Preparation and storage of pancreatic islets

• US 5,919,703 A
• Filed: 06/06/1996
• Issued: 07/06/1999
• Est. Priority Date: 04/10/1995
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Kenmochi, Takashi, Mullen, Yoko
• Primary Examiner(s): Naff, David M.
• Assistant Examiner(s): Ware, Deborah K.
• Application Number:
  US08/660,746
• Time in Patent Office: 1,125 Days
• Field of Search: 435/325, 435/378, 435/381, 435/394
• US Class Current: 435/381
• CPC Class Codes: A01N 1/02 Preservation of living parts
• CPC Class Codes: A01N 1/0226 Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

A method, a solution and a chamber for the preparation and storage of pancreatic islets. The method includes contacting a pancreas with a warm collagenase solution, digesting the pancreas in the warm collagenase solution to form warm digest, adding cold preservative solution to the warm digest, agitating the warm digest/cold preservative solution at a temperature between about 0° and 15° C., to thereby further digest the partially digested pancreas included in the warm digest, to form cold digest and collecting liquid from the cold digest to form isolated islets. The cold preservative solution and a pancreatic islet preservative solution of the present invention include D-mannitol, K-lactobionate and a buffer.

Claims

1. A method of isolating islets from a pancreas, the method comprising:

at least partially digesting the pancreas in a warm collagenase solution to form a warm digest;

adding a cold preservative solution to the warm digest to form a mixture;

the cold preservative solution comprising D-mannitol, at least 80 mM K-lactobionate, and a buffer;

agitating the warm digest and preservative solution mixture at a temperature of about 0°

C. to about 15°

C. to cool and form a cold digest comprising solid and liquid material which comprises the pancreatic islets; and

separating said islets from the cold digest.

2. The method of claim 1 wherein the step of at least partially digesting the pancreas in the warm collagenase solution is carried out at a temperature of about 35°

C. to about 38°

C.

3. The method of claim 1 wherein the warm collagenase solution comprises Collagenase P, about 2% (v/v) heat-inactivated newborn bovine serum, about 1 mg/ml CaCl₂ and 0.4 mg/ml DNase in a buffer selected from the group consisting of 5.6 mM glucose, 15 mM KH₂ PO₄, 0.33 mM Na₂ HPO₄, 0.82 mM MgSO₄, 5.4 mM KCl, 137 mM NaCl and 1.3 mM CaCl₂, pH 7.2;

30 mM raffinose, 100 mM K-lactobionate, 15 mM KH₂ PO₄, 5 mM MgSO₄, 5 mM adenosine, 3 mM glutathione, 1 mM allopurinol and 8 mM dexamthazone, pH 7.4;

30 mM raffinose, 100 mM K-lactobionate, 15 mM KH₂ PO₄, 5 mM MgSO₄, 5 mM adenosine, 3 mM glutathione, 1 mM allopurinol, 8 mM dexamthazone and 5% (w/w) hydroxyethyl starch, pH 7.4; and

30 mM D-mannitol, 100 mM K-lactobionate, 15 mM KH₂ PO₄, 5 mM MgSO₄, 30,000 units/1 superoxide dismutase and 5 mM nicotinamide.

4. The method of claim 1 wherein the cold preservative solution further comprises a membrane stabilizer, a radical scavenger, and nicotinamide.

5. The method of claim 1 wherein the cold preservative solution further comprises MgSO₄, superoxide dismutase, and nicotinamide.

6. The method of claim 1 wherein the cold preservative solution comprises about 25 mM to about 50 mM D-mannitol, about 80 mM to about 120 mM K-lactobionate, and about 15 mM KH₂ PO₄.

7. The method of claim 6 wherein the cold preservative solution further comprises about 1 mM to about 5 mM MgSO₄, up to about 30,000 units/liter superoxide dismutase, and up to about 5 mM nicotinamide.

8. The method of claim 1 wherein the step of at least partially digesting the pancreas comprises:

contacting the pancreas with a first portion of the warm collagenase solution;

digesting the pancreas in the first portion of the warm collagenase solution to form a first portion of the warm digest and undigested pancreas;

separating the undigested pancreas from the first portion of the warm digest;

contacting the undigested pancreas with a second portion of the warm collagenase solution;

digesting the pancreas in the second portion of the warm collagenase solution to form a second portion of the warm digest and indigested pancreas;

separating the undigested pancreas from the second portion of the warm digest;

repeating the contacting, digesting, and separating steps until the undigested pancreas comprises undigested ductal structures; and

combining ail of the portions of the warm digest.

9. The method of claim 1 wherein said separating of the pancreatic islets from the cold digest comprises:

separating the solid material from the liquid material in the cold digest;

wherein the liquid material comprises isolated islets in the form of cell clumps less than 15 mm in size;

adding a cold preservative solution to the solid material;

agitating the cold preservative solution and solid material at a temperature of about 0°

C. to about 15°

C. to form a second cold digest comprising solid material and liquid material;

repeating the separating, adding, and agitating steps until the solid material comprises no pancreatic islets.

10. The method of claim 1 wherein the step of adding said cold preservative solution comprises adding the preservative solution at a temperature of about 0°

C. to about 15°

C. to the warm digest.