WO1997035958A1

WO1997035958A1 - Storage articles for prolonged viability and function of living cells

Info

Publication number: WO1997035958A1
Application number: PCT/US1997/001731
Authority: WO
Inventors: Patrick Soon-Shiong; Neil P. Desai; Molly Moloney; Qiang X. Yao
Original assignee: Vivorx Pharmaceuticals, Inc.
Priority date: 1996-03-26
Filing date: 1997-01-30
Publication date: 1997-10-02
Also published as: AU1855297A

Abstract

An article has been developed which unexpectedly prolongs the viability of living cells or cell aggregates and which maintains the biological function and viability of the stored cells for very long periods of time. Also provided is a novel method of storing and culturing cells which is especially useful for accumulating large numbers of cells for therapeutic purposes, including transplantation into human patients to alleviate disease processes. The invention method involves surrounding cells or cell aggregates with a suitable cell-protective layer, thereby providing articles of various sizes and shapes.

Description

Storage Articles for Prolonged Viability and Function of Living Cells

Field of the Invention

The present invention relates to methods and compositions useful for the long-term maintenance of living cells in liquid culture, and for the preservation of the specific biological function of said cells, including the production of substances useful for therapeutic purposes.

Background of the Invention

Living cells produce a variety of substances (e.g. , insulin) which are necessary for maintaining a normal healthy condition. When the patient's own cells have been damaged by physical injury or by disease processes (e.g., diabetes), the resulting abnormal state can be treated by transplantation of the appropriate exogenous cells or cell aggregates (e.g. , pancreatic islets) into the patient's body under conditions which permit them to function normally. For example, pancreatic islets have been transplanted into patients to achieve independence from insulin injections. In view of these successes, the inadequate supplies of fresh islets have necessitated the development of improved methods for collecting and preserving islet cells. In the long run, transplantation of individual cells or cellular communities (including human or porcine pancreatic islets, hepatocytes, keratinocytes, chondrocytes, acinar cells, chromaffin cells, and the like) for therapeutic purposes will depend upon establishing a long-lasting supply of functional living cells. Such cells can be used in experimental models as well as in human therapy.

One major obstacle to routine therapeutic transplantation is the difficulty of amassing large quantities of viable and functional cells, especially in view of the long period of time required to achieve the cell volume required for effective treatment of the disease. While many strategies have been employed for long-term culture, generally these methods significantly decrease the number of viable cells available for transplantation. For example, conventional methods of cell growth and storage, such as tissue culture in T-flasks, have been inadequate because cell viability and function is not maintained for more than a few days. In addition, surviving cells exhibit a lower secretory responsiveness and a generally lower basal biosynthetic activity. This diminished activity correlates with various morphological modifications which occur during the culture period.

While standard cultures of such cells as pancreatic cells or chromaffin cells on culture-treated plastic dishes permit the cells to rapidly establish wide monolayers, such cells exhibit a quick and definitive decline in product secretion, with survival not exceeding several days. Moreover, in standard tissue culture, the primary cells (e.g. , islet cells or chromaffin cells) are rapidly overgrown by accompanying proliferating cells

(e.g., fibroblasts or endothelial cells). As a consequence, the primary cells lose function and show a rapid decline in viability (typically within about 7 days) .

While cryopreservation provides slight improvement, a major limitation to the use of this technique is the occurrence of physical damage to living cells as a result of the freeze-thaw process. Indeed, as little as half of the cells remain viable after one or two weeks of storage. Cryopreservation therefore still does not facilitate the collection of a large volume of therapeutically active cells from various sources over an extended period of time. Accordingly, -there is still a need in the art for means to stably store large volumes of therapeutically active cells obtained from various sources over extended periods of time.

Summary of the Invention

In accordance with the present invention, we have made the surprising discovery that cells stored within articles prepared employing a cell-protective layer and maintained in an appropriate growth-promoting culture medium can be kept alive and functional for very long periods of time (e.g., at least about 15 up to 600 days or longer) . Thus, invention treatment allows cell storage for much longer than the brief times (e.g., 7 days) typically observed using conventional tissue culture methods.

Brief Description of the Figures

Figure 1 illustrates the maintenance of insulin secretion rate by human islets after "storage" according to the invention for 79 days.

Figure 2 illustrates the maintenance of insulin secretion rate by human islets after "storage" according to the invention for 627 days.

Detailed Description of the Invention

In accordance with the present invention, natural tissue (e.g., pancreatic tissue, adrenal tissue, hepatic tissue, and the like) is dissociated using suitable dissociation methodology (e.g., a collagenase digestion procedure) to yield aggregates of living cells or free individual cells (e.g., islets of Langerhans, chromaffin cells, and the like) . These cell aggregates or free cells are then enclosed within an article prepared employing a suitable cell-protective layer.

