Method for preparing an expanded culture and clonal strains of pancreatic, thyroid or parathyroid cells

• US 5,646,035 A
• Filed: 06/07/1995
• Issued: 07/08/1997
• Est. Priority Date: 04/08/1993
• Status: Expired due to Fees

• Current Assignee: Human Cell Cultures, Inc.
• Original Assignee: Human Cell Cultures, Inc.
• Inventors: Ambesi-Impiombato, Francesco Saverio, Curcio, Francesco, Coon, Hayden G.
• Primary Examiner(s): Rollins, John W.
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US08/480,149
• Time in Patent Office: 762 Days
• Field of Search: 435/240.2, 435/240.21, 435/240.3, 435/240.31
• US Class Current: 435/378
• 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 for producing an expanded, enriched, non-transformed cell culture of mammalian pancreatic endocrine cells, comprising the steps of:

(a) selecting said pancreatic endocrine cells from pancreatic tissue;

(b) concentrating said selected cells;

(c) resuspending said concentrated cells in a culture medium;

(d) culturing said resuspended cells for a time and under conditions to effect sustained cell division; and

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

wherein said culture medium is Coon'"'"'s 4506.035 or Coon'"'"'s 4506.07, and said culture is substantially free of fibroblast, macrophage, and capillary endothelial cells.

2. The method of claim 1, wherein said cell culture has undergone at least seven population doublings.

3. The method of claim 1, wherein said pancreatic endocrine cells are selected from the group consisting of alpha cells, beta cells, delta cells, pluripotent cells, and mixtures thereof.

4. The method of claim 3, wherein said pancreatic endocrine cells comprise at least two different types of pancreatic endocrine cells selected from the group consisting of alpha cells, beta cells, and delta cells.

5. The method of claim 1, wherein said pancreatic endocrine cells comprise human pancreatic endocrine cells.

6. The method of claim 5, wherein said cell culture has undergone at least seven population doublings.

7. A method for preparing clonal strains of an expanded non-transformed cell culture of mammalian pancreatic endocrine cells, comprising the steps of:

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

(b) growing said culture to confluence;

(c) dissociating said cells;

(d) inoculating said disassociated cells into a culture vessel that contains a pancreatic endocrine cell conditioned Coon'"'"'s 4506.035 or Coon'"'"'s 4506.07 medium 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) passaging the cells of step (f) periodically.

8. The method of claim 7, wherein said cell culture has undergone at least seven population doublings.

9. The method of claim 7, wherein said pancreatic tissue is human.

10. The method of claim 9, wherein said pancreatic endocrine cells are selected from the group consisting of alpha cells, beta cells, delta cells, pluripotent cells, and mixtures thereof.

11. The method of claim 10, wherein said pancreatic endocrine cells comprise at least two different types of pancreatic endocrine cells selected from the group consisting of alpha cells, beta cells and delta cells.

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

13. A method for producing an expanded, non-transformed cell culture of mammalian thyroid or parathyroid cells, comprising the steps of:

(a) selecting said thyroid or parathyroid cells from thyroid or parathyroid tissue;

(b) concentrating said selected cells;

(c) resuspending said concentrated cells in a culture medium;

(d) culturing said resuspended cells for a time and under conditions to effect sustained cell division; and

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

wherein said culture medium is Coon'"'"'s 4506.035 or Coon'"'"'s 4506.07, modified so that the concentration of MgCl₂ is about 0.48 mM, the concentration of hydrocortisone is 3.5 ng/ml, the concentration of selenous acid is about 2 ng/ml, the triiodothyronine concentration is about 3 pg/ml, the final concentration of bovine hypothalamus extract is about 75 μ

g protein/ml, and the final concentration of bovine pituitary extract is about 5 μ

g protein/ml, and said culture is substantially free of fibroblast, macrophage, and capillary endothelial cells.

14. The method of claim 13, wherein said cell culture has undergone at least seven population doublings.

15. A method for preparing clonal strains of an expanded, non-transformed cell culture of mammalian thyroid or parathyroid cells, comprising the steps of:

(a) culturing the cell culture obtained according

to the method of claim 13;

(b) growing said culture to confluence;

(c) dissociating said cells;

(d) inoculating said dissociated cells into a culture vessel that contains a thyroid or parathyroid cell conditioned Coon'"'"'s 4506.035 or Coon'"'"'s 4506.07 medium for a first plating and culturing said inoculated cells to produce colonies of cells;

(e) harvesting individual colonies of cells;

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

(g) passaging the cultured cells of step (f) periodically.

16. The method of claim 15, wherein said mammalian thyroid or parathyroid cell is human thyroid or parathyroid cell.