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WO1997035958A1 - Articles de stockage permettant de prolonger la duree de vie et les fonctions de cellules vivantes - Google Patents

Articles de stockage permettant de prolonger la duree de vie et les fonctions de cellules vivantes Download PDF

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Publication number
WO1997035958A1
WO1997035958A1 PCT/US1997/001731 US9701731W WO9735958A1 WO 1997035958 A1 WO1997035958 A1 WO 1997035958A1 US 9701731 W US9701731 W US 9701731W WO 9735958 A1 WO9735958 A1 WO 9735958A1
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WIPO (PCT)
Prior art keywords
cells
article
cell
protective layer
living cells
Prior art date
Application number
PCT/US1997/001731
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English (en)
Inventor
Patrick Soon-Shiong
Neil P. Desai
Molly Moloney
Qiang X. Yao
Original Assignee
Vivorx Pharmaceuticals, Inc.
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Publication date
Application filed by Vivorx Pharmaceuticals, Inc. filed Critical Vivorx Pharmaceuticals, Inc.
Priority to AU18552/97A priority Critical patent/AU1855297A/en
Publication of WO1997035958A1 publication Critical patent/WO1997035958A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0613—Cells from endocrine organs
    • C12N5/0614—Adrenal gland
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C12N5/0677—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/126—Immunoprotecting barriers, e.g. jackets, diffusion chambers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars

Definitions

  • 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.
  • Living cells produce a variety of substances (e.g. , insulin) which are necessary for maintaining a normal healthy condition.
  • substances e.g. , insulin
  • 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.
  • pancreatic islets have been transplanted into patients to achieve independence from insulin injections.
  • the inadequate supplies of fresh islets have necessitated the development of improved methods for collecting and preserving islet cells.
  • transplantation of individual cells or cellular communities including human or porcine pancreatic islets, hepatocytes, keratinocytes, chondrocytes, acinar cells, chromaffin cells, and the like
  • Such cells can be used in experimental models as well as in human therapy.
  • pancreatic cells or chromaffin cells 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
  • the primary cells lose function and show a rapid decline in viability (typically within about 7 days) .
  • 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.
  • Figure 1 illustrates the maintenance of insulin secretion rate by human islets after "storage” according to the invention for 79 days.
  • FIG. 2 illustrates the maintenance of insulin secretion rate by human islets after “storage” according to the invention for 627 days.
  • natural tissue e.g., pancreatic tissue, adrenal tissue, hepatic tissue, and the like
  • suitable dissociation methodology e.g., a collagenase digestion procedure
  • aggregates of living cells or free individual cells e.g., islets of Langerhans, chromaffin cells, and the like
  • cell aggregates or free cells are then enclosed within an article prepared employing a suitable cell-protective layer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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) .
  • proteins are also preferred for use in the practice of the present invention.
  • proteins especially proteins which have been modified so as to be capable of crosslinking, as described in United States Patent Application Serial No.
  • 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.
  • the matrix of polymeric material applied to cells or aggregates of cells in accordance with the present invention serves several purposes.
  • the matrix of polymeric material allows long-term maintenance of such cells in in vitro culture.
  • 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
  • a metabolic function e.g., hepatocytes
  • 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.
  • these properties are provided by the cell-protective layer itself, optionally modified to contain a semi-permeable membrane thereon.
  • 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.
  • 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.
  • maintenance of invention articles under typical culture conditions i.e., at temperatures in the range of about 20 up to 45"C, is contemplated.
  • temperatures typically employed for relatively short-term storage of cells i.e., temperatures which fall in the range of at least 0 ⁇ C, but less than about 20°C.
  • 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.
  • 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.
  • VRXMED liquid culture medium 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 (HC0 3 ' ) ions, glucose
  • 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
  • invention articles can vary substantially.
  • 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.
  • 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
  • the largest dimension of invention articles will fall in the range of about 10 up to 10,000 micrometers.
  • 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.
  • 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.
  • articles 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • chronic syndromes e.g., inflammatory arthritis and peripheral neuropathy following nerve constriction injury; see, for example, Hama and Sagen, Pain 52:223-231 (1993)
  • these symptoms are markedly reduced or completely eliminated for the duration of the neuropathic disorder in animals with adrenal medullary or chromaffin cell transplants.
  • 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 ⁇ -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.
  • chromaffin cells may provide an ideal combination of neuroactive substances for long-term pain alleviation.
  • the chromaffin cells surrounded by a cell- protective layer designed to protect the transplanted cells from the host immune response.
  • 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.
  • temporary articles are formed from a polysaccharide gum (either natural or synthetic) which can be gelled by exposure to a change in conditions.
  • the gum is alkali metal alginate, specifically sodium alginate, although other water-soluble gums may be used.
  • 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.
  • 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.
  • 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) .
  • 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.
  • 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
  • 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.
  • LGl 3.3 mM glucose
  • HG incubation in high glucose medium, representative of stimulatory condition
  • 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) .
  • LG2 i.e., subsequent incubation in low glucose medium, representative of return to the basal condition
  • 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 ⁇ IU/islet/60 min) in response to 16.5 mM glucose (300 mg/dl) by insulin output (expressed as ⁇ IU/islet/60 min) in response to 3.3 M glucose (60 mg/dl).
  • the insulin release values were varying but maintained over the entire incubation time.
  • free islet cells in standard tissue culture lose function within about 7 days, and are rapidly overgrown with fibroblast-like and endothelial-like cells.
  • 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.
  • 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.
  • DTZ dithizone
  • 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.
  • the islet cells were incorporated into a matrix of a chemically modified alginate (a polysaccharide) , thereby providing a cell-protective layer.
  • a chemically modified alginate a polysaccharide
  • 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.
  • ionic crosslinking inherent to alginates
  • covalent crosslinking incorporated by chemical modification of the alginate
  • 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) .
  • SI stimulation index
  • 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 were 17 and 5, respectively, for islets at 42 and 51 days. The results are summarized in Table 2.
  • DTZ+ indicates positive staining for insulin with DTZ
  • 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 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.
  • ionic crosslinking inherent to alginates
  • covalent crosslinking introduced by chemical modification of the alginate
  • 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.

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Abstract

La présente invention concerne un article qui prolonge de manière inattendue la durée de vie de cellules vivantes ou de groupes de cellules et qui maintient les fonctions biologiques et la viabilité des cellules entreposées pendant des laps de temps très longs. La présente invention concerne aussi un nouveau procédé de stockage et de culture de cellules, particulièrement avantageux pour l'accumulation de quantités importantes de cellules à usages thérapeutiques, y compris la transplantation sur des patients humains dans le but de diminuer les processus pathologiques. Le procédé de la présente invention consiste à envelopper des cellules ou des groupes de cellules au moyen d'une couche protectrice appropriée, ce qui permet de produire des articles de différentes tailles et de formes diverses.
PCT/US1997/001731 1996-03-26 1997-01-30 Articles de stockage permettant de prolonger la duree de vie et les fonctions de cellules vivantes WO1997035958A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107904190A (zh) * 2017-12-19 2018-04-13 南京工业大学 一种用于细胞表面功能涂层修饰的方法
CN111418682A (zh) * 2020-05-22 2020-07-17 吴成祥 一种提升人体免疫功能的sod聚能香袋泡茶及其制备方法

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CN107904190B (zh) * 2017-12-19 2021-10-15 南京工业大学 一种用于细胞表面功能涂层修饰的方法
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