Cell cultures of and cells culturing method for nontransformed parotid cells

• US 5,849,584 A
• Filed: 06/07/1995
• Issued: 12/15/1998
• 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): Lankford, Jr., Leon B.
• Assistant Examiner(s): TATE, CHRISTOPHER ROBIN
• Application Number:
  US08/485,650
• Time in Patent Office: 1,287 Days
• Field of Search: 435/240.2, 435/240.21, 435/240.3, 435/325, 435/366, 435/378, 435/382, 435/383, 435/391, 623/1.1
• 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 parotid cells and other glandular cells such as pancreatic, thyroid, and parathyroid, and 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. The nontransformed cells are cultured in a growth medium comprising a suitable basal medium supplemented with effective concentrations of hypothalamus and pituitary extracts, and serum.

Claims

1. A method for producing an expanded, non-transformed human cell culture of human parotid cells, comprising the steps of:

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

(b) concentrating said selected cells;

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

(d) culturing said resuspended cells in said growth medium to effect sustained cell division; and

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

wherein said growth medium comprises a combination of a suitable basal medium and supplements of ingredients including hypothalamus and pituitary extracts, and serum, which ingredients of the growth medium are adjusted in amounts to provide a medium which is selective for said human cells in which said cultured parotid cells are selectively proliferated without being transformed and differentiated functions are retained through periodic passaging, providing an expanded human cell culture of non-transformed, proliferated, functionally differentiated human parotid cells that is substantially free of passenger cells including fibroblast, macrophage, and capillary endothelial cells.

2. The method of claim 1, wherein said expanded cell culture has undergone at least seven population doublings (7 PDL .

3. The method of claim 2 wherein said expanded cell culture contains aggregated cells, non-aggregated cells or mixtures thereof.

4. The method of claim 3, wherein said aggregated cells or non-aggregated cells are embedded in a biocompatible matrix.

5. The method of claim 4, wherein said aggregated cells or non-aggregated cells are embedded in a biocompatible gel-matrix.

6. The method of claim 1 wherein said growth medium ingredients further include one or both of human placental extract or human placental lactogen.

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

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

(b) growing said culture;

(c) disassociating said cells;

(d) inoculating said disassociated cells into a culture that contains a parotid cell conditioned 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 obtained according to the method of claim 1 has undergone at least seven population doublings (7 PDL).

9. The method of claim 8, wherein said cell culture contains aggregated cells, non-aggregated cells, or mixtures thereof.

10. The method of claim 9, wherein said aggregated cells or non-aggregated cells are matrix-embedded.

11. The method of claim 10, wherein said aggregated cells or non-aggregated cells are embedded in a gel-matrix.

12. Clonal strains of an expanded, non-transformed cell culture of human parotid cells produced according to the method of claim 7.

13. An expanded, non-transformed human cell culture of human parotid cells produced by the method of claim 1.

14. The expanded, non-transformed human cell culture of claim 13, wherein said cells have undergone at least seven population doublings (7PDL).

15. The expanded, non-transformed human cell culture of human parotid cells of claim 14, wherein said cells are embedded in a biocompatible matrix.

16. The expanded, non-transformed human cell culture of human parotid cells of claim 15, wherein said cells are embedded in a biocompatible gel.

17. The method of claim 1 wherein said cells are obtained from an adult.