MXPA01002927A - Compositions of restricted cells capable of rapid growth which produce proliferation suppressive materials, and uses thereof - Google Patents

Abstract

Compositions of matter are described which contain restricted proliferative cells. When so restricted, the cells produce an unexpectedly high amount of material which suppresses cell proliferation. The phenomenon crosses cell type and species lines. Processes for making these compositions, and their use, are also described.

Description

COMPOSITIONS OF RESTRICTED CELLS CAPABLE OF A FAST GROWTH WHICH PRODUCES SUPRESSIVE PROLIFERATION OF MATERIALS, AND USES OF THEMSELVES DESCRIPTION OF THE INVENTION This application is a continuation in part of the US patent application Serial No. 09 / 188,476 filed on November 9, 1998, which is a continuation in part of the application for U.S. Patent No. 08 / 745,063, filed November 7, 1996, now U.S. Patent No. 5,888,497 which is a continuation in part of copending application Serial No. 08 / 625,595, filed on April 3, 1996 , abandoned, each of which is incorporated for reference. The present invention relates to the restriction of the proliferation of cells in or capable of rapid growth in the log phase which, when their proliferation is physically restricted in a semipermeable-compatible material, produces normally not expressed or increasing amounts of a factor or factors normally expressed capable of inhibiting the growth of other rapidly proliferating cells of the same or different types and / or origins. These restricted cells are referred to below as proliferating cells. A non-limiting list of proliferating cells belonging to this category include neoplastic cells, cancer cells, and non-cancerous cells including, but not limited to, embryonic cells, stem cells, and cells in a regenerative post-wound phase of tissue that includes epatocytes, fibroblasts and epithelial cells. The structures that are a feature of the invention can be used "as is" and to produce material such as concentrates with a defined approximate molecular weight that also has an anti-proliferative effect on rapidly proliferating cells associated with diseases or conditions such as for example , cancer or to control the growth of cells to prevent certain medical problems that may arise from free cell growth such as the formation of post-operative scars. The encapsulation of various biological materials into biologically compatible materials, which is well documented in the literature, is a technique that has been used for some time, although with limited success. Examples of the technique are US Patents Nos. 5,227,298 (eber, et al.); 5,053,332 (Cook, et al.); 4,997,443 (Walthall, et al.); 4,971,833 (Larsson, et al.); 4,902,295 (Walthall, et al.); 4,798,786 (Tice, et al.); 4,673,566 (Goosen, et al.); 4,647,536 (Mosbach, et al.); 4,409,331 (Lim); 4,392.909 (Lim); 4, "352, 883 (Lim), and 4,663, 286 (Tsang, et al.) Also to be noted is US Patent No. ,643,569 to Jain, et al., Incorporated herein by reference. Jain, et al. Discuss, in some detail, the encapsulation of islets in various biocompatible materials. The islets produce insulin, and the use of the materials described by Jain, et al. in the treatment of diabetes is taught in it. U.S. Patent No. 5,888,497 to Jain, et al. describes coated implantable agarose, the agarose beads having cancer cells restricted therein, which produce more than one material that suppresses the growth of unrestrained cancer cells than an equal number of the same cancer cells without restriction. The patents of Jain, et al. discuss, in some detail, the previous proposals made by the technique in transplant therapy. These are also summarized in this. Five main proposals are known to protect the transplanted tissue from the host immune response. All involve attempts to isolate the transplanted tissue from the host's immune system. Immuno-isolation techniques used to date include: extravascular diffusion chambers, intravascular diffusion chambers, intravascular ultrafiltration chambers, micro encapsulation and macro encapsulation. There are many problems associated with prior art methods, which include a host fibrotic response to the implant material, instability of the implant material, limited nutrient diffusion through semipermeable membranes, secretagogue and product permeability, and diffusion in time lag through the semipermeable membrane barriers. For example, a microencapsulation procedure for enclosing viable cells, tissues, and other labile membranes within a semipermeable membrane was developed by Lim in 1978 (Lim, Research report to Damon Corporation (1978)). Lim used microcapsules of alginate and poly L-lysine to encapsulate islets of Langerhans (referred to as "islets" below). In 1980, the first successful in vivo application of this new technique in diabetes research was reported (Lim, et al., Science 210: 908 (1980)). The implantation of these microencapsulated islets resulted in a sustained euglycemic state in diabetic animals. Other researchers, however, repeating these experiments, found that alginate causes a tissue reaction and were unable to reproduce the results of Lim, et al. (Lamberti, et al Applied Biochemistry and Biotechnology 10: 101 (1984); Dupuy, et al., J. Biomed. Ma terial and Res. 22: 1061 (1988); Weber, et al., Transplantation 49: 396 (1990); and Doon-shiong et al., Transplantation Proceedings 22: 754 (1990)). The water solubility of these polymers is now considered responsible for the limited stability and biocompatibility of these microcapsules in vivo (Dupuy, et al., Supra, Weber et al., Supra, Doon-shiong, et al., Supra, and Smidsrod , Faraday Discussion of Chemical Society 57: 263 (1974)). Iwata et al., (Iwata, et al., Biomedical Journ.

Ma terial and Res. 26: 967 (1992)) used agarose for the microencapsulation of allogeneic pancreatic islets and found that it could be used as a means for the preparation of microbeads. In their study, 1500-2000 islets were individually microencapsulated in 5% agarose and implanted into diabetic mice induced with streptozotocin. The graft survived for a long period of time, and the recipients maintained normoglycemia indefinitely. His method, however, suffers from a number of disadvantages. It is uncomfortable and imprecise. For example, many beads remain partially coated and several hundred empty agarose beads are formed. Thus additional time is required to separate the encapsulated islets from the empty accounts. In addition, most implanted micro-counts meet in the pelvic cavity, and a large number of islets in fully coated individual beads are required to achieve normoglycemia. In addition, transplanted bills are difficult to remove, tend to be fragile, and release islets easily with light damage. A macroencapsulation procedure has also been tested. Macrocapsules of various different materials, such as poly-2-hydroxyethyl-methacrylate, polyvinyl-c-acrylic acid, and cellulose acetate were made for the immunoisolation of islets. (See Alt an, et al., Diabetes 35: 625 (1986); Altman, et al., Transplantation: American Society of Artificial In ternal Organs 30: 382 (1984); Ronel, et al. Jour. Biomedical Ma Terial Research 17: 855 (1983); Klomp, et al., Jour.Medical Biomedical Research 17: 865-871 (1983)). In all these studies, only a transient normalization of the glycemia was achieved. Archer, et al., Journal of Surgi cal Research 28:77 (1980), used acrylic copolymer hollow fibers to temporarily prevent rejection of islet xenografts. They reported the long-term survival of neonatal murine pancreatic grafts dispersed in hollow fibers which were transplanted into diabetic hamsters. Recently Lacy, et al., Science 254: 1782-1784 (1991) confirmed its results, but found the euglycemic state which is a transient phase. They found that when the islets are injected into the fiber, they are added into the hollow tube with resultant necrosis in the central portion of the islet masses. The central necrosis prevents the extension of the graft. To solve this problem, they used alginate to disperse the islets in the fiber. However, this experiment has not been repeated extensively. Therefore, the role of the membrane as a means of transplantation of islets in humans is questionable. The Jain patent, et al. discussed supra reports that secretory cells encapsulated in a permeable hydrophilic gel material results in a functional non-immunogenic material, which can be transplanted into animals, can be stored for long periods of time, and is therapeutically useful in vivo. The macroencapsulation of the secretory cells provides a more effective and manageable technique for the transplantation of secretory cells. The patent does not discuss in any way the incorporation of cells capable of rapid proliferation. A study of the literature on cell encapsulation reveals that, after encapsulation, cells almost always produce less of the materials they produce when they are not encapsulated. See Lloyd-George, et al., Bioma t. Art . Cells & Immob. Biotech 21 (3): 323-333 (1993); Schinstine, et al., Cell Transplant 4 (1): 93-102 (1995); Chicheportiche, et al., 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. 