As employed herein, the term "cell-protective layer" refers to any material which can be applied to cells in accordance with the present invention to protect the cells from the normal environmental influences which cause loss of viability. The term "cell-protective layer" embraces the use of both naturally occurring and synthetic polymers which are not detrimental to biological materials with which they come in contact, as well as combinations of two or more of such polymers. Examples of polymeric materials from which the cell-protective layer contemplated for use in the practice of the present invention can be prepared include polysaccharides, lipids, polyamides (e.g., protein), polyesters (e.g., polyglycolic acid, polylactic acid or polycaprolactone) , polynucleic acids (e.g., DNA or RNA) , polyalkylene oxides, polyvinyl alcohols, polyhydroxyalkyl (meth)acrylates, polyacrylic acids, polyalkyloxazolines, polyacrylamides, polyvinyl pyrrolidinones, and the like, as well as combinations of any two or more thereof. Optionally, polymeric materials from which the cell-protective layer contemplated for use in the practice of the present invention can be prepared can be modified with a moiety containing a carbon-carbon double bond or triple bond capable of free-radical polymerization.

Presently preferred materials for use in the practice of the present invention are materials which are both ionically and/or covalently crosslinkable. Suitable ionically and/or covalently crosslinkable polymeric materials which can be employed for the preparation of the cell-protective layer contemplated for use in the practice of the present invention are described in the following publications: PCT International Publication No. WO 93/09176, published May 13, 1993; PCT International Publication No. WO 94/15589, published July 21, 1994; United States Patent No. 5,334,640, issued August 2, 1994; United States Patent No. 5,439,686, issued August 8, 1995; and United States Patent Application Serial No. 08/484,724, filed June 7, 1995; each of which are hereby incorporated by reference herein in their entirety.

Presently preferred materials for use in the practice of the present invention include polysaccharides (especially alginate polymers, typically crosslinked by calcium) . Especially preferred materials employed in the practice of the present invention for the preparation of cell-protective layers are alginate polymers high in alpha- L-guluronic acid content. As used herein, the terminology "alginate polymers high in alpha-L-guluronic acid content" refers to alginate materials wherein greater than half of the sugar residues are guluronic acid residues (as opposed to mannuronic acid residues, which commonly predominate in alginate materials) .

Also preferred for use in the practice of the present invention are proteins, especially proteins which have been modified so as to be capable of crosslinking, as described in United States Patent Application Serial No.

08/484,724, filed June 7, 1995, which is hereby incorporated by reference herein in its entirety. Especially preferred proteins contemplated for use in this aspect of the invention include albumins, collagens, gelatins, immunoglobulins, hemoglobins, transferrins, caesins, pepsins, trypsins, chy otrypsins, fibronectins, vitronectins, laminins, Upases, lysozymes, fibrinogens, 1actalbumins, ovalbumins, amylases, and the like.

Throughout this specification and appended claims, it is understood that the singular embraces the plural and vice versa. The matrix of polymeric material applied to cells or aggregates of cells in accordance with the present invention serves several purposes. For example, the matrix of polymeric material allows long-term maintenance of such cells in in vitro culture. Ultimately, the matrix of polymeric material allows the storage and accumulation of sufficient cells to enable the therapeutic use of the cells, e.g., in a transplantation modality wherein the matrix-encased cells serve as a source of therapeutic agent (e.g. , adrenaline, angiotensin, colony stimulating factor, dopamine, erythropoietin, Factor VIII, Factor IX, gamma interferon, heparin, insulin, metacephalin, nerve growth factor, norepinephrine, proinsulin, somatostatin, streptokinase, superoxide dismutase, tissue plasminogen activator, urokinase, and the like) , or perform a metabolic function (e.g., hepatocytes) .

For example, in the treatment of diabetes employing implanted islet cells, in order for the implanted islet cells to function upon implantation, the matrix surrounding the cells must serve to prevent rejection by the immune system of the host, and must control diffusion of molecules into and out of the article. In accordance with the present invention, these properties are provided by the cell-protective layer itself, optionally modified to contain a semi-permeable membrane thereon. Thus, where articles of the invention are prepared from material which itself undergoes a sufficient level of crosslinking to provide a matrix of controlled porosity, no further modification of the cell-protective layer is required. However, in those instances where articles of the invention are prepared from polymeric material which does not itself undergo a sufficient level of crosslinking to provide a matrix of controlled porosity, it is desirable to modify the resulting article to apply a semi-permeable coating thereon. Where the optional application of a semi¬ permeable coating on invention articles is indicated, the cells or cell aggregates having a cell-protective layer thereon are then further surrounded by a biocompatible semi-permeable membrane which is permeable to small molecules but is impermeable to the core material and to potentially deleterious large molecules. Such biocompatible semi-permeable membranes can readily be applied to invention articles employing methodology which is well known in the art. See, for example, PCT International Publication No. WO 94/15589, published July 21, 1994, incorporated by reference herein in its entirety, as well as the other references cited herein.

