of culture. Culture containing Epo (only Epo, IL-3+Epo and IL-3+GM-CSF+Epo), the product is collected cells were erythroid on the third week of cultivation. If present, the combination of IL-3+Epo+GM-CSF, culture continued to produce erythroid cells for 16 weeks, with about 5-15% of the non-adhesive cells were typed as erythroid. Thus, the presence of IL-3+Epo promoted erythropoiesis.

IL-3Epo gave the population of the non-adhesive cells, which at the fifth week, mostly accounted for (60-70%) of Mature granulocytes (LG). The percentage LG gradually decreased up until 18 weeks is not reached about 20%. The production of macrophages, respectively, were increased. When IL-3Epo added GM-CSF, a high percentage LG remained for 18 weeks. Thus, the combination of IL-3+GM-CSF promoted granulepos for 18 h in culture, and the addition of Epo also supported erythropoiesis. Photomicrography control and IL-3+GM-CSF+Epo-supplemented cultures 5.5 weeks showed a sharp increase in the density of cultures and diversity of the produced cells.

The kinetics of production of the non-adhesive precursor cells

The production of progenitor cells was increased when adding multiple quantities of HGF. The production of colony-stimulating units granulocyte-macrophage CFU-GM in raw contatami, obtained using fast perfesional LTBMC without the addition of HGF. CFU-GM produced in IL-3+GM-CSF and IL-3+EpoGM-CSF-cultures approximately 10 times higher than in the control environments within 3-5 weeks.

Earlier it was reported that the production of erythroid burebasaga units in LTBMC quickly decreases and stops (Coutinho and others, Blood (1990) 75 11: 2118-2129). Bystroperemennyi untreated control cultures showed a rapid decline in the production of BFU-E, although low levels of BFU-E was producirovanie for 17 weeks in culture. Adding one Epo had no significant effect on the number of produced BFU-E. IL-3 stimulates one easy short-term increase production BFU-E in 3-5 weeks. On the other hand, IL-3+ or Epo or GM-CSF induce 10-20-fold increased levels of non-adhesive BFU-E compared to control levels within 3 to 5 weeks of culture.

III. Transformation of human stem cells

Materials and methods

Cells: cells of human bone marrow was obtained from represiroannyh aspiration biopsies taken from the bone marrow of the iliac crest volunteers who gave this informed consent in accordance with the procedure validated by the University of mosti Ficolla-Paque (Pharmacia), and cells with a low density collected (<of 1.077 g/cm3and three times washed with IMDM. Between the second and third washes the cells were counted. For experiments with gene transfer of bone marrow was obtained from patients with deficiency of CD18 and informed consent.

Selection of bone marrow cells with a negative response to differentiation (Lin-)

Mature mononuclear cells were isolated from the above cell preparations by incubation of these cells with a mixture of monoclonal antibodies (MAb) after the third washing IMDM. 107cells were incubated in 1 ml of MAb cocktail on ice for 1 hour, stirring gently every 10 to 15 minutes Used MAb cocktail shown in table 4.

Cells were washed 3 times with excess ice IMDM and centrifuged at 4oC. the Appropriate amount of magnetic goat antimisting Ig (Biomag; Collaborative Research Corp., cat. N 74340-50, 1 mg/ml, 5108particles/ml) were washed 3 times in ice IMDM and centrifuged. The cells were respendable in Biomag at 50 part./class. and was placed in a flask with tissue culture T-25 or T-75, and then incubated on ice for an hour, shaking occasionally. After incubation, the flask was placed on a flat magnet, pre collected. Described incubation with shaking, the location on the magnet, the establishment of vertical and collection of the supernatant was repeated 2 more times. After that, cells were counted and were sown on 6-hole tablets for the cultivation of tissues (Costar N 3406).

Cultural environment. In the experiments used the IMDM medium (Gibco Laboratories, cat. N 430-2200) containing 10% amniotic calf serum (Hyclone Laboratories), 10% horse serum (Hyclone Laboratories), 1% penicillin/streptomycin (Sigma, 10000 u/ml penicillin G and 10 mg/ml streptomycin, cat. N P3539) and 10-5M hydrocortisone (17-hydroxycorticosterone, Sigma cat. N H0888).

