KR20000005246A - Implantable agarose and collagen beads containing cells which produce a diffusible biological product, and uses thereof - Google Patents

Abstract

PURPOSE: Implantable beads which are made of agarose and collagen, and/or coated with agarose have incorporated within samples of cells which produce diffusible biological products. CONSTITUTION: The cells produce diffusible biological products. The beads may be used as implants to modulate a recipient's immune response. The beads can also be used in an in vitro context to encourage specific types of cells to grow, to produce desirable products in culture, or to suppress growth of certain cells. The implants can also suppress growth of certain cells following administration to a subject.

Description

Implantable Agarose-Collagen Beads Containing Diffuse Biological Product Producing Cells, and Uses thereof

<Related application>

This application is a partial continuing application of co-pending application serial number 08 / 625,595, filed April 3, 1996, which is incorporated herein by reference.

<Field of invention>

The present invention relates to encapsulation of cells in agarose and collagen followed by agarose coating, methods of treatment using cells of such materials and methods for their preparation.

〈Background and Prior Art〉

Encapsulation of various biological materials in biologically suitable materials is a technique that has been used for some time, although with limited success. Examples of such techniques are described in US Pat. Nos. 5,227,298 to Weber, et al .; No. 5,053,332 to Cook, et al .; No. 4,997,443 to Walthall, et al .; 4,971,833 to Larsson, et al .; No. 4,902,295 to Whalal; No. 4,798,786 to Tees, et al .; No. 4,673,566 to Goosen, et al .; No. 4,647,536 to Mosbach, et al .; No. 4,409,331 to Lim, et al .; No. 4,392,909 to Rim, and 4,352,883 to Rim; And No. 4,663,286 to Tsang, et al. Also pending is co-pending application serial number 08 / 483,738, which is incorporated herein by reference and filed on June 7, 1995 by Jain, et al., Et al. Zain et al. Discussed in detail the encapsulation of secretory cells in various biocompatible materials. As discussed in Zain et al., Secretory cells are cells that secrete biological products. In general, secretory cells have at least some properties of endocrine cells and can be treated on an equal basis with cells that are usually in fact endocrine glands. In co-pending applications, for example, the encapsulation of insulin producing cells is preferably encapsulated with agarose-collagen beads, also coated with agarose, preferably in the form of islets. The products produced are useful for treating the symptoms of patients in need of insulin treatment, such as diabetes.

Zain et al. Discuss in more detail the prior studies accepted in the art in transplantation therapy. This study is also summarized herein.

Five major methods for protecting the transplanted tissue from the host's immune response are known. All methods relate to attempts to isolate transplanted tissue from the host's immune system. Immunoisolation techniques used to date include extravascular diffusion chambers, intravascular diffusion chambers, intravascular ultrafiltration chambers, microencapsulation, and macroencapsulation. However, all of these methods, however, include a host's fibrosis response to the graft material, instability of the graft material, restriction of nutrient diffusion through the semipermeable membrane, secretagogue and secretory permeability, and diffusion delay-time through the semipermeable membrane boundary ( failed due to one or more problems of lag-time).

For example, a microencapsulation method for sealing live cells, tissues, and other labile membranes in a semipermeable membrane was developed in 1978 by Lim (Lim, Research Report of Damon Corporation (1978)). Rim used microcapsules of alginate and poly L-lysine to encapsulate Langerhans islets. In 1980, the first successful in vivo use of this new technique for diabetes was reported (Lim, et al., Science 210: 908 (1980)). Transplantation of these microencapsulated islets led to a maintenance of normal blood glucose in diabetic animals. However, other investigators who repeated these tests found that alginate caused tissue reactions and could not reproduce the results of the rim (Lamberti, et al., Applied Biochemistry and Biotechnology 10: 101 ( 1984); Duffy, Dupuy, et al., J. Biomed.Material and Res. 22: 1061 (1988); Weber, Weber, et al., Transplantation 49: 369 (1990); And Dun-shiong, et al., Transplantation Proceedings 22: 754 (1990). The water solubility of these polymers is currently believed to be responsible for the limited stability and biocompatibility of these microcapsules in vivo (see Duffy, Weber, Dun-Schong, and Smithsrod, Smidsrod, Faraday). Discussion of Chemical Society 57: 263 (1974).

