Method of culturing, cryopreserving and encapsulating pancreatic islet cells

• US 6,303,355 B1
• Filed: 03/22/1999
• Issued: 10/16/2001
• Est. Priority Date: 03/22/1999
• Status: Expired due to Term

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Opara, Emmanuel C.
• Primary Examiner(s): Naff, David M.
• Application Number:
  US09/273,407
• Time in Patent Office: 939 Days
• Field of Search: 435/178, 435/182, 435/260, 435/374, 435/375, 435/382, 435/384, 435/395, 435/404, 424/93.7, 424/423, 424/489
• US Class Current: 435/178
• CPC Class Codes: A61K 2035/128 capsules, e.g. microcapsules
• CPC Class Codes: C12N 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

Cells, preferably isolated pancreatic islet cells, are treated with a compound or a combination of compounds selected from an antioxidant, an anti-cytokine, an anti-endotoxin and an antibiotic. The compound or combination of compounds is in a medium for culturing the cells before microencapsulation, in a medium for cryopreserving the cells by freezing followed by thawing and microencapsulation, in a medium for culturing the cells after microencapsulation, or in a medium for culturing the cells before microencapsulation and in a medium for culturing the cells after microencapsulation. In a preferred method, isolated pancreatic islet cells are cultured for about 12 to about 36 hours in a medium containing an antioxidant as the compound, cryopreserved by freezing in a medium containing the compound or combination of compounds, thawed and microencapsulated. The microcapsule contains a hydrogel core such gelled alginate and a semipermeable outer membrane such as formed with polylysine. Pancreatic islet cells are provided for transplantation that have enhanced glucose-stimulated insulin production.

Claims

1. A method of treating isolated pancreatic islet cells, comprising:

(a) culturing said isolated pancreatic islet cells for from about 12 to about 36 hours in the presence of an antioxidant;

then

(b) cryopreserving said cells by freezing the cells in a cryopreservation medium comprising at least one compound selected from the group consisting of an antioxidant, an anti-cytokine, an anti-endotoxin, and an antibiotic;

then

(c) thawing said cells, and

(d) encapsulating said cells in a biocompatible microcapsule comprising a core and a semipermeable outer membrane, to provide a microcapsule containing living cells therein.

2. A method according to claim 1, wherein said cryopreservation medium contains at least two compounds selected from the group consisting of an antioxidant, an anti-cytokine, an anti-endotoxin, and an antibiotic.

3. A method according to claim 1, wherein said cryopreservation medium contains at least three compounds selected from the group consisting of an antioxidant, an anti-cytokine, an anti-endotoxin, and an antibiotic.

4. A method according to claim 1, wherein said cryopreservation medium contains at least one each of an antioxidant, an anti-cytokine, an anti-endotoxin, and an antibiotic.

5. A method according to claim 1, wherein said antioxidant is selected from the group consisting of glutathione, glutathione monoester, N-acetylcysteine, superoxide dismutase, catalase, vitamin E, α

-tocopherol, lipoic acid, lazaroids, butylated hydroxyanisole (BHA), and vitamin K.

6. A method according to claim 1, wherein said anti-endotoxin is selected from the group consisting of L-N^G-Monomethylarginine (L-NMMA), lactoferrin, N-acetylcysteine (NAC), adenosine receptor antagonists and anti-lipopolysaccharide compounds.

7. A method according to claim 1, wherein said anti-cytokine is selected from the group consisting of dimethylthiourea, citiolone, pravastatin sodium;

L-N^G-Monomethylarginine (L-NMMA), lactoferrin and 4-methylprednisolone.

8. A method according to claim 1, where said microcapsule comprises a polysaccharide gum surrounded by a semipermeable membrane.

9. A method according to claim 1 where said microcapsule comprises alginate and polylysine.

10. A method according to claim 1 wherein said microcapsule has an internal cell-containing core of alginate.

11. A method according to claim 10 wherein said internal cell-containing core of alginate is gelled.

12. A method according to claim 10 wherein said internal cell-containing core of alginate is not gelled.

13. A method according to claim 1 wherein said microcapsule has a diameter of from about 50 μ

m to about 2 mm.

14. A method according to claim 1 wherein said microcapsule has a diameter of from about 200 μ

m to about 1000 μ

m.

15. A method according to claim 1 wherein said microcapsule has a diameter of from about 300 μ

m to about 700 μ

m.