The composition subjected to restriction analysis of cells capable of rapid growth, which produce substances that inhibit cell proliferation, and their use
The invention relates to medicine. The claimed compositions, which contain limited in reproduction proliferating cells. Under this restriction, the cells produce an unexpectedly high amount of a substance that suppresses the proliferation of cells and has a molecular weight of about 30 KD. This phenomenon does not depend on cell type, nor of their species. Disclosed are methods of making such compositions and their use. The invention provides the possibility of using cells other than cancer, for the treatment of cancer and other conditions. 16 N. and 94 C.p. f-crystals, 22 PL.
The present invention relates to the limitation of cell proliferation, which is on a logarithmic growth phase, or their ability to rapid growth in logarithmic phase, which are at the physical limit of their breeding biocompatible semi-permeable material, produce normal expressively or increased amounts usually expressed by the factor or factors that can inhibit the growth of other rapidly proliferating cells of the same or a different type and/or origin. Those affected by the restriction of the cells here are prolif who engages tumor cells, cancer cells and non-cancerous cells, including (but without limiting them) cells of the embryo, stem cells and cells in the recovery phase after tissue injury, including hepatocytes, fibroblasts and epithelial cells. Patterns, which are one of the features of this invention can be used by themselves or to obtain a material, for example, concentrates established with approximately molecular weight, which also have an antiproliferative effect on proliferating cells associated with the disease or associated conditions such as cancer, or for monitoring the growth of cells to prevent some medical problems that can occur when uncontrolled growth of cells, for example, in the formation of scars.
Background of invention and prior art
Encapsulation of various biologically active substances in a biologically compatible material which is adequately described in the literature, is a technique that has been used for some period of time, albeit with limited success. Examples from this area can serve US bach et al.); 4409331 (Lim); 4392909 (Lm); 4352883 (Lm) and 4663286 (Tsang et al.). It should also be noted US patent 5643569 issued by Jain et al. Jain et al., in detail discuss the encapsulation of islets in various biocompatible materials. The islets produce insulin, and because there is disclosed the application of the described Jain et al. products for the treatment of diabetes. In US patent 5888497 issued by Jain et al., describes agarose beads coated with a layer suitable for implantation of agarose containing cancer cells, subjected to the restriction that produce more of the substance that suppresses the growth is not subjected to the restriction of cancer cells, compared with the same quantity of the same (previously mentioned), but not subjected to the restriction of cancer cells.
In patents Jain et al. rather it discusses the methodology used previously in transplant practice. In addition, they summarized.
Famous five main approaches to the protection of the transplanted tissue from the immune response of the host. They are all based on the desire to isolate transplantirovannam fabric from the immune system of the host. Methods for immunotolerance today usually include extravascular diffusion chambers, intravascular diffusion chambers, intravascular St is provided with the previously used methods, including fibrotic reaction of the host to the implanted material, the instability of the implantable material, the limited diffusion of nutrients through the semi-permeable membrane, the permeability of secretion stimulator and product and lag-phase diffusion through a semi-permeable membrane filters.
For example, a method of microencapsulation for encapsulation of viable cells, tissues, and other labile membrane inside the shell, representing a semipermeable membrane, was developed Lim in 1978 (Lim, Research report to Damon Corporotion (1978)). Lim used the microcapsules of alginate and poly-L-lysine to encapsulate the islets of Langerhans (here called "Islands"). In 1980 it was reported the first successful application in vivo of this new method in the study of diabetes (Lim, et al., Science 210:908 (1980)). Implantation of these microencapsulating Islands supported euglycemic condition in animals with diabetes. However, when the repetition of these experiments, other researchers have found that alginate causes tissue reaction, and could not reproduce the results Lim, et al. (Lamberti et al. Applied Biochemistry and Biotechology 10:101 (1984); Dupuy, et al., J. Biomed. Material and Res. 22:1061 (1988); Weber, et al., Transplantation 49:396 (1990); Doon-shiong, et al., Transplantation Proceedings 22:754 (1990)). In nastojashemu these microcapsules in vivo (Dupuy, et al., see above; Weber et al., see above; Doon-shiong, et al., see above; Smidsrod, Faraday Discussion of Chemical Society 57:263 (1974)).
Iwata et al., (Iwata, et al., Jour. Biomedical Material and Res. 26:967 (1992) used the agarose to microencapsulate allogeneic pancreatic islets and found that it can be used as a material for the manufacture of microgranules. In this study, 1500-2000 islets were microencapsulated individually in 5% agarose and implanted mice with streptozotocin - induced diabetes. The graft was kept alive for a long period of time and recipients indefinitely continued normoglycemia.
However, this method has a number of disadvantages. It is time-consuming and inaccurate. For example, many granules are not completely covered, and a few hundred pellets remain in the form of free agarose. Thus, additional time is required to separate microencapsulation islets from "Unallocated" (blank) pellets. In addition, most of the implanted microcapsules accumulates in the pelvic cavity and in order to achieve normoglycemia, require a large number of individual granules, fully covered islets. Moreover, transplanted granules not be in the How macroencapsulation was also investigated. For immunovaccine islets were made microcapsule from a variety of materials, for example, poly-2-hydroxyethyl-methacrylate, polyvinyl chloride - acrylic acid, and cellulose acetate (See. Altman, et al., Diabetes 35:625 (1986); Altman, et al., Transplantation: American Society of Artificial Internal Organs 30:382 (1984); Ronel, et al., Jour. Biomedical Material Research 17:855 (1983); Klomp, et al., Jour. Biomedical Material Research 17:865-871 (1983)). In all these experiments was achieved only transient normalization of glycemia.
Archer, et al., Journal of Surgical Research, 28:77 (1980), used a hollow fiber of an acrylic copolymer in order to prevent the rejection of islet xenografts. They reported lengthening life expectancy is included in the hollow fiber neonatal rat pancreatic grafts that were transplanted hamsters with diabetes. Recently Lacy, et al., Science 254:1782-1784 (1991) confirmed these results, but it was found that euglycemic state is temporary. The authors found that when the islets are injected into the fiber, they aggregate inside a hollow fiber channel, which leads to necrosis in the core area of the islet mass. This necrosis in the center prevents the prolongation of life of the transplant. To resolve this issue raised many times. Therefore, the role of the membrane as a means for transplantation of islets in humans is questionable.
In the patent Jain et al., mentioned above, it is reported that the microencapsulation of secretory cells in permeable hydrophilic gel results in functionally useful non-immunogenic material that can be implanted animals, can be stored for a long time and is useful in therapeutic plan in vivo. Macroencapsulation secretory cells provided more effective and improved technique for transplantation of these secretory cells.
In the patent, in all its text does not discuss the inclusion of cells capable of rapid proliferation.
The literature review for encapsulating cells suggests that due to encapsulate cells almost always produce less nutrients than they produce without encapsulation. Cm. Lloyd - George, et al., Biomat.Art. Cell & Immob. Biotech. 21(3): 323-333 (1993); Schinstine, et al., Cell Transplant 4 (I): 93-102 (1995); Chicheportiche, etal., Diabetologica 31:54-57 (1988); Jaeger, et al., Progress In Brain Research 82:41-46 (1990); Zekorn, et al., Diabetologica 29:99-106 (1992); Zhou, et al., Am. J. Physiol. 274: C. 1356-1362 (1998); Darquy, et al., Diabetologica 28:776-780 (1985); Tse, et al., Biotech. & Bioeng. 51:271-280 (1996); Jaeger, et al., Neurol. 21:469-480 (1992); Hortelano, et al., Blood 87(12): to a rapid proliferation, inside the structure, which absorbs them and at the same time limits their growth, but nevertheless provides the diffusion of substances inside the structure and outside of it.