Recently, Iwata, et al., Jour. Biomedical Material and Res. 26: 967 (1992) used agarose for microencapsulation of allogeneic pancreatic islets and found that it could be used as a medium for microbead preparation. In this study, 1500-2000 pancreatic islets were microencapsulated in 5% agarose, respectively, and transplanted into streptozotocin-induced diabetic mice. The graft survived for a long time and the recipient maintained normal blood glucose indefinitely.

However, this method has many drawbacks. This method is difficult to handle and inaccurate. For example, many beads remain partially coated and hundreds of empty agarose forms of beads are formed. Thus, additional time is required to separate the encapsulated islands from the empty beads. Moreover, most of the implanted microbeads accumulate in the pelvic cavity, and large amounts of islets are required in each fully coated bead to obtain normal blood glucose. In addition, the implanted beads are difficult to recover and tend to be brittle, which will easily release the islands even with some minor damage.

In addition, macroencapsulation procedures are also being tested. Macrocapsules of various different materials, such as poly-2-hydroxyethyl-methacrylate, polyvinylchloride-c-acrylic acid, and cellulose acetate, have been prepared for immune isolation of islands of Langerhans (see, eg, Altman's literature). Altman, et al., Diabetes 35: 625 (1986); Altman, et al., Transplantation: American Society of Artificial Internal Organs 30: 382 (1984); Ronel, et al., Jour.Biomedical Material Research 17: 855 (1983); see Klomp, et al., Jour.Biomedical Material Research 17: 865-871 (1983). In all these studies, only transient normalization of glycemia was achieved.

Archer, et al., Journal of Surgical Research 28: 77 (1980) used acrylic copolymer hollow fibers to temporarily prevent rejection of islet xenografts. Arker reported long-term survival of pancreatic tissue grafts of murine fetuses dispersed in hollow fibers implanted in diabetic hamsters. Recently, Lacy et al., Science 254: 1782-1784 (1991) confirmed such results, but found that normal blood glucose status was short term. When Lacy injected the fibers into the fibers, they found that the islands aggregated in the hollow tube causing necrosis in the central portion of the island colony. Central necrosis prevented graft prolongation. To solve this problem, lash was used to distribute the islands in the fiber using alginate. However, the test was not repeated intensively. Thus, the function of the membrane as islet transplantation medium in humans has been questioned.

Thus, it was necessary to achieve secretory cell transplantation and, in particular, pancreatic islet allograft and xenograft survival without the use of chronic immunosuppressants.

In the study discussed above, the inventors reported that encapsulating secretory cells in hydrophilic gel materials to produce functional non-immunogenic materials that can be transplanted into animals and stored for long periods of time. Encapsulation of secretory cells has provided a more effective and manageable technique for secretory cell transplantation. Encapsulation techniques have been described as useful for encapsulating other biological substances, such as enzymes, microorganisms, recombinantly produced nutritional substances, cytotoxic substances, and chemotherapeutic agents. Encapsulated biological materials have been discussed as useful for treating symptoms known to respond to biological materials.

The application does not discuss to any extent the incorporation of cells that produce diffuse biological materials useful for treatment. Herein is the difference between secretory cells and cells producing diffuse biologics. According to the examples set forth in the design of gynecologic secretory cells usually represent products commonly thought of as biological "messengers" such as hormones, cell signaling substances and the like. In contrast, diffusible biological material refers to materials such as MHC-providing peptides, cell expression regulators such as inhibitors, promoters, inducers, and the like. Such differences can be seen in the field of oncology as discussed below.

