Method of obtaining homogenous population of stem cells and its application

FIELD: medicine.

SUBSTANCE: described is method of obtaining population of stem cells, which come from dental follicle of human, named FENC (coming from follicles stem cells of embryonic nerve crest), which include: a) collection of follicle sac in sterile conditions, splitting, growing and grafting of initial culture; b) optional amplification; c) sorting out on FAC - sorter by the following stem characteristics: SSEA-4, TRA 1-60, TRA 1-81, OCT-4+; d) analysis of RNA with respect to positivity by transcriptional factors Nanog and Rex-1.

EFFECT: invention makes it possible to obtain homogenous population of stem cells.

10 cl, 6 ex

 

The technical field to which the invention relates.

The invention relates to a new method of selection from the dental follicle of the person of a new subpopulation of stem cells with ambrina-like antigenic characteristics, and how to build them in culture: selected cells can be characterized and stored in cell collections for experimental or therapeutic purposes.

More specifically, the invention describes: isolation of stem cells from periodontal (dental) follicular tissue donor; their capacity in vitro, in particular in the culture conditions under which you may receive ambrina-like cells; storage to maintain viability;

their differentiation into different types of cells; the application of these cell types for cell therapy and/or tissue engineering in respect of the donor or HLA-compatible individual, in order to regenerate damage of different origin.

The level of technology

The possibility of obtaining stem cells from human tissue controversial and widely discussed today. The very definition of stem cells capable of self-renewal and differentiation into all cell types of the organism from which they originate, is not consistent with the antigenic and functional characteristics of adult trunks of the x cells, at the moment we get from people after birth. From the point of view of "self-renewal", in fact, adult stem cells, obtained at the moment, do not demonstrate an unlimited proliferative potential and, on the other hand, the ability to differentiate struggling to overcome multipotential (the ability to differentiate into all cell types that originate from the same embryonic leaf) or pluripotency (the ability to differentiate into all cell types that originate from other embryonic leaves), not reaching totipotential (the ability to differentiate into any cell type); the latter is a property of the zygote and the cells of the blastocyst, determining the first stages of human development. On the other hand, the possibility of manipulating cells derived from human embryos for therapeutic or research purposes are often at odds with the technical, ethical and legal principles. For these reasons, the possibility of obtaining undifferentiated cell types from adults not only provides an interesting experimental model of cellular differentiation and the model for solving problems related to Oncology, but also an important therapeutic tool for the treatment of diseases which are based on degenerat the fabrics I, caused by quantitative and qualitative defects.

Stem cells are types of cells, characterized by:

1) unlimited self-renewal;

2) capable of differentiation into many cell types.

With regard to the prior art, presents WO 03/066840, including follicular serotype, this invention has the following differences:

1) Antigenic pattern and method of selection. WO 03/066840 does not disclose any selection, while the method presented in this invention, uses a selection of cytofluorimetry embryonic stem cells by detection of such antigens as SSEA-4, TRA 1-60, TRA 1-81, CD133, CD90, flk-1. In this way it is possible to obtain a homogeneous population of embryonic cells.

2) methods of cultivation, cell proliferation and differentiation. In fact, WO 03/066840 describes the methodology of collection cell population, which contains only a small proportion of adult stem cells, that as a method of selection according to the present invention allows to obtain a homogeneous population ambrina-like stem cells.

Disclosure of invention

The invention includes a method of separation of follicular bag new, not hematopoietic, mesenchymal stem cell subpopulations, then here called FENC (originating from FD is Mikulov stem cells embryonic neural crest), through selection FAC-sorted, after appropriate staining with specific antibodies.

The invention also provides methods of culturing and proliferation of FENC. Cells obtained by the method according to the invention, are able to differentiate into all tissues derived from the three embryonic leaves, thus presenting FENC as the population of totipotent/multipotent cells. FENC can be stored with the maintenance of viability so that it becomes possible establishment of cell banks for storage of stem cells collected from adult patients. The invention also provides methods for cell and/or genomic therapy by FENC, as well as clinical therapeutic use of cells and tissues derived from differentiated FENC.

