Encapsulation of pancreatic cells derived from human pluripotent stem cells

Abstract

The present invention relates to methods for encapsulating pancreatic progenitors in a biocompatible semi-permeable encapsulating device. The present invention also relates to production of human insulin in a mammal in response to glucose stimulation.

1 - 11 . (canceled) 
     
     
         12 . A method for cryopreserving an in vitro cell population comprising:
 a. obtaining cells to be cryopreserved;   b. incubating the cells to be cryopreserved in a freezing medium comprising dimethyl sulfoxide solution (DMSO) for longer than 5 minutes; and   c. decreasing the temperature of the cells to be cryopreserved to less than 0° C. following incubation in DMSO.   
     
     
         13 . The method of  claim 12  wherein the cells to be cryopreserved are human pancreatic progenitor cells. 
     
     
         14 . The method of  claim 12  wherein the cells to be cryopreserved are human pancreatic progenitor cell aggregates. 
     
     
         15 . The method of  claim 12  wherein the freezing medium further comprises compounds selected from the group comprising Dulbecco's Modified Eagle's Medium (DMEM), Xeno-free Knockout Serum Replacement, (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid) (HEPES) and combinations thereof. 
     
     
         16 . The method of  claim 14  wherein the freezing medium further comprises DMEM with 30% Xeno-free Knockout Serum Replacement, 25 mM HEPES and 10% DMSO solution. 
     
     
         17 . The method of  claim 12  wherein the cells to be cryopreserved are incubated in the freezing media for about 60 minutes. 
     
     
         18 . The method of  claim 12  wherein the cells to be cryopreserved are incubated in the freezing media for about 15 minutes at ambient temperature and then 45 minutes at 4° C. 
     
     
         19 . The method of  claim 12 , wherein the cells to be cryopreserved are loaded into an implantable semi-permeable device prior to step b. 
     
     
         20 . The method of  claim 12  wherein the temperature of the cells to be cryopreserved in step c is decreased to at least −20° C. 
     
     
         21 . The method of  claim 12  wherein the temperature of the cells to be cryopreserved in step c is decreased to at least −90° C. 
     
     
         22 . The method of  claim 12  wherein the temperature of the cells to be cryopreserved in step c is decreased to at least −130° C. 
     
     
         23 . The method of  claim 12  wherein the temperature of the cells to be cryopreserved in step c is decreased to at least −130° C. at a slow rate. 
     
     
         24 . The method of  claim 23 , wherein the decreasing the temperature of the cells to be cryopreserved in step c comprises decreasing the temperature from 0° C. to −9° C. at a rate of 2° C. per minute. 
     
     
         25 . The method of  claim 24 , further comprising manually seeding the cells to be cryopreserved at −9° C. 
     
     
         26 . The method of  claim 25 , further comprising holding the cells to be cryopreserved at −9° C. for about 10 minutes. 
     
     
         27 . The method of  claim 26 , further comprising decreasing the temperature of cells to be cryopreserved to −40° C. at a rate of 0.2° C. per minute following manually seeding and holding at −9° C. 
     
     
         28 . The method of  claim 27 , further comprises decreasing the temperature of the cells to be cryopreserved to about −150° C. at a rate of 25° C. per minute. 
     
     
         29 . The method of  claim 28 , further comprising storing the cells to be cryopreserved in the vapor phase of a liquid nitrogen storage freezer. 
     
     
         30 . A population of cryopreserved pancreatic progenitors which were incubated in DMSO for longer than 5 minutes prior to freezing. 
     
     
         31 . A method for producing insulin in vivo in a mammal, said method comprising:
 a. providing an in vitro human pancreatic and duodenal homeobox gene 1 (PDX1)-positive pancreatic progenitor cell population into an implantable semi-permeable device;   b. implanting the device containing the progenitor cell population into a mammalian host; and   c. maturing the progenitor cell population in said device in vivo such that there is no cell-to-cell contact between the progenitor cell within the device and the host cell in the mammal and the resulting cell population comprises endocrine and acinar cells, wherein at least some of the endocrine cells are insulin secreting cells that produce insulin in response to glucose stimulation in vivo, thereby producing insulin in vivo to the mammal.