Some theory considering cancerophobia education, likens this kind of education, for example, tumors with normal bodies. Healthy organs, such as the liver, grow to a certain size and then the growth stops, however, if part of the liver to remove it to a certain extent. The same phenomenon is observed with respect to tumors. In conclusion, it should be noted that if the tumor be removed, the cells in the remaining part of the tumor will begin to multiply very quickly, until the tumor reaches a certain size, after which reproduction is declining and stops. This suggests that there is some internal mechanism for regulating the growth of cancer cells.
The invention, the essence of which is disclosed in this description shows that when cells are able to multiply quickly, physically limited, for example, by including any structure, their rate of reproduction decreases significantly and they produce unexpectedly Bo is quickly growing inhibits the propagation of these have not been subject to the constraint cells. The possibility of inhibition of proliferation of cancer cells is the original purpose of Oncology. Therefore, to be understood by therapeutic usefulness of this invention in the treatment of cancer and other diseases and conditions caused by rapid cell proliferation, and this will be explained in detail hereinafter. Apparently, produced substance is not limited by the type of proliferating cells used or the species of animal from which derived proliferating cells. Further, this effect, apparently, is not species-specific, as subjected to the restriction of the cells of the first type produce a substance inhibiting the reproduction is not subjected to restriction analysis of cells of the second kind. Further, with regard to cancer, apparently, this effect is not dependent on the type of cancer, as subjected to the restriction of the cells of the same type of cancer produce a substance that inhibits the reproduction is not subjected to restriction analysis of cells of another type of cancer.
This effect does not require also, apparently, and immune response. Antiproliferative effect seen in vitro systems using non-immune cells. Hence, it is clear that antium, a preferred variant of the invention relates to compositions of matter, having a biocompatible limiting reproduction, selectively permeable structure. This structure limits the proliferating cells, which as a result produce more of the substance that suppresses rapid cell proliferation, compared with an equal number of such proliferating cells, but not further restriction.
Another preferred variant of the present invention relates to a method for creating a biocompatible, limiting the reproduction of the selectively permeable structure through the formation of this structure by the interaction of proliferating cells with biocompatible limit reproduction substance to provide the above patterns and cultivation of these structures over an extended period of time in order to limit proliferating cells so that they have produced the substance, the vast proliferation proliferating cells, which must be suppressed as compared with an equal number are not affected by the restriction analysis of proliferating cells of the same type.
Another preferred variant of izobreteny the working cells, including the limitation of proliferating cells in a biocompatible, selectively permeable structure and the cultivation of proliferating cells so that they have produced the above substance. When placing these structures in the culture medium mentioned substance leaves these structures and enters into the culture medium. The resulting cultural environment is also a sign of the present invention.
In addition, it was found that a strong antiproliferative effect can be achieved by filtering the conditioned medium obtained by culturing structures of the present invention in a culture medium. Concentrates have an extremely strong antiproliferative properties.
The substance in question, air-conditioned environment and/or concentrates derived from it, can also be useful for inducing the production of the specified antiproliferative substances not subject to restriction proliferating cells.
In preferred embodiments, subjected to the restriction of the cells are cancer cells, but the benefits of using cells other than cancer, for cancer treatment and the other features of the invention will be understood from the following description.
A detailed description of the preferred options
In this example, followed by the use RENCA cells. This cell spontaneous adenocarcinoma of the kidney from BALB/C mice, which are widely available, stored in cultures in vitro and in vivo. Cm. Franco, et al., Cytokine Induced Tumor Immunoqenecity, 181-193 (1994).
Samples frozen RENCA cells were allowed to thaw at C and then transferred into a flask with a modified medium Dulbecco (D-MEM), which was added 10% bovine serum, penicillin (100 u/ml) and streptomycin (50 μg/ml) with the receiving environment, which will be referred to as "complete medium".
Cells were grown to confluence and then treated them with trypsin followed by washing in a balanced salt solution Hanks, and then the environment, the above "complete medium".
In order to determine how effectively produce RENCA tumor cells, two BALB/C mice intraperitoneally injected these cells in an amount of 106. The animals were observed over a period of 3-4 weeks. In the first two weeks by clinical signs they looked healthy and showed normal activity. Then the clinical manifestations of cancer became apparent. One mouse died after 23 days, and the second 25 days. After the death of m which were bleeding.
A sample from a single tumor, taken from one of the mice, fixed in 10% formalin for further histological validation.
After it was shown that RENCA cells grow in vivo, began the study aimed to identify, grow and whether these cells when they are limited in the structure according to the invention.
RENCA cells were grown to confluence as described above, were treated with trypsin and washed as described above. Then the prepared samples with a content of from 60000 to 90000 cells. Cells are then centrifuged at 750 rpm and the liquid was removed. The cells are then suspended in solutions of 1% atelocollagen in phosphate-saline buffer solution at pH 6.5.
Prepared 1% solution of agarose with a low viscosity at the minimum medium (MEM), kept at 60C and then 100 μl of this solution was added to the suspension RENCA cells and atelocollagen described above. Then this mixture was immediately transferred in the form of one large drops of sterile mineral oil at room temperature. This mixture formed a single smooth semi-solid granule. This procedure was repeated to obtain several granules.
A moment later, the pellets are moved in a complete environment, which openantivirus, listed above. The granules are then kept overnight at 37°C in a humidified atmosphere of air containing 5% CO2. After incubation, the pellets, the now solid was transferred to a sterile spoon, which contained 1 ml of 5% agarose in MEM. Granules rolled 2-3 times in the solution to achieve uniform application of agarose. Before the agarose hardens, the pellet was transferred into a mineral oil to get a smooth outer surface. After 60 seconds, the pellets washed five times in complete medium at S to remove the oil. After that exercised restraint during the night when C in humidified air with 5% CO2.
Received RENCA containing granules used in further experiments.
Before you carry out in vivo studies, it was necessary to determine whether RENCA cells grow inside the granules, prepared as described above.
For this purpose, the granules obtained as explained in example 2, were incubated in the medium described in example 2, for three weeks under the above conditions. Three pellets were cut into small pieces and cultured in standard culture flasks, allowing direct contact with the bulb, Macartney RENCA colony. This proves that the cells in the pellet remained viable.
Then were brought in vivo studies. In these experiments, the pellets were incubated for 7 days at 37°C. Experimental animals introduced granules. To this end, each of the four mice did midline incision, performed intraperitoneally. Three pellets, each of which contained 60000 RENCA cells were transplanted. The incisions were then stitched (double-layer closure) using absorbable suture. Four mice (BALB/C) were normal (mouse - males, weighing 24-26 g) and seemed healthy. Were made 2 series of controls. In the first series two mice were injected 3 granules without RENCA cells, two mice not treated with anything.
Later, 3 weeks after implantation, all mice received intraperitoneal injections of 106RENCA cells. 18 days later one control mouse died. All remaining mice were then euthanized and examined for the presence or absence of a tumor.
The control mice were numerous tumors, while mice that received implants encapsulated in granule cells showed only small nodules throughout the cavity.
These encouraging results formed the basis of the plan of the experimental the diversified types of cancer by introducing RENCA cells under the renal capsule of each of the six BALB/C mice. After 15 days, mice were divided into two groups. Three mice in the first group introduced three pellets as described in example 4 above. The second group (control) was injected pellets not containing RENCA cells.
In the first 4-5 days of a mouse that received implants containing RENCA cells, looked sluggish, apathetic, wool they have puffed up.
After they returned to normal. Animals of the control group remained full of energy, their hair has not changed.
10 days after implantation (25 days after injection of RENCA cells), however, mice from the control group were slow, the abdomen is swollen. One of three control mice died on the 14th day after transplantation granules. Then the mice were euthanized.
In the body cavity to the control mice was found profusely bleeding with multiple tumors throughout the digestive tract, liver, stomach and lungs.