DETAILED description of the INVENTION

The invention relates to a method of setting a new cytotype of embryonic stem cells collected from adults, in particular allocation method of these cells, including their collection of follicular sack. Dental follicle, as tissue complex surrounding the tooth during its formation before the eruption, is a preferred source of undifferentiated cells for two reasons:

1) Each tooth, permanent or milk, is an unfinished organdie birth. He brings in place for the development of undifferentiated cells, which, after proliferation, will be of odontogenic cells. Embryonic origin from the neural crest explains their immaturity. Also at the end of odontogenesis these undifferentiated cells remain inside the follicle until the eruption, since they will be transformed into the cells of the root sheath and root apex of the tooth, which develop after the eruption. At the moment, certainly the presence of adult stem cells in the periodontal ligament (Gronthos, Lancet 2004), and dental pulp (Gronthos, PNAS 2000; Miura, PNAS 2003; the laino, JBMR 2005).

2) Dental follicle is a biological niche, easily accessible surgically, as well as has a high content of undifferentiated stem cells in the collected volume of tissue. The amount of fabric sacrifice for collecting stem cells, minimally, as the extraction of wisdom teeth is often, and it requires the collection of follicular sacs. Derived from these biological samples stem cells with characteristics of embryonic, can be stored for research and autologous therapeutic applications.

These cells originate from the neural ectoderm and are capable of differentiation inherent in citation - derivative of this layer.

The invention for the first time before what is the method of obtaining from adults homogeneous cell population with embryonic characteristics.

This strategy has the following advantages: 1) little invasive surgery with local anesthesia; 2) low pain the place of intervention; 3) high count of clonogenic mesenchymal culture, due to the lack of hematopoietic fraction; 4) obtaining embryonic cells from adults.

Getting FENCstem cells derived from follicular SAC directly formed from mesenchymal cells derived from the neural crest during organogenesis. Their origin explains their plasticity. For isolation of stem cells from follicular sack, the latter must be intact, not to have contact with the oral cavity.

The patient, a week before the operation, must follow appropriate Protocol oral hygiene, such as rinsing the mouth CHX 0.12, or a similar drug, at least twice a day. To collect follicular sacs, after anesthesia, incision flap of fabric and will have access to the bone to achieve impacted (unerupted) of the tooth, surrounded by follicular bag. Collected under sterile conditions the specimen shall be immersed in digestively ("perevalochny") solution for 1 hour at 37°C. At the end of the first hour, digestively the solution is filtered to remove cell aggregates and ECM particles. After this, the process of farms is ntatives splitting stops, and the cells are cultured in the medium Mega Cell (SIGMA, Milan, Italy). Alternatively can be used medium for embryonic stem cells (ES). Observations under the microscope, starting with the first day of doing culture, identify a large number of cells attached to the bottom of the culture vial, which give rise to clones. After 5 days in the bottle 75 ml can be up to 60 clones.

On day 9-10 supernatant was removed, the cells are sent to FAC for sorting and selection of clones positive for stem tokens.

Selected FENC differentiated, with/without stimulation in different cell types. There is a possibility to receive proliferation without differentiation by adding to the culture medium βFGF. Upon reaching the appropriate number of cells, they can be frozen and stored in liquid nitrogen at -80°C.

Summary of procedure:

1) Collection of follicular sacs under sterile conditions, enzymatic cleavage and cultivation;

2) Amplificate primary culture, as necessary;

3) Sort on FAC;

4) Amplification, freezing and/or cell differentiation;

5) Analysis at FAC;

6) Maintaining conditions that do not allow differentiation;

7) Tissue engineering of a differentiated cell.

All types of cells derived from FENC differentiation can be applied not only in KL is accurate therapy, but also to build various tissues.

For example, FENC can be used to obtain osteoplastic precursors expressing RUNX-2, of which are osteoblasts expressing HLA-1, CD44, RUNX-2e, CD54 and osteocalcin.