Intensive studies on cancer include heterogeneous cell extracts, and studies of various cellular components. Using monoclonal antibodies, related cancers associated with antigens such as GM2, TF, STn, MUC-1 and various epitopes derived therefrom have been identified in the art. Current theory assumes that epitopes derived from these various tumor markers bind noncovalently with MHC molecules, thus forming soil types by specific cytolytic T cells. This mechanism is different from the various mechanisms involved in the biological response to viral infections. In this regard, Van der Bruggen, et al., Science 254: 1643-1647 (1991); See, for example, Boon, et al., 5,405,940 and 5,342,774, both of which are incorporated herein by reference.

Another study paralleling the study of the so-called identification of cancer epitopes focuses on the control of cancer proliferation via, for example, inhibitory action or, more generally, bioregulation. See, eg, Mitchell, J. Clin. Pharmacol 32: 2-9 1992; Maclean, Maclean, et al., Can. J. Oncol. 4: 249-254 (1994). The aim of encompassing all of the various studies on cancer is to modify the host's immune response to improve the patient's condition.

The key to all these studies lies in the activity of one or more diffuse biological products that work in tandem with other substances to modulate the immune response. Boun and Van der Brügen et al. Disclosed small peptide molecules. Mitchell discussed larger molecules that function like inhibitors.

The problem with all treatment methods using these materials is to transport them in a safe and effective form. This is not easy to achieve. Surprisingly, it has now been found that techniques such as Zyne, which are very useful in the development of therapies for symptoms requiring secretory cell products, are now available in other areas.

The method for achieving this is the object of the present invention, which is described in detail below.

<Detailed Description of the Preferred Embodiments>

<Example 1>

In this example and the following examples, RENCA cells were used. These cells are naturally occurring renal adenocarcinoma cells of BALB / C mice that are widely obtained because they are maintained in both in vivo and ex vivo cultures. See, eg, Franco, et al., Cytokine Induced Tumor Immunogenecity, 181-193 (1994).

Frozen Lenka cell samples were thawed at 37 ° C., followed by Dulbecco's Modified Medium (D-MEM) fed 10% bovine serum, penicillin (100 u / ml) and streptomycin (50 μg / ml). Into the containing tissue culture flask, a medium was referred to herein as "complete medium".

Cells were allowed to grow, then treated with trypsin and washed with Hank's Balanced Salt Solution and then complete media as mentioned above.

To determine if the Lenka cells efficiently produced tumor cells, two BALB / C mice were injected intraperitoneally with 10 ⁶ of these cells. Mice were observed over a 3-4 week period. Clinically, mice appeared healthy during the first two times and were normal in activity. Later, the emergence of clinical cancer became evident. One mouse died 23 days later and the second died 25 days later. After death, mice were examined and examined for many tumors of various sizes. In addition, some tumors showed bleeding.

One tumor sample removed from one mouse was then immobilized in 10% formalin for histology.

<Example 2>

After knowing that the Lenka cells had grown in vivo, a test was conducted to determine if these cells had grown in the beads according to the invention.

Lenka cells were cultured to combine as described above, treated with trypsin and washed as described above. Subsequently, samples of 60,000 to 90,000 cells were prepared. Cells were then centrifuged at 750 RPM and fluid removed. The cells were suspended in 1% atelocollagen solution in phosphate buffered saline solution at pH 6.5.

A low viscosity 1% solution of agarose was prepared in minimal essential medium (MEM) and maintained at 60 ° C., then 100 μl of this solution was added to the above-described Lenka cell and atelocollagen suspensions. The preparation was then immediately dropped as a large drop of droplets into sterile mineral oil at room temperature. The mixture formed a single smooth semisolid bead. This process was repeated to produce many beads.

After 1 minute, the beads were transferred to the complete medium described above at 37 ° C. The beads were then washed three times with the minimum essential medium containing the antibiotic described above. The beads were then incubated overnight at 37 ° C. under humid air and 5% CO ₂ atmosphere. After incubation, the solidified beads were transferred to a sterile spoon containing 1 ml of 5% agarose in minimal essential medium. The beads were rolled 2-3 times in solution and uniformly coated with agarose. The beads were transferred to mineral oil before agarose hardened to obtain a smooth exterior. After 60 seconds, the beads were washed four times at 37 ° C. with complete medium to remove oil. It was then incubated overnight (37 ° C., humid air and 5% CO ₂ atmosphere).

