Claims for Patent: 5,079,160

Summary for Patent: 5,079,160

Title:	Method to isolate clusters of cell subtypes from organs
Abstract:	A method to obtain purified, well-defined cell populations from intact organs uses digestion of the distended organ with suitable proteolytic enzymes and harvest of the cell subpopulation by screening the effluent from treatment of the organ with physiologically compatible medium by a filtration screen permitting the passage of the desired cells or clusters of cells, but preventing the passage of larger particles. In this manner, physical/mechanical disruption of the cell population is unnecessary, and the cells or clusters are eased out of their structural matrix and harvested directly. Application of this method to the preparation of purified Islets of Langerhans from intact pancreas is described.
Inventor(s):	Lacy; Paul E. (St. Louis, MO), Scharp; David W. (St. Louis, MO), Ricordi; Camillo (20121 Milano, IT)
Assignee:
Application Number:	07/059,125
Patent Claims:	1. A method to obtain a designated subpopulation of cells from an intact vertebrate organ, which method comprises, with no more than gentle agitation, the following steps: (a) distending the intact organ with a physiologically compatible medium containing an amount of protease effective to free the cells contained in said subpopulation; (b) continuously recirculating physiologically compatible medium through and past the distended organ and past a segregation means, which segregation means permits the passage of the cells or clusters thereof of said subpopulation, but not the organ, for a time sufficient to dislodge the cells and cause them to exit the segregation means; (c) after the cells commence passage through the segregation means, circulating fresh physiologically compatible medium through and past the organ, and through the segregation means into a collecting means; and (d) collecting the subpopulation of cells which pass through the segregation means. 2. The method of claim 1 wherein the organ is the pancreas and the subpopulation of cells is Islets of Langerhans. 3. The method of claim 2 wherein the pancreas is a human pancreas. 4. The method of claim 1 wherein the temperature of the medium in contact with the organ during recirculation in (b) is brought to and maintained at 28-38.degree. C. 5. The method of claim 1 wherein the temperature of the fresh medium in (c) is maintained at 4-15.degree. C. 6. The method of claim 1 wherein the protease is collagenase. 7. The method of claim 1 wherein the organ is distended by cannulation and injection of collagenase-containing medium through the cannula. 8. The method of claim 1 wherein additional protease is added to the organ during the recirculation of (b). 9. The method of claim 1 wherein the segregation means is a filter. 10. The method of claim 9 wherein the pore size of the filter discriminates between the desired cells and larger particles. 11. The method of claim 1 which further includes density separation of the subpopulation of cells obtained in (d). 12. The method of claim 11 wherein the density separation is conducted using Ficoll centrifugation. 13. The method of claim 1 wherein the organ is agitated during the recirculation of (b) and/or the circulating of fresh medium of (c). 14. A method to separate Islets of Langerhans from an intact pancreas which comprises, with no more than gentle agitation, the following steps: (a) inserting a cannula into the pancreatic duct and injecting about 50-300 ml of physiologically compatible medium containing about 0.5-3 mg/ml collagenase through the cannula into the pancreas via the duct so as to distend the pancreas; (b) placing the distended pancreas into a digestion chamber, which chamber contains physiologically compatible medium having additional collagenase, which medium is of volume sufficient to immerse the pancreas, said chamber having an inlet means and outlet means; the passage of medium through said outlet means being regulated by a size discrimination means which permits the passage of islets, but does not permit the passage of substantially larger particles; and the outlet means being connected to a cooled supply/collector flask; (c) continuously recirculating physiologically compatible medium through the chamber with gentle agitation through said inlet and outlet means and through and into said cooled collector, while maintaining the temperature in the chamber at 28-38.degree. C. until islet cells are passed through the outlet means; (d) after said islet cells begin to be passed through the outlet means, harvesting the islets obtained by passing fresh physiologically compatible medium through the digestion chamber and through the outlet means into a collector without recirculation. 15. The method of claim 14 wherein the harvest of the islets is conducted at a temperature of 4-15.degree. C. 16. The method of claim 14 wherein the chamber further contains agitation enhancers. 17. The method of claim 16 wherein the agitation enhancers are marbles. 18. The method of claim 14 wherein the physiologically acceptable medium is Hank's solution containing fetal bovine serum. 19. The method of claim 14 wherein the medium used in the process of (a) has a temperature of 24-37.degree. C. 20. The method of claim 14 wherein the pancreas is a human pancreas.

Details for Patent 5,079,160

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2039-03-29