Articles of the invention are maintained in a culture vessel in tissue culture medium for a sufficient period of time until adequate cell mass has been accumulated for transplantation. Assays for viability and for biological function (e.g., insulin secretion) can be carried out periodically to assure proper long-term maintenance. In accordance with the present invention, it has been found that living cells enclosed within these articles are able to maintain their long-term in vivo activity for much longer periods of time than is provided by any form of conventional tissue culture.

Articles of the present invention can be stored and/or maintained under a wide variety of conditions, from below zero to well above room temperature. Thus, maintenance of invention articles under typical culture conditions, i.e., at temperatures in the range of about 20 up to 45"C, is contemplated. Also contemplated are temperatures typically employed for relatively short-term storage of cells, i.e., temperatures which fall in the range of at least 0^βC, but less than about 20°C. Moreover, temperatures typically employed for long-term storage of cells, i.e., temperatures of less than 0°C (i.e., cryogenic storage) are also contemplated for use in the practice of the present invention.

As employed herein, "nutrient medium" refers to tissue-culture medium suitable for long-term maintenance of cells or cell aggregates. Those of skill in the art can readily identify suitable nutrient media, which will vary depending on the types of cells being treated.

An exemplary nutrient medium is referred to herein as VRXMED liquid culture medium, which is established at a pH of 7.4, an osmolarity between 270 and 320 mOsMol, a temperature of 37"C, and surface tension sufficiently low to prevent formation of air bubbles. VRXMED contains an effective cell-growth-promoting concentration of water, sodium (Na⁺) ions, potassium (K^*) ions (0.23 g/1), calcium (Ca**) ions (between 0.37 and 1.1 mM) , magnesium (Mg⁺⁺) ions, zinc (Zn⁺⁺) ions, chloride (Cl^") ions, sulfate (S04^") ions, bicarbonate (HC0₃ ^') ions, glucose

(1500 mg/L), all essential amino acids, cysteine, tyrosine, glutamine (between 2 and 7 mM) , water-soluble vitamins, nicotinamide, coenzymes, and inorganic trace elements. Glucose is preferably present at 0.8 to 1.2 mg/mL. The culture medium contains a source of an aqueous mixture of lipoprotein, chloresterol, phospholipids, and fatty acids with low endotoxin. A broad-spectrum antibiotic (e.g., gentamicin) may be included in the culture medium to prevent contamination by bacteria, yeast, or fungi.

As readily recognized by those of skill in the art, the size and shape of invention articles can vary substantially. For example, invention articles can be formed in the shape of a cylinder (i.e., a geometrical solid generated by the revolution of a rectangle about one of its sides), a sphere (i.e., a solid geometrical figure generated by the revolution of a semicircle around its diameter), a disc (i.e., a generally flat, circular form), a flat sheet (i.e., -a generally flat polygonal form, preferably square or rectangular), a wafer (i.e., an irregular flat sheet), a dog-bone (i.e., a shape that has a central stem and two ends which are larger in diameter than the central stem, such as a dumbell) , a lacy structure, a noodle, a teabag, a thread, a worm, or the like. Typically, the largest dimension of invention articles will fall in the range of about 10 up to 10,000 micrometers. In one aspect of the invention, relatively small articles are prepared, wherein the largest dimension of such articles falls in the range of about 10 up to 1000 micrometers, with articles having a maximum dimension in the range of about 200-900 micrometers being presently preferred.

In another aspect of the invention, relatively large articles are prepared, wherein the largest dimension of such articles is greater than about 1000, but less than about 10,000 micrometers. Articles having a maximum dimension in the range of about 2000-8000 micrometers are presently preferred.

In yet another aspect of the invention, articles are provided for the storage and maintenance of living cells, wherein said article consists of a plurality of first matrices, wherein each of the first matrices consist of a core of living cells substantially completely surrounded by a cell-protective layer, wherein the largest dimension of such first matrices falls in the range of about 10 up to 1000 micrometers, and wherein said plurality of first matrices are further physically contained within a second matrix having a diameter in the range of 500 up to 10,000 micrometers.

As readily recognized by those of skill in the art, a variety of cell types can be incorporated into invention articles. Useful cells can be obtained from a variety of sources, typically from eukaryotic species. For example, the living cells employed in the practice of the invention can be endocrine cells. Additional examples include chromaffin cells, epithelial cells, hepatocytes, hematopoietic cells, keratinocytes, muscle cells, neural cells, pancreatic islet cells, thyroid cells, tumor cells, stem cells, cells of the immune system, and the like. Presently preferred cells for incorporation into invention articles are pancreatic islet cells.