21 A: C1356-1362 (1998); Darquy, et al., Diabetologica 28: 776-780 '(1985); Tse, et al., Biotech. & Bioeng. 51: 271-280 (1996); Jaeger, et al., J. Neurol. 21: 469-480 (1992); Hortelano, et al., Blood 87 (12): 5095-5103 (1996); Gardiner, et al., Transp. Proc. 29: 2019-2020 (1997). None of these references deals with the incorporation of cells capable of rapid proliferation within a structure that traps them and restricts their growth, but nevertheless allows the diffusion of materials inside and outside the structure. A theory that refers to the growth of cancerous masses compares such masses, for example tumors, with normal organs. Healthy organs, for example, the liver, grow to a particular size, and then grow no bigger; however, if a portion of the liver is removed, it will regenerate to a certain degree. This phenomenon is also observed with tumors. For resumix, it has been noted that if a portion of a tumor is removed, the cells in the remaining portion of the tumor will begin to proliferate very rapidly until the resulting tumor reaches a particular size, after which the proliferation decreases or ceases. This suggests that there is some internal regulation of cancer cells. The invention, which will be seen in the following description, shows that when cells capable of rapid proliferation are physically restricted such as being trapped, their rate of proliferation decreases markedly, and produces unexpectedly high amounts of material, which, when applied to unrestricted rapidly proliferating cells, inhibits the proliferation of these unrestricted cells. The ability to slow the proliferation of cancer cells has been a goal of oncology since its inception. Accordingly, the therapeutic utility of this invention with respect to its treatment of cancer and other conditions of affection and conditions caused by rapid cell proliferation will be clear and will be elaborated herein. The material produced does not seem to be limited by the rapidly proliferating cell type used, nor by the animal species from which the rapidly proliferating cells originate. Additionally, the effect does not appear to be species-specific, since restricted cells of a first species produce material that inhibits the proliferation of cells without restriction of a second species. Also, with respect to cancer, the effect does not appear to be specific to the type of cancer, since cells restricted from a first type of cancer produce material that inhibits the proliferation of cells without restriction of another type of cancer. Neither the effect seems to require an immune response. The antiproliferative effect is seen in in vitro systems, where immune cells are not used. Consequently, the antiproliferative effect can not be attributed to classical immunological responses.

Thus, a preferred embodiment of the invention relates to a composition of material having a selectively permeable, proliferation-restrictive, biocompatible structure. The structure restricts rapidly proliferating cells which then produce more than one material that suppresses rapid cell proliferation compared to an equal number of the same rapidly proliferating cells when they are unrestricted. Another preferred embodiment of the present invention relates to a process for preparing a selectively permeable, proliferative-restrictive, proliferative structure by forming a structure by contacting rapidly proliferating cells with proliferating, biocompatible, restrictive material to form the structure, and culture the structures for a sufficient period of time to restrain the rapidly proliferating cells so as to produce a material that suppresses the proliferation of the rapidly proliferating cells to be suppressed compared to an equal number of rapidly proliferating cells without restriction of the same type. Yet another preferred embodiment relates to a method for increasing the production of material that suppresses the cell growth of rapidly proliferating cells, which comprises restricting cells that rapidly proliferate in a restrictive, selectively permeable, biocompatible, proliferating structure and rapidly culturing the cells proliferating so that they produce the material. When the referred structures are placed in a culture medium, the referred material leaves the structure and enters the culture medium. The resulting culture medium is also a feature of the invention. It has also been found that a powerful anti-proliferative effect can be achieved by filtering the conditioned medium obtained by culturing the structures of the invention in a culture medium. The resulting concentrates have extremely strong anti-proliferative effects. The material, the conditioned medium, and / or the concentrates derived therefrom may also be useful for inducing the production of the anti-proliferative material by other rapidly proliferating non-restricted cells. In preferred embodiments, the restricted cells are cancer cells, but the advantages of using non-cancerous cells to treat cancer and other conditions provides additional benefits readily apparent to those skilled in the art. These and other features of the invention will be understood from the description that follows.

Example 1 This example, and those that follow, employ RENCA cells. These are spontaneous renal adenocarcinoma cells from BALB / c mice, which are widely available, having been maintained in in vitro and in vivo cultures. See Franco, et al., Cytokine Induced Tumor Immunogenecity, 181-193 (1994). Samples of frozen RENCA cells were thawed at 37 ° C, and then placed in tissue culture flasks containing modified Dulbecco's medium (D-MEM), which had been supplemented with 10% bovine serum, penicillin (100 u / ml) and streptomycin (50 ug / ml), to give what will be referred to as "complete medium" below. The cells were grown to confluence, and then they were trypsinized, followed by washing with Hank's balanced salt solution, and then with the complete medium referred to supra. To determine if RENCA cells produced tumors efficiently, two BALB / C mice were injected, intraperitoneally, with 106 of these cells. The mice were observed for a period of 3-4 weeks. Clinically, they seemed healthy during the first two weeks, and showed normal activity. After that, the clinical manifestations of cancer became evident. One mouse died after 23 days, and the second after 25 days. After dying, the mice were examined, and numerous tumors of various sizes were observed. Some of the tumors also presented hemorrhage. A sample of a tumor, taken from one of the mice, was fixed in 10% for alina for further histological examination. Example 2 After showing that RENCA cells grew in vivo, studies were conducted to determine if these cells grew when restricted in the structure of the invention. The RENCA cells were grown to confluence, as described supra, they were trypsimized, and washed, also as described above. Samples of between 60,000 and 90,000 cells were then prepared. The cells were then centrifuged at 750 RPMs, and the fluid was removed. The cells were then suspended in solutions of 1% atelocollagen, in phosphate-buffered saline, at a pH of 6.5. A 1% solution of low viscosity agarose was prepared in minimal essential medium (MEM), maintained at 60 ° C, and then 100 ul thereof was added to the suspension of RENCA and atelocollagen cells, described supra. The materials were then transferred, immediately, as a single large droplet, in sterile mineral oil at room temperature. The mixture formed a semisolid, smooth, simple account. This procedure was repeated to produce a number of accounts. After one minute, the beads were transferred to a complete medium as described above, at 37 ° C. The beads were then washed three times in a Minimum Essential Medium (MEM) containing the antibiotics listed above. The beads were then incubated overnight at 37 ° C, in an atmosphere moistened with air and 5% C02. After incubation, the beads, now solid, were transferred to a sterile spoon containing 1 ml of 5% agarose in MEM. The beads were rotated in the solution 2-3 times to coat them uniformly with agarose. The beads were transferred to mineral oil before the agarose solidified, to produce a smooth outer surface. After 60 seconds, the beads were washed five times with a complete medium at 37 ° C to remove the oil. Followed by