Osteomy matrix, called LAB (live autologous bone)can also be derived from osteoblasts, accumulated and stored in the freezer. LAB made from samples of bone tissue, with a thickness of more than 1 cm and a volume of more than 1 cm3. The formation of the LAB containing the osteoblasts, which actively produce bone, is characterized by: 1) the formation of clusters, which secrete into the Central area of inorganic crystals, collagen fibers and glycoproteins; 2) 3D organization of this structure when building the mineralized bone matrix.

Specified LAB is formed when the growth of cells at 37°C in an atmosphere of 5% CO2in the medium α-MEM, supplemented with 20% FBS, 100 μm 2-phospho-ascorbic acid, 2 mm L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin. The formation of the LAB is blocked only by lack of nutrients.

Specified LAB can be produced continuously, and osteopenia tissue can be preserved, while maintaining osteoblasts at 4°C or temperatures below 0°C, conventional techniques for preservation of cells by cryopreservation.

Also what can be derived 3D matrix, contains LAB, where osteoblasts grow in the presence of biocompatible 3D matrix populated by cells. Specified biocompatible matrix can be absorbed or notin vivo: examples of suitable matrix can serve as the copolymer of lactic and glycolic acids, synthetic collagen, HA, biocoral, calcium sulfate, while it is not absorbed include polymethacrylates, PTF, titanium, compact HA biphasic HA (50% HA and 50% β-TCP)ceramics.

According to further variants of execution of the invention, FENC can also be differentiated in cultured myoblasts, with the aim of obtaining smooth-muscle cells, using culture medium Mega Cell with 2% FBS, 100 μm 2-phospho-ascorbic acid, 2 mm L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 10 ng/ml TGFβ.

FENC can also be differentiatedin vitroin cultured myoblasts, with the aim of obtaining striated muscle, using co-cultivation with SS mouse muscle tubes. Co-culturing is carried out at a ratio of 1:10 for 7 days, using the following culture medium: modified method of Dulbecco Wednesday Needle (DMEM, Invitrogen), 4 mm L-glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/l glucose and 1 mm sodium pyruvate, supplemented with 10% FBS.

FENC can also be differentiatedin vitroin neurons and glial cells, with the use of cultivation in use the e 15-30 days in Neurobasal A (Invitrogen, Milan, Italy)supplemented with protein B27 (Invitrogen, Milan).

Obtained by differentiation of FENC glial cells can be sorted from neurons by FACS using an anti-GFAP antibody for glial cells.

FENC can also be differentiated into adipocytes, when grown for 30 days in culture medium α-MEM, supplemented with 20% FBS, 100 μm 2-phospho-ascorbic acid, 2 mm L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 10-8M dexamethasone.

Differentiation FENC in chondrocytes, with the aim of obtaining cartilage, can be obtained by culturing for 30 days in culture medium α-MEM, supplemented with 20% FBS, 100 μm 2-phospho-ascorbic acid, 2 mm L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, at lower Rho2below 40 mm RT. Art.

The invention provides autologous use FENC in cell therapy protocols.

In particular, autologous use FENC and cells derived from them, may be useful in the following applications:

- when the pathologies of the skeletal system, such as defects in bone by osteoblast - derived FENC.

when muscle tissue degenerations, both smooth and striated by cultured myoblasts derived FENC.

- when the pathologies of the Central nervous system by neurons derived FENC.

- by degeneration of myelin by glial cell - derived FENC.

in plastic surgery, disorders of the formation of adipose tissue, by adipocyte - derived FENC.

- in the areas of Oncology and plastic surgery, by chondrocytes derived FENC.

Tests for carcinogenesis- to eliminate the possibility of carcinogenesis, FENC and differentiation of these cells were injected in Nude mice (mutant genenude), after infection with lentiviruses III (Invitrogen, Milan, Italy) vector containing the cDNA of green fluorescent protein GFP. After transplantation, animals were euthanized after 30 days. Histological observation of the places transplantation, and major organs showed no tumor development.

Cells were analyzed for the presence of cancer markers and defects in cell cycle: all cells were euploid, without tumor characteristics.

Some examples of the practical application of FENC below.

