EP0190337A1 - A process for proliferation of wholly or partially differentiated beta-cellls - Google Patents

A process for proliferation of wholly or partially differentiated beta-cellls

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Publication number
EP0190337A1
EP0190337A1 EP19850904430 EP85904430A EP0190337A1 EP 0190337 A1 EP0190337 A1 EP 0190337A1 EP 19850904430 EP19850904430 EP 19850904430 EP 85904430 A EP85904430 A EP 85904430A EP 0190337 A1 EP0190337 A1 EP 0190337A1
Authority
EP
European Patent Office
Prior art keywords
islets
cells
culture medium
growth hormone
human
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19850904430
Other languages
German (de)
French (fr)
Inventor
Jens Hoiriis Nielsen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nordisk Gentofte AS
Original Assignee
Nordisk Gentofte AS
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Filing date
Publication date
Application filed by Nordisk Gentofte AS filed Critical Nordisk Gentofte AS
Publication of EP0190337A1 publication Critical patent/EP0190337A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • 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
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/305—Growth hormone [GH], aka. somatotropin

Definitions

  • the present invention relates to a process of the type defined in the introductory portion of claim 1 for pro ⁇ liferation of wholly or partially differentiated beta- cells .
  • the type I diabetes ellitus disease is usually accompanied by progressive destruction of the insulin producing so-called beta-cells in the islets of Langerhans.
  • Patients suffering from this disease are normally treated with daily injections of insulin re ⁇ covered from the pancreas from animals, such as beef or pig, or produced by engineering.
  • this mode of treatment is still incomplete, and complications at the advanced stages of the disease result in a high mortality rate among diabetics.
  • a considerably better regulation of the glucose content of the blood may be obtained by transplantation of tissue from pancrease or isolated islets of Langerhans, which is believed to reduce the risk of the mentioned complications considerably.
  • this treatment it is required that sufficient amounts of insulin producing tissue are provided
  • the number of available human pancrease organs is by and large the same as the number of kidney donors since the pancrease organs are obtained from the same deceased persons.
  • Another source is pancrease from fetal tissue from abortions, but this source, too, is limited. There ⁇ fore, the ability of proliferating the insulin producing cells by cell cultivation is very important (ref. 1).
  • beta-cell division Even with rat islets of Langerhans, only limited beta-cell division has been observed, which has led to the general assumption that the postnatal division capacity qf the beta-cell is poor, while some neoformation and/or division of beta-cells in vitro seems to occur in the embryonic and very late fetal state (see ref. 4). Only few cases of stimulation of beta-cell division in vitro have been reported, and it has generally been believed that glucose is the most important factor in cell division both in vivo and in vitro (ref. 4).
  • the conventional method of cultivating cells is performed by depositing the cells on the surface of a solid substrate, e.g. of a plastics material. Such deposit is promoted by the presence of serum, e.g. 10% fetal calf serum, which is therefore widely used in the cultivation of insulin producing cells (ref. 6). Under these conditions, no effect of growth hormone was observed, either on the DNA synthesis or the insulin production (ref. 7). Increased insulin production is described (ref. 8), and an increased DNA synthesis without an increase in insulin secretion is likewise described (ref. 9 and ref. 10). While considerable, but time-limited stimulation of both insulin production and DNA synthesis has been demonstrated in the intact islets (ref. 5), this has so far not been demonstrated in monolayer cultures.
  • serum e.g. 10% fetal calf serum
  • the object of the present process is to provide an efficient and rational method of proliferating wholly or partially differentiated beta-cells in large amounts.
  • Another object of the invention is to proliferate , cells which produce insulin in considerable amounts and which are useful for implantation in humans.
  • a further object of the invention is to produce human insulin.
