Method for producing preparations of mature and immature pancreatic endocrine cells, the cell preparation and its use for treatment of diabetes mellitus

• US 6,686,197 B2
• Filed: 04/12/2001
• Issued: 02/03/2004
• Est. Priority Date: 04/12/2000
• Status: Expired due to Term

• Current Assignee: Beta-Cell NV
• Original Assignee: Beta-Cell NV
• Inventors: Pipeleers, Daniel
• Primary Examiner(s): Lankford, Jr., Leon B.
• Application Number:
  US09/834,096
• Publication Number:
  US 20020177228A1
• Time in Patent Office: 1,027 Days
• Field of Search: 435/325, 435/366, 435/371
• US Class Current: 435/325
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

A method for preparing a preparation of mammalian pancreatic endocrine cells comprising the steps: dissociating intact pancreatic tissue into a cell suspension comprising single cells and cell aggregates; enriching said cell suspension with regard to the content in endocrine cells by separating single cells and cellular aggregates with size <100 μm; and removing contaminating non-endocrine cells by density centrifugation.

Claims

1. A method for preparing a preparation of mammalian pancreatic endocrine cells comprising:

dissociating intact pancreatic tissue into a cell suspension comprising single cells and cellular aggregates;

removing single cell and cellular aggregates larger than 100 μ

m from said cell suspension;

enriching said cell suspension with regard to the content in endocrine cells and cellular aggregates with size ≦

100 μ

m; and

removing contaminating non-endocrine cells by density centrifugation, wherein the resulting preparation of mammalian pancreatic endocrine cells consists essentially of endocrine cells and cellular aggregates having sizes ≦

100 μ

m.

2. The method according to claim 1, wherein said cell suspension is being enriched by separating single cells and cellular aggregates with size <

100 μ

m.

3. The method according to claim 2, wherein said cell suspension is being enriched by separating single cells with size <

15 μ

m.

4. The method according to claim 2, wherein said cell suspension is being enriched by separating cellular aggregates with a size of 15 to 100 μ

m.

5. The method according to claim 2, wherein said cell suspension is being enriched by counterflow centrifugation or elutriation and wherein said cell suspension is being enriched by separating single cells with a size of 6-15 μ

m.

6. The method according to claim 2, wherein contaminating acinar cells are removed by density gradient centrifugation using a layer with a density of at least 1.075 g/l.

7. The method as claimed in claim 6, wherein the intact pancreatic tissue is dissociated by sequential shaking incubations, first in the presence of collagenase and second in a calcium-free medium.

8. The method according to claim 7, wherein the separatino comprises centrifugation through a layer with a density <

1.05 g/ml in order to enrich in viable cells.

9. The method according to claim 8, wherein said separated cells or aggregates with a size <

100 μ

m are used to further purify beta cells with a mature or immature phenotype, glucagon-containing alpha cells, and/or non-granulated cells containing precursor endocrine cells, by fluorescence activated cell sorting (FACS).

10. The method according to claim 9, wherein immature pancreatic endocrine cells survive, and are further enriched, by suspension culture in serum-free medium containing glucocorticoids and nicotinamide.

11. The method according to claim 10, wherein immature endocrine cells are maintained during culture with serum.

12. The method according to claim 11, wherein the pancreatic tissue is obtained from fetal pig.

13. The method according to claim 12, wherein the fetal pig is obtained from a pregnant sow of minimally 108 days of gestation.

14. The method according to claim 13, wherein the cell suspension depleted in single immature endocrine cells is used to obtain a cell preparation enriched in mature endocrine cells.

15. The method according to claim 1, wherein minimally 10⁷ endocrine cells are isolated per mammalian pancreas at a higher than 80% purity with minimally 40% insulin immunoreactive cells.

16. The method according to claim 15, wherein mammalian beta cell preparations are produced with a potential for growth in vitro and in vivo, following transplantation.

17. The method according to claim 16, wherein mammalian alpha cell preparations are produced at minimally 65% purity.

18. The method according to claim 17, wherein mammalian beta cell preparations can be cultured for minimally four weeks while retaining an insulin biosynthetic activity of more than 10 fmol/10³ beta cells/hr.

19. A preparation of viable endocrine cells obtainable by a method according to claim 11.

20. The preparation according to claim 19, comprising more than 40% insulin containing beta cells.

21. The preparation as claimed in claim 20, for use in the treatment of diabetes mellitus.