Hematopoietic growth factors. Used hematopoietic growth factors have been described above. Concentration was 1 ng/ml or 0.4 u/ml IL-3 (obtained from genetics Institute, Cambridge, MA), 1 ng/ml GM-CSF (obtained from genetics Institute, Cambridge, MA), 50 u/ml IL-1a (Genzyme Corp.), 0.1 units/ml Epo (Terry Fox Labs, Vancouver, Canada), 10 ng/ml MGF (growth factor fat cells, c-Kit-ligand, Immunex Corp., Seattle, WA), and 2.0 ng/ml Hibikino ([P1XY321] Immunex Corp., Seattle, WA).

Analysis of hematopoietic precursor cells

The non-adhesive hematopoietic cells isolated from cultures during the weekly sampling, GF, GM-CSF and Epo at 50 ng/ml, 20 ng/ml and 2 u/ml, respectively. Then the colonies were counted using an inverted microscope, and colonies of more than 50 cells was determined as GM-colony forming units (CFU-GM), erythroid burebasaga unit (BFU-E) or colony-forming unit granulocyte-erythrocyte-macrophage-megakaryocytes (CFU-GEMM).

Cell lines retroviral producers.

Cell line producing retrovirus were obtained from the laboratory of Dr. Eli Gilboa Memorial Cloan Kettering Cancer Center, new York, NY. This cell line produces apotrope viral particles which contain the NEO gene, producing neomycinphosphotransferase that confer resistance to the neomycin analogue G418 mammals. Both cell lines also produce retroviral particles that do not contain genes required for retrovirus, so cells infected with this retrovirus unable to produce infectious virus.

SAX-containing cell line with a defect in the packaging is a cell line-based 3T3, which contains the modified virus murine leukemia, Malone (MoMuLV). SAX provirus contain the NEO gene, SV40-activated gene adenosine-deaminase, site ranscripts) and env (envelope proteins). This second retroviral particle contains a double copy of alien DNA, and these retroviral particles were labeled DC-29 (dvuhkabinnye 29th clone). Provirus DC-29 contain 2 copies of the NEO gene and other retroviral and alien DNA in the cell line 3T3.

For CD18-experiments used apotrope defective packaging cells Psi-Crip infected with retroviral vector containing human full (CD19-cDNA, Wilson and others, Science (1990) 248 1413-1416). This retrovirus full CD18 cDNA human cloned in the BamHI site of the vector, which expresses a recombinant gene from a heterologous sequences located in the 5'region of the gene-actin chicken, denoted by BA-CD18. Sequence from 5' to predrainage gene (IE) of human cytomegalovirus was subclinically with PUC19, and the area containing the sequence IE enhancer was deleted, starting with XhoI (from polylinker and NcoI (-220 gene IH-fragment). Synthetic linkers used for the conversion of the NcoI-XhoI site in the site, and the modified fragment was cloned into the unique XhoI site BA-CD18, located in the region 5' to the promoter-actin. This new vector was designated CMV-BA-CD18.

Production of retroviral particles

SAX-retroviral particles were recip is mutant. Retroviral particles DC-29 and CD18 were produced by growing lines with defective DC-29 and CD18-virus package to a state close to continuity in the flask T-75, with the replacement of the entire environment, incubation of cells within 12-15 hours, and subsequent collection medium containing viral particles. The virus-containing supernatant is then centrifuged to remove cells with viable packaging, after which the medium was removed and frozen in aliquot at -80oC.

LTBMC with supernatant containing SAX retroviruses, DC-29-retroviruses or CD18-retroviruses

Cultures were incubated at 37oC in humidified atmosphere containing 5% CO2/95%. During the first two weeks of culture from each well with the culture of daily removed two-thirds of the medium (1 ml), and the medium was replaced with an equivalent volume of medium containing HGF (0,85 ml) and the supernatant containing the cell-producer of the virus (0.15 ml). The medium containing the retroviral supernatant was thawed immediately before use, and if the environment must be used immediately, it was placed on ice in the refrigerator. Wednesday, remote from the cultures were centrifuged, the medium decantation, and the cells were returned to the original holes.

LTBMC, cultivated together with glue is up to approximately 10% of continuity in flasks T-25 (Costar, N 3056), and then subjected to irradiation at 2000 rad. To irradiated cells producing virus was added hematopoietic cells, obtained as described above, and cultured with daily change 50% of the environment within 2.5 weeks, all of the cells were returned to the wells after the change of environment. After 2.5 weeks of cultivation in the flask was added a 0.5 mm solution add to remove hematopoietic cells, left stroma. These remote hematopoietic cells were added to 3 wells of a 6-hole tablets with vegetablegrowing cells (1000 per well) fibroblasts bone marrow.