  • the process of the invention which is characterized by the features stated in the characterizing portion of claim 1, is based on the surprising finding that wholly or partially differentiated beta-cells develop strongly with formation of monolayers by cultivation on a solid substrate and in contact with a nutrient medium containing both serum and growth hormone in the stated concentration ranges, and when the nutrient medium is repeatedly exchanged during a cultivation period of preferably several weeks .
  • the nutrient medium used in the proliferation of the cells may expediently have a relatively high content of glucose, such as 1/2 - 10 g/1 , preferably 1.5 - 5 g/1, with 2 g/1 as the optimum value.
  • the content of human serum is expediently 1/2 - 1% , preferably 1/2 - 3% , with about 2? ⁇ as the optimum value.
  • Growth hormone of animal or human origin may be replaced by hormones having similar properties, such as prolactin or placental lactogen.
  • the preferred concentration of growth hormone or the hormone having similar properties in the culture medium is 10 to 1000 ng/ml, such as about 100 ng/ l.
  • a consider ⁇ able effect may also be obtained, however, at lower con ⁇ centrations, such as down to about 1 ng/ml.
  • Larger amounts of growth hormone may also be used, such as up to 1000 ng/ml culture medium or more, but, usually, no consider ⁇ ably improved cell formation is obtained at higher concentrations than 200 ng/ml.
  • the process of the invention permits proliferation of insulin producing cells of any type, also in the form of host cells into which one or more other genes which are to be expressed in an.i ⁇ ral or human cells have been intro ⁇ quizzed. Examples of this are cells in which DNA fragments which code for Factor VIII, growth hormone or interferon have been introduced.
  • Pancrease from 15 three days old rats is excised, treated with collagenase, and the islets are isolated as described in ref. 5. 1000 islets are placed in culture medium RPMI 1640 containing 10? ⁇ serum from newborn calves, distributed with 100 islets in 5 ml medium in Petri dishes. The dishes stand at 37 °C for 2 days as described in ref. 5. Then the islets are treated with a mixture of EGTA, DNase and trypsin and are aspirated until a cell suspension has been obtained, as described in ref. 11. The cells are suspended in medium RPMI 1640, and 10 cells in 5 ml medium are placed in cell cultivation dishes, as described in ref. 9. The dishes stand for 2 days at 37 °C, and the medium is then changed to RPMI 1640 admixed with 2% normal human serum and 100 ng/ml human growth hormone (Nanormon,
  • the dishes stand at 37 °C, and the medium is changed once a week. Insulin is measured by radioimmunoassay , as described in ref. 5.
  • the cells proliferate to colonies, and after 5 months the colonies have spread so that they cover almost the entire dish. They are treated with a mixture of EGTA, DNase and trypsin, and the resulting cell suspension is placed in new dishes with the media RPMI 1640 admixed with.2% normal human serum and 100 ng/ml human growth hormone. Again the cells proliferate to colonies and spread in the dish. The insulin production increases again gradually.
  • curve I refers to the experiment performed in this example.
  • Curve II refers to a comparative example performed in the same manner, except that no growth hormone was added.
  • a cell suspension is produced as described in example 1.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Endocrinology (AREA)
  • Microbiology (AREA)
  • Diabetes (AREA)
  • General Engineering & Computer Science (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Un procédé de prolifération de cellules bêta entièrement ou partiellement différenciées consiste à déposer des îlots ou des cultures d'îlots de pancréas d'origine humaine ou animale sous forme de monocouches sur un substrat solide et à les conserver en contact avec un milieu nutritif à base de glucose et contenant de 0,5 à 7% de sérum humain et de 1 à 1000 ng d'une hormone de croissance par ml de milieu nutritif. La culture des cellules bêta dans de telles conditions assure une prolifération continue ou à long terme des cellules et une forte augmentation simultanée de la production d'insuline.A method of proliferating fully or partially differentiated beta cells consists in depositing islets or cultures of islets of pancreas of human or animal origin in the form of monolayers on a solid substrate and in keeping them in contact with a nutritive medium based on glucose and containing 0.5 to 7% human serum and 1 to 1000 ng of growth hormone per ml of nutrient medium. Culturing beta cells under such conditions ensures continuous or long-term proliferation of cells and a large simultaneous increase in insulin production.

