Compositions of restricted cells capable of quick growth and producing agents having cell proliferation inhibiting activity and uses thereof

FIELD: medicine, in particular composition containing proliferating cells with limit growth.

SUBSTANCE: invention relates to cells producing unexpected high yield of substance, which inhibits cell proliferation. Such property is independent neither cell type nor genus thereof.

EFFECT: new compositions for cell proliferation inhibition.

21 cl, 18 ex, 15 tbl

 

Scope

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 istropolitana cells of the same or different types and/or origin. Those affected by the restriction of the cells are referred to here as proliferative cells. Limitiruyuschie list of proliferative cells belonging to the specified category includes 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, such as concentrates from around the established molecular weight, which also have an antiproliferative effect on proliferating cells associated with a disease or relevant SOS is ojaniemi, for example 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 patents 5227298 (Weber et al.); 5053332 (Cook et al.); 4997443 (Walthall et al.); 4971833 (Larsson et al.); 4902295 (Walthall et al.); 4798786 (Tice et al.); 4673566 (Goosen et al.); 4647536 (Mosbach 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 what kolichestvo the same (previously mentioned), but not subjected to the restriction of cancer cells.

In patents Jain et al. detail obsujdayte techniques 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 ultrafiltration camera, microencapsulation and macroencapsulation. There are many problems associated with 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 for incapsulate the Oia of 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 (Lira, et al., Science 210:908 (1980)). Implantation of these microencapsulating Islands supported autocamionale condition in animals with diabetes. However, 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)). Now suppose that the solubility of these polymers in water due to the limited stability and biocompatibility of 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 the poison weaknesses. 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 are not subject to restoration and recovery, is quite fragile and the slightest damage to the islets are easily disconnected.

How macroencapsulation was also investigated. For immunovaccine islets were made microcapsule from a variety of materials, such as poly-2-hydroxyethyl-methacrylate, polyvinyl chloride - acrylic acid, and cellulose acetate (See. Altman, et al., Diabetes 35:625 (1986); Altaian, 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 UD is ininii life expectancy is included in the hollow fiber neonatal rat pancreatic grafts, which 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 solve this problem using alginate dispersion of Islands in the fiber. However, this experiment was not reproduced repeatedly. 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.

A literature review on Encaps is the place 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(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. 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): 5095-5103 (1996); Gardiner, et al., Transp. Proc. 29:2019-2020 (1997). None of these links does not mention the inclusion of cells capable of 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 the multiplication is the and decline and stops. This suggests that there is some internal mechanism for regulating the growth of cancer cells.

The invention

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 large number of substances which, when applied to cells not subjected to restriction and fast-breeding, inhibits the reproduction 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, h is concerns about 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 the antiproliferative effect cannot be attributed to classical immunological reactions.

Thus, the 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 structure and kultivirovanie structures over an extended period of time, 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 the invention relates to a method for increasing production of a substance that inhibits the growth of cells proliferating 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 is 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 the treatment of cancer and other conditions provides additional benefits, as is obvious to a person skilled in the field.

These and other features of the invention will be understood from the following description.

A detailed description of the preferred options

Example 1.

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 37°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 the components. 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 the mice were examined and were found numerous tumors of different sizes. In addition, some of the tumors were bleeding.

A sample from a single tumor, taken from one of the mice, fixed in 10% formalin for further histological validation.

Example 2.

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), maintained at 60°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 is deficient in oil to room temperature. This mixture formed a single smooth semi-solid granule. This procedure was repeated to obtain several granules.

A moment later, the pellets were transferred to complete medium, as described above, at a temperature of 37°C. the Pellets were then washed three times in minimum essential medium (MEM)containing antibiotics listed above. The granules are then kept overnight at 37°C in 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 37°to remove the oil. After this has been aging over night at 37°C in humidified air with 5% CO2.

Received RENCA containing granules used in further experiments.

Example 3.

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 environment description is authorized in example 2, within 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, and cultural environment.

Observation of these cultures showed that cells grow and form a standard RENCA colony. This proves that the cells in the pellet remained viable.

Example 4.