overnight incubation (37 ° C, humid atmosphere of air, 5% C02). These accounts containing RENCA were used in the experiments that follow. Example 3 Before carrying out in vivo investigations, it was necessary to determine if the RENCA cells would grow in beads prepared in the manner described above. To do this, beads prepared as discussed in example 2 were incubated in the medium described in example 2, for a period of three weeks, under the conditions described. Three of the beads were then cut into small pieces and cultivated in standard culture flasks, producing direct contact with the flask and the culture medium. Observation of these cultures indicated that the cells grew and formed standard RENCA colonies. This indicated that the cells had remained viable in the accounts. Example 4 In vivo experiments were then carried out. In these experiments, the beads were incubated for 7 days at 37 ° C. Then the subject mice received account transplants. To do this, each of four mice received a midline incision, carried out intraperitoneally. Three beads were transplanted, each of which contained 60,000 RENCA cells. The incisions were closed afterwards (two-layer closure), using an absorbable suture. All four mice (BALB / C) were normal, male mice weighing between 24-26 grams, and appeared to be healthy. Two sets of controls were fixed. In the first set, two mice received three beads that did not contain RENCA cells, and in the second, two mice were not treated with anything. Three weeks after implantation, all mice received intraperitoneal injections of 106 RENCA cells. Eighteen days later, a mouse ~ control died. All the remaining mice were sacrificed after, and were evaluated for the presence or absence of tumors. The control mice showed numerous tumors, whereas the mice that received the encapsulated cell implant on account showed only small nodules isolated through the cavity. These favorable results suggested the design of the experiments indicated in the following example. Example 5 In these experiments, established cancers were simulated by injecting RENCA cells under a kidney capsule of each of six BALB / C mice. Fifteen days later, the mice were divided into two groups. The three mice of the first group each received three beads, as described in Example 4, supra. The second group (the control group) received accounts that did not contain RENCA cells. During the initial 4-5 days, the mice that had received implants containing RENCA cells seemed lethargic, and their skin had become bristly. After that, they went back to normal. The control group remained energetic, with no change in the condition of the skin. Ten days after the implant (25 days after injection of RENCA cells), however, the control mice became lazy and showed distended abdomens. One of the three control mice died fourteen days after the account transplant. He followed the sacrifice of the mice. The body cavities of the control mice showed profuse hemorrhage, with numerous tumors in all parts of the alimentary canal, liver, stomach and lungs. All the organs of the abdominal cavity had become indistinguishable due to the unbridled tumor growth. The mice that had received beads with encapsulated RENCA cells, however, did not show hemorrhage, and only a few nodules on the alimentary canal. In addition, the comparison of the test and control groups showed that in the test group, the nodules had not progressed beyond their initial growth under the kidney capsule and before macro-counting. Example 6 In vitro, the growth of freely inoculated RENCA cells is inhibited when such cells are incubated together with encapsulated RENCA macrobead cells. An additional set of experiments was carried out to determine if this effect was observable with other cells. An adenocarcinoma cell line, ie, MMT (mouse mammary tumor), was obtained from the American Type Culture Collection. The encapsulated MMT cells were prepared, as described, supra with MMT cells, to produce beads containing 120,000 or 240,000 cells per count. After the preparation of the beads, they were used to determine if they would inhibit the proliferation of RENCA cells in vi tro. Specifically, two six-well petri dishes were prepared by inoculation with 1 x 10 4 RENCA cells per well in 4 ml of medium. In each plate, three wells served as control, and three as a test. One of the three control wells in each plate received an empty account. Each of the other wells received two or three empty accounts. The second set of wells was treated similarly, with wells receiving one, two or three beads containing 120,000 or 240,000 MMT cells. The wells were incubated at 37 A for one week, after which the RENCA cells were trypsinized, washed, and counted, using a hemocytometer. The results are shown in Table 1: TABLE 1 Example 7 Following the results of Example 6, the same experiments were carried out using 1 x 104 cells of MMT co or inoculum (ie, free cells) more than RENCA cells. The experiment was carried out precisely as the e-j. 6. The results are indicated in Table 2 below. TABLE 2 These results favor an in vivo experiment. This is presented in Example 8. Example 8 The mouse mammary tumor cell (MMT) line described supra was used. Using the indicated protocols, supra, implants containing 120,000 cells per account and 240,000 cells per account were prepared. The experimental model used was the mouse model, supra. Twenty-two mice were divided into groups of 4 (control), 9 and 9. The first group, ie the controls, were further divided into three groups: two received implants from an empty account, one received two empty accounts and one received three accounts empty Within experimental group A (9 als), the beads contained 120,000 cells, while in experimental group B, the beads contained 240,000 cells. Within Groups A and B, there were three subdivisions, each of which contained three mice. The subgroups received one, two, or three accounts containing MMT cells. During the first few days, the mice in Groups A and B were lethargic, with bristly hair. This persisted for approximately five days, after which normal behavior was observed. Twenty-one days after implantation, all als received injections of 40,000 RENCA cells. After another twenty days, the control mice showed distended abdomens, and extremely bristly hair. A control mouse died twenty-five days after injection, while the remaining control mice appeared terminal. All mice were sacrificed, and tumor development was observed. These observations are recorded in Table 3 below: TABLE 3 These results show that, of eighteen treated mice, thirteen did not show disease. Of the mice in Group A, one mouse had a few small nodules (+), and another mouse showed a few tumors (++). Within Group B, a mouse that had received an account, and a mouse that received two counts showed a few tumors, entangled with the intestine. One of the mice that received three beads had developed a large solid tumor and was apparently very ill (+++). All the control mice had numerous tumors (++++). The results showed that encapsulated mouse mammary tumor cells inhibited tumor formation. Example 9 As suggested, supra, the practice of the invention results in the production of the material that inhibits and / or prevents the proliferation of tumor cells. This was explored further in the experiment that follows. Additional accounts were made, as described above in Example 2, except that atelocollagen was not included. Consequently, these accounts are agarose / agarose beads. RENCA cells, as described, supra, were incorporated into these accounts, again as described supra. Two sets of three six-well plates were then used as control and experimental groups. In the control group, the wells were filled with "4 ml of RPMI complete medium (10% fetal calf serum and 11 ml / 1 penicillin)." Each well control group was then inoculated with 10,000 RENCA cells. experimental group, the RPMI complete medium was conditioned, adding insured material. to incubate ten beads containing RENCA (120,000 cells per count), in a 35 x 100 mm petri dish containing 50 ml of RPMI complete medium. After incubation, the medium was collected from these plates, and four of them were placed in each test well.These wells were then inoculated with 10,000 RENCA cells in each.All plates (control and experimental) were incubated at 37 ° C. C for five days After the incubation period The cells were trypsinized, washed, pooled and counted using a hemocytometer The results are shown in Table 4: TABLE 4 These results show that the cells, when restricted in, for example, the accounts of the examples, produce some material that results in the suppression of the proliferation of tumor cells. EXAMPLE 10 The experiment indicated above showed that the