A wide range of therapeutic products are contemplated for delivery employing invention articles. Indeed, any naturally occurring or recombinant products which can be used as a therapeutic agent are contemplated, such as, for example, anticoagulants, clotting factors, cytokines, endocrine hormones, enzymes, fibrinolytic agents, growth factors, immunologically active factors, interferons, neurotransmitters, opiates, vasopressors, and the like. Examples of such products include adrenaline, angiotensin, colony stimulating factor, dopamine, erythropoietin, Factor VIII, Factor IX, gamma interferon, heparin, insulin, metacephalin, nerve growth factor, norepinephrine, proinsulin, somatostatin, streptokinase, superoxide dismutase, tissue plasminogen activator, urokinase, and the like. A presently preferred therapeutic product which can be delivered emloying invention articles is insulin.

Thus, for example, the transplantation of islet cells isolated from the pancreas can alleviate the symptoms of diabetes. Injection of these cells into the diabetic patient has been shown to be capable of effecting a cure. The use of such cells, however, runs the risk of rejection by the host, unless some sort of cell-protective layer is applied to the cells. By surrounding living islets with a protective layer that allows insulin to be secreted, yet prevents antibodies from reaching the islets, rejection of the injected cells can-be inhibited. This protective layer protects the islet from rejection and allows insulin to be secreted through its "pores", thereby enabling one to maintain a diabetic subject in normal glucose control.

Similarly, the transplantation of adrenal medullary tissue or chromaffin cells isolated from the adrenal gland can alleviate pain resulting from terminal cancer, chronic syndromes (e.g., inflammatory arthritis and peripheral neuropathy following nerve constriction injury; see, for example, Hama and Sagen, Pain 52:223-231 (1993)). Notably, these symptoms are markedly reduced or completely eliminated for the duration of the neuropathic disorder in animals with adrenal medullary or chromaffin cell transplants.

While the mechanism for the beneficial effects of adrenal medullary transplants is unclear, it appears to be two-fold: a direct mechanism via release of pain-reducing neuroactive substances, and a secondary long-term mechanism via intervention in the cascade of excitotoxicity in the spinal cord initiated by peripheral damage. Potentially important pain-reducing neuroactive substances released from transplanted chromaffin cells include the opioid peptides and the catechola ines. These agents, via activation of host opioid or α-adrenergic receptors in the spinal cord, can produce analgesia in a variety of species, including humans. Adrenal medullary transplants in the spinal subarachnoid space provide continual release of catecholamines and opioid peptides for prolonged periods. Thus, chromaffin cells may provide an ideal combination of neuroactive substances for long-term pain alleviation. To avoid the possibility of rejection of transplanted neural tissue, the chromaffin cells surrounded by a cell- protective layer designed to protect the transplanted cells from the host immune response. In accordance with a particular embodiment of the present invention, the enclosure of living cells within a matrix formed by the cell-protective layer is accomplished as follows. Core materials (such as living tissue, individual cells, or cell aggregates) are enclosed within a matrix in the form of a hydrogel. The material to be enclosed is suspended in a physiologically compatible medium containing a water-soluble substance which can be reversibly gelled to provide a temporary protective environment for the cells. The medium is formed into droplets containing the cells and gelled to form a temporary article, which is thereafter treated in one or more of several ways to form a matrix having controlled permeability about the cells. The semi-permeable nature of the matrix permits nutrients and oxygen to flow to the core of the resulting article and permits metabolic products to flow out, while stably retaining the core material within the article.

The temporary articles may be formed from any non-toxic water-soluble substance which can be gelled to form a shape-retaining mass by a change of conditions in the medium. Preferably, temporary articles are formed from a polysaccharide gum (either natural or synthetic) which can be gelled by exposure to a change in conditions. Most preferably, the gum is alkali metal alginate, specifically sodium alginate, although other water-soluble gums may be used.

Where covalently crosslinkable materials are employed for the preparation of invention articles, the temporary article can then be subjected to appropriate crosslinking conditions, as described, for example, in PCT International Publication No. WO 93/09176, published May 13, 1993, incorporated by reference herein in its entirety. Alternatively, when the formation of a permanent semi-permeable membrane about the temporary articles is desired, this is preferably effected by ionic reaction between free acid groups in the surface layer of the gelled gum and biocompatible biopolymers containing acid-reactive groups (e.g., amino groups), typically in a dilute aqueous solution of the selected polymer. Suitable biocompatible biopolymers containing acid-reactive groups include polylysine and other polyamino acids. The resulting semi- permeable membrane is then treated with a non-toxic biocompatible water-soluble polymeric material which is capable of ionic reaction with free amino groups to form the outer negatively-charged coating about the membrane. The material used to form the outer coating is preferably the same material used to form the temporary articles, preferably a polysaccharide gum, more preferably an alkali metal alginate (e.g. , sodium alginate) .