Example 1. The selection of cells and cultivation

Every healthy patient, starting a week before the surgery was prescribed rinsing the oral cavity of CNH 0.12 (weight/volume) twice a day. To collect follicular sacs, after local anesthesia, adrenals flap fabric and opened access to the bone to reach retenir the bath (unerupted) of the tooth, surrounded by follicular bag. Assembled, kuratau Gracie or alveolar spoon, in sterile conditions, the sample was immersed in digestively ("perevalochny") solution for 1 hour at 37°C. as digesting solution was used PBS (phosphate saline buffer pH 7.4, 1 M)containing 3 mg/ml collagenase type I and 4 mg/ml dispute, 100 units/ml penicillin, 100 μg/ml streptomycin and 500 μg/ml of clarithromycin. The volume of solution depended on the volume of the harvested follicular SAC and ranged from 8 to 15 ml of the Samples were kept in the solution for 1 hour at 37°C. At the end of the first hour, digestively the solution was filtered through a 70 μm filter Falcon®to remove fragments of ECM, cell or large cell aggregates. The sample was subjected to enzymatic cleavage again within 30 minutes, if still present tissue fragments. To stop the enzymatic cleavage, the sample was added 10 volumes to the volume digesting solution, then the sample was centrifugals 10 min at 140, as the culture medium used was medium Mega Cell (SIGMA, Milan, Italy), supplemented with 10% fetal bovine serum (FBS), 100 μm 2-phospho-ascorbic acid, 2 mm L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin. After centrifugation, the sediment from the bottom of the test tube was transferred to a 25 ml suspension, cultivation in the cult of the social mattresses 50 ml at 37°C and 5% CO 2with the change of environment twice a week. Observations under the microscope, starting with the first day of doing culture, has shown attached cells, which give rise to clones.

Example 2. Expansion and characterization

After 5 days of cultivation in the environment described above, or in the medium for embryonic stem cells (ES medium, Invitrogen, Milan, Italy), in bottles of 75 ml was counted to 60 clones.

9-10 day, after removal of the supernatant and washed with sterile PBS, cells were detached from the bottom of the culture of the mattress. After conditioning in 4 ml EDTA 0,02%, diluted in free ions from Mg/Ca-PBS solution for 10 min at 37°C, were sorted by FACS and the collection of clones of cells positive for stem markers. Cells were precipitated for 10 min at 140 g), washed in 0.01% BSA (Bovine Serum Albumin) in PBS solution at 4°C and were incubated for 30 min at 4°C for staining 10 ál of stock solution of antibody. After incubation, cells were washed with 1 ml of 0.1% BSA in PBS to remove unreacted or non-specific antibodies, and analyzed by positive staining for the following antibodies: SSEA-4, TRA 1-60, TRA 1-81, CD133, CD90, flk-1 (Santa Cruz, CA, USA). 70% of the cells were positive for SSEA-4 and 80% TRA 1-60 and TRA 1-81. Namely, SSEA-4+cells were also positive as TRA 1-60 and TRA 1-81. SSEA-4, TRA 1-60 and TRA 1-81 are surface molecules that are present only n the undifferentiated cells, either embryonic stem cells (totipotent ES) and embryonic germ cells (EG pluripotent). After sorting a small sample was analyzed for the presence of three transcription factors, Nanog, OCT-4 and Rex-1, which is usually detectivey only in undifferentiated embryonic cells. Positivity in the previous antigens confirms the embryonic nature of the cells (ES) (Zhang Nature 2003). Positive for OCT-4 was 100% in all the sorted cells. To determine Nanog and Rexl, after receiving the appropriate number of cells, one of them was isolated RNA was analyzed using RT-PCR.

Example 3. Differentiation by type (bone, smooth muscle and cartilage)

FENC, isolated and characterized, as shown in examples 1 and 2, can be differentiated, with or without stimulation, or to proliferate with the suppression of differentiation, adding 8 ng/ml βFGF into the culture medium.

For osteogenic differentiation, cells can be cultured in the medium α-MEM, supplemented with 20% FBS (weight/volume), 100 μm 2-phospho-ascorbic acid, 2 mm L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin. Cells differentiate into osteoblasts for about 25 days, forming immature bone tissue (positive for antibodies to collagen type I and III, osteocalcin, osteonectin, BAP).