growth of RENCA cells, in a conditioned medium, was about half the growth of the cells in the control medium. The experiments set forth herein examined whether the suppression or proliferation would continue after the conditioned medium froze. The RENCA conditioned medium was prepared by incubating ten beads containing RENCA for five days. The incubation was in 35 x 100 mm petri dishes, with 50 ml of RPMI complete medium, at 37 ° C. After incubation, the medium was collected and stored at -20 ° C. The conditioned medium was prepared by incubating beads containing MMT cells (mouse mammary tumor). The accounts contained 240,000 cell per account; otherwise all the conditions were the same. The frozen medium was thawed at 37 ° C, and then used in the following tests. Three six-well plates were used for each treatment, ie, (i) RPMI control medium, (2) RENCA frozen conditioned media, and (3) MMT frozen conditioned media. A total of 4 mis of medium was dispersed within each well. All wells were then inoculated with 10,000 RENCA cells, and incubated at 37 ° C, for five days. After incubation, two sample plates were taken from each well, trypsinized, washed, pooled and counted in a hemocytometer. After eight days, the three remaining plates of each well were tested in the same way: The results follow: TABLE 5 When these results are compared with those of Example 6, supra, it will be seen that, while the frozen / thawed RENCA conditioned medium did not suppress proliferation to the same extent as did the frozen / thawed MMT conditioned medium (compare examples 6 and 7). ), however, proliferation was suppressed. EXAMPLE 11 The experiments indicated supra showed that the conditioned medium frozen from macrobeads containing RENCA- or MMT- inhibits the proliferation of RENCA cells in vi tro. The experiments indicated in the present review, whether from a RENCA- or MMT- macrocontainer medium, prepared as 30 kd or 50 kd concentrates by filtration, inhibited the proliferation of RENCA cells in vi tro. The effects of the macrocontainer medium were compared with the effects of the conditioned medium in the presence of cells without RENCA restriction and MMT growing in monolayer cultures, to determine if unrestrained tumor cells grown to confluence also proliferate the regulatory material. For these experiments, 10 macrobeads, each containing 120,000 RENCA or MMT cells (i.e., a total of 1.2 x 106 cells) were used to condition the medium (complete RPMI) for a period of 5 days. In parallel, 1.2 x 10 6 RENCA or MMT cells, ie the same number of cells, were seeded in a culture box and allowed to proliferate as a monolayer for a period of 4 days in a complete RPMT medium. The medium was changed afterwards, and this medium was collected twenty-four hours later. The reason for the different amount of exposure time of the beads and the cells without restriction was the difference in cell numbers in the monolayers vs. the beads (3- to 5-fold more cells in the monolayers) at the end of the 5-day period. In other words, the cells without restriction grew so much faster than the encapsulated cells, that there were 3-5 times more cells. 30 kd and 50 kd filtrates were used to prepare concentrates from the conditioned medium that presumably would contain the active material, and also eliminate toxic metabolic and / or waste materials as confounding factors in the experiments. These pollutants, which are well known, they are too small to hold on a 30 kd filter. The filtrates were also tested, but any interpretation of the results with this material is complicated by the presence of cellular waste products. A serum free medium (AIM V) was also used in some experiments to ensure that any effect of the serum by itself was controlled. Essentially, the conditioned medium was collected, three to five days after the macrobeads had been added to it, or twenty-four hours after the new medium had been added to the cells without restriction. The medium was then placed in a filtered test tube with an appropriate filter (a 30 kd or 50 kd filter), and centrifuged for 90 minutes. The material that remains on the filter is referred to as the "concentrate", while the one that passes through the filter and is collected at the bottom of the tube is the filtrate. The results, summarized in Tables 6, 7 and 8 below, show that when the conditioned medium resulting from the RENCA cells restrained in the macrobeads was used, it inhibited the proliferation of RENCA cells by approximately 52% in two separate experiments. The 50 kd concentrate inhibited proliferation by approximately 99%, in both cases, while the 30 kd concentrate inhibited proliferation by approximately 97%.

TABLE 8 Inhibition of RENCA Cell Growth in a Medium of Macro Concentrated Conditioning RENCA and Concentrate (Medium AIM V) An important point of the experiment is that MMT cells and RENCA cells, when trapped and restricted in the macro accounts both suppress the proliferation of RENCA cells, indicating that the restrictive effect of proliferation is not specific for the type of tumor. These experiments confirm those of Example 8 in which macrobeads containing MMT suppressed the proliferation of RENCA cells in vivo. In addition, they extended the findings to indicate that the material released from the macrobeads within the medium contains molecules that are at least 30kd in molecular weight that are responsible, in part, for the restrictive proliferation effect. Finally, these experiments show that RENCA and MMT cells from restricted macro accounts produce much more of the material that suppresses proliferation than the same cells grown to confluence in monolayer cultures. EXAMPLE 12 The experiments indicated above show that the macrocontact medium conditioned by MMT and RENCA contains material released from the proliferation cells restricted in the macro account which can inhibit the proliferation of RENCA cells in vivo and in vitro. Importantly, the experiments show that the inhibitory effect of proliferation is not specific to the type of tumor. The experiments indicated herein examine whether the effect is also independent of the species in which the tumor originally arose. Here, the inhibitory effects of tumor cell proliferation of a human breast cancer-derived cell line on RENCA cells (using macrobeads and macrocontact medium conditioned) and also MMT cells (using only macrocontact medium conditioned) were examined. vi tro. The methodologies for these in vitro studies were similar to those described in the previous examples. 100,000 MCF-7 (human breast cancer cells) were encapsulated in macrobeads, and the resulting MCF-7 macrobeads were incubated with RENCA cells (10,000 per well) for 5 days to evaluate the inhibitory effects of macrobead proliferation. In addition, MCF-7 macrocontainer medium was prepared during a 5-day incubation period and tested in RENCA and MMT cells. Cell proliferation was measured over a period of 5 days. The results are indicated below: TABLE 9 MCF-7 MACROCUENT RESULTS ON RENCA OBJECTIVE CELLS TABLE 10 RESULTS OF MEDIUM CONDITIONING MCF-7 ON RENCA OBJECTIVE CELLS TABLE 11 RESULTS OF MEANS CONDITIONED MCF-7 ON OBJECTIVE CELLS MMT The results show that MCF-7, a human breast adenocarcinoma cell line, when macrobiotic proliferation is restricted produces a material that inhibits the proliferation of mouse renal adenocarcinoma cells and tumor cells of breast cancer to a significant degree (30-70%) as demonstrated by the macro accounts in themselves and the conditioned media derived from them. This indicates that the inhibitory effect of proliferation of growth-restricted cancer cells is independent of the type of tumor and the species of tumor origin, i.e., mouse and human. EXAMPLE 13 The experiments indicated above demonstrate that a human-derived breast adenocarcinoma cell line (MCF-7), when growth is restricted in macrobeads, produces inhibition of proliferation of mouse and renal mouse adenocarcinoma cells. in vitro The experiments set forth herein examine whether there is a parallel effect of macro beads containing MCF-7 on tumor growth of RENCA cells in vivo. Eighteen Balb / c mice were injected with 20,000 RENCA cells intraperitoneally. After three days the mice were divided into two groups. Group 1 had six mice and Group 2 had the remaining twelve mice. The mice of Group 1, the controls were transplanted with three empty macro accounts each. Group 2 mice each received three macrobeads containing MCF-7 (100,000 cells per count). After twenty-five days, 2 mice of Group 1 and three mice of Group 2 were sacrificed. The same number was sacrificed on day twenty-six and the remaining mice were sacrificed on day twenty-seven. In necroscopy, the peritoneal cavities of the control mice