These Lenka cell-containing beads were used in the following tests.

<Example 3>

Prior to performing the in vivo test, it was necessary to test whether the lenka cells grew in the beads prepared in the manner described above.

To this end, the beads prepared as discussed in Example 2 were incubated in the medium described in Example 2 for 3 weeks under the conditions described above. Three beads were then cut into small pieces and incubated in a standard medium flask that could be in direct contact with both the flask and the culture medium.

Observation of these cultures showed that the cells grew to form standard Lenka colonies. This means that the cells are alive in the beads.

<Example 4>

In vivo testing was performed. In these tests, the beads were incubated at 37 ° C. for 7 days. Subject mice were then given a bead graft. For this purpose, the centerline of each of the four mice was dissected and administered intraperitoneally. Three beads each containing 60,000 Lenka cells were implanted. The incision was then sutured (two-layer suture) using an absorbent suture. Four mice (BALB / C) were normal male mice weighing 24-25 g and appeared healthy. Two groups were made as a control. In the first group, two mice received three beads that did not contain lenka cells, and in the second group, two mice received no treatment.

Three weeks after transplantation, all mice received 10 ⁶ Lenka cells by intraperitoneal injection. After 18 days, one of the mice in the control group died. All remaining mice were then killed and examined.

Mice receiving grafts of bead-encapsulated cells showed only few nodules throughout the body cavity, whereas control mice showed many tumors.

This positive result led to the proposal of the test design described in the Examples below.

<Example 5>

In this test, cancer development was simulated by injecting Renka cells into one kidney capsule of each of six BALB / C mice. After 15 days, mice were divided into two groups. Three mice of the first group were each given three beads as described in Example 4 above. The second group (control) was given beads that did not contain lenka cells.

After 4 to 5 days, mice receiving lenka cell-containing grafts showed coma and their hairs became stiff, while the controls were active with no change in hair condition.

However, control mice 10 days after transplantation (25 days after infusion of Lenka cells) began to dull activity and showed abdominal swelling. One of three control mice died 14 days after the bead transplant. The other mouse was then killed.

The body cavity of control mice showed a lot of bleeding with many tumor cells throughout the digestive tract, liver, stomach and lungs. Tumor cell growth is so prevalent that the entire abdominal cavity cannot be distinguished. However, mice receiving beads containing encapsulated Lenka cells had no bleeding and had only a few nodules in digestive system tumor cells. Comparing the test and control group, it can be seen that no nodules in the test group.

<Example 6>

Freely inoculated Lenka cell growth is inhibited when incubated with encapsulated Lenka cells. Further testing was performed to determine if these results also occurred in other cells.

Adenocarcinoma cell line, MMT (mouse breast cancer), was obtained from American Type Culture Collection. Encapsulated MMT cells were prepared from MMT cells as described above to prepare beads containing 120,000-240,000 cells per bead. After the beads were prepared, they were used to determine if they inhibited Lenka cell proliferation in vitro. Specifically, two 6-well Petri plates were prepared by inoculating 4 ml of medium with 1 × 10 ⁴ Lenka cells per well. In each plate, three wells were used as controls and three as test groups. One bead was given to one of three control wells in each plate. Each other well was given two or three empty beads. The second well was treated identically to the wells that provided one, two or three beads containing 120,000-240,000 MMT cells. After wells were incubated for one week at 37 ° C., Lenka cells were treated with trypsin, washed and counted using a hemocytometer. The results are shown in the table below.

Petri dish # 1 (empty giant) cell counts after 1 week Petri dish # 2 (MMT cell-containing macrophage) cell count counted after 1 week Well number Control Empty bead 120,000 MMT Cells 240,000 MMT Cells One 2.4 × 10 ⁵ 2.8 × 10 ⁵ 1.4 × 10 ⁵ 1 × 10 ⁵ 2 2.0 × 10 ⁵ 3.5 × 10 ⁵ 1.2 × 10 ⁵ 7 × 10 ⁴ 3 4.4 × 10 ⁵ 2.5 × 10 ⁵ 1.25 × 10 ⁵ 9 × 10 ⁴

<Example 7>

According to the results of Example 6, the same test was performed using 1 × 10 ⁴ MMT cells instead of Lenka cells. The test was carried out as in Example 6 above. The results are shown in the table below.