The invention will now be described in greater detail by reference to the following non-limiting examples.

Example 1

Human islets encased in invention articles were maintained in vitro in T75 tissue culture flasks in groups of 1000/flask with culture Medium #3 (RPMI-1640 containing 11 mM glucose, 10% vol/vol heat inactivated fetal bovine serum, 2 mM L-glutamine, 0.1 mg/ml penicillin and 0.1 mg/ml streptomycin) with regular media changes twice a week. To assess the in vitro functional capabilities of these articles after various periods of incubation (2, 14, 20, 25, 30, 44, 55, 71, 88, 98, 121, 211, 345, 401, 534 and 855 days) insulin secretory response to glucose stimulation was studied in a static glucose stimulation (SGS) assay.

SGS assay is carried out as follows: 30 articles according to the invention containing islet cells were incubated in RPMI containing 3.3 mM glucose (60 mg/dl; also referred to herein as "LGl", i.e., initial incubation in JLow glucose medium, representative of the basal condition) and 0.5% BSA for 60 minutes, basal insulin secretion assessed, then islet-containing articles were transferred to RPMI with 16.5 mM glucose (300 mg/dl; also referred to herein as "HG", incubation in high glucose medium, representative of stimulatory condition) and 0.5% BSA for 60 minutes to stimulate insulin secretion. The assay was finished with repeated incubation in 3.3 mM glucose medium (60 mg/dl; also referred to herein as "LG2", i.e., subsequent incubation in low glucose medium, representative of return to the basal condition) to assess normal regulation (suppressed insulin release in presence of low glucose medium) .

The insulin concentration in culture supematants was measured with radioimmunoassay (Coat-A-Count Insulin RIA, Diagnostic Product Corporation, Los Angeles) . The stimulation index (SI) was calculated by dividing insulin output (expressed as microunits of insulin per islet per 60 minutes, or μIU/islet/60 min) in response to 16.5 mM glucose (300 mg/dl) by insulin output (expressed as μIU/islet/60 min) in response to 3.3 M glucose (60 mg/dl).

Table 1 shows the results (average of n=2 samples at each time point) of insulin secretion in seven different batches of islet cells treated according to the invention. The insulin release values were varying but maintained over the entire incubation time. In contrast, free islet cells in standard tissue culture lose function within about 7 days, and are rapidly overgrown with fibroblast-like and endothelial-like cells. T ble i

SGS of invention articles containing human islets

Insulin secretion

Total /ulU/islet/80 min Days Days

ID# in in LG1(60)¹ HG(300)² LG2(60)³ SI⁴ Culture matrix

HD- 2 1 1.70 19.00 2.80 11.18

131/1 25 24 1.21 21.06 6.18 17.40

71 70 0.49 10.24 0.60 20.90

79 78 0.73 7.60 1.61 10.41

88 87 0.42 4.00 0.59 9.52

98 97 0.50 12.33 2.07 24.66

121 120 0.49 9.97 1.99 20.35

211 210 1.31 11.86 1.99 9.05

345 344 1.67 11.70 1.97 7.01

401 400 1.21 13.40 7.30 11.07

534 533 1.28 33.58 2.49 26.23

855 854 0.48 1.22 0.10 2.54

HD- 2 1 4.10 23.40 4.16 5.71

131/2 25 24 1.23 10.59 3.54 8.61

96 97 0.58 10.17 1.65 17.53

345 344 1.29 18.50 2.00 14.34

401 400 0.73 17.15 9.57 23.49

534 533 1.23 40.45 1.81 32.89

855 854 1.00 6.50 2.75 6.50

HD- 2 1 0.65 15.03 1.30 23.12

132 20 19 0.91 7.71 1.52 8.47

48 45 0.45 9.80 1.47 21.78

75 74 1.08 10.34 1.33 9.57

100 99 0.83 9.05 2.06 10.90

HD- 2 1 1.91 41.20 9.54 21.57

133 21 20 1.04 16.30 1.44 15.67

44 43 0.48 22.37 2.04 46.60

HD- 2 1 1.84 23.49 2.86 12.77

134 20 19 1.72 11.44 6.03 6.65

24 23 2.02 6.39 3.83 3.16

30 29 5.44 26.92 13.42 4.95

44 43 0.86 10.74 3.26 12.49

63 62 0.91 12.67 1.49 13.92

HD- 2 1 0.75 20.21 2.12 26.95

136 14 13 0.79 8.85 1.83 11.20

17 16 1.57 7.14 2.36 4.55

37 36 0.62 18.20 2.03 29.35

55 54 0.81 11.20 2.99 13.83

HD- 4 3 1.64 19.42 6.63 11.84

138 25 24 0.77 8.45 2.02 10.97

44 43 0.81 5.19 1.20 6.41 LG1(60) = initial incubation in low glucose medium (i.e., 60 mg/dl), representative of basal condition HG (300) = incubation in high glucose medium

