WO1986001530A1 - Procede de proliferation de cellules beta entierement ou partiellement differenciees - Google Patents

Procede de proliferation de cellules beta entierement ou partiellement differenciees Download PDF

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Publication number
WO1986001530A1
WO1986001530A1 PCT/DK1985/000083 DK8500083W WO8601530A1 WO 1986001530 A1 WO1986001530 A1 WO 1986001530A1 DK 8500083 W DK8500083 W DK 8500083W WO 8601530 A1 WO8601530 A1 WO 8601530A1
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WIPO (PCT)
Prior art keywords
cells
islets
beta
culture medium
proliferation
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Application number
PCT/DK1985/000083
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English (en)
Inventor
Jens Ho^/iriis NIELSEN
Original Assignee
Nordisk Gentofte A/S
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Publication date
Application filed by Nordisk Gentofte A/S filed Critical Nordisk Gentofte A/S
Publication of WO1986001530A1 publication Critical patent/WO1986001530A1/fr

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Classifications

    • 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.
PCT/DK1985/000083 1984-08-23 1985-08-23 Procede de proliferation de cellules beta entierement ou partiellement differenciees WO1986001530A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK402984A DK402984D0 (da) 1984-08-23 1984-08-23 Fremgangsmaade til formering af insulin-producerende celler
DK4029/84 1984-08-23

Publications (1)

Publication Number Publication Date
WO1986001530A1 true WO1986001530A1 (fr) 1986-03-13

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ID=8129448

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Application Number Title Priority Date Filing Date
PCT/DK1985/000083 WO1986001530A1 (fr) 1984-08-23 1985-08-23 Procede de proliferation de cellules beta entierement ou partiellement differenciees

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

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0605428A1 (fr) * 1991-06-24 1994-07-13 Pacific Biomedical Research, Inc. Cellules de secretion d'hormones maintenues en culture a long terme
WO1995029988A1 (fr) * 1994-04-28 1995-11-09 University Of Florida Research Foundation, Inc. Croissance in vitro d'ilots fonctionnels de langerhans et utilisations in vivo de ces ilots
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
US5821121A (en) * 1991-06-24 1998-10-13 Pacific Biomedical Research, Inc. Hormone-secreting cells maintained in long-term culture
US6001647A (en) * 1994-04-28 1999-12-14 Ixion Biotechnology, Inc. In vitro growth of functional islets of Langerhans and in vivo uses thereof
EP1301589A1 (fr) * 2000-06-30 2003-04-16 Amcyte, Inc. Culture de cellules embryonnaires pancreatiques presentant un stade de developpement intermediaire specifie
KR20030033638A (ko) * 2001-10-24 2003-05-01 (주)한국췌도이식연구소 인공 췌도 세포 및 이의 용도
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
DE10313291A1 (de) * 2003-03-25 2004-10-14 Universität Leipzig Mediumzusammensetzung für das Tissue engineering vaskularisierter Gewebe und von Blutgefäßen
US7202080B2 (en) 2001-03-29 2007-04-10 Ixion Biotechnology, Inc. Method for transdifferentiation of non-pancreatic stem cells to the pancreatic differentiation pathway
WO2011126921A2 (fr) 2010-04-06 2011-10-13 University Of Kansas Ilots de langerhans à matrice et petits amas d'îlots de langerhans pour le traitement du diabète
EP3498300A1 (fr) 2011-07-27 2019-06-19 University of Kansas Cellules d'îlots créées à partir de modèles et petits amas de cellules d'îlots pour le traitement du diabète

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2757169A1 (de) * 1977-12-22 1979-07-05 Hoechst Ag Verfahren zur gewinnung insulin produzierender tierischer zellen
US4225671A (en) * 1977-06-10 1980-09-30 Battelle-Institut E.V. Process for the in-vitro biosynthesis of hormones, especially insulin
GB2083826A (en) * 1980-07-30 1982-03-31 Hayashibara Biochem Lab Process for the production of human insulin