All the abdominal organs become unrecognizable due to lesion of rapidly growing tumor. In mice which were injected pellets encapsulated with RENCA cells, was not observed, however, the bleeding, but only a few knots in the digestive tract. In addition, comparison of experimental and kontrolpaketes capsule and prior to implantation of microgranule.
In vitro growth of freely insulinaemic RENCA cells inhibited when these cells are incubated along with the encapsulated into microgranule RENCA cells.
The next series of experiments was performed in order to determine, does this effect other cells.
Line adenocarcinoma cell, i.e., MMT (tumor of the mammary gland of the mouse) was obtained from the American type culture collection. As described above prepared encapsulated MPM cells based on MPM cells, obtained granules containing from 120000 to 240000 cells in the granule. After receiving the pellets used them to determine whether they inhibit the proliferation of RENCA cells in vitro. In particular, prepared two 6-hole Petri dishes and were inoculable on 1x104RENCA cells in 4 ml of medium in the well. In each Cup three wells were used as control and three were experienced. In one of the three control wells in each plate were placed one empty granule. In each of the other holes were placed or 2, or 3 empty granules. The second series of holes were processed similarly with the introduction in wells 1, 2 and 3 pellets containing and 120000 240000 MPM cells. The cups were incubated at S within one week, after which RENCA cells were treated with trypsin Example 7
After receiving the results of example 6, the same experiments were done using as inoculum 1104MPM cells (i.e. empty cells) instead of RENCA cells. The experiment was carried out exactly as in example 6.
The results are shown in table 2.
These results confirmed the in vivo experiment. These data are presented in example 8.
Used a cell line of breast cancer mouse (MMT), described above. Using the above techniques have prepared implants containing 120,000 cells per pellet and 240,000 cells in the granule.
Used an experimental mouse model. 22 mice were divided into groups consisting of 4 (control), 9 and 9 individuals. The first group (i.e. control) was still divided into three groups: two received the implant from one free granules, one got 2 free granules and one received 3 free granules.
Within the experimental group (9 animals) granules contained 120,000 cells, whereas in the experimental group In the granules contained 240,000 cells. Within groups a and b were three subgroups, each of which had 3 mice. Subgroups of mice received one, two or three granules containing MMT cells.
After another 20 days in control mice increased abdominal cavity and greatly useralias coat. One control mouse died 25 days after injection, while the rest of the control mouse was already dead. All mice were dissected for observation developed a tumor. These observations are given in table 3.
These results show that, of the 18 treated mice 13 are not sick. As for the mice in group a, one mouse had a few small knots (+), and another mouse - multiple tumors (++).
Within the group In one mouse that received one pellet and one mouse that received 2 pellets had multiple tumors in the intestine. One of the mice that received 3 pellets, developed a large solid tumor, and she was clearly very sick (+++). All control mice had numerous tumors (++++).
The results showed that the encapsulated cells of the mouse mammary tumor inhibit the formation of tumors.
According to the aforementioned assumption, the implementation of the present invention results in a prophetic is xperimenta, which is described above.
As described above in example 2, additionally prepared pellets, but without the inclusion of atelocollagen. Therefore, these granules had the structure of agarose - agarose. RENCA cells, as described above, were included inside these granules, again respectively visitandome description.
Two series of three 6-hole plate was used as a control and experimental group. In the control group in holes made in 4 ml RPMI complete medium (10% fetal calf serum and 10 ml/l penicillin). Each cell of the control group was then inoculable 10000 RENCA cells.
In the experimental group RPMI complete medium was conditioned by adding substances resulting from incubation of ten RENCA containing granules (120,000 cells per 1 grain) in a Petri dish size 35100 mm containing 50 ml of RPMI complete medium. After five days of incubation environment with these cups were collected and 4 ml was placed into each pilot hole. These wells are then incubated, making 10000 RENCA cells in each.
Both plates (and the control and experimental) were incubated with S within five days. At the end of incubation cells were treated with trypsin, washed, collected and counted using hemocytometer inclusion, for example, the granules according to the examples, produce a substance that inhibits the multiplication of tumor cells.
Experiment description of which are described above, showed that the growth of RENCA cells in air-conditioned environment is approximately two times less than the growth of cells in the control environment. Using the described experiments examined whether to continue the suppression of reproduction, if an air-conditioned environment frozen.
RENCA-conditioned medium was prepared by incubating ten RENCA containing granules within 5 days. Incubation was performed in 35100 mm Petri dishes with 50 ml RPMI complete medium at S. After incubation the medium was collected and stored at-20C. Conditioned medium was prepared by incubating the pellet containing cells MMT (tumor of the mammary gland of the mouse). Pellets were kept 240,000 cells each; in all other respects the terms of the experiences were the same.
Frozen environment, allowed to thaw at S and then used in the following experiments. Three 6-hole plate was used for each treatment, i.e., (1) RPMI control environment, (2) RENCA frozen air-conditioned environment, (3) MMT frozen air-conditioned environment. In General, the rule of law 5 days. After incubation, samples were taken from each cell of the two plates, were treated with trypsin, washed, collected and counted using hemocytometer. After eight days the contents of each cell of the three remaining plates were investigated in the same way.
The results are given in table.5.
If we compare these results with those in example 6, above, it will become clear that, despite the fact that frozen and thawed RENCA air-conditioned environment and does not suppress proliferation to the same degree as frozen and thawed MMT air-conditioned environment (compare examples 6 and 7), it is, however, inhibits proliferation.
The experiments described above showed that the frozen air-conditioned environment, and RENCA-and MMT-containing granules, inhibits the proliferation of RENCA cells in vitro. In the experiments described now, checking whether to inhibit the reproduction of RENCA cells in vivo environment, air-conditioned RENCA - or MMT-microgranuloma. Effects of air-conditioned microgranuloma environment compared with the effects of the environment, air-conditioned in the presence not subjected to restriction RENCA and MPM cells growing in monolayer cultures, in order to determine whether not aliteracy.
For these experiments, 10 microgranule, each of which contained 120000 RENCA or MPM cells (i.e., 1,2106cells only), used for conditioning medium (complete RPMI) during the 5-day period. In parallel 1,2106RENCA or MPM cells, i.e., the same number of cells were added to the culture Cup and then they multiplied in the form of a monolayer in complete RPMI medium for 4 days period of time. The medium then was replaced by another and the medium was collected after 24 hours the Reason for different durations of incubation, pellets and not subjected to restriction analysis of cells was the difference between the amounts of cells in monolayers compared with granules (3-5-fold excess of the number of cells in monolayers) at the end of the 5-day period. In other words, not subjected restrictive cells grew much faster than the encapsulated cells, so they were 3-5 times more.
To obtain concentrates air-conditioned environment, which were supposed to contain the active substance with the exception of toxic metabolites and/or waste cells as co-factors of the experiment, used filters with a pore size of 30 kilodaltons and 50 kilodaltons. Contaminating substances to which any interpretation of the results with this material is complicated by the presence of waste cell metabolism. Serum-free medium (AIM V) was also used in some experiments in order to ensure that any effects due to the presence of serum, can themselves be controlled.
Basically, conditioned medium was collected or 3-5 days after microgranule were added thereto, or after 24 h after addition of fresh medium to the cells not subjected to restriction analysis. The environment then was placed in a filter for the experiments in the form of tubes with an appropriate filter (or 30 KD, 50 KD), and centrifuged for 90 min Mass, which remained on the filter, called the "concentrate", whereas that which has passed through the filter and gathered at the bottom of the tube, was the filtrate.
The results are summarized in tables 6, 7 and 8; they show that when used conditioned medium obtained from RENCA cells subjected to restriction in microgranular, it inhibited the proliferation of RENCA cells in two separate experiments by about 52%. Concentrate 50 KD inhibited proliferation by about 99% in both cases, while the concentrate 30 KD inhibited proliferation by approximately 97%.