Sampling infected LTBMC:

Starting from the second week of cultivation, after adding retroviruses and termination of co-culture, in the cultures was replaced 50% of the daily environment, and the non-adhesive cells in the replacement medium was removed once for analysis. The non-adhesive cells were removed from the cultures during the daily exchange environment, mononuclear cells were counted, and the wells were added with fresh medium. In the remaining six days of the week, when the cells were not counted, 50% of medium from each well with the culture was replaced with fresh medium remote medium was centrifuged, after which the environment decantation with cellular osedlal bone marrow were sown at 0, of 0.4, and 0.8, and 1.2, 1.6 and 2.0 mg/ml G418 curve of inhibition in order to determine the concentration of G418, which are cultivated postinfusion cells. Cells isolated from crops sown in methylcellulose with G418 at concentrations of 0.0, 0.8 and 1.6 mg/ml After two weeks the number of colonies of progenitor cells in methylcellulose were calculated. Then individual colonies were collected from the methylcellulose and analyzed for the presence of retroviral DNA using polymerase chain reaction (PCR).

Statistical analyses: probability of significant differences between groups of cultures was determined by comparing the cumulative quantities of produced cells from experimental samples with the corresponding control cultures using double sided T-test. Statistical significance was considered at level 5%.

Results

Retroviral infection using SAX retroviruses

The kinetics of production of cells in LTBMC infected SAX-retrovirus.

Producing cells in cultures infected with a retrovirus is an indicator of the likelihood of being infected with retroviral infection, and therefore can be used as a parameter for measuring the level of infected is the cause of increased crop productivity increases the probability of mitosis of stem cells, consequently, increases the probability of retroviral infection. The highest level of productivity occurs in cultures containing supernatant with the virus, and supplemented with IL-3+GM-CSF and IL-3+GM-CSF+IL-1 that produce an increasing number of cells within 4 weeks of cultivation. LTBMC, cultured together with cells Packed SAX-virus, has produced a greater number of cells than cultures with the addition of supernatant for 2 weeks, although two weeks producing cells decreased.

Analysis of retroviral infection in LTBMC with the addition of the supernatant, containing SAX-virus

The percentage of surviving cells-precursors at high [G418] varied from 2 to 50% in cultures supplemented with IL-31GM-CSF or IL-3+GM-CSF+IL-1 during the first 4-6 weeks of culture. After 10 weeks of culture (8 weeks after adding virus) 43% of the number of precursor cells that were capable of cloning in hematopoietic colony could survive when exposed to G418. This suggests that these precursor cells had acquired resistance to G418 due to the content of the gene of resistance to G418 transferred by the retrovirus in stem cells, Puri, which have not been added HGF, had on average 12% of survivors of progenitor cells with high [G418] in the period from 8 to 11 weeks of cultivation. After completion of cultivation after 11 weeks stromal layer of the cultures with added IL-3+GM-CSF was trypsinization, and 17% of the precursor cells were associated with surviving the stroma at high [G418]. This suggests that a significant percentage of the adhesive precursor cells was also with SAX-virus.

Analysis of retroviral infection in LTBMC, cultured together with irradiated cells with SAX viral packaging

The percentage of progenitor cells surviving in G418 by co-cultivation with irradiated SAX cells varied between 0 and 36%. Culture, which was not added HGF, was produced CFU-GM, which survived at high [G418] only during weeks 4-7. After 7 weeks in these cultures did not producirovanie CFU-GM, which survived at high [G418], suggesting that stem cells were only slightly infected or were not infected. LTBMC, supplemented with IL-3+GM-CSF and cultured together with irradiated SAX cells within 2.5 weeks, has produced a high percentage of CFU-GM, which would survive 0,8 Metsa resistance CFU-GM to G418. This suggests that in these cultures, stem cells are few infected or who are not infected, or that the stem cells can differentiate or die.

Retroviral infection using retrovirus DC-29

The kinetics of production of cells in LTBMC infected with retrovirus DC-29

The number of cells produced in cultures infected with supernatant containing retrovirus DC-29, is HGF-dependent. Culture, supplemented with IL-3+GM-CSF+Epo was produced 1,5-4106cells based on weekly assessment within 10 weeks of culture. Culture, supplemented with IL-3+GM-CSF+Epo+MGF, were more productive, and culture, supplemented Hibriten+Epo, gave the highest level of cell production. It is interesting to note that of the control cultures supplemented with IL-3+GM-CSF+Epo, but which has not been added DC-29-retroviral supernatant were less productive than a similar culture with the addition of DC-29-retroviral supernatant. Producing cells in IL-3+GM-CSF+Epo, IL-3+GM-CSF+Epo+MGF-and hibriten+Epo-cultures (with the addition of virus) was significantly higher than in the control culture (IL-3+GM-CSF+Epo, without adding virus), at levels nachinayushego supernatant may be due to the presence of growth factors, such as MGF (c-Kit ligand), which, as is well known, is produced by the cell line with defective packaging on 3T3.