Well number Control Empty giant (1) MMT Giant (2) MMT Giant One 3.1 × 10 ⁶ 2.8 × 10 ⁶ 1.6 × 10 ⁶ 1.3 × 10 ⁶ 2 3.3 × 10 ⁶ 2.6 × 10 ⁶ 1.0 × 10 ⁶ 1.1 × 10 ⁶ 3 3.0 × 10 ⁶ 2.8 × 10 ⁶ 6.0 × 10 ⁵ 5.0 × 10 ⁶

These results led to the use of in vivo tests. This is shown in Example 8.

<Example 8>

Renka cells as used in the previous examples were kidney cancer cells. To more fully demonstrate the general efficacy of the present invention, studies were conducted using different types of cancer cells. In particular, adenocarcinoma cells were used.

Breast cancer cell line (MMT) of mice was obtained from American Type Culture Collection. Using the test method described above, grafts containing 120,000 cells per bead and 240,000 cells per bead were prepared.

The test model used was a mouse model as above. 22 mice were divided into 4, 9 and 9 groups. The first group, the control group, was further divided into two, one and three groups. The first subgroup received grafts of one cell-free bead. One mouse was given two empty beads and the other one was given three empty beads.

Test group A (9) contained 120,000 cells in the beads, while group B contained 240,000 cells in the beads. There were three subgroups in the "A" and "B" groups, and each group contained three mice. Subgroups received one, two or three beads containing MMT cells.

At 21 days after transplantation, all animals received 40,000 Lenka cells. Immediately after infusion, the mice became coma with stiff hairs. After this condition lasted for about 5 days, normal behavior was observed.

After 20 days, abdominal swelling of control mice appeared and the hairs became very stiff. One control mouse died on day 25 post-injection, while the other control mice displayed late. All mice were killed to observe the development of the tumor. These observations are reported below.

Number of Giants given to mouse Control Test group A Test group B One ++++ - - One ++++ - - One + ++ 2 ++++ - - 2 - - 2 ++ ++ 3 ++++ - - 3 - - 3 - +++

These results show that 13 of 18 mice tested were disease free. One of the group A mice had some nodules and the other mice had some tumors. Some tumors were found in one mouse receiving two beads.

In group B, one mouse receiving one bead and one mouse receiving two beads showed some tumors and bowel problems. One mouse, given three beads, grew a large, hard tumor and seemed very sick in appearance. Nevertheless, the overall results showed that encapsulated mouse breast tumor cells inhibited tumor formation.

<Example 9>

As suggested above, the practice of the present invention results in the production of several substances or factors that inhibit and / or prevent tumor cell proliferation. This was further attempted in the following test.

Beads were further prepared as described in Example 2, except that it did not contain atelocollagen. Therefore, these beads were agarose / agarose beads. Lenka cells were incorporated into these beads as described above.

Subsequently, two sets of three six well plates were used as control and test groups. In the control group, 4 ml of RPMI complete medium (10% fetal calf serum and penicillin 11 ml / l) was added to the wells. Each control well was then inoculated with 10,000 Lenka cells.

In the test group, the RPMI complete medium was controlled by addition of the settled material by incubating 10 immune isolated Lenka cell-containing beads (120,000 cells per bead) in a 35 × 100 mm Petri dish containing 50 ml of RPMI complete medium. It was. After 5 days of incubation, media was collected from these plates, 4 ml of which were placed in each test well. These wells were then inoculated with 10,000 Lenka cells.

All plates (both control and test) were incubated at 37 ° C. for 5 days. After the incubation period, cells were trypsinized, washed and counted using a hemocytometer. Cells in each well of the plate were collected after trypsin treatment and counted, and the results are shown in the table below.

