Islet isolation process

• US 4,868,121 A
• Filed: 09/09/1985
• Issued: 09/19/1989
• Est. Priority Date: 02/07/1985
• Status: Expired due to Term

• Current Assignee: Neurotech S.A. (LivaNova Plc), Mcdonnell Douglas Corporation (The Boeing Co.), Washington University In St Louis
• Original Assignee: Mcdonnell Douglas Corporation (The Boeing Co.), Washington University In St Louis
• Inventors: Scharp, David W., Lacy, Paul E., Finke, Edward H., Poteat, Thomas J.
• Primary Examiner(s): Kepplinger, Esther M.
• Application Number:
  US06/773,459
• Time in Patent Office: 1,471 Days
• Field of Search: 435/1, 435/268, 435/240, 424/110
• US Class Current: 435/268
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36

Abstract

A method of producing intact islets of Langerhans in an insulin producing condition uses a mixture of Hank'"'"'s solution and 10% by volume fetal calf serum to ductally distend the human pancreas. The exocrine tissue of the pancreas is digested at about 37° C. by an enzyme preparation of collagenase, trypsin and proteolytic enzyme present in the mixture at a level of about 0.2% by weight. The digestion is monitored at regular intervals during the process. The digested pancreas is comminuted, filtered and intact islets of Langerhans are recovered. The recovered islets retain their insulin producing properties.

Claims

1. The method of recovering intact islets of Langerhans from pancreas comprising ductally distending an intact pancreas with a mixture of Hank'"'"'s solution and 10% by volume fetal calf serum containing 0.2% by weight of an enzyme preparation containing collagenase, and incubating the pancreas to digest the exocrine tissue of the pancreas, monitoring the pancreas at regular intervals during the digestion to empirically determine the point of maximum islet separation, the combination of physical disruption of the distension and the enzymatic digestion being effective to free viable islets of Langerhans from the pancreatic tissue while retaining the insulin producing properties of the islets, removing the intact islets from the remaining pancreatic tissue and digested product and recovering the intact islets of Langerhans in their insulin producing condition, the recovery being performed by first chopping the digested pancreas and macerating the digested pancreas by mechanical grinding, the grinding being effective to reduce the size of the pancreatic tissue to about the size of an intact islet while retaining the maximum number of islets in their whole, insulin producing condition, and second, separating the intact islets from the exocrine tissue of the pancreas by filtration and concentration, the recovered concentrate containing intact islets in a condition effective to produce insulin.

6. The method of claim 1 wherein a human pancreas is used and the pancreas is digested for between about 45 and about 90 minutes.

7. The method of claim 1 wherein a pig pancreas is used and wherein the pancreas is digested for between about 14 and 16 minutes.

2. The method of claim 19 wherein the pancreas is distended with between about 150 and about 200 ml of the mixture.

3. The method of claim 19 including partially distending the pancreas, checking for leaks, suturing the leaks closed and fully distending the pancreas for incubation.

4. The method of claim 19 wherein the step of filtration and concentration comprises washing the macerated pancreatic tissue through a series of successively smaller screens with Hank'"'"'s solution to remove particles larger than about 500 microns.

5. The method of claim 4 wherein the step of filtration and concentration includes a second filtration, the second filtration comprising washing the once filtered pancreatic tissue through a series of successively smaller filters, the smallest being about 53 microns, with Hank'"'"'s solution and backwashing the filters with Hank'"'"'s solution and fetal calf serum to recover the filtered islets.

8. A process for isolating intact islets from a pancreas, comprising the steps of ductally distending the pancreas with a solution containing collagenase and incubating the pancreas in a solution containing collagenase until an effective separation of islets from the other pancreatic tissue is observed;

chopping the pancreas into large fragments and macerating the large fragments by mechanical grinding, the grinding being effective to reduce the size of the pancreatic tissue to about the size of an intact islet while retaining the islets in their whole condition;

forcing the macerated pancreas fragments through successively smaller screens to filter the other pancreatic tissue from the islets;

and purifying the islets.