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 we have is her received implants encapsulated in granule cells showed only small nodules throughout the cavity.

These encouraging results formed the basis of the plan of the experiments described in the following example.

Example 5.

In these experiments simulated in advance certain 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 day 14 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 organs of the abdominal cavity steel newsnew is feasible as a result of losing a 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 the experimental and control groups showed that in the experimental group nodules were not increased compared with their initial growth under the renal capsule and prior to implantation of microgranule.

Example 6.

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 1×104RENCA 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 is emali 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 120000-240000 MPM cells. The cups were incubated at 37°C for one week, after which RENCA cells were treated with trypsin, washed and cheated by using gamopetalae.

The results are shown in table 1.

Table 1
Cup 1Cup 2
The number of cells recovered after one weekThe number of cells recovered after one week
Hole No.Control (blank microgranule)120000 MPM cellsControl (blank microgranule)240000 MPM cells
12,4×1051,4×1052,8×1051×105
22,0×1051,2×1053,6×1057×105
34,4×1051,25×1052,5×1059×105

Example 7

After receiving the results of example 6 are the same on the ity was done using as inoculum 1× 104MPM cells (i.e. empty cells) instead of RENCA cells. The experiment was carried out exactly as in example 6. The results are shown below in table 2.

Table 2
Cup 1Cup 2
Hole No.Control (blank microgranule)120000 MPM cells in microgranuleControl (blank microgranule)240 000 MPM cells in microgranule
13,1×1061,6×1062,8×1061,3×106
23,3×1061,0×1062,6×1061,1×106
33,0×1066,0×1052,8×1065,0×105

These results confirmed the in vivo experiment. These data are presented in example 8.

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 times the Helen group, 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.

During the first few days, mice in groups a and b were sluggish with disheveled hair. It lasted about five days, and then observed normal behavior of mice. After 21 days after implantation, all animals entered 40000 RENCA 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.

Table 3
The number of microgranule in miceControlExperimental group aExperienced group
1++++--
1++++--
1+++
2++++--
2--
2++++
3++++--
3--
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 this breath is retene leads to the production of substances inhibiting and/or preventing the multiplication of tumor cells. This was studied further in the experiment, 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 given above.

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 35×100 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 at 37°C for five days. At the end of incubation cells were treated with trypsin, washed, collected and counted using hemocytometer.

The results are shown in table 4:

Table 4
Experience the Well # RENCA cells with control mediumRENCA cells with conditioned medium
17×1053×105
28×1052,5×105
37×1053,4×105

These results show that cells while limiting their inclusion, for example, granules according to the examples, produce a substance that inhibits the multiplication of tumor cells.

Example 10.

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 35×100 mm Petri dishes with 50 ml RPMI complete medium at 37°C. After incubation the medium was collected and stored at -20°C. the Conditioned medium was preparation, is in the incubation granules, containing MPM cells (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 37°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, in 4 ml of medium was added to each cell. All cells were inoculable 10000 RENCA cells and incubated at 37°C for 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 as follows:

Table 5
Cup 5-dayControl environmentFrozen air-conditioned environment RENCAFrozen air-conditioned environment MPM
16×1055×1058×105
26,8×1054,2×1058,5×104
8 day
32,8×1062×1068×104

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.

Example 11.

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 subjected to the restriction of tumor cells, grown to confluence, also to develop a substance that regulates proliferation.

For these experiments, 10 microgranule, each of which contained 120 000 RENCA or MPM cells (i.e 1,2× 106cells only) used for conditioning medium (complete RPMI) during the 5-day period. In parallel 1,2×106RENCA 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, which are well known, too small to hold a 30 KD filter. The filtrates were also investigated, but any interpretation of the results with this material is complicated by the presence of waste CL is the exact 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 monitored.

Basically, conditioned medium was collected or 3-5 days after microgranule were added thereto, or 24 hours 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 or 50 KD), and centrifuged for 90 minutes. The 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%.