Analysis of retroviral infection in LTBMC with the addition of DC-29-retroviral supernatant

The efficiency of retroviral infection was estimated using the level of survival of CFU-GM at 1.6 mg/ml G418, a concentration that killed all the bone marrow cells to infection by the retrovirus. All infected cultures (see table 5), the average percent surviving for 8 weeks (6 weeks after infection) CFU-GM was high.

As it turned out, adding MGF to the combination of IL-3+GM-CSF+Epo increased the efficiency of infection by 8-10 week cultivation, and one of the cultures contained 7.7% of the G418-resistant colonies. Data received on the 8th week, suggest that culture, which were added Hibriten+Epo, have a high efficiency of infection, although the data obtained for 10 weeks, indicate the absence of infection. As for the data obtained for 10 weeks, it should be noted that the reduction in the number of CFU-GM, isolated from several cultures, to a very small number or to the complete lack of CFU-GM to be expected when the level of infection privatevideo, it is quite sufficient to suppress the effectiveness of the transfected gene resistance to G418 when even a very small suboptimal levels. Thus, the efficiency of gene transfer into hematopoietic stem cells in these cultures in some samples was at least 7.7 percent, and perhaps 45% or higher.

The kinetics of production of the non-adhesive precursor cells

Because the analysis of retroviral infection in these experiments depends on the assessment of progenitor cells in order to conclude about the presence of stem cells, infection and changes of cells, it is important to assess the effect of HGF on the production of cells in culture. In addition, in this experiment with the retrovirus, MGF and Hibriten used in combination with other HGF. Both MGF and Hibriten was not used in the fast perfoirmance HGF-supplemented LTBMC, and it was therefore necessary to evaluate their effects on haematopoiesis.

Production of precursor cells depended on the addition of HGF. The number of CFU-GM, isolated from the cultures shown in table 6.

Every second week the samples taken from cultures was assessed by CFU-GM, and unrated values were determined by linear interpolation on the values of the total number of CFU-GM, isolated from the culture.

Adding retroviral supernatant contributed to the increase of precursor cells was 2.2 times compared with those cases where this add-on did not. The selection of crops, which added retroviral supernatant and IL-3+GM-CSF+Epo+MGF or Hibriten+Epo, 4.2 and 3.8 times the number of CFU-GM from uninfected control cultures, respectively. The number of remote CFU-GM was statistically greater at 1% level of significance in all cultures with the addition of the virus than the number of inoculated CFU-GM.

Production of precursor cells in culture

The ratio of populations in the distribution of CFU-GM shows that the addition of MGF or Hibikino to cultures supplemented with IL-3+GM-CSF+Epo gives a significant positive effect on CFU-GM pool. Adding MGF to the combination of IL-3+GM-CSF+Epo increases allocated CFU-GM 1.9 times, and differentiation is 0.5 times the value compared to similar cultures, which was not added MGF (see table 7).

Every second week the samples taken from cultures was assessed by CFU-GM, and unrated values were determined by linear interpolation between two data points. Known, R of the culture.

The combination Hibriten+Epo had a very significant impact on the production and differentiation of CFU-GM. Hibriten+Epo gave a 1.8-fold excess of the number of remote CFU-GM and more than 3-fold excess differentiation of CFU-GM in comparison with the previously optimal cultures supplemented with IL-3+GM-CSF+Epo. In addition, Hibriten+Epo contributed to producing almost twice the number of granulocytes and macrophages than it was in the case of the combination of IL-3+GM-CSF+Epo+MGF. This suggests that Hibriten is a potent inducer line differentiation of granulocytes and macrophages.

Analysis of neutrophils produced from stem cells, infected CD18 encoding a retrovirus.