The important point in this experiment is the fact that MPM cells and RE RENCA cells, demonstrating that limit reproduction the effect is not specific to the type of tumor. These experiments confirm that the experiments of example 8, in which the IMT-containing microgranule inhibited the proliferation of RENCA cells in vivo. In addition, they have expanded the data obtained and showed that the substance is released from microgranule on Wednesday, consists of molecules with a mass of at least 30 KD, which to some extent are responsible for limiting the proliferation effect. Finally, these experiments show that the limited inclusion in microgranule RENCA and MPM cells produce significantly more vast proliferation of matter than the same cells grown as a monolayer.
These experiments show that air-conditioned as MMT and RENCA cells, the medium contains a substance released from cells included in microgranule and suffered as a result of this limitation in reproduction. This substance can inhibit the reproduction of RENCA cells in vivo and in vitro. It is important that the experiments show that the effect of inhibition of reproduction is not specific in relation to the type of tumor. The described experiments reveal whether this effect is independently researched and, does cancer cell line human breast suppressing proliferation effect in vitro on RENCA cells (when using microgranule and air microgranuloma environment) and the IMT-cells (using only air-conditioned microgranuloma environment).
Methodologically, these in vitro studies are similar to the experiments described in examples above. 1000000 MCF-7 cells (breast cancer cells human) was encapsulated in microgranule, and the resulting MCF - 7 microgranule incubated with RENCA cells (10000 per cell) for 5 days in order to evaluate the inhibiting proliferation effect of microgranule. In addition, within 5 days of incubation was prepared environment, air-conditioned MCF - 7 microgranuloma; it was tested and RENCA, and on MPM cells. Cell proliferation was determined after 5 days.
The results are shown in table.9-11.
These results show that cells MCF-7 belonging to the line of adenocarcinoma of the breast of a man being included in microgranule and limited in relation to reproduction, produce a substance that inhibits the proliferation of cells of renal adenocarcinoma mouse cells and mammary tumor mouse sachimi environment. This shows that the effect of inhibition of reproduction shown limited growth of cancer cells, does not depend on tumor type or the species of animal has a tumor, i.e., mouse and human.
The experiments described above demonstrate that the cell line, isolated from adenocarcinoma of human breast (MCF-7), being limited in growth when included in microgranule, provides inhibition of cell multiplication renal adenocarcinoma mouse and adenocarcinomas of the mammary gland of the mouse in vitro. The described experiments aim to test whether the in vivo parallel effect MCF-7 containing microgranule on the growth of RENCA tumor cells.
Eighteen Balb/c mice intraperitoneally introduced 200000 RENCA cells. After 3 days, mice were divided into two groups. In group 1 there were six mice, and in group 2 - the remaining 12 mice. Mice from group 1, control, were transplanted every three empty microgranule. Each mouse of group 2 transplanted three MCF - 7 - containing granules (100,000 cells per pellet). After 25 days, two mice from group 1 and three mice from group 2 were euthanized. The same number of mice was killed on the twenty-sixth day and the remaining mouse OneNote tumors of the abdominal cavity, and normal bodies were unrecognizable. This tumor saturation was classified as+++++ (100%). In mice subjected to processing, tumor saturation was + (10-20%).
These results show that microgranule containing cell adenocarcinoma of the breast of man, is able to inhibit the growth of cells adenocarcinoma tumors in mice, Reaffirming that the effect of inhibiting the growth of tumor/cancer cell proliferation is independent of tumor type or from a specific species, the source of its origin.
The above experiments demonstrate that the effect of inhibition of cell proliferation/growth under the action of tumors whose growth is limited by microgranuloma, does not depend on the type of tumor, or from species of its origin. The described experiments aimed to examine whether the cell adenocarcinoma of the mammary gland of the mouse, limited in reproduction inclusion in microgranule, to inhibit the growth of spontaneous mammary gland tumors, and tumors resulting from injection of MPM cells.
In experimental animals mice have a very high probability of developing breast cancer throughout jisne five of these seven mice implanted by 4 MMT of microgranule, containing 100,000 cells each. The other two control mice received 4 empty microgranule each. Two control mice developed large tumors and they died within three months after implantation of pellets. Mouse treated, were killed and 11 months after implantation of the IMT of microgranule. Retrieved microgranule, organs and tumors were examined macroscopically and histologically. Staining MMT of microgranule hematoxylin and eosin revealed viable cells. Existing tumors was not increased in size and it was obvious the lack of development of any new tumors.
We also conducted experiments by subcutaneous introduction of MMT tumor cells in the breast area. Fourteen experimental animals mice were divided into 2 groups. Five mice from the control group implanted by 3 empty microgranule each. Nine treated mice received three MMT - containing microgranules (240,000 cells) each. Later, 3 weeks after implantation, all fourteen mice subcutaneously introduced in the area of the breast on 200000 MPM cells each.
Between the 25th and 30th day five control mice became sick with the apparent formation of tumors, and they all died to the 35th day after Ynez any obvious sign of cancer or no loss of health. After 10-12 months after injection, tumors in four of the nine treated mice appeared nodules and in these "foci" got the coat. The remaining five mice again implanted three MMT of microgranule 13 months after the first injection of the tumor. One mouse died 3 days after the operation, but at autopsy, no tumor was not found. Four surviving mice were euthanized after 8 months after the second implantation microgranule. The autopsy showed minimal or no tumor growth.
Further observation of these experiments was that the pellets extracted after the first implantation, contained viable tumor cells that is based on histology and their ability to resume aggressively growing tumor patterns in tissue culture after extraction of the granules.
The results of these experiments show that the effects of limited granules of cancer cells by inhibition of cell proliferation/growth of the tumor in relation to the cells of adenocarcinomas of the mammary gland of the mouse can affect the development and growth of spontaneously arising tumors, and experimentally induced tumors resulting from injection of tumor cells into the military in microgranule and limited reproduction of cancer cells in relation to cell proliferation/growth of the tumor, which manifests itself regardless of tumor types and regardless of species sources of these cells, as well as against both spontaneous and artificially induced tumors. The described experiments are designed to extend the obtained results to study the effects included in microgranule and bounded multiplication of cells of adenocarcinoma of the prostate man (ARCap 10), renal cell adenocarcinoma (RENCA cells) mouse (Balb/c) and cell adenocarcinoma of the mammary gland (MMT), mouse (AWN) on the proliferation ARCaP10 tumor cells and tumor growth ARCaP10 naked mice (Nu/Nu).
In the first series of experiments, fifteen Nu/Nu mice were introduced subcutaneously in the flank of 2,5106ARCaP10 cells. On the twentieth day after the injection, when the average maximum size of tumors reached 0.5 cm, mice were divided into 2 groups. Nine mice implanted for 4 ARCaP10 microgranule (1,0105cells on microgranule), and six control mice received 4 empty microgranule.
After 10 weeks after implantation of five control mice were found very large vascularization tumors (average 2.5 cm in diameter) and one mouse had a tumor, a smaller (less than 0.5 cm). In group s was without tumors up to killing eight months. Two mice revealed the absence of tumor growth, i.e., tumors were of the same maximum diameter, which they had during implantation microgranule, and two mice had tumors that grew from the moment of implantation microgranule.
These results [volume and tumor size (DL. x Shire. x h)] the experiment, which RENCA - containing microgranules (1,2105) were implanted 4 Nu/Nu mice after 18 days after subcutaneous injection each animal in the side on 3,0106ARCaP10 tumor cells, shown in the table.12, 13.
In another experiment 10 Nu/Nu mice were injected on 2,5106Arsar cells, and six of them have been observed tumor development after 64 days after injection. Three of these mice were introduced on 4 MMT of microgranule (2,4105cells), and three empty microgranule. The results are shown in table.14, 15.