Description

A process for proliferation of wholly or partially differentiated beta-cells
The present invention relates to a process of the type defined in the introductory portion of claim 1 for pro¬ liferation of wholly or partially differentiated beta- cells .
The type I diabetes ellitus disease is usually accompanied by progressive destruction of the insulin producing so-called beta-cells in the islets of Langerhans. Patients suffering from this disease are normally treated with daily injections of insulin re¬ covered from the pancreas from animals, such as beef or pig, or produced by engineering. However, this mode of treatment is still incomplete, and complications at the advanced stages of the disease result in a high mortality rate among diabetics.
A considerably better regulation of the glucose content of the blood may be obtained by transplantation of tissue from pancrease or isolated islets of Langerhans, which is believed to reduce the risk of the mentioned complications considerably. However, for this treatment to be used on a larger scale, it is required that sufficient amounts of insulin producing tissue are provided The number of available human pancrease organs is by and large the same as the number of kidney donors since the pancrease organs are obtained from the same deceased persons. Another source is pancrease from fetal tissue from abortions, but this source, too, is limited. There¬ fore, the ability of proliferating the insulin producing cells by cell cultivation is very important (ref. 1).
It is possible to preserve functional islets of Langerhans, isolated from human pancreas, for months under ordinary tissue culture conditions (see ref. 2 and ref. 3). However, these known processes involve no increase in the number of beta-cells in cultures of human islets of Langerhans from adults.
Even with rat islets of Langerhans, only limited beta-cell division has been observed, which has led to the general assumption that the postnatal division capacity qf the beta-cell is poor, while some neoformation and/or division of beta-cells in vitro seems to occur in the embryonic and very late fetal state (see ref. 4). Only few cases of stimulation of beta-cell division in vitro have been reported, and it has generally been believed that glucose is the most important factor in cell division both in vivo and in vitro (ref. 4). However, experiments with mice and rats have demonstrated that growth hormone and related hormones, such as prolactin and placental lactogen, may exert a direct stimulating effect on the Langerhans's islets DNA synthesis and insulin production in vitro (ref. 5). However, this effect diminished as the cultivation period progressed, just as the islet growth in vivo decreases with the age of the animal.
The conventional method of cultivating cells is performed by depositing the cells on the surface of a solid substrate, e.g. of a plastics material. Such deposit is promoted by the presence of serum, e.g. 10% fetal calf serum, which is therefore widely used in the cultivation of insulin producing cells (ref. 6). Under these conditions, no effect of growth hormone was observed, either on the DNA synthesis or the insulin production (ref. 7). Increased insulin production is described (ref. 8), and an increased DNA synthesis without an increase in insulin secretion is likewise described (ref. 9 and ref. 10). While considerable, but time-limited stimulation of both insulin production and DNA synthesis has been demonstrated in the intact islets (ref. 5), this has so far not been demonstrated in monolayer cultures.
The object of the present process is to provide an efficient and rational method of proliferating wholly or partially differentiated beta-cells in large amounts.
Another object of the invention is to proliferate , cells which produce insulin in considerable amounts and which are useful for implantation in humans.
A further object of the invention is to produce human insulin.
The process of the invention, which is characterized by the features stated in the characterizing portion of claim 1, is based on the surprising finding that wholly or partially differentiated beta-cells develop strongly with formation of monolayers by cultivation on a solid substrate and in contact with a nutrient medium containing both serum and growth hormone in the stated concentration ranges, and when the nutrient medium is repeatedly exchanged during a cultivation period of preferably several weeks .
The nutrient medium used in the proliferation of the cells may expediently have a relatively high content of glucose, such as 1/2 - 10 g/1 , preferably 1.5 - 5 g/1, with 2 g/1 as the optimum value. The content of human serum is expediently 1/2 - 1% , preferably 1/2 - 3% , with about 2?ό as the optimum value. Growth hormone of animal or human origin may be replaced by hormones having similar properties, such as prolactin or placental lactogen.