For trim is Interoute in smooth muscles, FENC must be cultivated in an environment Mega Cell (SIGMA) with 2% FBS, 100 μm 2-phospho-ascorbic acid, 2 mm L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, with the addition of 10 ng/ml TGFβ. Under these conditions, cells differentiate within 4-5 days and become positive staining of SMA, which is a marker of differentiation into smooth muscle.

For differentiation into cartilage, FENC were cultured in the medium α-MEM, supplemented with 20% FBS, 100 μm 2-phospho-ascorbic acid, 2 mm L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, at lower Rho2below 40 mm RT. Art., at 37°C with 5% CO2. Build cartilage, due to the high cell concentration (500,000/ 15 ml), takes about 30 days and can be confirmed by analysis of the expression of collagenase type II.

Example 4. Differentiation by type b ( neurons and glial cells)

Follicular cells can differentiate either spontaneously or with the use of medium Neurobasal A (Invitrogen, Milan, Italy), with the addition of protein B27 (Invitrogen, Milan, Italy). Culture is 5-7 days. In both cases there were a large number of differentiated neurons and glial cells. Then, use cytofluorimetric analysis with anti-GFAP antibodies may be given the opportunity to select and highlight the cells of two types. In fact, the differentiated cells are:

- glial, if they Deposit account opened is wny for antibody to GFAP;

- neurons, if they are positive for the antibody anti-TuJl, anti-neurofilament, anti-Bra3A (transcription factor);

- peripheral neurons, if they are positive for the antibody anti-peripherin and anti-p75.

Example 5. Differentiation by type c (striated muscle, adipocytes)

Differentiation FENC in striated muscle cells can be achieved by co-cultivation with SS mouse muscle tubes. Co-culturing is carried out at a ratio of 1:10, by known methods (for example, the laino et al., 2006), during the week in the following culture medium: modified method of Dulbecco Wednesday Needle (DMEM, Invitrogen), 4 mm L-glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/l glucose and 1 mm sodium pyruvate, and 10% FBS. After culturing, the percentage of FENC, fused with murine muscle tubes, it has been estimated by means of anti-human nuclear laminin. Usually, the percentage of mergers was about 15-20%. Merged muscular tubes can be used for transplantation.

Differentiation into adipocytes was received within 30 days by adding 10-8M dexamethasone to the culture medium α-MEM, supplemented with 20% FBS, 100 μm 2-phospho-ascorbic acid, 2 mm L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin. At the end, were observed visiting the creation of adipocytes, which can be stained with Sudan black, or, again, the oil red. Fat cells can be used in plastic surgery.

Example 6. Tissue engineering and clinical applications

Differentiated FENC can also be used for tissue repair through roller cultures, in the apparatus for rotating flasks. Cells differentiated into osteoblasts, for example, can be planted in a matrix of a copolymer of lactic and glycolic acid (85: 15) (which resolves within 15 days). Roller culture (1/5 sec) was created in 5% CO2incubator for 30 days. As soon as he had reached a thickness of 1 cm, was observed 3D tissue structure, and evaluated using techniques of histological, immunohistochemical and scanning electron microscope.

Bone tissue can be used for regeneration of the defective areas. If these cells can be obtained from patients, the matrix can be modeled and created the culture of the three-dimensional structure. After 20-30 days, the complex matrix of cells ready for implantation in the body of the patient.

The transplant can be done using standard surgical techniques. Preparation of the body for transplantation involves creating a locus of the open bleeding and cuts in the outer integument. Thus, the integration of whom the Lex matrix cells in the host organism will be easier and then transplantation.

Best integration was achieved in 40-60 days.

Repair of tissues can be achieved with autologous this technique with the use of stem cells.