The results of these experiments confirm again does not depend on the species of cell source and nature of the tumor suppressing tumor growth effect of the restriction of reproduction provided for tumors of various types. In addition, these experiments demonstrate the ability of the limited reproduction of cancer cells by Nichola in vivo.
The experiments described above have shown that cancer cells, reproduction is limited, derived from different tumor types from different species can inhibit the proliferation of cancer cells of the same or other types of cancer in vitro and to prevent the formation of both spontaneous and induced tumors and prevent tumor growth and induce regression of tumors in vivo, irrespective of the species of the source of such cells or cancer type. The experiment described here concerns the dissemination of these findings with regard to other species of animals (rabbit), and tumors of rabbits caused, as you know, viruses (VX2).
In this experiment, a new Zealand white rabbit (2.5 lb) was intramuscularly injected in one hip (at different points) 0.5 ml in each point VX2 tumor suspension (which can be characterized by the fact that she had the ability to pass through the needle 26-gauge). 3.5 weeks on the dorsal side of the femur was found a tumor size of 5 2,5 cm (length x width) and on the ventral thigh two tumors with a diameter of 3 see At this time were implanted intraperitoneally 211 microgranule (108 granules with RENCA cells, 63 granules with MPM cell is ilaci about 50%; however, two ventral tumor has not decreased. Animal killed 10 days after implantation microgranule. At autopsy, it became clear difference between the dorsal and ventral tumors, namely, that the first was much smaller than it was during implantation microgranule, while two ventral tumors were bleeding, and necrotic.
This experiment extends the conclusions regarding the effectiveness of the restriction of reproduction of different types of cancer cells to prevent, suspend and even regression of tumors in other species of animals, particularly rabbits. In addition, it complements the list of types of tumors, in respect of which shows this response, tumors of viral origin, and, finally, confirms the cross in relation to the tumor and the species of origin of tumor cells in nature inhibiting the growth of cancer cells effect, as used microgranule with cells of kidney cancer mouse breast cancer mouse and breast cancer person. In addition, in this experiment used a model developed on a larger animal that is very important to demonstrate in vivo effective is opisanie experiment shows the restriction of the multiplication of tumor cells of different types leads to their ability to inhibit the growth of cells of the same or of different types in vitro and to prevent, inhibit the growth of, or cause regression of various types of tumors in vivo and found that the effects do not depend on tumor type or origin of cancer cells. In the experiments described now evaluated the long-term viability is limited in terms of reproduction RENCA cancer cells inside the agar/agarose microgranule maintained in culture for 1 month, 6 months, 2 years and 3 years using histological, kulturalny and in vivo methods. MMT - containing microgranule kept in culture for up to 6 months. In addition, RENCA - and MMT-containing microgranule extracted from Balb/c and AWN mice, respectively, after a period of from 2 to 8 months after implantation, were tested for the presence of viable cancer cells as histological and cultural methods.
For these experiments was prepared by the agar/agarose of microgranule with either 1,2x105RENCA cells, or with 2,4105MPM cells. They were verified histologically (hematoxylin + eosinophile staining) and Kemeny. As for RENCA of microgranule, the number of cells increased approximately 3 - 5 times for the first month followed by an additional doubling in 6 months. After 1 year there has been a continual increase in cell mass, but the rate of cell proliferation was reduced. 2 years later amorphous material began to accumulate in the center of the granules and, apparently, the cell mass/number of cells did not increase, although the signs of mitosis were still evident. Three years later, in the centre of the granules apparently was still some more amorphous material, but the mass of cells/number of cells was stable. For the MMT microgranuloma watched only 6 months, but the early nature of cell proliferation and the appearance of the granules were such as to RENCA cells.
To assess the viability and biological properties of RENCA and MPM cells in the ranges listed above, broke 10 pellets and sown them in 2 or more than 25 cm2flask for tissue culture in complete RPMI medium. Then watched the growth of cells in flasks. The passage of time from 1 to 6 months, the number of viable cells isolated from granules increased. To year the number of RENCA cells, grown from crushed pellets, was the same as for 6-month. The second and third googo number, the obtained pellet (i.e., able to limit the growth) after three years of cultivation.
To evaluate the extracted RENCA and the IMT of microgranule (periods 1-4 for RENCA of microgranule and up to 8 months for the IMT of microgranule) still used histological methods. Nature cell proliferation and mass cells are very similar to those in the pellets maintained in culture within the relevant time periods, i.e., cells increase in number, at least up to 4 months for RENCA and 8 months for MMT.
For other types of lines of cancer cells such as MCF-7 and ARCaP10, pellet samples of the viability similar to those for RENCA and MMT.
These experiments show that cancer cells can be maintained in vitro for periods of time up to 3 years and in vivo for at least 8 months in the environment, limiting reproduction, and that they retain their viability during these periods of time, clearly demonstrating the increasing number of cells including at least one year. This is important not only in order to be able to create and maintain funds for cancer treatment, but also for the ability of cells to limit breeding to develop teplokrov, it will be necessary for successful treatment of experimental or naturally arising cancer.
The above experiments show that the cancer cells of different types can be supported and kept in conditions that limit reproduction in a long time (up to 3 years) while maintaining their ability to proliferate to form tumors and to allocate inhibiting the proliferation of cells and prevents tumor growth suppressing and destroying its substance. Describing now the experiment for the evaluation of the possible toxicity of long-term (one-year) implanted Balb/c mice the agar/agarose microgranules containing cancer cells.
Seven Balb/c mice implanted 3 RENCA of microgranule each (1,2105cell pellets). Immediately after surgery, the mouse began to feel sick (sticking out of the coat and apathy) in a few days, but then again they looked healthy. All mice survived in a good state of health during the period of time of at least 1 year, and one mouse died being old, and the other died of other causes. All mice were euthanized. At autopsy, there has been no anomalies, such as fibrosis, peritonitis to the serosal surfaces of the intestines, especially where there were intestinal loops. No interference with the normal function or structure of the intestine were observed.
These results show that the agar/agarose of microgranules containing cancer cells, are well tolerated by the animals in the experiment during the period of time over 1 year. These findings show that granules with cancer cells with constraint propagation can be used in vivo for the prevention, suppression and regression of cell reproduction, including in vivo tumors of various types.
The following examples provide additional evidence that, in connection with the present invention, immobilized stem cells can effectively inhibit the growth of stem cells (even stem cells other lines), and that immobilized cancer cells inhibit the growth of stem cells.
Two different murine embryonic stem (ES) cell lines (i.e., ES-D3 and SCC-PSA1, which is publicly available) obtained from the American type culture collection ("ATSS").
Both lines are grown in standard culture conditions, which include cultivation in the form of atonic "STO" embryonic fibroblast feeder cells monolayer. These cells is whose serum, factor, leukemia inhibitory (LIF), and (-mercaptoethanol (common name for the environment "Environment And"). Cells, which after receiving kept in the cold (cryopreserved), thawed and defined as crops, at least after 3 passages before to cultivate, as described above.
After three days culture ES cells at 70-80% is a monolayer, it is treated with trypsin, and then immobilized on the agarose pellets. Use agarose Sigma XII, with an initial concentration of about 1.0%. An aliquot of 100:1 agarose solution is added to 34:1 cell suspension. The resulting granules contain 2,0105±1,5104mouse embryonic stem cells. The granules are covered with a second layer of agarose with a concentration of about 5%. Pellets were cultured in the above medium, except that no LIF or viable STO nutritious cells ("Environment").
Cell viability in granules in time evaluate conventional histochemical studies and research under the microscope, as well as the standard MTT assays using cells isolated from granules or remaining in the granules, at different points in time.