(i.e., 300 mg/dl), representative of stimulatory condition LG2 (60) = return to basal condition by return to incubation in low glucose medium (i.e.,

60 mg/dl) SI = stimulation index

Example 2

Culture of Human Islets With and Without

Biocompatible Matrix

Human islet cells obtained by standard methods of collagenase digestion and purification were cultured in vitro in T75 tissue culture flasks in tissue culture medium described above. Tissue culture medium was changed every 2-3 days and the viability of these cells was carefully observed. In addition to monitoring viability (by acridine orange-propidium iodide staining) , the islet cells were stained with dithizone (DTZ) to note the presence of insulin within the islet cells, within the first few days of culture it was noted that a population of 'non-islet' cells, predominantly fibroblastic in nature, adhered and proliferated within the culture vessel. The viability of the islet cells showed a continuous decline from about 90% at the time of plating to about less than 30% at 7 days. By 7 days, a large number of proliferating fibroblast-like cells seemed to predominate, coupled with a concomitant rapid decline in the viability of the islet cell population.

As a comparison to the free islet cell culture described above, the islet cells were incorporated into a matrix of a chemically modified alginate (a polysaccharide) , thereby providing a cell-protective layer.

This chemically modified alginate has the dual property of ionic crosslinking (inherent to alginates) and covalent crosslinking (incorporated by chemical modification of the alginate) by free-radical polymerization initiated in the presence of suitable catalysts and exposure to suitable wavelength of visible or ultra-violet radiation. Such materials and methods for the use thereof have been described previously (see PCT International Publication No. WO 93/09176, published May 13, 1993; incorporated by reference herein in its entirety) .

The viability of human islet cells disposed in the above-described crosslinked polysaccharide matrix (as spherical beads of diameter in the range of 500 microns to about 5000 microns) was monitored over time. It was surprisingly discovered that not only was the viability of the matrix-disposed islet cells at 7 and 14 days substantially higher than observed ar comparable times with free cell culture (i.e., 75-85%), in addition, no overgrowth or proliferation of fibroblast-like and endothelial-like cells was observed.

The matrix-disposed islet cells were maintained in culture with periodic culture media changes. At 42 and 51 days following the initiation of culture, the matrix- disposed islet cells still maintained a viability of approximately 70%. Static glucose stimulation (described in Example 1) at 26 days in culture for the matrix-disposed human islet cells gave a stimulation index (SI) of 9.8, which is considered in the normal range for healthy functioning islets (ratios of greater than 3 are acceptable for healthy functioning islet cells) . In addition to viability, and static glucose stimulation, the matrix- disposed cells were subjected to a perifusion test to assess islet function. This method is equivalent to a static glucose stimulation, but is performed in a flow system and allows for the minute-to-minute observation of insulin secretion by the islet cells. Results of the perifusion showed a ^{^}typical insulin secretion profile, indicating the presence of healthy functioning islets. The perifusion indices (PI) of these stimulations (ratio of peak insulin output to basal insulin output) were 17 and 5, respectively, for islets at 42 and 51 days. The results are summarized in Table 2.

Table 2

Free Matrix-Disposed Islet Cells Islet Cells

Days in Viability General Viability General Culture (%) Observations (%) Observations

1 90 DTZ+¹ 90 DTZ+

7 <30 Overgrowth 85 No competing of other cells, DTZ+ cells

14 0 Severely 75 No competing Overgrown cells, DTZ+

26 — — — SI=9.8, DTZ+

42 — — 70 PI=17, DTZ+

51 70 PI=5, DTZ+

DTZ+ indicates positive staining for insulin with DTZ

Thus, it is surprisingly noted that the viability, function and purity of a culture of islet cells can be maintained over a long-term when these cells are maintained in a matrix storage article as described herein. Example 3

Culture of Bovine Chromaffin Cells With and Without

Biocompatible Matrix Storage Device

Bovine adrenal glands were obtained from a local slaughterhouse, and chromaffin cells were isolated therefrom by perfusion with collagenase. The medullary tissue was then dissected free from cortical tissue, minced, and filtered through nylon. The cells were purified on a Percoll gradient and plated in 1:1 DMEM:F12 media containing 5% fetal bovine serum in T-75 tissue culture flasks. Media changes were performed every 3 days and the viability and morphology of the cells were monitored periodically.