The preferred concentration of growth hormone or the hormone having similar properties in the culture medium is 10 to 1000 ng/ml, such as about 100 ng/ l. A consider¬ able effect may also be obtained, however, at lower con¬ centrations, such as down to about 1 ng/ml. Larger amounts of growth hormone may also be used, such as up to 1000 ng/ml culture medium or more, but, usually, no consider¬ ably improved cell formation is obtained at higher concentrations than 200 ng/ml.
The process of the invention permits proliferation of insulin producing cells of any type, also in the form of host cells into which one or more other gens which are to be expressed in an.iπral or human cells have been intro¬ duced. Examples of this are cells in which DNA fragments which code for Factor VIII, growth hormone or interferon have been introduced.
The process of the invention will be illustrated more fully below by means of some working examples.
EXAMPLE 1
Pancrease from 15 three days old rats is excised, treated with collagenase, and the islets are isolated as described in ref. 5. 1000 islets are placed in culture medium RPMI 1640 containing 10?ό serum from newborn calves, distributed with 100 islets in 5 ml medium in Petri dishes. The dishes stand at 37 °C for 2 days as described in ref. 5. Then the islets are treated with a mixture of EGTA, DNase and trypsin and are aspirated until a cell suspension has been obtained, as described in ref. 11. The cells are suspended in medium RPMI 1640, and 10 cells in 5 ml medium are placed in cell cultivation dishes, as described in ref. 9. The dishes stand for 2 days at 37 °C, and the medium is then changed to RPMI 1640 admixed with 2% normal human serum and 100 ng/ml human growth hormone (Nanormon,
Nordisk Gentofte, Denmark). The dishes stand at 37 °C, and the medium is changed once a week. Insulin is measured by radioimmunoassay , as described in ref. 5.
The cells proliferate to colonies, and after 5 months the colonies have spread so that they cover almost the entire dish. They are treated with a mixture of EGTA, DNase and trypsin, and the resulting cell suspension is placed in new dishes with the media RPMI 1640 admixed with.2% normal human serum and 100 ng/ml human growth hormone. Again the cells proliferate to colonies and spread in the dish. The insulin production increases again gradually.
The results are shown in figure 1 in which curve I refers to the experiment performed in this example. Curve II refers to a comparative example performed in the same manner, except that no growth hormone was added.
EXAMPLE 2
A cell suspension is produced as described in example 1.
10 cells suspended in 100 /ul serum-free medium RPMI 1640 are placed in cell cultivation dishes, and immediately following this are added 5 ml medium RPMI 1640 containing 0.5?ό, \% , 2% , 5% and 10?ό human serum, respectively, as well as 1 ,ug/ml human growth hormone. The dishes stand at 37 °C, and the medium is changed once a week. The measured insulin amount, released to the medium, is depicted in figure 2 as a function of serum concentration and cultivation time. Maximum insulin production is obtained after 3 weeks cultivation, as shown, at a serum REFERENCES
1. Fortune, August 6, 1984, p. 40-43: "Closing in on a Cure for Diabetes" by A.H. Moore.
2. Diabetologia 16, 97-100 (1979): "Preservation of Beta Cell Function in Adult Pancreatic Islets for Several Months in Vitro", by J.H. Nielsen et al.
3. Acta biol. med. germ., vol. 40, p. 55-60 (1981): "Beta-cell function in isolated human pancreatic islets in long-term tissue culture"., by J. Høiriis Nielsen.
4. Diabetologia 26: 393-400 (1984): "The life story of the pancreatic B cell", by C. Hellerstrδm.
5. Endocrinology, 110: 600-606 (1982): "Effects of Growth Hormone, Prolactin, and Placental Lactogen on Insulin Content and Release, and Deoxyribonucleic Acid Synthesis in Cultured Pancreatic Islets", by Jens Høiriis Nielsen.
6. US Patent Specification No. 4 439 521, Archer et al .
7. Diabetes 22: 687-693 (1973): "Beta cell replication in rat pancreatic onolayer cultures. Effects of glucose, tolbuta ide glucocorticoid, .growth hormone and glucasin." by W.L. Chick.
8. Diabetologia 22: 134-137 (1982): "Effects of hypo- physectomy and growth hormone on cultured islets of Langerhans of the rat." by J. Pierluissi et al.
9. Cold Spring Harbor Conferences on Cell Proliferati'on , vol. 9, p. 501-506: "Growth Hormone as a Growth Factor for Normal Pancreatic Islet Cells in Primary Culture", by J. Høiriis Nielsen et al. Published by Cold Spring Harbor Laboratory 1982.
10. Diabetes 32: 307-312 (1983): "Growth hormone stimu¬ lates islets B-cell replication in neonatal rat pancreatic monolayer cultures." by A. Rabinovitch et al . 11. Cell Separation: Methods and Selected Applications, vol. 2, chapter 7: "Purification of Islets and Cells from Islets", by J. Høiriis Nielsen et al. Published by Academic Press, 1983.