were observed to be completely packed with tumor, and the normal organs were difficult to identify. This was classified as tumor intensity ++++ (100%). In the treated mice, the tumor intensity was classified as + (10-20%). These results show that macrobeads containing human breast adenocarcinoma cells are able to inhibit the tumor growth of renal cell adenocarcinoma in mice, confirming once again that the inhibitory effect of cancer cell proliferation / tumor growth is not of the specific type nor of specific species. EXAMPLE 14 The experiments indicated above demonstrate that the inhibitory effect of cell proliferation / tumor growth of macro growth-restricted growth tumors is neither specific to the type of tumor or the species. Experiments initiated herein examine whether restricted breast proliferation (macroaccount) mouse breast adenocarcinoma cells can inhibit the growth of spontaneous mammary tumors and tumors resulting from the injection of MMT cells. C3H mice have a very high incidence of mammary tumor development during their lifetime. Seven mice at risk of developing such tumors showed tumors at 16 months of age. At this time, five of the seven mice were implanted with four MMT macrobeads containing 100,000 cells each. The remaining two control mice received four empty macro accounts each. The two control mice developed large tumors and died within three months after the counting implants. The treated mice were sacrificed eleven months after MMT macroaccount implants. The macro-accounts, organs and tumors removed were examined grossly and histologically. Hernotoxilin and Eosin staining of the MMT macrobeads showed viable cells. The pre-existing tumors had not increased in size and there was no evidence of any new tumor development. Experiments were also performed in which MMT tumor cells were injected subcutaneously into the thoracic region. Fourteen C3H mice were divided into two groups. The five control group mice were implanted with three empty macro accounts each. The nine treated mice received three macrobeads containing MMT (240,000 cells) each. Three weeks after implantation, all fourteen mice were injected subcutaneously into the mammary area with 20,000 MMT cells each. Within twenty-five to thirty days, the five mice of the control group became ill with evident tumor formation and all died thirty-five days after the injection. The nine treated mice, observed weekly, continued without any evidence of tumor formation or ill health during this period. Ten to twelve months after the tumor injection, four of the nine treated mice developed protrusions and lost their skin in patches. The remaining five mice were implanted again with three MMT macrobeads thirteen months after the initial tumor injection. A mouse died three days after this surgery, but at necropsy it was completely free of tumors. The four surviving mice were sacrificed eight months after the second macroaccount implant. The necropsy showed minimal or no tumor proliferation. A further observation from these experiments was that the beads removed from the first implant contained viable tumor cells based on histology and their ability to resume aggressive tumor growth patterns in tissue culture after the withdrawal of the bill. The results of these experiments show that the effects of inhibition of cell proliferation / tumor growth of cancer cells restricted by macro account, in this case mouse mammary adenocarcinoma cells, can influence the development and growth of spontaneously arising tumors and experimentally induced tumors that arise from the injection of tumor cells within the mammary area. EXAMPLE 15 The experiments set forth above demonstrate an inhibitory effect of tumor cell proliferation / tumor growth of cancer cells restricted from macroabout proliferation which is characterized by its effectiveness through tumor types and through species, as well as in spontaneous and artificially induced tumors. . The experiments described herein extend these findings to examine the effects of cells derived from human prostate adenocarcinoma (ARCaplO), mouse renal adenocarcinoma cells (RENCA cells) (Balb / c), and mammary adenocarcinoma cells (MMT) of mouse (C3H) restricted proliferation, trapped in macroaccount on the proliferation of tumor cells ARCaPlO and tumor growth ARCaPlO in nude mice (Nu / Nu). In the first series of experiments, fifteen Nu / Nu mice were injected with 2.5 x 10 6 ARCaPlO cells subcutaneously in the side. On day twenty-one after the injection, at which time the maximum average tumor diameter was 0.5 cm, the mice were divided into two groups. Nine were implanted with four ARCaPlO macro accounts (1.0 x 105 cells per macro account) each, and six control mice received four empty macro accounts each. Ten weeks after implantation, five of the control mice had very large vascularized tumors (average 2.5 cm in diameter) and one mouse showed a slightly smaller tumor (less than 0.5 cm). In the treated group, five mice showed complete regression of the initial tumors, and all remained tumor-free until sacrifice at eight months. Two mice did not show tumor growth, that is, their tumors had the same maximum diameter as they had at the moment of the macrobeads implant, and two mice showed tumors that had enlarged since the implantation of the macrobeads. The results (volume and tumor size (1 xwxh)) of an experiment in which macrobeads containing RENCA (1.2 x 105) were implanted eighteen days after subcutaneous side injection of 3.0 x 10 6 ARCaPlO tumor cells per animal in 4 Nu / Nu mice are indicated below: TABLE 12 SIZE OF TUMORS OBSERVED IN TREATED MICE (in mm) TABLE 13 VOLUME OF THE TUMORS OBSERVED IN THE TREATED MICE In another experiment 10 Nu / Nu mice were injected with 2.5 x 10 6 APCaPlO cells, with six of the mice showing tumor development sixty-four days after injection. Three of these mice were given four MMT macro counts (2.4 x 10 cells) each and three received empty macro accounts. The results are indicated below: TABLE 14 SIZE OF TUMORS OBSERVED IN TREATED MICE (in mm) TABLE 15 SIZE OF TUMORS OBSERVED IN THE CONTROL MICE (in mm) The results of these experiments further confirm the cross-species nature, cross-tumor inhibitory effect of tumor growth restriction proliferation in tumors of various types. In addition, these experiments demonstrate the ability of cancer cells restricted to proliferation not only to suppress tumor growth and to prevent tumor formation, but also to cause the current regression of tumors in vivo. Example 16 The experiments indicated above show that cancer cells restricted from proliferation of various types of tumors and species can inhibit the proliferation of same and different types of cancer cells in vi tro and prevent the formation of spontaneous and induced tumors, prevent growth of tumors, and cause the tumors to return in vivo in an effect that is independent of the species and type of cancer. The experiment indicated here describes the extent of the discoveries to another species (rabbit) and a rabbit tumor known to have been virally induced (VX2). In this experiment, a New Zealand White Rabbit (2.5 pounds) was injected intramuscularly in one thigh (two sites) with 0.5 ml of a VX2 tumor suspension (characterized by being able to pass through a # 26 gauge needle) in each site. At 3.5 weeks, a 5 cm x 2.5 cm (lxw) tumor had appeared on the dorsal thigh and two 3 cm diameter tumors were present on the ventral thigh. At this point, 211 macrobeads were implanted intraperitoneally (108 counts of RENCA cells, 63 counts of MMT cells, and 40 cancer cells of human breast containing MCF-7). Within two days, the tumor on the dorsal thigh had shrunk by approximately 50%; however, the two ventral tumors did not change. The animals were sacrificed ten days after the implant of the macro account. At necropsy, there was a clear difference between the dorsal and ventral tumors in that the former was much smaller than it had been at the time of the macro account, while the two ventral tumors were hemorrhagic and necrotic. This experiment extends the discoveries of the effectiveness of the restriction of proliferation of various types of cancer cells in relation to the prevention, interruption and even regression of tumor growth in other species, the rabbit adds a tumor of known viral origin to the list of types of cancer, and additionally supports the cross-tumor and cross-species nature of the growth inhibitory effect, since a combination of mouse kidney, mouse breast and human breast cancer cells containing macroaccount was used. In addition, the experiment adds a larger animal model to the in vivo test of the effectiveness of restricting the proliferation of cancer cells for the treatment of cancer.