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4225671A (en) * 1977-06-10 1980-09-30 Battelle-Institut E.V. Process for the in-vitro biosynthesis of hormones, especially insulin
DE2757169A1 (de) * 1977-12-22 1979-07-05 Hoechst Ag Verfahren zur gewinnung insulin produzierender tierischer zellen
GB2083826A (en) * 1980-07-30 1982-03-31 Hayashibara Biochem Lab Process for the production of human insulin

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Chemical Abstracts Vol 88 (1978), abstract 83813h, J.Endocrinol. 1977, 75(3), p 49-50. *
Diabetes Vol 32 p 307-12, published 1983 (Rabinovitch A. et al) "Growth Hormone Stimulates Islet B-Cell Replication in Neonatal Ray Paneratic Monolayer Cultures". see especially tabel 2 and fig. 1B. *
Dissertation Abstracts Int. B Vol. 42 (1982), no 12, part 1, 4646. *
Growth of Cells in Hormonally Defined Media, Cold Spring Harbor Conf. on Cell Proliferation Vol 9, published 1982 (Ed. Sato G.H. et al ) by Cold Spring Harbor Laboratories, see pages 501-506, especially p 502. "Cell Culture". *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0605428A4 (fr) * 1991-06-24 1995-01-11 Pacific Biomed Res Inc Cellules de secretion d'hormones maintenues en culture a long terme.
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
US5821121A (en) * 1991-06-24 1998-10-13 Pacific Biomedical Research, Inc. Hormone-secreting cells maintained in long-term culture
US5928942A (en) * 1991-06-24 1999-07-27 Pacific Biomedical Research, Inc. Hormone-secreting cells derived from pancreatic islet maintained in long-term culture
EP0605428A1 (fr) * 1991-06-24 1994-07-13 Pacific Biomedical Research, Inc. Cellules de secretion d'hormones maintenues en culture a long terme
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
WO1995029988A1 (fr) * 1994-04-28 1995-11-09 University Of Florida Research Foundation, Inc. Croissance in vitro d'ilots fonctionnels de langerhans et utilisations in vivo de ces ilots
US5834308A (en) * 1994-04-28 1998-11-10 University Of Florida Research Foundation, Inc. In vitro growth of functional islets of Langerhans
US6001647A (en) * 1994-04-28 1999-12-14 Ixion Biotechnology, Inc. In vitro growth of functional islets of Langerhans and in vivo uses thereof
EP1301589A1 (fr) * 2000-06-30 2003-04-16 Amcyte, Inc. Culture de cellules embryonnaires pancreatiques presentant un stade de developpement intermediaire specifie
EP1301589A4 (fr) * 2000-06-30 2004-06-16 Amcyte Inc Culture de cellules embryonnaires pancreatiques presentant un stade de developpement intermediaire specifie
US7202080B2 (en) 2001-03-29 2007-04-10 Ixion Biotechnology, Inc. Method for transdifferentiation of non-pancreatic stem cells to the pancreatic differentiation pathway
KR20030033638A (ko) * 2001-10-24 2003-05-01 (주)한국췌도이식연구소 인공 췌도 세포 및 이의 용도
DE10313291A1 (de) * 2003-03-25 2004-10-14 Universität Leipzig Mediumzusammensetzung für das Tissue engineering vaskularisierter Gewebe und von Blutgefäßen
WO2011126921A2 (fr) 2010-04-06 2011-10-13 University Of Kansas Ilots de langerhans à matrice et petits amas d'îlots de langerhans pour le traitement du diabète
EP3498300A1 (fr) 2011-07-27 2019-06-19 University of Kansas Cellules d'îlots créées à partir de modèles et petits amas de cellules d'îlots pour le traitement du diabète

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EP0190337A1 (fr) 1986-08-13
DK402984D0 (da) 1984-08-23
AU4805185A (en) 1986-03-24

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