Claims

P a t e n t C l a i m s
1. A process for proliferation of wholly or partially differentiated beta-cells, comprising suspending islets or islets cultures isolated from human or animal pancrease in a culture medium and contacting them with a solid substrate until the islets or islet cultures have been deposited on the substrate, and cultivating the islets or islet cultures deposited on the solid substrate in a culture medium under conditions where the beta-cells form monolayers during their growth, c h a r a c t e r i z e d by culturing in a nutrient medium containing growth hormone or a hormone having similar properties in an amount of 1 - 1000 ng/ml and serum in an amount of 1/2 - 7?ό, and exchanging the culture medium repeatedly during a cultivation period of several days, preferably several weeks.
2. A process according to claim 1, c h a r a c t e r ¬ i z e d by performing the cultivation for a period of at least 2 weeks, and exchanging the culture medium several times, such as once a week.
3. A process according to claims l or 2, c h a r a c ¬ t e r i z e d in that the culture medium used for the cultivation of the cells deposited on the solid substrate contains glucose in an amount of 1/2 - 10 g per litre, preferably 1.5 - 5 g per litre, serum in an amount of 1/2 - 7?ό, preferably 1/2 - 3?ό, as well as growth hormone or a hormone having similar properties in an amount of 10 - 1000 ng/ml.
4. A process according to any of claims 1-3, c h a r a c t e r i z e d by using human serum and human growth hormone.
5. A process according to any of claims 1-4, c h a r a c t e r i z e d in that the cells used in the proliferation originate from pancrease from fetal tissue.
EP19850904430 1984-08-23 1985-08-23 A process for proliferation of wholly or partially differentiated beta-cellls Withdrawn EP0190337A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK402984A DK402984D0 (en) 1984-08-23 1984-08-23 PROCEDURE FOR PROMOTING INSULIN-PRODUCING CELLS
DK4029/84 1984-08-23

Publications (1)

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EP0190337A1 true EP0190337A1 (en) 1986-08-13

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EP (1) EP0190337A1 (en)
AU (1) AU4805185A (en)
DK (1) DK402984D0 (en)
WO (1) WO1986001530A1 (en)

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JPH06508527A (en) * 1991-06-24 1994-09-29 パシフイック　バイオメデイカル　リサーチ　インコーポレイテッド Hormone-secreting cells maintained in long-term culture
US5821121A (en) * 1991-06-24 1998-10-13 Pacific Biomedical Research, Inc. Hormone-secreting cells maintained in long-term culture
US5747341A (en) * 1991-06-24 1998-05-05 Pacific Biomedical Research, Inc. Culture media having low osmolarity for establishing and maintaining hormone-secreting cells in long-term culture
US6703017B1 (en) 1994-04-28 2004-03-09 Ixion Biotechnology, Inc. Reversal of insulin-dependent diabetes by islet-producing stem cells, islet progenitor cells and islet-like structures
US6001647A (en) * 1994-04-28 1999-12-14 Ixion Biotechnology, Inc. In vitro growth of functional islets of Langerhans and in vivo uses thereof
US5834308A (en) * 1994-04-28 1998-11-10 University Of Florida Research Foundation, Inc. In vitro growth of functional islets of Langerhans
WO2002002750A1 (en) * 2000-06-30 2002-01-10 Amcyte, Inc. Culturing pancreatic stem cells having a specified, intermediate stage of development
JP2004526449A (en) 2001-03-29 2004-09-02 イクシオン・バイオテクノロジー・インコーポレーテッド Transdifferentiation of non-pancreatic stem cells into pancreatic differentiation pathway
KR20030033638A (en) * 2001-10-24 2003-05-01 (주)한국췌도이식연구소 Artificial pancreatic islet cell and the use thereof
DE10313291A1 (en) * 2003-03-25 2004-10-14 Universität Leipzig Medium for engineering of vascularized tissue and blood vessels, useful for making tissue for reconstructive surgery, includes somatotropin to improve stability or formation of a capillary-like network
US8735154B2 (en) 2006-10-30 2014-05-27 The University Of Kansas Templated islet cells and small islet cell clusters for diabetes treatment
WO2013016544A2 (en) 2011-07-27 2013-01-31 University Of Kansas Templated islet cells and small islet cell clusters for diabetes treatment

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2726313C3 (en) * 1977-06-10 1980-02-07 Battelle-Institut E.V., 6000 Frankfurt Process for the in vitro biosynthesis of hormones, in particular of insulin
DE2757169A1 (en) * 1977-12-22 1979-07-05 Hoechst Ag METHOD OF OBTAINING INSULIN-PRODUCING ANIMAL CELLS
JPS5729294A (en) * 1980-07-30 1982-02-17 Hayashibara Biochem Lab Inc Preparation of human insulin

Non-Patent Citations (1)

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Title
See references of WO8601530A1 *

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WO1986001530A1 (en) 1986-03-13
DK402984D0 (en) 1984-08-23
AU4805185A (en) 1986-03-24

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