Example 17 The experiments indicated above show that the restriction of proliferation of various types of tumor cells results in their ability to inhibit the growth of cells of the same or different type in vi tro and to prevent the formation of, suppress the growth of, or cause the regression of various types of tumors in vivo and that the effects seen are independent of the type and species of tumor. The experiments reported herein evaluated the long-term viability of proliferation-restricted RENCA cancer cells in macro agarose-agarose beads maintained in culture for 1 month periods., 6 months, 2 years, and 3 years using histological, culture, and in vivo techniques. The macro-accounts containing MMT were kept in culture for up to 6 months. In addition, macrobeads containing RENCA- and MMT- removed from Balb / c and C3H mice respectively after periods of 2 to 8 months after implantation were examined by viable tumor cells by histological and culture techniques.

For these experiments the agarose-agarose macrobeads were prepared with 1.2 x 10 5 RENCA cells or 2.4 x 10 5 MMT cells. They were examined histologically (staining by hermatoxylin and eosin) and by culture techniques for cell viability and tumor characteristics at the intervals described above. For the RENCA macro accounts, cell numbers increased approximately 3 to 5 times during the first month with an additional subsequent folding in six months. After one year, there was a continuous increase in cell mass, but the rate of cell proliferation had decreased. After two years, the amorphous material had begun to appear in the center of the count, and the cell mass / quantities did not seem to increase, although the mitotic figures were still evident. After three years, there seemed to be some more amorphous material at the center of the count, but the cell mass / amount was stable. The MMT macro accounts have been followed for only six months, but the early pattern of cell proliferation and appearance of the account is similar to that of RENCA. For evaluation of the viability and biological behavior of the RENCA and MMT cells at the intervals described above, ten beads were crushed and plated in two or more 25 cm2 tissue culture flasks in a complete RPMI medium. The flasks were then observed by cell growth. At intervals of one and six months, the number of viable cells removable from the accounts was increased. In one year, the number of RENCA cells growing from squashed counts appeared to be similar to those of six months. At two and three years, the proportion of viable cells seemed to be somewhat less, falling to approximately 20% of the maximum number that they reached in the account (ie in their restricted state) after three years in culture. For the evaluation of the RENCA and MMT macro accounts that were withdrawn after the in vivo implant (periods of 1-4 years for the RENCA macro accounts and up to 8 months for the MMT macro accounts), histological techniques have been used to date. The proliferation and cell mass patterns are very similar to those of the accounts maintained in culture during the corresponding periods of time, that is, the cells increase in number at least up to 4 months for RENCA and 8 months for MMT. For the other cancer cell lines used, such as MCF-7 and ARCaPlO, the viability patterns in macro-accounts are similar to those observed for RENCA and MMT. These experiments show that cancer cells can remain in place for periods of up to 3 years and in vivo for periods of at least 8 months in a proliferation restriction environment and maintain their viability during these periods with clear demonstration of increasing amounts of cancer. cells for at least a year. This is important not only because of the ability to create and store cancer treatment materials, but also because of the ability of proliferation-restricted cells to remove material that suppresses tumor growth in warm-blooded animals during prolonged continuous periods, probably necessary for the successful treatment of experimental or naturally occurring cancer. Example 18 The experiments indicated above show that cancer cells of various types can be maintained under restricted conditions of proliferation for long periods of time (up to 3 years) with retention of their ability to proliferate, to form tumors, and to release materials that inhibit cell proliferation and prevent tumor growth, suppressive and even regressive. The experiments indicated herein evaluate the possible long-term (one year) implant toxicity of macro agarose-agarose beads, which contain cancer cells in Balb / c mice. Seven Balb / c mice were implanted with 3 ma.crocuentas of RENCA each (1.2 x 105 cells per account). Immediately after the surgery the mice looked sick (bristling skin and lethargy) for a few days, but they became healthy again after this. All mice survived in apparent good health for a period of at least one year, with one mouse dying of old age and one of unrelated causes. All mice were sacrificed. At necropsy, no abnormalities were observed, such as fibrosis, peritonitis, or tumor growth. All the organs observed appeared normal, although some adherence of the beads to the serosal surfaces of the intestines was observed, especially where there were intestinal loops. No interference has been observed with the normal function or structure of the intestines. These results show that agarose-agarose macroaccounts containing cancer cells are well tolerated in experimental animals over a period of one year. These findings show that cancer cell counts restricting proliferation can be used in vivo for the prevention, suppression and regression of cell proliferation including in vivo tumors of various types. The above examples describe the invention, which includes, inter alia, compositions of matter that can be used to produce material that suppresses or controls the proliferation of rapidly proliferating cells, particularly the suppression of cancer cells. These compositions comprise cells in a log growth phase (rapid proliferative phase) trapped in a selectively permeable material to form a structure that restricts the proliferation of trapped cells. As a result of being restricted, the cells produce unexpectedly high amounts of material that suppresses the proliferation of rapidly proliferating cells, such as cancer cells. Restricted cells produce more of the material than comparable amounts of unrestricted cells. The material used to make the structures of the invention include any biocompatible material that restricts the growth of rapidly proliferating cells, thereby inducing them to produce larger amounts of material that suppresses cell proliferation / growth. The structure has a suitable pore size such that the above suppressor material can diffuse to the external environment, and prevent the entry of the products or cells of the host immune system into the structure and cause the rejection of the cancer cells and deteriorate otherwise. its ability to survive and continue producing the desired material. The material used to form the structure will also be able to maintain viable cells (restricted from proliferation, but survivors) in vi tro and in vivo, preferably for periods of up to several years by providing the input of appropriate nutrients with the elimination of waste products. cellular and a compatible intrastructural physicochemical environment. The material used to prepare the structure is preferably well tolerated when anted in vivo, more preferably for the full duration of the ant in the host. A non-limiting list of materials and combinations of materials that could be used includes alginate-poly- (L-lysine); alginate-poly- (L-lysine) -alginate; alginate-poly- (L-lysine) -polyethylenimine; chitosan-alginate; polyhydroxyethyl methacrylate methyl methacrylate; carbonylmethylcellulose; moss from Ireland-K; chitosan; agarose-polyethersulfone-hexadi-metirine bromide (Polibrene); ethyl cellulose; silica gels; and combinations thereof. The structures comprising the compositions of matter can take many forms, such as a bead, a sphere, a cylinder, a capsule, a sheet or any other form that is suitable for implantation in a subject, and / or culture in an environment in vitro The size of the structure may vary, depending on its eventual use, as will be clear to the skilled artisan. The structures of the invention are selectively permeable, such that nutrients can enter the structure, and so that the material that inhibits proliferation as well as cell debris can leave the structure. For in vivo use, it is preferred that the structures prevent entry of products or cells from a host immune system which could cause rejection of the restricted cells, or otherwise impair the ability of the cells to produce the suppressive material of the host. proliferation. Another aspect of the invention includes compositions that are useful for suppressing the proliferation of cancer cells. These compositions are prepared by culturing restricted cells as described supra in an appropriate culture medium, followed by recovery of the resulting conditioned medium. The concentrates can then be formed from the conditioned medium, for example, by separating fractions having molecular weights greater than 30 kd or greater than 50 kd, which have antiproliferative effects on the cancer cells. The invention is not limited to any particular type of proliferative cell. Any rapidly proliferating cell as defined supra can be used according to the invention which includes, but is not limited to neoplastic cells, embryonic cells, stem cells, cells in a regenerative stage such as post-wound or trauma, particularly hepatocytes, fibroblasts and epithelial cells. Cancer cells are a preferred type of cell, and effects of cancer cell type that can be used include kidney cancer cells, breast cancer cells, prostate cancer cells, choriocarcinoma