It is observed that immobilized stem cells is wow, as cells die due to apoptosis, reaching the lowest point in the metabolic activity of about 21 days. However, after this low point the surviving cells slowly proliferate, and there is a gradual increase in overall metabolic activity on a 35 day after immobilization and forth. These results are consistent with observations on cancer cells immobilized.
Morphologically there is a noticeable difference between the colonies formed in the inner layer of agarose pellets cancer cells, and colonies formed by stem cells. Although both types of colonies are ovoid shape, the colonies formed by cancer cells that are characterized by external area of viable cells (typically, the thickness of the two or three cells) with the Central area eosinophilic cellular debris. On the other hand, the colonies formed by stem cells, fully engaged in viable cells, and the Central area of the cell debris is missing.
In this experiment validate the inhibitory effect of stem cells on the proliferation of other stem cells.
The viability of a ten-day stem cells contained in the agar/agarose GURNEE 6 days. After 6 days the environment condition using immobilized stem cells. In the future, it is called "Medium, conditioned by stem cells" (SCM).
After 6 days of SCM is transferred into a 6-hole tablets containing fresh cells SCC-PSA-1. Each of these tablets contains h5STO nutritious cells, covered with 1,5x104SCC-PSA1 cells. The STO cells treated with mitomycin C to prevent proliferation. There are three control wells, i.e. wells that contain Environment (reconditionnement environment), and three wells that contain SCM.
After 3 days the contents of all wells treated with trypsin and consider the total number of cells using standard methods. The correct results in the calculation of the 910 nutritious cells. The results are shown in table.16.
In an analogous experiment, the following results are obtained (see tab.17).
Further, the effect is not specific regarding cell lines, as shown by the results obtained when adding in the cell environment ES-D3 (see tab.18).
In Example 20 shows that the inhibitory effect of stem cells on the proliferation is not specific in relation to cell lines. In the OPI is where cancerous cells.
In these experiments, use RENCA tumor cells. In each well seeded 15000 tumor cells. Use SCM (air-conditioned or SCC-PSA-1 or ES-D3), as described above, as a control use Environment In the same way as described above.
As for the SCM, the conditioning is carried out for 5 days. The analysis is performed within 32 weeks. Inhibition of RENCA cells determined by fixing the cells with 100% methanol, followed by staining with neutral red, lysis with SDS and scanning by using a spectrophotometer to determine the amount of neutral red in the cell lysate, which is proportional to the number of cells per well.
The results are combined in tables 19, 20, which shows the effect of ES-D3 and SCC-PSA 1 stem cells, respectively. The results for weeks 1-3 correlate with the results discussed in Example 1, i.e., the death of immobilized stem cells reaches the lowest point on day 21 followed by regeneration.
In previous experiments verified and confirmed by the ability of the immobilized stem cells to inhibit the proliferation of stem cells and cancer cells. Digislide the radio stem cells.
Stem cells are placed in tablets and cultured as described above. Granules containing RENCA cells, obtained as described in U.S. patent 6303151; 6224912 and 5888497, cultivated in the Environment with a view to its conditioning for 5 days. Then, this air-conditioned with RENCA medium (RCM) add in tablets of stem cells and after 3 days the stem cells is considered. The results presented above represent the data for the cells of ES-D3, and then cells SCC-PSA1 (see tab.21, 22).
These results indicate that immobilized cancer cells inhibit the proliferation of stem cells.
The above examples describe the invention, which includes, inter alia, compositions that can be used to obtain a substance that inhibits or regulates the proliferation proliferating cells, in particular, provides suppression of cancer cells. These compositions contain cells in the logarithmic growth phase (phase of rapid reproduction) included in the selectively permeable material forming a structure that restricts the reproduction of the included cells. The resulting cells undergoing this limitation, produce unexpectedly high the cancer cells. Cells subjected to restriction (restriction), produce more of this substance than comparable quantities of cells not subjected to the restriction.
The material used for the formation of structures of this invention, is any biocompatible material, which limits the growth of proliferating cells, stimulating them to produce large quantities of the substance that suppresses the growth and reproduction of cells. This structure has a suitable pore size, so the above suppressive substance can diffuse outwards into the external environment, and protects the produced substance and cells from the immune system of the host, from the ingestion of this structure any products and cells and induction of exclusion need of cancer cells or, in other words, weakening their ability to survive and continue to produce the desired substance. The material used to create this structure must also ensure the preservation of viability (limited to the reproduction, but surviving) cells both in vitro and in vivo, preferably for periods of time up to several years, supporting the flow of relevant nutrient ELA. The material used to obtain this structure, preferably, is well compatible material for implantation in vivo, and most preferred is well tolerated by the owner throughout the implant period.
Limitiruyuschie list of materials and combinations of materials that can be used include alginate-poly(L-lysine); alginate-poly(L-lysine)-alginate; alginate-poly(L-lysine)-polyethyleneimine; chitosan-alginate; polyhydroxyethyl-methacrylate-methyl methacrylate; carbonylmethyl; K-carrageenan; chitosan; agarose-polyethersulfone-hexade-meterin-bromide (Polybrene); ethylcellulose; silica gels and combinations thereof.
Patterns that contain the composition of substances, can have different forms, for example, granules (grains), spheres, cylinders, capsules, wafers, or any other form which is suitable for implantation of the individual and/or in culture medium in vitro. The size of the structure can vary depending on its potential use, that should be clear to a skilled specialist.
Structures of this invention have selective permeability, so that the nutrients can penetrate into the structure, and inhibiting the proliferation of the s these structures were guarding "the entrance" products or cells of the immune system of the host, what would cause a rejection subjected to restriction analysis of cells or, in other words, impair the ability of cells to produce the vast multiplication of substance.
Another aspect of the invention includes compositions that can be applied to suppress the proliferation of cancer cells. These compositions are prepared by culturing subjected to restriction analysis of cells, as described above, in an appropriate culture medium, followed by separation of the resulting air-conditioned environment. The air-conditioned environment can then be obtained concentrates, for example, the Department of fractions having a molecular weight of more than 30 KD or more than 50 KD, which exert strong antiproliferative effects on cancer cells.
The invention is not limited to any particular type of proliferative cells. Any proliferating cell, in accordance with defined above, can be used according to the invention, including (but not limited to, tumor cells, cells of the embryo, stem cells, cells in the recovery phase, for example after damage or injury, in particular, hepatocytes, fibroblasts and epithelial cells. Cancer cells are predpochtitelnye breast cancer, prostate cancer cells, cell choriocarcinoma and so on. Cancer cells may be epithelial, mesothelial, or endothelial cells of embryonic origin and include cancer cells that do not form solid tumors, for example, leukemia cells.
As should be clear from this description, another aspect of the invention are therapeutic methods for treatment of persons suffering from hyperproliferative diseases such as cancer, inflammation, fibrosis and trophoblasts. The subject of the invention is to control the growth of cells in the proliferative phase, so that they were reproduced in a controlled way. This is particularly useful, for example, for controlling the formation of keloids or scar tissue, preventing adhesion after abdominal surgery, or perhaps in hyperproliferative skin diseases, e.g. psoriasis. When used in therapeutic purposes, as will be proved below, the type of cells that are limited in growth by inclusion in a specified structure may not necessarily be the type of cells that causes (or may cause) of suffering of the patient, although it may be so. One method includes the introduction, craissati suppression of cell proliferation. Preferably, this method is applicable for the treatment of cancer, and the individual is a man, although it applies to animals, such as Pets, farm animals or any other animal that has cancer.