Table 6

Inhibition of growth of RENCA cells in the environment, air-conditioned microgranuloma with RENCA cells, and in the selected concentrates
latina No. Reconditionnement RPMI mediumWednesday, air-conditioned microgranuloma with RENCA cells30 KD concentrate in this environment50 KD concentrate in this environment
The number of cellsThe number of cellsInhibitionThe number of cellsInhibitionThe number of cellsInhibition
11,6×1067,8×10551,3%4,2×10497%2,0×10499%
21,65×1068,0×10551,5%5,0×10497%2,0×10499%
Table 7

Inhibition dew RENCA cells in medium conditioned by culture RENCA cells, and in the selected concentrates
Plate No.Reconditio Nirvana RPMI mediumWednesday, conditioned culture RENCA cells30 KD concentrate in this environment50 KD concentrate in this environment
Quantities of the cell The number of cellsInhibitionThe number of cellsInhibitionThe number of cellsInhibition
11,6×1061,3×10618,8%1,1×10631,3%9,0×10543,8%
21,6×1061,2×10625,0%1,0×10637,5%a 9.5×10540,6%

Table 8

Inhibition of growth of RENCA cells in the environment, air-conditioned microgranuloma with RENCA cells, and the concentrate (AIM V medium)
Plate No.AIMV control environmentAir-conditioned environment30 KD concentrate50 KD concentrate
The number of cellsinhibitionthe number of cells% inhibitionThe number of cells% inhibition
11,3×1066,0×10554% -5,0×10496%-4,0×10497%
21,3×1065,5×10558%-5,0×10496%-4,0×10497%

The important point in this experiment is the fact that MPM cells, and RENCA cells being included in microgranule and subjected as a result of this restriction, inhibit the reproduction 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 invivo. 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.

Example 12.

These experiments show that air-conditioned as MMT and RENCA cells among the and contains the substance released from the 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 independent of the form of an animal, which naturally arose tumor.

In this experiment tested, does cell line of breast cancer human inhibitory 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 obtained when the topic can be found below.

Table 9

The effects of MCF-7 microgranule on RENCA target cells
Cell No.Control (blank microgranule)MCF-7 microgranule
18,4×1054,4×105
28,0×1054,4×105
37,4×1053,8×105

Table 10

The effects of MCP-7 air-conditioned environment on RENCA target cells
PlateRPMI Control environmentRPMI medium, air-conditioned MCF-7
19,0×1055,0×105
28,8×1054,8×105
Table 11

The effects of MCF-7 air-conditioned environment on MMT target cells
PlateRPMI Control environmentRPMI medium, air-conditioned MCF-7
15,0×1051,5×105
26,0×1051,8× 105

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 and tumor cells of the mammary gland of the mouse to a significant extent (30-70%), as was demonstrated with the help of microgranule and their air-conditioned 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.

Example 13.

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 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 mice euthanized at the twenty-seventh day.

If Necroscope control mice was observed absolute fullness of the abdominal tumors and normal organs become unrecognizable. This tumor saturation was classified as a +++++ (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.

Example 14.

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 are the spruce to check can cells adenocarcinoma breast cancer 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 life. Seven mice with the risk of developing these tumors these tumors appeared in the sixteenth month of life. At this time, five of these seven mice implanted by 4 MMT of microgranules 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 on postie 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 35-th day after injection. Nine treated mice monitored weekly during this period were alive without 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 with the touch 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 in the chest area.

Example 15.

The above experiments demonstrate the inhibiting effect is included in microgranule and limited reproduction of cancer cells in relation to cell proliferation/growth of the tumor, which is manifested 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 a 2.5×106ARCaP10 cells. On the twentieth on the HB after 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 (1,0×105cells 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 the group treated mice, five mice were observed complete regression of the initial tumors, and all the rest were no tumors up to killing eight months. Two mice revealed the absence of tumor growth, i.e. their 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. × Shir. × h)] the experiment, which RENCA-containing microgranules (1,2×105) were implanted 4 Nu/Nu mice after 18 days after subcutaneous injection each animal in the side by a 3.0×106ARCaP10 tumor cells, is shown below.