cells, etc. The cancer cells may be cells of epithelial, mesothelial, endothelial or germinal origin, and include cancer cells that do not generally form solid tumors such as leukemia cells. As will be clear from this description, a further aspect of the invention are therapeutic methods for treating individuals suffering from hyperproliferative diseases such as cancer, inflammation, fibrosis, and trophoblastosis. It is also an aspect of the invention to control the growth of cells in a proliferative phase so much that they reproduce in a controlled manner. This is particularly useful, for example, to control the production of keloids and scar tissue by preventing adhesions after abdominal surgery, and possibly in hyperproliferative skin diseases such as psoriasis. When used in a therapeutic context, as will be elaborated below,. The type of cells restricted in the structure does not need to be the same type of cells that cause (or can cause) the suffering of the patient, although they can be. Such a method involves inserting at least one of the structures of the invention within the subject, in an amount sufficient to cause the suppression of cell proliferation in the subject. Preferably the method is for the treatment of cancer and the subject is a human being, although it is applicable to other animals such as domestic animals, farm animals or any type of animal suffering from cancer. The composition of the present invention can be used as a primary therapy in the treatment of hyperproliferative diseases, and as an adjunct treatment in combination with other therapies. For example, patients may be treated with compositions and methods described herein, along with known therapies such as radiation therapy, chemotherapy, treatment with other biologically active materials, such as cytokines, antisense molecules, steroid hormones, gene therapy, and Similar. The compositions and methods of the invention may also be used in conjunction with surgical procedures to treat cancer, for example by implanting the macrobeads after recession in a tumor to prevent regrowth and metastasis. Cancers that are present in an inoperable state can be made operable by treatment with the antiproliferative compositions of the invention. The compositions of the invention can also be used prophylactically in individuals at risk of developing cancer, for example in the presence of individual risk factors, generally family members with a history of cancer, family with a history of cancer of a specific type (for example, breast cancer). ) and exposure to occupational carcinogens or other agents that promote cancer. For prophylaxis against cancer, a prophylactically effective amount of the structures of the invention is administered to the individual with identification of one or more risk factors. As indicated in the examples, supra, the antiproliferative effect is not limited to the type of proliferative cell used, nor by the species from which the proliferative cell originates. Accordingly, one can administer structures containing, for example, cancer cells of a first type to a subject with a second, different type of cancer. Additionally, proliferating cells from a different species of the species being treated can be used in the structures administered. For example, mouse cancer cells can be restricted in the structures of the invention, and then administered to a human. Of course, the structures may contain cancer cells of the same species being treated. Still further, the proliferating cells may be taken from the individual to be treated, trapped and restricted, and then administered to the same individual. In other words, the patient's own cancer cells can be restricted and used to suppress the same cancer. Yet another aspect of the invention is the use of concentrates, as described herein, as therapeutic agents. These concentrates can be treated as described herein and then administered to a subject with a hyperproliferative disease such as cancer, or to a patient in need of controlling the rate of cell growth, such as a postoperative patient having a wound in a site where the excessive healing tissue would be detrimental or simply not desired. All the modalities described above can be used to prepare the concentrates. For example, after the in vitro culture of structures containing mouse cancer cells, the concentrates can be prepared and then administered to humans. Similarly, the structures may contain human cells and even cells from the same individual. Also, as discussed above, the type of cancer cell used to prepare the concentrate may be, and does not need to be the same type of cancer as the subject suffers. Accordingly, murine mammary cancer cells can be used, for example to prepare a concentrate to be used to treat a human with melanoma, or an individual with prostate cancer can obtain some of their prostate cancer cells removed, trapped in a structure of the invention, cultured in an appropriate medium, and then having the resulting conditioned medium filtered to produce a concentrate. It must be borne in mind that the conditioned medium resulting from cultures within the structures of the invention is also a part of the invention. The processes for making the structures of the invention, as well as the concentrates of the invention are also a part of the invention. In the case of concentrates, one simply cultivates the structures of the invention for a time sufficient to produce a sufficient amount of antiproliferative material and then separate the desired portions of the resulting conditioned medium, for example by filtration with a filter having a point of appropriate cut, such as 30 kilodaltons or 50 kilodaltons. Other facets of the invention will be clear to the skilled artisan, and do not need to be set forth herein. The terms and expressions that have been used are used as terms of description and not limitation, and there is no intention in the use of such terms and expression to exclude some equivalents of the characteristics shown and described or portions of them, recognizing that they are possible various modifications within the scope of the invention.

Claims (65)

1. CLAIMS 1. A composition useful for suppressing cell proliferation, produced by trapping a sample of rapidly proliferating cells in a selectively permeable, proliferative-restrictive, biocompatible structure, by culturing the structure in a culture medium for a sufficient time to restrict proliferation. ration of the trapped cells, characterized in that the trapped cells produce a cell proliferation suppression material which suppresses the proliferation of the cells and filters the medium through a filter separating the material having a molecular weight of at least about 30. kd of the material having a molecular weight of less than 30 kd and recovering the material having a molecular weight of at least about 30 kd.
2. 2. The composition according to claim 1, characterized in that the trapped cells are selected from the group consisting of neoplastic cells, cancer cells, embryonic cells, stem cells, hepatocytes, fibroblasts and epithelial cells.
3. 3. The composition according to claim 2, characterized in that the trapped cells are epithelial cells.
4. 4. The composition according to claim 2, characterized in that the trapped cells are cancer cells.
5. 5. The composition according to claim 4, characterized in that the trapped cells are selected from the group consisting of breast cancer cells, renal cancer cells, prostate cancer cells and choriocarcinoma cells.
6. 6. The composition according to claim 1, characterized in that the trapped cells are human cells.
7. 7. The composition according to claim 1, characterized in that the trapped cells are mouse cells.
8. The composition according to claim 1, characterized in that the structure contains from about 10,000 to about 500,000 cells.
9. 9. The composition according to claim 1, characterized in that the structure contains from about 30,000 to about 250, 0, 0 cells.
10. 10. A process for stimulating the production of a material having an effect that inhibits cell proliferation, characterized in that it comprises culturing a selectively permeable structure restrictive to biocompatible proliferation which has rapidly proliferating cells trapped therein in a medium for a time sufficient for the cells to produce proliferation suppression material, and to filter the medium through a filter that separates the material having a molecular weight of at least about 30 kd from the material having a molecular weight of less than 30 kd, and recover the material having a molecular weight of at least about 30 kd.
11. The process according to claim 10, characterized in that the trapped cells are selected from the group consisting of neoplastic cells, cancer cells, embryonic cells, stem cells, hepatocytes, fibroblasts and epithelial cells.
12. 12. The process according to claim 11, characterized in that the trapped cells are epithelial cells.
13. 13. The process according to claim 11, characterized in that the trapped cells are cancer cells.
14. 14. The process in accordance with the claim 13, characterized in that the cancer cells are selected from the group consisting of breast cancer cells, renal cancer cells, prostate cancer cells, and choriocarcinoma cells.
15. 15. The process according to claim 10, characterized in that the medium is free of serum.
16. 16. The process according to claim 10, characterized in that the trapped cells are human cells.
17. 17. The process in accordance with the claim 10, characterized in that the trapped cells are mouse cells.
18. 18. The process according to claim 10, characterized in that the structure contains from about 10,000 to about 500,000 cells.
19. 19. The process according to claim 18, characterized in that the structure contains from about 30,000 to about 250,000 cells.
