Claims for Patent: 8,252,589

Summary for Patent: 8,252,589

Title:	Method for isolating cellular products by cryopreservation
Abstract:	Methods of isolating cellular products, such as pancreatic islets, may be used in diabetes research and therapeutic transplantation. The methods may involve providing a tissue having desired cells that are less prone to destructive freezing and undesired cells that are more prone to destructive freezing, or pre-treating a tissue to have such characteristics. The methods may involve freezing the tissue, disrupting the tissue, warming the tissue, and separating the desired cells from undesired cellular material to obtain the cellular product. The methods may thereby provide an enzyme-free or reduced-enzyme method of isolating a cellular product that is more consistent, reliable and less toxic than conventional methods. The methods may also yield an optimum quantity of cellular product that retain sufficient functional integrity to be useful as a transplantation resource.
Inventor(s):	Taylor; Michael J. (Mount Pleasant, SC), Pegg; David E. (York, GB)
Assignee:
Application Number:	12/654,147
Patent Claims:	1. A method of isolating a cellular product, comprising: pre-treating a tissue such that desired cells are less prone to destructive freezing and undesired cells are more prone to destructive freezing, freezing the tissue, disrupting the tissue, warming the tissue, and separating the desired cells from undesired cellular material to obtain a cellular product, wherein pre-treating the tissue comprises: providing to the desired cells a cryoprotectant solution comprising a cryoprotective agent, and infusing the tissue with an aqueous solution via a ductal system. 2. The method of claim 1, wherein the tissue is pancreatic tissue and the cellular product is pancreatic islets. 3. The method of claim 2, wherein pre-treating the pancreatic tissue comprises: infusing the pancreatic islets with the cryoprotectant solution comprising a cryoprotective agent via a vascular system, and infusing acinar tissue with the aqueous solution via the ductal system. 4. The method of claim 2, further comprising treating the pancreatic islets with a digestive enzyme. 5. The method of claim 1, wherein the cryoprotective agent is selected from the group consisting of dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, sucrose and trehalose. 6. The method of claim 1, wherein the aqueous solution is water or isotonic saline. 7. The method of claim 1, wherein pre-treating the tissue comprises infusing the tissue with cryoprotectant solution until the desired cells equilibrate thermodynamically with the cryoprotective agent. 8. The method of claim 3, wherein infusing the pancreatic islets with the cryoprotectant solution is performed at a temperature of from about 2.degree. C. to about 35.degree. C. and for a period of about 20 to about 70 minutes. 9. The method of claim 3, wherein infusing the acinar tissue with an aqueous solution is continued until the pancreatic tissue is visibly distended. 10. The method of claim 1, wherein the cryoprotective agent is present in the cryoprotectant solution at a concentration of from about 0.5 molar to about 3.0 molar. 11. The method of claim 2, wherein freezing the pancreatic tissue comprises freezing the pancreas to a temperature of from about -10.degree. C. to about -200.degree. C. 12. The method of claim 2, wherein freezing the pancreatic tissue occurs at a cooling rate of from about 1.degree. C./min. to about 20.degree. C./min. 13. The method of claim 2, wherein warming the pancreatic tissue occurs at a warming rate of more than about 12.degree. C./min. 14. The method of claim 2, wherein warming the pancreatic tissue is achieved by direct immersion of the tissue in a warm osmotically-buffered medium. 15. The method of claim 2, wherein disrupting the pancreatic tissue is achieved by a technique selected from the group consisting of mechanical stress, thermo-mechanical stress induced by differential expansion, thermo-mechanical stress induced by steep temperature gradients, and thermo-mechanical stress induced by volume change upon freezing, and combinations thereof. 16. The method of claim 2, wherein disrupting the pancreatic tissue is achieved while the pancreatic tissue is frozen or while the pancreatic tissue is thawing. 17. The method of claim 3, further comprising eluting the cryoprotective agent from the pancreatic islets and applying osmotic buffering to the pancreatic tissue during the elution of the cryoprotective agent from the pancreatic islets. 18. The method of claim 2, further comprising adding a collagenase to the pancreatic tissue at a concentration of less than 1.4 mg/ml. 19. A method of isolating pancreatic islet tissue that retains sufficient functional integrity to be useful as a transplantation resource, the method comprising: surgically preparing an ex vivo pancreas for vascular and ductal cannulation; cooling the pancreas to from about 2.degree. C. to about 35.degree. C.; equilibrating the islet tissue with a cryoprotective agent; infusing the pancreas with distilled water or saline by ductal flush to promote extensive ice formation upon freezing; freezing the pancreas to a temperature from about -10.degree. C. to about -200.degree. C.; mechanically disrupting the pancreas while keeping the pancreas frozen; thawing the pancreas by immersion in medium to dilute out the cryoprotective agent; filtering the pancreas; washing the pancreas with a medium containing a protease inhibitor and a deoxyribonuclease; and gradient purifying or culturing the islet tissue.

Details for Patent 8,252,589

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2028-12-12