Table 12

The sizes of the tumors observed in treated mouse on the y (in mm)
The treated mouseThree days before transplantationThe day of transplantation (3/6/98)3 days after. transplantation (3/9/98)6 days after transplantation10 days after transplantation14 days after transplantation
(3/3/98)(3/12/98)(3/16/98)(3/20/98)
13,5×3×6,2×5,44×4×endangered00
flatflatflat
23×3×1,55,1×2,2×24×2×0,53×3×0,42×2×0,32×2×0,3

33×to 2.5×13,1×3,3×13×2×0,53×2×0,23×2×0,23×2×0,2
42,5×to 2.5×3,2×3,4×0the spot under000
flat5leather
Table 13

The volume of tumors observed in treated mice
The treated mouse No.Three days before transplantation (3/3/98)The day of transplantation (3/6/98)3 days after transplantation (3/9/98)6 days after transplantation (3/12/98)10 days after transplantation (3/16/98)14 days after transplantation (3/20/98)
1was 2.768,811,68about00
27,1011,812,101,890,630,63
33,955,381,580,630,630,63
41,642,860000

In another experiment 10 Nu/Nu mice were administered 2.5×106Arsar 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 (2,4×105cells), and three empty microgranule. The results are shown below:

Table 14

The sizes of the tumors observed in treated mice (in mm)
The treated mouse No.5 days before transplantation (2/5/98)The day of transplantation (2/10/98)18 days after transplantation (2/28/98)22 days after transplantation (3/4/98)2? days after transplantation (3/9/98)30 days after transplantation (3/12/98)
12×2×13×3×1,51×1×0,5000
23×2×13×to 2.5×12×2×<1 mm<0.8 mm<0.8 mm
flat
34×4×1,56×6×1,56×2×4×1×3×1×3×1×
flatflatflatflat

Table 15

The sizes of the tumors observed in the con the control mice
Control mouse No.5 days before transplantation (2/5/98)The day of transplantation (2/10/98)18 days after transplantation22 days after transplantation27 days after transplantation30 days after transplantation
(2/28/98)(3/4/98)(3/9/98)(3/12/98)
14×4×1,55×5×26,5×6×36,5×6×36,5×6×37×7×3
23×2×14×6×34,5×7×35×8×311×12×513,3×13,3×
6,5
2nd
tumor
6൶× 1
35×4×15×4×25x4,6×2,55×5×2,56×6×2,57×7×2,5
a lot2nd2nd
Dolnytumortumor
HHHH,5

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 not only inhibit the growth of tumors and to prevent its formation, but also effective to cause regression of tumors in vivo.

Example 16.

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 tipo is cancer in vitro and to prevent the formation of the 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 cm × 2.5 cm (length (width), and the ventral part of thighs 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 cells and 40 pellets containing MCF-7 breast cancer cells human). 2 days later the tumor on the dorsal part of thigh decreased by 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 m is crosrol, while the 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 the efficiency of cancer cells with the restriction of the multiplication in the treatment of cancer.

Example 17.

The above experiment shows that 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 effective depend on the 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, culture 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 or 1,2×105RENCA cells, either with 2.4×105MPM cells. They were verified histologically (hematoxylin + eosinophile staining and culture method on cell viability and tumor characteristics in the above time intervals. As for RENCA of microgranule, the number of cells increased approximately 3-5 times the first month, and then further 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 skiplists is 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 year, the ratio of viable cells was slightly lower, falling by approximately 20% from the maximum number obtained in the pellet (i.e. in a state of growth restrictions) 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 which ranulf, maintain 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 the MPM.

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 in warm-blooded animals continuously and over a long period of time, substances that suppress tumor growth that will be necessary for successful treatment of experimental or naturally arising cancer.

Example 18.

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, about Asociate tumors and highlight 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 (1,2×105cell 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 or a growing tumor. All the organs looked quite normal, although there was a slight adhesion of the granules 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 to prevent, is davleniya and regression of cell reproduction, including the in vivo tumor types.