Terminology

Mega cell = culture medium for cells (Sigma-Aldrich, Milan, Italy)

ES = culture medium (Invitrogen, Milan, Italy)

βFGF = fibroblast growth factor β

α-MEM = minimal supportive environment alpha (Invitrogen)

anti-GFAP = antibodies to glial fibrillar acidic protein

GFAP = glial fibrillar acidic protein

SMA = antibody to smooth muscle

TGFβ = transforming growth factor beta

BAP = bone alkaline phosphatase

βFGF = fibroblast growth factor β

RT-PCR = reverse transcription - polymerase chain reaction

Nanog = transcription factor in embryonic cells during early development

Rexl = transcription factor in embryonic cells during early development

OCT-4 - octamer 4

SSEA-4 = stagespecific embryonic antigen 4

TRA 1= antigen exclusion tumor 1

CD = differencirovany cluster

flk-1 = fetal liver kinase 1

HA = hydroxyapatite

TCP = tricalcium phosphate

1. A method of obtaining a population of stem cells that originate from the dental follicle of human rights and called FENC (originating from follicles stem cells C is radisheva neural crest), including:
a) harvesting a follicular bag under sterile conditions, cleavage, growth and expansion of primary culture;
b) optional amplification;
c) sorting on FACS-sorting device for the following stem markers: SSEA-4, TRA 1-60, TRA 1-81, OCT-4+;
d) analysis of RNA in respect of positivity for the transcription factors Nanog and Rex-1.

2. The method according to claim 1, where the cleavage is carried out with the use of proteolytic enzymes and antibodies.

3. The method according to claim 2, where the enzymatic cleavage is carried out by means of an aqueous solution containing 3 mg/ml collagenase type I and 4 mg/ml dispute, 100 units/ml penicillin, 100 μg/ml streptomycin and 500 μg/ml of clarithromycin in PBS.

4. The method according to claim 2, where the enzymatic cleavage is carried out for 1 h at 37°C with shaking.

5. The method according to claim 1, where the growth and capacity of the primary culture is performed with the use of culture medium, specialized for stem cells.

6. The method according to claim 5, where the cultural environment consists of the Mega Cell, supplemented with 10% fetal bovine serum (FBS), 100 μm 2-phospho-ascorbic acid, 2 mm L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin.

7. The method according to claim 5, where the incubation is at 37°C in an atmosphere of 5% CO2within 15-20 days, with change of medium every 3 days.

8. The method according to any one of claims 1 to 7, in which the sorting on FACS-sorting device is carried out with the use of the group of antibodies in addition to CD133, CD90 and flk-1 as markers of stem cells.

9. The use of a population of stem cells obtained by a method according to any one of claims 1 to 8, to obtain the osteogenic precursors, osteoblasts, cultured myoblasts, chondrocytes, adipocytes, neurons, glial cells.

10. The use according to claim 9, where the osteogenic precursors, osteoblasts, cultured myoblasts, chondrocytes, adipocytes, neurons, glial cells are designed to obtain autologous drugs used in cell therapy protocols for the treatment of pathologies of the skeletal system, muscle tissue degeneration, pathologies of the Central nervous system, degeneration of myelin, cancer and plastic surgery.



 

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13 cl, 14 ex, 1 tbl, 16 dwg

FIELD: biology, genetic engineering.

SUBSTANCE: invention relates to preparing immortalized cellular lines from health human skin tissues and can be used in immunological, pharmacological, photo- and chemical-toxicological analysis of cutaneous response, for expression of heterologous genes and for construction of artificial skin. Keratinocytes are immortalized by infection of keratinocytes of health human. The human skin sample is isolated and prepared its for culturing in vitro. Keratinocytes are prepared from this prepared human skin sample and plated in serum-free medium for growing keratinocytes in cultural plates with cover alleviating attachment and growth of cells. In the process for culturing keratinocytes the serum-free medium is replaced to provide preparing the optimal confluent growth of cells in culture with continuous maintenance of cup cover. Keratinocytes are transferred in selective serum-free medium in cultural cups with cover and infected with vectors pLXSHD + SV40(#328) and pLXSHD + E6/E7. Then prepared immortalized keratinocytes are transferred in cultural cups with cover to useful medium for proliferation. Then prepared proliferated keratinocytes are transferred in medium with high calcium content for differentiation in cultural chambers with cover. Invention provides preparing the human keratinocyte cellular line that has no oncogenic property and retains capacity for differentiation and expression of proteins and enzymes expressing by normal differentiated keratinocytes being even after increased number of passages in culture. Also, this cellular line forms lamellar and polarized epithelium with keratinized layer (stratum corneum) consisting of ortho-keratinocytes in the process for culturing in organotypical culture in serum-free medium and without layer of feeding cells.