The above-described treatment of adrenal glands results in the removal of a large quantity of non- chromaffin cell types such as fibroblasts, endothelial cells, etc. However, since it is virtually impossible to completely remove extraneous cell types from the cell mixture, within a few days of initiating culture, the rapidly dividing fibroblasts and endothelial cells overtake the non-proliferating chromaffin cells. Indeed, within 7 days of initiating culture, a substantial population of all cells within the T-flask comprise fibroblast-like and endothelial-like cells. Over the same timeframe, the non- dividing chromaffin cells begin to show a reduction in viability (by trypan blue exclusion assay) from about 95% to less than 70%. Within 14 days, the fibroblasts and endothelial cells completely overtake the chromaffin cell culture, and the viability of the chromaffin cells drops to below 30%.

As a comparison to the free chromaffin cell culture described above, the isolated chromaffin cells were incorporated into a matrix of a chemically modified alginate (a polysaccharide) . This chemically modified alginate has the dual property of ionic crosslinking (inherent to alginates) and covalent crosslinking (introduced by chemical modification of the alginate) by free-radical polymerization initiated in the presence of suitable catalysts and exposure to suitable wavelength of visible or ultra-violet radiation. Such material and methods for the use thereof have been described previously (see PCT International Publication No. wo 93/09176, published May 13, 1993; incorporated by reference herein in its entirety) .

The viability of chromaffin cells disposed in the crosslinked polysaccharide matrix (as spherical beads having a diameter in the range of about 500 microns to about 5000 microns) was monitored over time. It was surprisingly discovered that not only was the viability of the matrix-disposed chromaffin cells at 7 and 14 days substantially higher than observed at comparable times with free cell culture (i.e., 90-95%), in addition, no overgrowth or proliferation of fibroblast-like and endothelial-like cells was observed.

The matrix-disposed chromaffin cells were then maintained in culture with periodic culture media changes. At 45 days following the initiation of culture, the matrix- disposed chromaffin cells still maintained excellent viability (approximately 90%, varying from 85-93%). The results are summarized in Table 3.

Table 3

I ''ree Matrix-Disposed

Chromaj .fin Cells Chromaffin Cells

Days in Viability General Viability General Culture (%) Observations % Observations

1 95 Healthy 95 Healthy cells cells

7 <70 Overgrowth 90 No competing of other cells cells

14 <30 Severely 90 No competing Overgrown cells

45 90 No competing cells

Thus, it is surprisingly observed that the viability and purity of a culture of chromaffin cells can be maintained over a long-term when these cells are maintained in a matrix storage article.

5 While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.

Claims

That which is claimedr

1. An article for the storage and maintenance of living cells, wherein said article comprises a core of living cells substantially completely surrounded by a cell- protective layer.

2. An article according to claim 1 wherein said living cells are eukaryotic.

3. An article according to claim 1 wherein said living cells are endocrine cells.

4. An article according to claim 1 wherein said living cells are selected from chromaffin cells, epithelial cells, hepatocytes, hematopoietic cells, keratinocytes, muscle cells, neural cells, pancreatic islet cells, thyroid cells, tumor cells, stem cells, or cells of the immune system.

5. An article according to claim 1 wherein said living cells are pancreatic islet cells.

6. An article according to claim 1 wherein said living cells produce a naturally occurring or recombinant product for use as a therapeutic agent.

7. An article according to claim 1 wherein said living cells produce an anticoagulant, a clotting factor, a cytokine, an endocrine hormone, an enzyme, a fibrinolytic agent, a growth factor, an immunologically active factor, an interferon, a neurotransmitter, an opiate or a vasopressor.

8. An article according to claim l wherein said living cells produce a therapeutic agent selected from adrenaline, angiotensin, colony stimulating factor, dopamine, erythropoietin, Factor VIII, Factor IX, gamma > interferon, heparin, insulin, metacephalin, nerve growth factor, norepinephrine, proinsulin, somatostatin, streptokinase, superoxide dismutase, tissue plasminogen activator or urokinase.

9. An article according to claim 1 wherein said living cells produce insulin.

10. An article according to claim 1 wherein said cell-protective layer is prepared from a naturally occurring or synthetic polymer, or combinations of two or more thereof.

11. An article according to claim 1 wherein said cell-protective layer is prepared from a polysaccharide, a lipid, a polyamide, a polyester, a polynucleic acid, a polyalkylene oxide, a polyvinyl alcohol, a polyhydroxyalkyl (meth)acrylate, a polyacrylic acid, a polyalkyloxazoline, a polyacrylamide, a polyvinyl pyrrolidinone, or combinations of any two or more thereof.

12. An article according to claim 1 wherein the polymeric material from which said cell-protective layer is prepared is modified with a moiety containing a carbon- carbon double bond or triple bond capable of free-radical polymerization.

13. An article according to claim 1 wherein said cell-protective layer is prepared from a polysaccharide.

14. An article according to claim 1 wherein said cell-protective layer is prepared from an alginate polymer.

15. An article according to claim 1 wherein said cell-protective layer is prepared from an alginate polymer high in alpha-L-guluronic acid content.