The composition of the present invention can be used for primary therapy of hyperproliferative diseases, as well as additional treatments in combination with other therapies. For example, patients can be treated using the compositions and methods described here, in addition to well-known therapies, such as radiation, chemotherapy, gene therapy, treatment with other biological active substances such as cytokines, substances, dulling the sensitivity (containing drugs), steroid hormones, etc., Compositions and methods of this invention can also be used in addition to surgical procedures, cancer treatment, for example, for implantation of microgranule after excision of the tumor to prevent its re-growth and metastasis. Cancer in inoperable condition can be turned into an operable, when used for the treatment of antiproliferative compositions of this izobreteniya cancer, for example, in the presence of individual risk factors, mainly at hereditary risk factors in General, when an inherited risk factor for cancer of a certain type (for example, breast cancer), exposure to impact professional or other carcinogens and substances that cause cancer. When cancer prevention prophylactically effective amount of structures of the invention is administered to face when establishing one or more risk factors.
As shown in the examples above, the antiproliferative effect is not determined by the type of proliferating cells or species of animal from which derived proliferating cells. However, you can enter structures that contain, for example, cancer cells of the first type, the individual with another, different from the first type of cancer. Further, the proliferating cells of species other than species that are subjected to treatment, can be used in the input structures. For example, cancer cells of the mouse can be included in the structure in accordance with the invention and is limited in reproduction and then introduced the man. Of course, the structure may contain cancer cells of the same species of animals, and that animals subjected to the treatment. More togoro, limited reproduction and then entered the same individual. In other words, their own cancer cells of the patient can be subjected to a limitation in reproduction and used to suppress the same type of cancer.
Another aspect of the invention reflects the use of concentrates, which are described here, as therapeutic agents. These concentrates can be prepared as described above, then be introduced to a subject with a hyperproliferative disease such as cancer, or a patient who needs to control the rate of cell multiplication, for example, a postoperative patient who suture the wound is in a place where excessive scar tissue will cause harm or simply unwanted. All the options described above can be used for making concentrates. For example, after in vitro cultivation structures containing cancer cells of a mouse, can be prepared concentrates and introduced to people. Similar patterns can contain human cells and even cells derived from the same person. In addition, as discussed above, the type of cancer cells used to obtain the concentrate may be, but not necessarily, the same smirker, to obtain a concentrate, which will be applied for the treatment of people with melanoma, or an individual with prostate cancer can be removed part of the tumor cells of the prostate, is included in the structure according to the invention, subjected to cultivation in an appropriate environment and then by filtering the resulting air-conditioned environment dedicated concentrate. It should be borne in mind that the conditioned medium obtained by culturing in vitro structures of the present invention, is also the subject of the invention.
Methods of obtaining structures of the present invention, as these concentrates are the subject of this invention. In the case of concentrates, you can just cultivate the structure obtained in accordance with the invention, over a period of time sufficient to produce the required number antiproliferative substances, and then to separate the desired part from the received air-conditioned environment, for example, filtration through a filter with an appropriate limit cut-off, for example, 30 kilodaltons or 50 kilodaltons.
Other aspects of the invention will be clear to the skilled technician and is not summarized here.
The terms and expressions to icepay and does not seek to exclude any equivalents disclosed and described signs or parts thereof, this implies that in the scope of the invention various modifications are possible.
1. Composition for suppressing cell proliferation obtained by including a sample of rapidly proliferating cells in biocompatible material with obtaining permeable structure, limiting the reproduction of these cells, culturing the specified patterns in culture medium for a time sufficient to limit the reproduction of these included cells, and these included cells produce this substance capable of inhibiting proliferation of cells, subsequent filtration through a filter that separates substances with a molecular weight of at least about 30 KD from substances with a molecular mass of less than 30 KD, and excretion of a substance with molecular weight, at least about 30 KD.
2. The composition according to p. 1, which included cells selected from the group comprising tumor cells, cancer cells, cells of the embryo, stem cells, hepatocytes, fibroblasts and epithelial cells.
3. The composition according to p. 2, which included cells are epithelial cells.
4. The composition according to p. 2, which included cells are cancerous, cldi, cells of kidney cancer, prostate cancer cells and cells choriocarcinoma.
6. The composition according to p. 2, which included cells are stem cells.
7. The composition according to p. 1, which included cells are human cells.
8. The composition according to p. 1, which included cells are mouse cells.
9. Composition under item 1, in which the structure contains from about 10,000 to about 500,000 cells.
10. The composition according to p. 9, in which the structure contains from about 30,000 to about 250,000 cells.
11. Composition under item 1, in which the biocompatible material is agarose.
12. Method of stimulating production of a substance that has the property to inhibit the proliferation of cells, involving the cultivation of biocompatible, limiting the proliferation of cells permeable structure together with included inside its rapidly proliferating cells in the medium for a time sufficient for these cells were produced vast proliferation of cells substance, and filtering the medium through a filter which separates a substance with a molecular weight of at least about 30 KD from substances with a molecular mass of less than 30 KD, and vydelennymi selected from the group including tumor cells, cancer cells, cells of the embryo, stem cells, hepatocytes, fibroblasts and epithelial cells.
14. The method according to p. 13, in which the cells are epithelial cells.
15. The method according to p. 13, in which the cells are cancer cells.
16. The method according to p. 15, in which cells are selected from the group comprising breast cancer cells, cells of kidney cancer, prostate cancer cells and cells choriocarcinoma.
17. The method according to p. 13, in which the cells are stem cells.
18. The method according to p. 12, in which this medium does not contain serum.
19. The method according to p. 12, in which the cells are human cells.
20. The method according to p. 12, in which cells are mouse cells.
21. The method according to p. 12, in which this structure contains from about 10,000 to about 500,000 cells.
22. The method according to p. 21, in which this structure contains from about 30,000 to about 250,000 cells.
23. The method according to p. 12, in which this structure is a granule.
24. The method according to p. 12, in which the biocompatible porous structure made of agarose.
25. The method of suppressing the reproduced is valid, limiting the proliferation of cells permeable structures that contain subjected to restriction in the reproduction of highly proliferating cells derived from a species different from the species belongs to the individual, with the specified subject constraint in the reproduction of cells produce a substance that inhibits the growth of cells in a quantity sufficient to inhibit cell multiplication specified individual with a molecular weight of at least about 30 KD.
26. The method according to p. 25, which is subjected to the constraint cells are cells other than the cell type associated with that condition that affects the individual.
27. The method according to p. 25, which is subjected to the constraint cells belong to the same type as the cells associated with that condition that affects the individual.
28. The method according to p. 25, in which this structure is a granule.
29. The method according to p. 25, in which this structure contains from about 10,000 to about 500,000 cells.
30. The method according to p. 29, in which this structure contains from about 30,000 to about 250,000 cells.
31. The method according to p. 25, which is subjected to limitation shall hepatocyte, fibroblasts and epithelial cells.
32. The method according to p. 31, which is subjected to the constraint cells are epithelial cells.
33. The method according to p. 31, which is subjected to the constraint cells are cancer cells.
34. The method according to p. 33, in which the cancer cells are selected from the group comprising breast cancer cells, prostate cancer cells, cells of kidney cancer and malignant cells choriocarcinoma.
35. The method according to p. 31, which is subjected to the constraint cells are stem cells.
36. The method according to p. 25, wherein said individual is a human.
37. The method according to p. 25, which is subjected to the constraint cells are mouse cells.
38. The method of suppressing the propagation of cells from the individual, introducing him therapeutically effective amount of a biocompatible, limiting the proliferation of cells permeable structures that contain subjected to restriction in the reproduction of highly proliferating cells derived from the same species, belongs to the individual, with the specified subject constraint in the reproduction of cells produce a substance that inhibits the reproduction of the current of the specified individual moreover, this substance having a suppressive ability, diffuses through these patterns.
39. The method according to p. 38, which is subjected to the constraint cells come from an individual other than the one specified.
40. The method according to p. 38, which is subjected to the constraint cells obtained from the same individual, who introduce these structures.