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 amount of a substance that suppresses the reproduction of highly proliferating cells such as 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 supressive substance can diffundere the TB out, in 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 nutrients, removal of products of cell metabolism and compatible physico-chemical conditions inside the structure. The material used to obtain this structure, preferably, is well compatible material for implantation in vivo and, most preferred, 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)-polietileno-min; chitosan-alginate; polyhydroxyethyl-methacrylate-methyl methacrylate; carbonylmethyl; K-carrageenan; chitosan; agarose-polyethersulfone-Gex is di-meterin-bromide (Polybrene); ethylcellulose; silica gels and combinations thereof.

Patterns that contain the composition of substances, can have different forms, such as 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 the skilled technician.

Structures of this invention have selective permeability, so that the nutrients can penetrate into the structure, and inhibiting the proliferation of a substance, as well as cellular waste can leave the structure. For in vivo use, it is preferable that these structures were guarding "the entrance" products or cells of the immune system of the host that 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 be p the volume 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 the preferred cell type and approximate types of cancer cells, which can be used include cancer cells, kidney, breast cancer cells, prostate cancer cells, cells 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, such as 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 invention is also con is the role of the growth of cells in the proliferative phase with the so 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 such as 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 of at least one of the described structures according to the invention to an individual in amounts sufficient to cause 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 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 is Tim forms of therapy, for example, irradiation, chemotherapy, gene therapy, treatment with other biological active substances such as cytokines, substances, dulling the sensitivity (containing drugs), steroids, etc. Compositions and methods of this invention can also be used in addition to surgical procedures, cancer treatment, such as 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 invention.

Compositions of the invention can be used prophylactically in individual cases at risk of developing 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 the notches, no 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. Moreover, proliferating cells can be obtained from the individual who undergoes treatment, included in the structure, limited to the 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 speed of reproduction of cells, n is the sample post-operative patient which 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 type of cancer from which the patient suffers. However, breast cancer cells mouse can be used, for example, 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 what I 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 which have been applied, are used to describe the invention and the application of these terms and expressions does not limit and does not seek to exclude any equivalents disclosed and described signs or parts thereof, it is understood that in the scope of the invention various modifications are possible.

1. The method of suppressing the propagation of cells from the individual, introducing him therapeutically effective amount of pellets covered with agar, incorporating subjected to limitation in reproduction in a high degree, proliferating cells, obtained 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 reproduced is the cell number, sufficient to suppress cell multiplication of the specified individual.

2. The method according to claim 1, which is subjected to the constraint cells are cells other than the cell type associated with that condition that affects the individual.

3. The method according to claim 1, which is subjected to the constraint cells belong to the same type as the cells associated with that condition that affects the individual.

4. The method according to claim 1, wherein the granule contains from about 10,000 to about 500,000 cells.

5. The method according to claim 4, in which the granule contains from about 30,000 to about 250,000 cells.

6. The method according to claim 1, which is subjected to the constraint cells are epithelial cells.

7. The method according to claim 1, which is subjected to the constraint cells are cancer cells.

8. The method according to claim 7, 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.

9. The method according to claim 1, which is subjected to the constraint cells are stem cells.

10. The method according to claim 1, wherein said individual is a human.

11. The method according to claim 1, which is subjected to the constraint cells are mouse cells.

12. The medium containing the substance, inhibiting the proliferation of cells obtained by the method including the following this is s:

(i) the inclusion of sample istropolitana cells in biocompatible material with obtaining permeable structure, limiting the reproduction of these cells;

(ii) the location of the structure obtained in the culture medium;

(iii) culturing the specified patterns in culture medium for a time and under conditions necessary for granting listrophoridae cells through the permeable structure in the culture medium of a substance capable of inhibiting proliferation of cells;

(iv) collection of the resulting environment.

13. Environment item 12, 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.

14. Environment item 13, which included cells are epithelial cells.

15. Environment item 13, which included cells are cancer cells.

16. Environment § 15, which included cells selected from the group comprising breast cancer cells, cells of kidney cancer, prostate cancer cells and cells choriocarcinoma.