EFFECT: improved immortalizing method, valuable biological properties of cellular line.

7 cl, 2 dwg, 4 ex

FIELD: organic chemistry, natural compounds, medicine, oncology.

SUBSTANCE: invention represents new saponin mixtures used for inhibition of initiation and activation of mammalian epithelial cell in pre-malignant or malignant state, for stimulation of apoptosis of mammalian malignant cell, prophylaxis of anomalous proliferation of mammalian epithelial cell, for treatment of inflammatory and regulation of angiogenesis in mammal. These mixtures are isolated form plants of species Acacia victoriae. Also, invention relates to methods for their applying. These compounds can comprise triterpene component, such as acacic or oleanolic acid to which oligosaccharides and monoterpenoid components are joined. Mixtures and compounds elicit properties associated with regulation of apoptosis and cytotoxicity of cells and strong anti-tumor effect with respect to different tumor cells.

EFFECT: valuable medicinal properties of compositions.

43 cl, 53 tbl, 50 dwg, 44 ex

FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

FIELD: biotechnology, molecular biology, medicine, genetic engineering, pharmacy.

SUBSTANCE: the hemopoietic protein comprises the amino acid sequence of the formula: R1-L1-R1, R2-L1-R1, R1-R2 or R2-R1 wherein R1 represents the modified ligand flt-3; R2 represents the modified human IL-3, the modified or unmodified colony-stimulating factor. Modification of R1 is carried out by addition of N-end with C-end directly or through linker (L2) that is able to join N-end with C-end to form new C- and N-ends. The modified human IL-3 is prepared by replacing amino acids at positions 17-123. The human G-CSF is modified by exchange of amino acids. The hemopoietic protein is prepared by culturing cells transformed with vector comprising DNA that encodes the hemopoietic protein. The hemopoietic protein stimulates producing hemopoietic cells and this protein is used as a component of pharmaceutical composition used in treatment of humans suffering with tumor, infectious or autoimmune disease. Invention provides preparing multifunctional hemopoietic proteins eliciting the enhanced activity with respect to stimulation of hemopoietic cells and eliciting the improved physical indices. Invention can be used for preparing chimeric multifunctional hemopoietic proteins.

EFFECT: improved preparing and producing method, valuable medicinal properties of protein.

22 cl, 19 dwg, 18 tbl, 117 ex

FIELD: cellular biology, medicine.

SUBSTANCE: invention relates to isolating and cryopreserving precursor-cells. Methods involve treatment of human liver tissue for preparing the essentially monocellular suspension containing precursor-cells and cells that are not precursor-cells, a single or more lines of cellular differentiation presenting in the human liver. Invention describes methods involving stage for separating cellular population resulting to reducing amount of cells that are not precursor-cells and providing preparing the separated suspension enriched with precursor-cells expressing one or more markers and associated with a single or more lines of the cellular differentiation. Also, invention describes a method for selection cells from the separated suspension wherein these cells or their progeny, or their more matured forms express one or more markers associated with lines of the cellular differentiation. These markers involve: CD14, CD34, CD38, CD45 and ICAM. Hepatic precursor-cells have diameter size 6-16 mc, they are diploid and show indices: glycoforin A-, CD45-, AFP+++, ALB+, ICAM+ and they comprise subpopulations varying with respect to expression of CD14+, CD34++, CD38++ and CD117++. These cells are useful for carrying out cellular and genetic therapy in liver treatment and for preparing artificial organs also.

EFFECT: valuable biological and medicinal properties of cells.

41 cl, 7 tbl, 13 dwg, 15 ex

FIELD: medicine, surgery, transplantology.

