Patent US5079160A successfully refreshed from Google Patents.
US5079160A
Expired / Lapsed
US
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Method to isolate clusters of cell subtypes from organs
Assignee: Neurotech Pharmaceuticals Inc
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Filing: 1987-06-08
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Granted: 1992-01-07
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Expires: 2007-06-08
Abstract
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.
Inventors
- 👤 Paul E. Lacy
- 👤 David W. Scharp
- 👤 Camillo Ricordi
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 20:08
CPC Classifications
C; C12; C12M; C12M45/00; C12M45/09
Claims Analysis
Total Claims: 22
Claim 1
Independent Claim
144 words
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.
Claim 2
Independent Claim
144 words
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.
Claim 3
Dependent Claim
20 words
The method of claim 1 wherein the organ is the pancreas and the subpopulation of cells is Islets of Langerhans.
Claim 4
Dependent Claim
12 words
The method of claim 2 wherein the pancreas is a human pancreas.
Claim 5
Dependent Claim
28 words
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° C.
Claim 6
Dependent Claim
19 words
The method of claim 1 wherein the temperature of the fresh medium in (c) is maintained at 4-15° C.
Claim 7
Dependent Claim
10 words
The method of claim 1 wherein the protease is collagenase.
Claim 8
Dependent Claim
20 words
The method of claim 1 wherein the organ is distended by cannulation and injection of collagenase-containing medium through the cannula.
Claim 9
Dependent Claim
18 words
The method of claim 1 wherein additional protease is added to the organ during the recirculation of (b).
Claim 10
Dependent Claim
12 words
The method of claim 1 wherein the segregation means is a filter.
Claim 11
Dependent Claim
20 words
The method of claim 9 wherein the pore size of the filter discriminates between the desired cells and larger particles.
Claim 12
Dependent Claim
18 words
The method of claim 1 which further includes density separation of the subpopulation of cells obtained in (d).
Claim 13
Dependent Claim
14 words
The method of claim 11 wherein the density separation is conducted using Ficoll centrifugation.
Claim 14
Dependent Claim
23 words
The method of claim 1 wherein the organ is agitated during the recirculation of (b) and/or the circulating of fresh medium of (c).
Claim 15
Independent Claim
219 words
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° 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.
Claim 16
Independent Claim
219 words
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° 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.
Claim 17
Dependent Claim
19 words
The method of claim 14 wherein the harvest of the islets is conducted at a temperature of 4-15° C.
Claim 18
Dependent Claim
12 words
The method of claim 14 wherein the chamber further contains agitation enhancers.
Claim 19
Dependent Claim
11 words
The method of claim 16 wherein the agitation enhancers are marbles.
Claim 20
Dependent Claim
17 words
The method of claim 14 wherein the physiologically acceptable medium is Hank's solution containing fetal bovine serum.
Claim 21
Dependent Claim
20 words
The method of claim 14 wherein the medium used in the process of (a) has a temperature of 24-37° C.
Claim 22
Dependent Claim
12 words
The method of claim 14 wherein the pancreas is a human pancreas.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1995-01-06 | 1995-07-08 | 1996-01-07 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1999-01-06 | 1999-07-08 | 2000-01-07 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2003-01-06 | 2003-07-08 | 2004-01-07 | $7700.00 | Window Closed / Lapsed |