Bone marrow with CD18-failure enriched for early hematopoietic cells, as described previously, and then were cultured for 14 days with daily replacement of 50% environment supernatant, supplemented with 1.0 ng/ml/day GM-CSF and 1.0 ng/ml/day of IL-3 and 40 u/ml/day of IL-1 and line CD18-retroviral producer. Starting from the 15th day of the cells cultured under the same conditions but without the addition of retroviral supernatant. The non-adhesive cells were removed from cultures on a weekly basis and analyzed for the presence of CD18 cell PA using standard methods (Updyke, etc. , Meth. Enzymol. (1986) 12: 717-725). The bone marrow cells with deficiency of CD18 were not able to Express the protein CD18 cell surface in this analysis, then, as neutrophils and monocytes, the number of which has increased in cultures with retroviral infection, expressed CD18 cell surface. In cultures with triple redundancy, the expression of CD18 on the cell surface of 3.5%/5%/2% at 6 weeks and 11%/28%/3% on the 11th week. Because neutrophils and monocytes present in the cultures after 11 weeks, contribute to the formation of progenitor cells CFU-GM only 10-14 days earlier, this suggests that hematopoietic stem cells are sufficiently and stably transfected with a recombinant retrovirus during the first two weeks of cultivation.

For interpretation of the obtained results it is important to understand that although some groups find the retrovirus gene transfer into hematopoietic precursor cells of the person, however, gene transfer into hematopoietic stem cells has not been demonstrated. Rapid decline in the production of cells in a traditional slow perfuziruemah human LTBMC is not possible to determine the degree of infection due to the lack of cell-ol which the studies were first assessed by cell culture, remote from LTBMC in methylcellulose in the presence of the antibiotic G418 in mammalian cells. Precursor cells infected with SAX or DC-29-retrovirus, and expressing the NEO-product must have the ability to survive and formation of colonies with high concentrations of G418, whereas uninfected cells should die at high concentrations of G418. In addition, for producing precursor cells that survive in high concentrations of G418 after 6 or more weeks after the addition of virus, it is necessary that these cells were only what differentiated from more primitive cells (stem cells), and therefore it can be assumed that stem cells were infected. Similarly for CD18-expression on the surface of neutrophils and monocytes produced during the 11th week of cultivation, it is necessary to predatirovaniya hematopoietic stem cell was infected during the first two weeks of cultivation, as all Mature cells, precursors, and count of clonogenic precursors which are present during the period of infection, died after 4-5 weeks of culture.

Analysis of retroviral infection used in this IP is offered by the expression of the NEO gene product. It was shown that the lack of expression of the product of the transferred gene creates some inconvenience for models of humans and primates. Therefore, in this study, the evaluation of the percentage of progenitor cells infected as described above, is probably underestimated.

The percentage of progenitor cells that survived at high [G418], was approximately 40% for 10 weeks in cultures infected with SAX-retroviral supernatant and supplemented with IL-3+GM-CSFIL-1. These preliminary results showed that a high percentage of stem cells with in cultures supplemented with retroviral supernatant, during the first two weeks of cultivation.

The percentage of progenitor cells, infected DC-29-retrovirus was high (0-21%) in IL-3+GM-CSF+Epo+MGF and Hibriten+Epo-cultures within the first four weeks after the addition of virus. This high level of infection of progenitor cells, probably due to direct infection of the predecessor and primitive cells by retroviruses. The percentage of progenitor cells surviving in high concentrations of G418, decreased 4 weeks after complete addition of the virus, but 2 weeks later, vos is predecessors, resistant to G418, probably gives an underestimation of the percentage of progenitor cells, infected DC-29-retrovirus. High concentration of G418 used for selection of positive colonies (1.6 mg/ml G418), twice the concentration used in the experiment with SAX-infection, and survival requires high levels of expression of NRO-gene product, neomycin-phosphotransferase.

It is interesting to note that in LTBMC, which added to the supernatant, containing SAX - or DC-29-retrovirus, HGF, IL-3+GM-CSFEpo, IL-1 or MGF, or a combination of hibriten+Epo, the percentage of progenitor cells surviving in the presence of G418 was increased after 6-8 weeks after completion of the addition of virus. Although the mechanism of increasing the percentage of progenitor cells surviving in the presence of G418, in the later stages of cultivation is not known, but we can assume that the stem cells that were infected during the first two weeks of cultivation, become more active in the process of cultivation. This fact contributes to an effective increase in the percentage of cells surviving in the presence of G418. As another explanation of this phenomenon can be assumed that the expression of the NEO gene product increases in glue, is the quiet are of a different and more expressing in the area of integration than precursor cells, transfected directly during the initial period of infection culture. Therefore, a high level of survival of progenitor cells in the late period of cultivation, although it can have several causes, but most likely it is due to the fact that stem cells were infected in these LTBMC.