SUBSTANCE: embryonic spleen should be sampled, washed in nutritive medium № 199 to be placed into fresh medium № 199 to obtain homogenate in teflon homogenizer followed by centrifuging; then one should isolate the upper, medium and inferior layers, suck off medium layer and the upper part of inferior layer; the cell mixture obtained should be diluted in nutritive medium № 199 to be then introduced by injections into mesentery of small intestine or rectus muscle of abdomen. The present innovation favors the activation of immune system in patients undergone splenectomic operation and in those in case of surgical immunodefficient state due to high functional and regenerating activity of transferred embryonic splenic cells.

EFFECT: higher efficiency of prophylaxis.

6 dwg, 2 tbl

FIELD: medicine, genetic engineering.

SUBSTANCE: invention relates to applying genetic engineering approaches for treatment of autoimmune diseases, in particular, for treatment of cerebrospinal sclerosis. This is achieved by incorporation of one or some recombinant genes encoding autoantigens that represent a target for autoimmune response. In particular, invention claims a method for designation of gene encoding encephalitogenous epitope of proteolipid protein and expression of gene product in vivo by using the recombinant retroviral vector. Expression and secretion of encephalitogenous epitope improves histopathological and clinical indices in experimental autoimmune encephalomyelitis in mice that is used as a model of cerebrospinal sclerosis. The advantage of invention involves the development of a method for recovery the tolerance in treatment of cerebrospinal sclerosis being without suppression of immune system.

EFFECT: improved and valuable method for treatment.

6 cl, 13 dwg, 3 ex

FIELD: biotechnology and pharmaceutical industry.

SUBSTANCE: title operations are accomplished by following way. Three-dimensional culture of stromal cells is cultured in piston flow bioreactor, in particular being introduced in fibrous matrix incorporated into substrate, which is placed in container constituting a part of bioreactor piston. Stromal cells are grown until density 5 x 106 cell/cm3 substrate is attained, whereupon non-differentiated hemopoietic cells are either sowed directly into piston flow bioreactor or cultured in conditioned medium of stromal cells obtained by gathering medium from indicated flow bioreactor. Non-differentiated hemopoietic cells obtained by cultivation in presence of three-dimensional culture of stromal cells or their conditioned medium are transplanted to into recipient.

EFFECT: enabled growth of large amounts of stromal cells within a relatively small volume to provide longer maintenance of vital activity and reproduction of non-differentiated hemopoietic stem cells or precursor cells.

77 cl, 9 dwg, 3 tbl

FIELD: biotechnology and pharmaceutical industry.

SUBSTANCE: title operations are accomplished by following way. Three-dimensional culture of stromal cells is cultured in piston flow bioreactor, in particular being introduced in fibrous matrix incorporated into substrate, which is placed in container constituting a part of bioreactor piston. Stromal cells are grown until density 5 x 106 cell/cm3 substrate is attained, whereupon non-differentiated hemopoietic cells are either sowed directly into piston flow bioreactor or cultured in conditioned medium of stromal cells obtained by gathering medium from indicated flow bioreactor. Non-differentiated hemopoietic cells obtained by cultivation in presence of three-dimensional culture of stromal cells or their conditioned medium are transplanted to into recipient.

EFFECT: enabled growth of large amounts of stromal cells within a relatively small volume to provide longer maintenance of vital activity and reproduction of non-differentiated hemopoietic stem cells or precursor cells.

77 cl, 9 dwg, 3 tbl

FIELD: biotechnology, molecular biology.

SUBSTANCE: method involves transfection of cells HKB with vector pCIS25DTR comprising a selective marker and a sequence encoding protein eliciting procoagulating activity of factor VIII. Cells are selected using the selecting agent and clones with high level for expressing protein eliciting procoagulating activity of factor VIII are isolated. Invention provides preparing the protein eliciting activity of factor VIII with high yield, and strain of cells HKB with improved production under protein-free conditions also. Invention can be used for preparing the protein eliciting activity of factor VIII in industrial scale.

EFFECT: improved preparing and isolating methods.

8 cl,, 6 dwg, 1 tbl, 5 ex

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