41. The method according to p. 38, wherein said individual is a human.
42. The method according to p. 38, in which this structure is a granule.
43. The method according to p. 38, in which this structure is made of agarose.
44. The method according to p. 38, which is subjected to the constraint cells selected from the group comprising tumor cells, cancer cells, cells of the embryo, stem cells, hepatocytes, fibroblasts and epithelial cells.
45. The method according to p. 44, which is subjected to the constraint cells are epithelial cells.
46. The method according to p. 44, which is subjected to the constraint cells are cancer cells.
47. The method according to p. 46, which is subjected to the constraint cells selected from the group comprising breast cancer cells, cells of kidney cancer and prostate cancer cells.
48. The method according to modernity limiting cells are types of cells, different from the type of cells which multiply the result of the condition that affects the individual.
50. The method according to p. 38, in which proliferating cells belong to the same type as the cells that cause a condition that affects the individual.
51. The method according to p. 38, in which this structure contains from about 10,000 to about 500,000 cells.
52. The method according to p. 51, in which this structure contains from about 30,000 to about 250,000 cells.
53. The method of suppressing the propagation of cells from the individual, introducing to him a sufficient amount of the composition under item 1 to suppress the reproduction of cells of a particular individual.
54. The method according to p. 53, in which the specified individual is a person.
55. The method according to p. 53, which is included in the composition of cells selected from the group comprising tumor cells, cancer cells, cells of the embryo, stem cells, hepatocytes, fibroblasts and epithelial cells.
56. The method according to p. 53, in which cells are epithelial cells.
57. The method according to p. 55, in which the cells are cancer cells.
58. The method according to p. 57, in which cells are selected from the group VCCI are stem cells.
60. The method according to p. 53, in which the cells are human cells.
61. The method according to p. 53, in which cells are mouse cells.
62. The method according to p. 53, in which the cells are human cells.
63. The method according to p. 53, in which cells belong to the same type of cells which multiply the result of the condition that affects the individual.
64. The method according to p. 53, in which cells derived from the same individual, which enter the specified structure.
65. The method according to p. 53, in which the structure contains from about 10,000 to about 500,000 cells.
66. The method according to p. 65, in which the structure contains from about 30,000 to about 250,000 cells.
67. The method according to p. 66, in which the cells are human cells.
68. A composition comprising biocompatible, limiting the proliferation of cells permeable structure, and this structure limits the multiplication of cells that under this restriction produce more substances with a molecular mass of at least 30 KD, which inhibits cell proliferation, compared with the same amount of the same is not subjected to the restriction of the cells, when teenycinema proliferation of cells permeable structure, providing the stage of structure formation by contacting cells with a biocompatible, limiting the proliferation of cells material and cultivation of such structures for a sufficient period of time in order to limit the above-mentioned cells so that they produce more substances with a molecular mass of at least 30 KD, which inhibits cell proliferation, compared with the same number are not subjected to the restriction of cells of the same type, with the specified substance diffuses through the specified structure.
70. Method of increasing the production of a substance that suppresses cell growth and has a molecular mass of at least 30 KD, including the limitation of proliferating cells forming the structure of the material with the formation of biocompatible porous structure containing proliferating cells, limited breeding and cultivation of such cells, which produce more substances with a molecular mass of at least 30 KD, compared with the same number are not subjected to the restriction of the cells, with the specified substance diffuses through the specified structure.
71. Composition for suppressing prevysuju proliferation of cells permeable structure, the cultivation of this structure in the culture medium for a time sufficient to limit the reproduction of these included non-cancer cells, and these included non-cancer cells produce a substance that is able to suppress cell proliferation.
72. Method of stimulating production of substances able to inhibit cell proliferation, involving the cultivation of biocompatible, limiting the proliferation of cells permeable structure, inside of which are incorporated rapidly proliferating non-cancer cell, in a period of time sufficient to produce the specified cells of the substance that suppresses the proliferation of cells.
73. The method of suppressing the propagation of cells from the individual, introducing him therapeutically effective amount of a biocompatible, limiting the proliferation of cells permeable structures that contain proliferating non-cancer cells derived from the same species, belongs to the individual, and specified, subjected to the restriction of non-cancer cells produce a substance that inhibits the proliferation of cells, in an amount sufficient to suppress Pruet through these patterns.
74. A method of suppressing proliferation of at least part of the population uninvolved cells, comprising culturing a specified population in the presence of the composition under item 1.
75. The method according to p. 74, characterized in that the said population is a population of stem cells.
76. The method according to p. 74, characterized in that the specified population refers to a type different than the origin of the cells contained in the composition.
77. The method according to p. 74, characterized in that this population belongs to the same species as the cells contained in the composition.
78. The method according to p. 74, characterized in that the said population is a population of cancer cells.
79. The method according to p. 74, characterized in that the said population is a population of mammalian cells.
80. The method according to p. 74, characterized in that the said population is a population of hyperproliferative cells.
81. The method according to p. 79, characterized in that the said population of mammalian cells is a population of human cells.
82. The method according to p. 79, characterized in that the said population of mammalian cells is a population for whom osili under item 1 in the environment over time, enough happened diffusion suppresses the proliferation of cells of the material in the specified environment, and contacting the specified environment population uninvolved cells in a period of time sufficient to suppress proliferation of a specified population.
84. The method according to p. 83, characterized in that the said population is a population of stem cells.
85. The method according to p. 83, characterized in that the specified population uninvolved cells and cells included in the composition, belong to different species.
86. The method according to p. 83, characterized in that the specified population uninvolved cells and cells included in the composition, belong to the same species.
87. The method according to p. 83, wherein the population of stem cells is a population of mammalian cells.
88. The method according to p. 87, wherein said population of mammalian cells is a population of human cells.
89. The method according to p. 87, characterized in that the said population of mammalian cells is a population of cells of mice.
90. The method according to p. 83, characterized in that it comprises contacting the specified environment with a subject in need of suppression prolifi the population of stem cells.
92. The method according to p. 90, characterized in that the specified population includes tumor cells.
93. The method according to p. 90, characterized in that is included in the composition of the cells and the cells of the subject belongs to a different species.
94. The method according to p. 90, characterized in that is included in the composition of the cells and the cells of the subject belong to the same species.
95. The method according to p. 94, wherein the cells are autologous cells of the subject.
96. The method of suppressing the proliferation of those that are not stem cells, comprising culturing the stem cells in the presence of a composition containing a sample of cancer cells included in a biocompatible, permeable structure, and this sample of cancer cells produces a material that suppresses the proliferation of stem cells.
97. The method according to p. 96, wherein the stem cells are mammalian cells.
98. The method according to p. 96, wherein the stem cells are embryonic stem cells.
99. The method according to p. 97, wherein the mammalian cells are human cells.
100. The method according to p. 97, wherein the mammalian cells are cells of the mouse.
102. The method according to p. 96, wherein the stem cells and cancer cells belong to different species.
103. The method of suppressing the proliferation of those that are not stem cells, comprising culturing a composition containing a sample of cancer cells included in a biocompatible, permeable structure, and this sample of cancer cells produces a material that suppresses the proliferation of stem cells, which penetrates into the culture medium, and contacting the culture medium with stem cells.
104. The method according to p. 103, wherein the stem cells are mammalian cells.
105. The method according to p. 103, wherein the stem cells are embryonic stem cells.
106. The method according to p. 104, wherein the mammalian cells are human cells.
107. The method according to p. 104, wherein the mammalian cells are cells of the mouse.
108. The method according to p. 103, wherein the stem cells and cancer cells belong to the same species.
109. The method according to p. 103, wherein the stem cells and cancer cells belong to different species.
110. The culture medium containing posebne to suppress the proliferation of cells.
with antitumor activity and low toxicity, which can be used in boron neutron capture therapy had cancer