Insulin-secreting cell lines, methods of production and use

• US 5,902,577 A
• Filed: 06/04/1996
• Issued: 05/11/1999
• Est. Priority Date: 09/30/1993
• Status: Expired due to Term

• Current Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Original Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Inventors: Asfari, Maryam, Czernichow, Paul
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): SANDALS, WILLIAM O
• Application Number:
  US08/619,612
• Time in Patent Office: 1,071 Days
• Field of Search: 424/93.21, 424/451, 424/490, 424/424, 435/69.1, 435/91.1, 435/172.3, 435/174, 435/240.22, 435/320.1, 435/101, 435/69.4, 435/284.1, 435/353, 435/382, 435/398, 514/3, 514/44, 530/303, 536/22.1, 536/23.51, 436/829
• US Class Current: 424/93.21
• CPC Class Codes: A61K 2035/126 Immunoprotecting barriers, e.g. jackets, diffusion chambers
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

The invention relates to the field of biology and, in particular, to the field of cellular biology. The invention concerns a novel glucose-sensitive cell line designated β cell line (INS-I) expressing glucokinase and the glucose carrier Glut 2 at levels comparable with those of normal β cells but which is, in addition, incapable of IGF-II expression-dependent proliferation because of genetic manipulation. The invention also concerns a method for the production of said novel cell line, its aggregation in the form of a pseudoislet, its immobilization in a biocompatible hydrogel and its hardening by means of a hardening solution. Application in insulin-secreting β-cell transplants.

Claims

1. A highly differentiated insulin-secreting β

-cell line comprising transformed β

-cells, wherein said β

-cells express insulin in response to glucose, and wherein expression of the insulin-like growth factor II (IGF-II) gene in said β

-cells is permanently inhibited.

2. A cell line of claim 1, wherein expression of the insulin-like growth factor II (IGF-II) gene in said β

-cell line is completely repressed.

3. A β

-cell line of claim 1, wherein the endogenous IGF-II gene is knocked out.

4. A β

-cell line of claim 1, wherein the highly differentiated insulin-secreting β

-cell line is an INS-1 cell line.

5. A β

-cell line of claim 1, wherein said β

-cell line is in the form of pseudoislet aggregates.

6. A method of producing a transformed β

-cell line of claim 1, comprising

transfecting β

-cells of a highly differentiated insulin-secreting β

-cell line with a plasmid carrying a modified IGF-II gene, wherein

said modified IGF-II gene has inserted, in phase in exon 4, a cassette encoding the

neomycin resistance gene, and, downstream of said neomycin gene, a cassette encoding viral thymidine kinase,

culturing said transfected β

-cells in the presence of neomycin, gancyclovir and IGF-II,

isolating transformed β

-cells which are resistant to neomycin and gancyclovir, whereby transformed β

-cells are obtained

wherein said modified IGF-II gene has been incorporated by homologous recombination,

whereby said β

-cells are dependent on the exogenous administration of IGF-II for proliferation.

7. A method of claim 6, wherein the endogenous IGF-II gene is knocked out.

8. A method of claim 6, wherein the highly differentiated insulin-secreting β

-cell line is an INS-I cell line.

9. A method of claim 6, wherein the plasmid is derived from pUC 119.

10. A method of claim 6, wherein the thus-produced β

-cell line is further made into aggregates in the form of pseudoislets by culturing of the cells in nonadhesive culture dishes.

11. A method of claim 10, wherein the pseudoislets are agglomerated in suspension in a gelling agent and immobilized in a biocompatible hydrogel by adding an immobilizing agent.

12. A method of claim 11, wherein the gelling agent is a sodium alginate.

13. A method of claim 11, wherein the immobilizing agent is an aqueous solution of a calcium salt.

14. A method of claim 11, wherein the agglomerated and immobilized pseudoislets contain a quantity of insulin equivalent to that found in 500 to 1,000 islets in adult rats.

15. A method of claim 11, wherein the pseudoislets immobilized by encapsulation in a biocompatible hydrogel which has been hardened by adding an aqueous solution of a calcium salt are then placed in tubular acrylic membranes which are permeable to molecules with a molecular weight of less than 50,000-80,000.

16. A method of claim 15, wherein the immobilized and hardened pseudoislets are incorporated into transplantable fibers.

17. A biocompatible transplantable device, comprising pseudoislets of a transformed insulin-secreting β

-cell line of claim 1, agglomerated and incorporated into tubular acrylic membranes which are permeable to molecules with a molecular weight of less than 50,000-80,000, and distributed in the form of fibers.

18. A transplantable device of claim 17, comprising 50,000 to 200,000 pseudoislets, whereby an effective amount of insulin for treating an insulin-dependent patient is produced.

19. A transplantable device of claim 17, adapted for transplant to a subcutaneous or intraperitoneal location.

20. A method of treating insulin deficiency in a patient in need of such treatment, comprising introducing into the body of said patient an effective amount of cells in the biocompatible transplantable device of claim 17.

21. A method of treating insulin deficiency in a patient in need of such treatment, comprising transplanting into the body of said patient a transplantable device of claim 17.