Well number Control group (cell count) Test group (cell count) One 7 × 10 ⁵ 3 × 10 ⁵ 2 8 × 10 ⁵ 2.5 × 10 ⁵ 3 7 × 10 ⁵ 3.4 × 10 ⁵

These results show that cells produce several factors that result in inhibition of tumor cell proliferation, for example when limited in the beads of the present invention. These limiting inhibitors are produced because the cells are trapped in the beads and are different from other substances, such as contact inhibitor factors, which are produced when the cells contact each other.

<Example 10>

The test described above shows that the lenca cell growth rate in titration medium is almost half the cell growth rate in control medium. The experiments described in this example examined whether growth inhibitory factor remained active even after freezing the conditioned medium.

Qualification medium of Lenka cells was prepared by incubating 10 immune isolated Lenka cell-containing beads for 5 days. 50 mL of RPMI complete medium in 35 × 100 mm Petri plates was incubated at 37 ° C. After incubation, the medium was collected and stored at -20 ° C. Titration medium was prepared by incubating immune isolated MMT (mouse breast tumor) cell-containing beads. This bead contained 240,000 cells per bead and all other conditions were the same.

The freezing medium was thawed at 37 ° C. and then used for testing. Three plates of six wells were used for each treatment: (1) RPMI control medium, (2) Lenka titration freezing medium, and (3) MMT titration freezing medium. 4 ml of total medium was dispensed into each well. All wells were then inoculated with 10,000 Lenka cells and incubated at 37 ° C. for 5 days. After incubation, samples of two plates were removed from each well, trypsinized, pooled together and counted with a hemocytometer. On day 8, each well of the remaining three plates was tested in the same manner.

The results are as follows.

Petri dishes on the 5th day Control badge Qualification of Lenka Cells Freezing Medium MMT Qualification Frozen Medium One 6 × 10 ⁵ 5 × 10 ⁵ 8 × 10 ⁴ 2 6.8 × 10 ⁵ 4.2 × 10 ⁵ 8.5 × 10 ⁴ Petri dishes on day 8 3 2.8 × 10 ⁶ 2 × 10 ⁶ 8 × 10 ⁴

Comparing these results with the results of Example 6, the frozen / thawed Lenka titration medium did not inhibit the growth to the same extent that the unfrozen medium (Examples 6 and 7) inhibited growth, but inhibited growth. You will know. Titration freezing media with MMT cells further inhibited growth rather than unfrozen MMT titration media. The results show that 13 of 18 test mice showed no disease. In group A mice, one mouse showed some nodules and the other mice showed some tumors.

The foregoing describes a method of making implantable beads containing one or more types of cells that produce a diffusive biological product as defined herein. Diffuse biological products are substances that affect the subject to which the beads are implanted. Preferably, this effect is to modulate an immune response, eg to stimulate an immune response, or to inhibit a response. For example, in the case of tumors, the diffuse bioproduct can be a peptide that binds with MHC molecules on tumor cells of the transplant subject to stimulate the CTL response to it and subsequently lowers the tumor content of the transplant subject. In addition, the proliferative product may be a tumor cell growth inhibitor. As for the form of treatment, although not necessarily preferred, the transplant beads can be placed in or near the identified tumor.

As used herein, “diffuse biological product” refers to substances such as, for example, proteins, glycoproteins, lipoproteins, carbohydrates, lipids, glycolipids, and peptides. More specifically, examples include substances such as antibodies, cytokines, hormones, enzymes, and the like, but are not limited to these types of substances. Substances known as "final products" of cellular processes, such as CO ₂ and H ₂ O, are excluded.

As shown in the above experiments, these implantable beads can also be used for prophylaxis. It is well known that at least some of the cancer patient populations tend to recur. The experiments described herein show that grafts can inhibit the occurrence or recurrence of cancer through biological effects on the metabolic system of the transplant subject that the diffusive product possesses.