In summary, it can be argued that the data obtained indicate that the culture conditions disclosed in this application, hematopoietic stem cells proliferate in these cultures that can be entered into these cells the genetic material carried by a retrovirus. Precursor cells are constantly and actively producirovanie of these stem cells, and many of these predecessors contained and expressed transfected genes. The data obtained showed that genetically modified hematopoietic stem cells of a person present and proliferated in these cultures.

IV. Experiments

I. Construction of transformants

Human growth factor GM-CSF (Wong. Science (1984) 228: 810-815) BBO is a fragment of pSP65 (Melton, Nucl. Acids. Res. (1984) 2: 7035-7056). The obtained plasmid was designated P65GM-CSF. The promoter of the mouse metallothionein (Glanville, Nature (1981), 292: 267-269) was digested EcoRI and BgIII, and a fragment of approximately 2 kb containing the promoter, was insertional into EcoRI-BamHI-fragment of pSP65 to get p65T. Then designed a plasmid pMT GM-CSF by digestion pSP65GM-CSF with EcoRI, filling in the "sticky" ends with a fragment maple DNA polymerase 1, followed by digestion of the obtained linearized DNA with HindIII to 700 p. O. fragment containing the coding region of GM-CSF. This fragment was subcloned into the SalI - filled HindIII site p65MT. Then the 2.7 kb fragment containing the promoter metallothionein and GM-CSF coding region, was isolated and introduced into pSV2neo (Southen and Berg. J. Mol. Appl. Genet (1982) 1:327), which was deleted promoter, SV-40. This led to the fact that SV-40 poly A signal was lower (5'--->3') from the GM-CSF coding sequence.

Neomycin-resistant gene, which was reported resistance to the antibiotic gentamicin (G418) were taken from pSV2neo by highlighting the PvuII-EcoRI fragment of approximately 3 kb and introduced EcoRI-linkers in the PvuII site. Gene neo-resistance with EcoRI ends was subcloned into the EcoRI site of GM-CSF-expressing plasmids to construct plasmids MTGM-CSFneo.n IL-3 Gibbons (monkeys) under control of the SV-40 promoter and poly-A site, was transferrable by electroporation of linearized DNA into the cell line CVI African green monkey cells and murine cell line NIH 3T3. Transformants were selected by selection in medium containing 500 mg/ml G418, were isolated and were skanirovali on the production of GM-CSF or IL-3 using bioanalysis of supernatants using cells AML-193 (Adams and others, Leukemia (1989) 3:314). Some positive lines were then used as stroma of bone marrow cells in dexterously culture.

In addition, normal cells mouse bone marrow was transferrable with the above plasmids using the calcium/phosphate method Okayama (Chen. Mol. Cell. Biol. (1987) 7: 2745-2752), and found that they effectively expressed the introduced genes.

It was investigated the secretion of GM-CSF and IL-3 transfected fibroblasts. Supernatant 72-hour culture, not containing serum was obtained from cells NIH-3T3 and analyzed for the secretion of hGF by3H-absorption in the target cells, inhibiting by neutralization with rabbit anti-GM-CSF or anti-IL-3 antibodies. Proliferation induced by 20 mg/ml of GM-CSF, was established as a 100% GM-CSF, and proliferation induced by 10 ng/ml IL-3, was set as 100 units of IL-3. These sowmya Luggage

The perfusion chamber was a glass cylinder with lids Derlin that allow autoclaving without deformation and biocompatibility. These caps are cylindrical grooves, which are inserted in the glass cylinder. At the bottom of the groove is placed a sealing ring for sealing an opening in the chamber. These caps have a few holes into which you insert fittings Leuer (Leuer Lok), and they are inserted line of the fluid and gas, as well as extension pipe leading to the Central section of the chamber for sampling the adhesive and/or non-adhesive cells. These covers are attached with three long bolts that are posted on the 120oand located outside of the glass cylinder, while for dense assemblies used o-rings and nuts.

This camera is attached to the side of the tank. This loop includes a pump, camera sensors, operating in real time, oxygenator and in addition to the side tank openings for sampling and injection. The environment in the side tank then slowly exchanged using a separate pump. This configuration allows to separately control the speed of the exchange environment and the flow rate of ceresi perfusion, and perfusion chamber can be used to control the pressure of dissolved oxygen and flow patterns in the chamber. Use polysulfone membrane with small holes allows a plug-flow mode flow and precise control of the supply of growth factors and other specific compounds that you must enter the bioreactor in very precise quantities.

