Method of altering blood sugar levels using non-transformed human pancreatic cells that have been expanded in culture

• US 5,780,299 A
• Filed: 06/07/1995
• Issued: 07/14/1998
• Est. Priority Date: 04/08/1993
• Status: Expired due to Fees

• Current Assignee: Ambesi-Impiombato F. Saverio, Francesco Curcio, Hayden G. Coon
• Original Assignee: Human Cell Cultures, Inc.
• Inventors: Ambesi-Impiombato, Francesco Saverio, Curcio, Francesco, Coon, Hayden G.
• Primary Examiner(s): Lankford, Jr., Leon B.
• Assistant Examiner(s): TATE, CHRISTOPHER ROBIN
• Application Number:
  US08/480,027
• Time in Patent Office: 1,133 Days
• Field of Search: 435/240.2, 435/240.21, 435/240.3, 435/325, 435/366, 435/382, 435/383, 435/391, 623/11
• US Class Current: 435/366
• 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: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61P 3/08 for glucose homeostasis pancreatic hormones A61P5/48
• CPC Class Codes: C12N 2500/16 Magnesium; Mg chelators
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/84 from mammals
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/395 Thyroid hormones
• CPC Class Codes: C12N 2502/22 pancreatic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0617 Thyroid and parathyroid glands
• CPC Class Codes: C12N 5/0633 Cells of secretory glands, e.g. parotid gland, salivary glands, sweat glands, lacrymal glands
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention provides a method for producing an expanded non-transformed cell culture comprising the steps of: (1) preparing partially purified, minced tissue; (2) concentrating the resulting cells and tissue pieces; (3) resuspending the concentrated tissue cells and pieces in a culture medium capable of supporting sustained cell division that is contained in a culture vessel; (4) incubating the cells; and (5) passaging the cells periodically. The present invention further provides clonal strains of cells derived from the above-mentioned cell culture, medium and conditioned medium designed for the culturing of such cells, including pancreatic, thyroid, parathyroid, and parotid cells, and the use of cultured pancreatic cells to form pancreatic pseudotissues composed of matrix-embedded aggregated (pseudoislets) or individual cells, to treat blood sugar disorders in mammals, and to test for cytotoxicity and autoimmune activities with reference to pancreatic endocrine cells.

Claims

1. A method of altering blood sugar levels of a mammal comprising administering expanded human pancreatic cells to the mammal, which expanded cells have been expanded in a cell culture and comprise enriched, non-transformed, serially passaged human pancreatic endocrine cells, wherein said cell culture is substantially free of fibroblast, macrophage, and capillary endothelial cells, and wherein said cell culture is prepared by a method comprising the steps of:

(a) selecting said pancreatic endocrine cells from a human tissue that comprises said cells;

(b) concentrating said selected cells;

(c) resuspending said concentrated cells in a culture medium, wherein said medium comprises a basal medium supplemented with (i) hypothalamus extract and (ii) pituitary extract;

(d) culturing said resuspended cells; and

(e) passaging said cultured cells periodically to expand said culture.

2. The method of claim 1, wherein said cell culture is a clonal cell culture of pancreatic endocrine cells prepared by a method comprising the steps of:

(a) culturing the cell culture obtained according to the method of claim 1;

(b) growing said culture;

(c) dissociating said cells;

(d) inoculating said disassociated cells into a culture vessel that contains medium conditioned by pancreatic endocrine cells for a first plating and culturing said inoculated cells to produce colonies of cells;

(e) harvesting individual colonies of cells;

(f) inoculating said colonies into a culture vessel for a second plating and culturing said inoculated cells; and

(g) serially passaging the cells of step (f).

3. The method of claim 1, wherein said cells are matrix-embedded.

4. The method of claim 3, wherein said cells are embedded in a gel matrix.

5. The method of claim 1, wherein said mammal is human.

6. A method of altering blood sugar levels of a human comprising administering expanded pancreatic cells of human origin to the human, which expanded cells have been expanded in a cell culture and comprise enriched, non-transformed, serially passaged pancreatic endocrine cells, wherein said cell culture is substantially free of fibroblast, macrophage, and capillary endothelial cells, and wherein said cell culture is prepared by a method comprising the steps of:

(a) selecting pancreatic endocrine cells from a human tissue that comprises said cells;

(b) concentrating said selected cells;

(c) resuspending said concentrated cells in a culture medium, wherein said medium comprises a basal medium supplemented with (i) hypothalamus extract, (ii) pituitary extract and (iii) one or both of human placental extract or human placental lactogen;

(d) culturing said resuspended cells; and

(e) passaging said cultured cells periodically to expand said culture.

7. A method of altering the blood sugar of a mammal comprising administering to said mammal non-transformed mammalian cells obtained from a non-transformed cell culture, wherein said cell culture comprises:

a growth medium comprising (i) a basal medium supplemented with (ii) hypothalamus extract, (iii) pituitary extract, and (iv) placental extract and/or a placental lactogen, and

an enriched, expanded, non-transformed population of human pancreatic endocrine cells.

8. The method of claim 7, wherein said pancreatic endocrine cells do not grow in soft agar, and do not form tumors in 45 days after about 10⁷ cells are implanted into SCID mice.

9. The method of claim 8, wherein said pancreatic endocrine cells are obtained from an adult.

10. The method of claim 9, wherein said culture is substantially free of fibroblasts, macrophages, and capillary endothelial cells.

11. The method of claim 10, wherein said pancreatic endocrine cells secrete insulin at a steady state and at a greater rate in response to glucose stimulation.

12. The method of claim 11, wherein said pancreatic endocrine cells are matrix embedded.

13. The method of claim 12, wherein said pancreatic endocrine cells are aggregated to form pseudoislets.

14. The method of claim 13, wherein said pseudoislets comprise pluripotent cells.