17. Environment item 13, which included cells are stem cells.

18. Environment item 12, which included cells are human cells.

19. Environment item 12, which included cells are mouse cells.

20. Environment p is 12, in which the structure contains from about 10,000 to about 500,000 cells.

21. Environment according to claim 20, in which the structure contains from about 30,000 to about 250,000 cells.



 

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2 cl, 2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: the method deals with selecting primary raw material, followed by acidic-alkaline treatment, cutting, forming semi-finished ligatures, followed by drying, polishing and packing into ampoules or parcels, followed by sealing and sterilization. As primary raw material one should predominantly apply porcine viscera followed by elution of ballast substances with1.3 dimethylketone, treatment with succinic anhydride, then comes cutting for, at least, 2-4 parts to be sterilized in dry water-free state at no any preliminary conservation with predominantly radiation emission followed by material rehydration and packing into individual parcels, then comes conservation and sealing individual packing with sterile ligatures. Rehydration of ligatures should be performed, for example, for 16 h in 0.02% solution of (1.2-ethylene-bis N diethylcarbodecynoxymethyl) ammonium chloride. Power of radiation emission at sterilization corresponds to at least 15 kGy, as for polishing, it is predominantly fulfilled due to "drilling". Simultaneously one should perform group sterilization of ligatures without individual packing followed by packing the sterile material. Group sterilization of suture material should be performed without individual packing with gas method of ethylenoxide, the mixture of ethylenoxide with methyl bromide or beta-propiolactone followed by packing the sterile material. The method provides considerable decrease of production cost price, decreased expenses of primary raw material, improved histocompatibility, decreased allergic and purulent-inflammatory complications and keeping strong parameters during sterilization.

EFFECT: higher efficiency.

5 cl, 3 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: the suggested multi-purpose heterogeneous collagen matrix for implantation is being flexible-elastic mass obtained out of two collagen sources, moreover, one source is a tissue of vertebrates of one and the same class, and another source - that of an animal of another class, moreover, collagen sources are animal tissues of, for example, mammalian class and avian class, matrix consists of two phases: solid phase - as microspheres out of mammalian tissue collagen, and liquid phase - out of denaturated avian tissue collagen at the ratio of phases being (1-10) : (1-10), at microspheres size being 100-300 mcm, as for final products of biodegradation they are represented by CO2 and H2O. Matrix, additionally, contains components of physiological culture media, and, also, additives that favor the growth and differentiation of cells and tissues, antibacterial and/or antiviral components, and, also, antiaggregation preparations in their efficient quantity, for example, additionally, it contains embryonic cells of nervous tissue. Another aspect of the present innovation deals with the method to obtain the matrix due to preparing mammalian collagen solution (MCS) and denaturated avian collagen solution (ACS), moreover, it is necessary to apply 0.3 M acetic acid, at final concentration for MCS being approximately 0.5-1.5%, and for ACS - approximately 3.0-5.0%, then MCS should be treated with γ -irradiation at the dosage of 1.0 Mrad to be further homogenized to obtain microspheres. Then both MCS and ACS should be washed off with distilled water up to pH of not less than 6.0 and with phosphate buffer solution to mix washed off mammalian collagen and avian collagen at 1:1 ratio at obtaining matrix. The matrix obtained should be additionally supplemented with antibacterial and/or antiviral components, and, also, stimulating agents for tissue regeneration and antiaggregation preparations. The matrix obtained should be sterilized due to γ -irradiation at the dosage of 0.5 Mrad/1 ml. The present innovation enables to obtain new heterogeneous collagen matrix which is considered to be a multi-purpose one applied for transplantology and substitution surgery of different tissues and organs in alive body in case of tissue lesions. Moreover, it is distinguished by controlled terms of biodegradation.

EFFECT: higher efficiency of application.

13 cl, 16 ex, 4 tbl

FIELD: biotechnology and pharmaceutical industry.

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

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

77 cl, 9 dwg, 3 tbl

FIELD: cellular biology, medicine.

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

EFFECT: valuable biological and medicinal properties of cells.

41 cl, 7 tbl, 13 dwg, 15 ex

The invention relates to medicine, in particular to clinical immunology

The invention relates to veterinary pharmacology

FIELD: cellular biology, medicine.

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

EFFECT: valuable biological and medicinal properties of cells.

41 cl, 7 tbl, 13 dwg, 15 ex

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