20. 20. The process according to claim 10, characterized in that the structure is an account.
21. 21. A method for suppressing cell proliferation in a subject in need thereof, characterized in that it comprises administering to the subject a therapeutically effective amount of selectively permeable structure restrictive of biocompatible proliferation, containing restricted highly proliferative cells of a species other than that of subject, wherein the restricted cells produce a material that suppresses cell proliferation, in an amount sufficient to suppress cell proliferation in the subject.
22. 22. The method according to claim 21, characterized in that the restricted cells are a cell type different from the cell type associated with the condition with which the subject is afflicted.
23. 23. The method according to the claim 21, characterized in that the restricted cells are of the same cell type associated with the condition with which the subject is afflicted.
24. 24. The method according to claim 21, characterized in that the structure is an account.
25. 25. The method according to claim 21, characterized in that the structure contains from about 10,000 to about 500,000 cells.
26. 26. The method according to claim 23, characterized in that the structure contains from about 30,000 to about 250,000 cells.
27. 27. The method according to claim 21, characterized in that the restricted cells are selected from the group consisting of neoplastic cells, cancer cells, embryonic cells, stem cells, hepatocytes, fibroblasts and epithelial cells.
28. 28. The method according to claim 27, characterized in that the restricted cells are epithelial cells.
29. 29. The method according to claim 27, characterized in that the restricted cells are cancer cells.
30. 30. The method according to claim 29, characterized in that the cancer cells are selected from the group consisting of breast cancer cells, prostate cancer cells, renal cancer cells, and choriocarcinoma cancer cells.
31. 31. The method according to claim 21, characterized in that the subject is a human.
32. 32. The method of compliance with the claim 31, characterized in that the restricted cells are mouse cells.
33. 33. A method for suppressing cell proliferation in a subject in need thereof characterized in that it comprises administering to the subject a therapeutically effective amount of selectively permeable, biocompatible proliferative restricting structures containing restricted highly proliferative cells of the same species according to the subject, wherein the restricted cells produce a material having a molecular weight of at least 30 kd which suppresses cell proliferation, in an amount sufficient to suppress cell proliferation in the subject, wherein the suppressor material diffuses through the structures.
34. 34. The method according to claim 33, characterized in that the restricted cells are from an individual different from the subject.
35. 35. The method according to claim 33, characterized in that the restricted cells are taken from the subject to which the structures are administered.
36. 36. The method according to claim 33, characterized in that the subject is a human.
37. 37. The method according to claim 33, characterized in that the structure is an account.
38. 38. The method of compliance with the claim 33, characterized in that the restricted cells are selected from the group consisting of neoplastic cells, cancer cells, embryonic cells, stem cells, hepatocytes, fibroblasts and epithelial cells.
39. 39. The method of compliance with the claim 38, characterized in that the restricted cells are epithelial cells.
40. 40. The method according to claim 38, characterized in that the restricted cells are cancer cells.
41. 41. The method according to claim 40, characterized in that the restricted cells are selected from the group consisting of breast cancer cells, renal cancer cells, and prostate cancer cells.
42. 42. The method according to claim 33, characterized in that the restricted cells are of a different type of cells, whose proliferation is associated with the condition with which the subject is afflicted.
43. 43. The method according to claim 33, characterized in that the highly proliferative cells are of the same type according to the cells that cause the condition with which the subject is afflicted.
44. 44. The method of compliance with the claim 33, characterized in that the structure contains from about 10,000 to about 500,000 cells.
45. 45. The method according to claim 44, characterized in that the structure contains from about 30,000 to about 250,000 cells.
46. 46. The method for suppressing the proliferation of cells in a subject in need thereof, characterized in that it comprises administering to the subject a sufficient amount of the composition according to claim 1 to suppress the proliferation of the cells in the subject.
47. 47. The method according to claim 46, characterized in that the subject is a human.
48. 48. The method according to claim 46, characterized in that the trapped cells are selected from the group consisting of neoplastic cells, cancer cells, embryonic cells, stem cells, hepatocytes, fibroblasts and epithelial cells.
49. 49. The method according to claim 48, characterized in that the trapped cells are epithelial cells.
50. 50. The method according to claim 48, characterized in that the trapped cells are cancer cells.
51. 51. The method of compliance with the claim 50, characterized in that the trapped cells are selected from the group consisting of renal cancer, choriocarcinoma, breast cancer, and prostate cancer.
52. 52. The method according to claim 46, characterized in that the trapped cells are not human cells.
53. 53. The method according to claim 46, characterized in that the trapped cells are mouse cells.
54. 54. The method of compliance with the claim 46, characterized in that the trapped cells are human cells.
55. 55. The method according to claim 46, characterized in that the trapped cells are of the same cell type, whose proliferation is associated with the condition with which the subject is afflicted.
56. 56. The method according to claim 46, characterized in that the trapped cells are cells taken from the subject to which the structure is administered.
57. 57. The method of compliance with the claim 46, characterized in that the structure contains from about 10,000 to about 500,000 cells.
58. 58. The method according to claim 46, characterized in that the structure contains from about 30,000 to about 250,000 cells.
59. 59. The method according to claim 58, characterized in that the trapped cells are human cells.
60. 60. A composition of matter characterized in that it comprises a selectively permeable structure restrictive of biocompatible proliferation, the structure of restrictive cells which when restricted produce more than one material having a molecular weight of at least 30 kd which suppresses cell proliferation compared to an equal number of the same cells when they are unrestricted, where the material diffuses through the structure.
61. 61. A process for preparing a selectively permeable structure restrictive of biocompatible proliferation characterized in that it comprises the steps of forming a structure by contacting cells with biocompatible proliferative restraining material to form the structure, and cultivating the structures for a sufficient period of time to restricting the cells in such a way as to produce more than one material having a molecular weight of at least 30 kd which suppresses cell proliferation as compared to an equal number of unrestricted cells of the same type, where the material diffuses through of the structure.
62. 62. A method for increasing the production of a material that suppresses cell growth and has a molecular weight of at least 30 kd, characterized in that it comprises restricting highly proliferative cells with a material that forms structure to form a structure that contains proliferative cells that are restrictive of selectively permeable biocompatible proliferation and grow the cells until they are restricted and produce more of the material at least 30 kd than an equal number of unrestricted cells, where the material diffuses through the structure.
63. 63. A composition useful for suppressing cell proliferation, produced by trapping a sample of rapidly proliferating non-cancerous cells in a selectively permeable structure restricting biocompatible proliferation, by culturing the structure in a culture medium for a sufficient time to restrict the proliferation of the non-cancerous cells trapped, characterized because the non-trapped cancer cells produce a material that suppresses cell proliferation, which suppresses the proliferation of the cells.
64. 64. A process for stimulating the production of a material having an effect that inhibits cell proliferation, characterized in that it comprises culturing a selectively permeable structure of biocompatible proliferation that has rapidly proliferating non-cancerous cells trapped therein in a medium for a time enough for cells to produce material that suppresses proliferation.
65. 65. A method for suppressing cell proliferation in a subject in need thereof characterized in that it comprises administering to the subject a therapeutically effective amount of selectively permeable biocompatible proliferative restrictive structures containing restricted highly proliferative non-cancerous cells of the same species according to the subject , wherein the restricted non-cancerous cells produce a material that suppresses cell proliferation, in an amount sufficient to suppress cell proliferation in the subject, wherein the suppressor material diffuses through the structures.