The discussion of the present invention focused on an in vivo perspective. It is also to be understood that the present invention has also been studied in vitro and some of them have been discussed herein. For example, it is well known that many cells producing the desired product require feeder cells when cultured in vitro. There is always a problem with supporting cells. Support cells can grow faster than the cells of interest, resulting in "strangulation" of the desired material. There may also be problems with various toxic products produced by the support cell layer. Implantable beads of the invention usually act as a cell incubator that protects the embedded cells, while allowing diffuse products to migrate, for example, to the culture medium in which they can be collected.

As indicated above, the preparation of implantable beads must first suspend the cells in a solution, preferably an aqueous collagen solution. Collagen is preferably a solution of about 0.5 to 2% in atelocollagen. Depending on the cell type used, at any given time, the number of cells in solution, and thus the number of cells in beads, will vary. Preferably, there are about 10,000 to 200,000 cells, more preferably about 30,000 to about 100,000 cells per bead. Most preferably, about 40,000 to about 60,000 cells are used.

After the cells are suspended in collagen solution, agarose solution is added. Preferably, this agarose solution may range from about 0.5% to about 5%, preferably about 1%. The beads are formed by dropwise addition of the mixture to an inert material such as TEFLON (trade name) or mineral oil. These beads are semisolid. The semisolid beads are then transferred to a sterile medium, preferably a medium containing antibiotics, washed and incubated to polymerize collagen. The polymerization of collagen is a well studied phenomenon and it will not be necessary to elaborate herein the conditions under which the polymerization takes place.

After solidification of the beads, the beads are coated with agarose, preferably by rolling in agarose solution. A preferred method of carrying out this procedure is a simple teflon-coated spoon, preferably containing 5% to 10% agarose solution.

The above discussion of diffusible biological products should not be construed as limited to wild type materials. For example, transformed or transfected host cells, e.g. eukaryotic cells (e.g., to produce heterologous proteins by treatment, or modified, for example through homologous recombination, to produce increased amounts of the desired biological product). , 283 cells, CHO cells, COS cells), or even prokaryotic cells (eg, E. Coli) can be incorporated very easily. Other materials, such as hybridomas, can also be used with the proliferative biological products that are monoclonal antigens.

Other features and aspects of the present invention will be apparent to those skilled in the art and need not be mentioned herein.

The terms and expressions used herein have been used for the purpose of description and not of limitation, and the use of such terms and expressions is not intended to exclude the equivalents or portions thereof of the features shown or described herein, and various modifications may be made to It will be appreciated that it is possible within the scope of the invention.

Claims (13)

A composition of matter comprising agarose coated solid agarose collagen beads containing cells producing a diffusive biological material. The composition of matter of claim 1, wherein said cells are cancer cells. The composition of matter of claim 2, wherein the cancer cells are kidney cancer cells. The composition of matter of claim 1, wherein the beads contain about 10,000 to about 200,000 cells. The composition of matter of claim 4, wherein the beads contain about 30,000 to about 100,000 cells. A method of administering a diffusive biological material to a transplant subject, comprising implanting the composition of the substance of claim 1 into the implant subject. The method of claim 6, wherein the transplant subject is a subject suffering from a pathological condition that is treatable with the diffusive biological material. 8. The method of claim 7, wherein said pathological condition is characterized by abnormal cell growth. The method of claim 8, wherein the pathological condition is cancer. (a) suspending cells producing a diffusive biological material in a collagen containing material, (b) adding agarose to the solution, (c) forming semi-solid beads of said collagen, agarose, and diffusible biological material-producing cells, (d) polymerizing collagen in the semi-solid beads to form solid, agarose-collagen beads containing the diffusive biological material-producing cells, (e) agarose coated solid agarose beads containing cells producing proliferative biological material, comprising coating said solid, agarose-collagen beads containing cells with agarose Method of preparation. The method of claim 10, wherein said cell is a cancer cell. The method of claim 10, comprising suspending about 10,000 to about 200,000 cells in the collagen containing solution. The method of claim 10, comprising suspending about 30,000 to about 100,000 cells in the collagen containing solution.