Transfected stromal cells seeded or over a layer of crushed collagen sponge, or plated on one side of a 5-μm porous polycarbonate filter, pre-coated with collagen, which allows stromal cells to contact the filter for several hours. Cells cultured in an appropriate nutrient medium up until the cells reach a state of continuity on one side, and cytoplasmic emissions was carried out through the pores. Then the bone marrow cells were sown on the other side of the membrane, and the stem cells are contacted with embedded cytoplasmic emissions passing through the pores.

After autoclaving chamber and components of the loop reactor was assembled under sterile conditions. Then the medium was passed through the side loop and gamecast bioreactor was then inoculable one or extracellular matrix, or pre-inoculated substrate for extracellular matrix, which contained stromal cells. Stromal cells can then be kept in the chamber for several days, and controlled their metabolic efficiency and/or reactivity of the growth factor, and if the results were satisfactory, the bone marrow was inoculable or immediately planted the camera bone marrow. In any case, the cell layer was kept at the bottom of the Central section of the perfusion chamber.

Cells were seeded on extracellular matrix and the cell layer was glued to the substrate. When using membrane Luggage can be inverted and the cellular layer will be placed on the flow center section. In this configuration, maturing cells settle to the bottom of the Central chamber, because they lose their adhesiveness relative to the stromal layer. The non-adhesive cells are then harvested by continuous cell stream caused by the pressure of the perfusion medium, and directed to the exhaust pipe.

In a typical camera inoculant cells NIH-3T3 on the first day of the experiment, sowing on a substrate of crushed collagen sponge. During the first 40 days correct speed perf is of about 20 days. On the 64th day the camera sow 33106the bone marrow cells. During the first 10 days the number of collected cells decreases until, until it settles at a stable level of about 7-8105cells every three days. Running cytometrics analysis showed that a constant fraction, about 20% of the collected cells was HLA-DP-positive. On the 90th day the pump was disconnected and the pH fell below 6,9 during the night. When the recovery rate of perfusion producing the non-adhesive cells were recovered and approached the previous sustainable level of production, if there was bacterial contamination. At this stage the study was completed.

The results showed that perfusion chamber allows hematopoiesis ex vivo hematopoiesis can be restored ex vivo after the fall of the pH, and the concentrations of glucose indicate that hematopoietic cells first develop aerobically on glucose as the glucose concentration decreases after inoculation, and the concentration of lactate thus does not increase, which suggests that oxygenation is limited. Obviously, glucose/lactate (anaerobic) metabolism primarily due to the stromal layer IH-3T3. Analogictech reduced that indicates that the absorption of glutamine by bone marrow cells is much less than the absorption of the stromal layer.

III. Control of metabolic products

The rate of absorption and the formation of glucose and lactate, and glutamine and ammonia were determined for transfected cells NIH-3T3. (Used medium: IMDM + 20% amniotic calf serum). Increase uptake of glucose was observed only for daily fed T-flasks, while recharging culture showed the same slow decrease absorption of glucose. Culture, in which the exchange mode with 50% changed daily 100% daily on the 18th day of cultivation, showed an immediate increase uptake of glucose with the same trend that was observed for cultures with 100% daily currency from the first day of cultivation. The rate of production of lactate were similar in nature, because their relationship was mostly constant (lactate/glucose = 0,9, indicating predominantly anaerobic stromal metabolism).

The concentration of glutamine and ammonia were the same in nature, as in the case of glucose-lactate metabolism. Using values, skorr the value of glutamine and glucose remains constant, about 1:8 = glutamine:glucose. This predicted optimum ratio varies depending on the rate of oxygen uptake (this ratio decreases with the increase of the optimal level of absorption).

Similar conclusions were made regarding the data for the metabolism of glucose/lactate obtained from normal stromal fibroblasts bone marrow. Frequent change of environment, culture first are anaerobic with constant high and quickly achieved levels of lactate. With more frequent changing environment, cellular metabolism becomes faster with the increase in the absorption of glucose and production of lactate. After data correction for spontaneous chemical decomposition, any significant absorption of glutamine was observed. Studies with 3T3 cells and normal bone marrow cells showed that the cells continued to divide and grow rapidly at a rate of exchange whey/environment, which exceeded obviously critical mode changing environment.