16. An article according to claim 1 wherein said cell-protective layer is prepared from a protein.

17. An article according to claim 1 wherein the largest dimension of said article falls in the range of about 10 up to 10,000 micrometers.

18. An article according to claim 1 wherein the largest dimension of said article falls in the range of about 10 up to 1000 micrometers.

19. An article according to claim 1 wherein the largest dimension of said article is greater than about 1000, but less than about 10,000 micrometers.

20. An article according to claim 1 wherein said article is formed in the shape of lace, a noodle, a teabag, a thread, a worm, a cylinder, a sphere, a disc, a flat sheet, a wafer or a dog-bone.

21. An article for the storage and maintenance of living cells, wherein said article consists of a plurality of first matrices, wherein each of said first matrices consist of a core of living cells substantially completely surrounded by a cell-protective layer, and wherein said plurality of first matrices are further physically contained within a second matrix having a maximum dimension in the range of 500 up to 10,000 micrometers.

22. Use of the article of claim 1 for transplantation into human patients for therapeutic purposes.

23. A method of storing and maintaining living cells, said method comprising: surrounding said cells with a cell-protective layer, thereby producing an article comprising a core of living cells substantially completely surrounded by said cell-protective layer, and maintaining said article in suitable medium.

24. A method according to claim 23 wherein said article is maintained at a temperature in the range of about 20"C up to 45°C.

25. A method according to claim 23 wherein said article is maintained at a temperature of at least 0°C, but less than 20'C.

26. A method according to claim 23 wherein said article is maintained at a temperature of less than 0*C.

27. A method according to claim 23 wherein said cells are stored and maintained for more than 7 days.

28. A method according to claim 23 wherein said living cells are eukaryotic.

29. A method according to claim 23 wherein said living cells are endocrine cells.

30. A method according to claim 23 wherein said living cells are selected from chromaffin cells, epithelial cells, hepatocytes, hematopoietic cells, keratinocytes, muscle cells, neural cells, pancreatic islet cells, thyroid cells, tumor cells, stem cells or cells of the immune system.

31. A method according to claim 23 wherein said living cells are pancreatic islet cells.

32. A method according to claim 23 wherein said living cells produce a naturally occurring or recombinant product for use as a therapeutic agent.

33. A method according to claim 23 wherein said living cells produce anticoagulants, clotting factors, cytokines, endocrine hormones, enzymes, fibrinolytic agents, growth factors, immunologically active factors, interferons, neurotransmitters, opiates or vasopressors.

34. A method according to claim 23 wherein said living cells produce a therapeutic agent selected from adrenaline, angiotensin, colony stimulating factor, dopamine, erythropoietin, Factor VIII, Factor IX, gamma interferon, heparin, insulin, metacephalin, nerve growth factor, norepinephrine, proinsulin, somatostatin, streptokinase, superoxide disrautase, tissue plasminogen activator or urokinase.

35. A method according to claim 23 wherein said living cells produce insulin.

36. A method according to claim 23 wherein said cell-protective layer is prepared from a naturally occurring or synthetic polymer, or combinations of two or more thereof.

37. A method according to claim 23 wherein said cell-protective layer is prepared from a polysaccharide, a lipid, a polyamide, a polyester, a polynucleic acid, a polyalkylene oxide, a polyvinyl alcohol, a polyhydroxyalkyl (meth)acrylate, a polyacrylic acid, a polyalkyloxazoline, a polyacrylamide, a polyvinyl pyrrolidinone, or combinations of any two or more thereof

38. A method according to claim 23 wherein the polymeric material from which said cell-protective layer is prepared is modified with a moiety containing a carbon- carbon double bond or triple bond capable of free-radical polymerization.

39. A method according to claim 23 wherein said cell-protective layer is prepared from a polysaccharide.

40. A method according to claim 23 wherein said cell-protective layer is prepared from an alginate polymer.

41. A method according to claim 23 wherein said cell-protective layer is prepared from an alginate polymer high in alpha-L-guluronic acid content.

42. A method according to claim 23 wherein said cell-protective layer is prepared from a protein.

43. A method according to claim 23 wherein the largest dimension of said article falls in the range of about 10 up to 10,000 micrometers.

44. A method according to claim 23 wherein the largest dimension of said article falls in the range of about 10 up to 1000 micrometers.

45. A method according to claim 23 wherein the largest dimension of said article falls is greater than about 1000, but less than about 10,000 micrometers.

46. A method according to claim 23 wherein said article is formed in the shape of lace, a noodle, a teabag, a thread, a worm, a cylinder, a sphere, a disc, a flat sheet, a wafer or a dog-bone.