To assess the relative importance of the speed of perfusion serum in relation to the rate of perfusion of nutrient medium were carried out the following experiments: 1) one series using T-flasks with Gedney, and the medium was changed every other day, and in the other series used 10% serum with daily exchange environment, 3) two series with T-flasks, where one contained 10% serum and the medium was changed every other day, and the other contained 5% serum with daily exchange environment, 4) two series with T-flasks, where one contained 5% serum with exchange medium through the day, and the other contained 2.5% serum with daily exchange environment. The exchange rate of serum in each group was similar, whereas the exchange rate of the nutrient medium was varied. The results of these series of experiments showed that the rate of exchange whey is a critical parameter. Although for experiment (1) glucose uptake was increased, and on the 4th day was leveled up to a level of about 9.5 mm per day, but in all other cases, the glucose uptake was lower than the initial uptake of glucose group (1) and fell approximately linearly regardless of whether you have used double the amount of serum, and daily or every other day changed the environment. The obtained result confirms the finding that the rate of perfusion serum or one or more of its components is a critical parameter that influences the nature metabolite can be successfully cultivated in the bioreactor. Stromal cells can be obtained from homologous or heterologous sources, and these stromal cells may be transfected with genes to provide important growth factors. Thus, to maintain cell growth does not necessarily add to the environment serum. Obtaining stromal cells, which are attached to the substrate so that hematopoietic cells can be separated from stromal cells, allows you to collect hematopoietic cells for use. By appropriate selection of the combinations of factors can be cultured hematopoietic cells with a specific direction of differentiation. In addition, if necessary, stromal cells can replenish transfairusa viruses for the introduction of genes into hematopoietic cells.

All cited in this application publications and patent applications are entered into the description by reference, unless otherwise noted.

It is obvious that the present invention can be made various changes and modification that does not extend, however, beyond being and scope of the invention stated in the claims.

1. Method of culturing stem cells CV physiologically acceptable terms, the replacement of the culture medium, characterized in that the replacement of the culture medium carried out at a speed sufficient to provide for the division of human stem cells ex vivo comprising 5 to 100% daily replacement at a density of cells 1 x 104- 1 x 1071 ml of medium.

2. The method according to p. 1, wherein the cultured stem cells are human hematopoietic system.

3. The method according to p. 1, wherein the cultured stem cells from human bone marrow.

4. The method according to p. 1, characterized in that use the medium containing IL-3, GM-CSF, steel factor, Epo, IL-1, IL-1, G-CSF, basic fibroblast growth factor, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, PDGF or EGF.

5. The method according to p. 1, characterized in that cultivate the composition of the hematopoietic stem cells of human origin.

6. The method according to p. 1, characterized in that it further includes the cultivation of human stem cells in the presence of the vector gene transfer and get a stable genetically transformed human stem cells.

7. The method according to p. 6, characterized in that cultured human stem cells found in the hematopoietic system.

8. The method according to p. 6, otlichuy the FDS on p. 6, characterized in that cultured human cells are mononuclear cells of peripheral blood, bone marrow cells, liver cells of the fetus, embryo stem cells, blood cells of the umbilical cord.

10. The method according to p. 8, characterized in that the specified hematopoietic human stem cells produce stable genetically transformed cell precursors.

11. The method according to p. 6, characterized in that the IL-3 and GM-CSF added continuously in the specified environment.

12. The method according to p. 6, characterized in that the steel factor or IL-1 added to the specified environment.

13. The method according to p. 12, characterized in that the steel factor and IL-1 added to the specified environment.

14. The method according to p. 6, characterized in that the vector gene transfer is a retrovirus.

15. The method according to p. 6, characterized in that cultivate these hematopoietic stem cells in the presence of supernatant of cell line, packing retrovirus.

16. The method according to p. 1, characterized in that the culturing hematopoietic stem cells in the presence of transformed stromal cells attached to the matrix and are able to secrete at least about the cells, moreover, the liquid culture medium is replaced daily and provides a result of the division of hematopoietic stem cells ex vivo.

17. The method according to p. 16, characterized in that the said hematopoietic cells are bone marrow cells, and the perfusion liquid culture medium and stromal cells support the division and the production of stem cells in human bone marrow, and then carry out their transfection.

18. The fibroblast line CVI African green monkeys producing GM-CSF, regulating proliferation and/or differentiation of hematopoietic progenitor cells, transformed with plasmid MTGM-CSFneo containing a DNA sequence encoding GM-CSF.

19. The method of separation of hematopoietic neoplastic cells from normal cells, including the impact on the population of cells by fluid flow, creating a shear stress sufficient to separate neoplastic cells, wherein the population gemopoeticheskoi cells combine with stromal cells, have limited mobility, and provide them a contact, and the shear stress creates equal to or more than 1.0 Dyne/cm2.

20. The method according to p. 19, the B. p. 19, characterized in that the generated shear stress is equal to 1.2 to 1.5 Dyne/cm2.

 

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