Automated method and apparatus for embryonic stem cell culture

Abstract

The invention concerns methods for automated culture of embryonic stem cells (ESCs) such as human ESCs. In some aspects, methods of the invention employ optimized culture media and limited proteolytic treatment of cells to separate cell clusters for expansion. Automated systems for passage and expansion of ESCs are also provided.

1 - 39 . (canceled) 
     
     
         40 . An apparatus for automated maintenance and expansion of pluripotent cells comprising:
 a) an incubator;   b) a liquid handler unit;   c) one or more reservoirs, said reservoirs comprising i) a viable population of pluripotent cells, ii) a defined media, the defined media being one in which pluripotent cells can be cultured and maintained in an undifferentiated state, and iii) one or more of a protease, protease inhibitor and a Rho-associated kinase (ROCK) inhibitor; and   d) a controller in communication with the liquid handler unit, wherein the controller is configured to direct the liquid handler unit to effect the automated expansion and maintenance of the pluripotent cells.   
     
     
         41 . The apparatus of  claim 40 , wherein the controller comprises a computer-readable medium including an operating program. 
     
     
         42 . The apparatus of  claim 40 , wherein the controller directs at least one of: the liquid handler unit, the one or more reservoirs, and the incubator to i) remove media from a pluripotent cell population; ii) contact pluripotent cells with a proteolytic enzyme; and iii) incubate pluripotent cells with a proteolytic enzyme to separate cell clusters. 
     
     
         43 . The apparatus of  claim 40 , wherein the apparatus further comprises a mechanical agitator and aspirator, and the controller further directs at least one of: the liquid handler unit, the one or more reservoirs, and the incubator to subject the pluripotent cells to mechanical agitation or aspiration to further separate cell clusters. 
     
     
         44 . The apparatus of  claim 40 , wherein the controller further directs at least one of: the liquid handler unit, the one or more reservoirs, and the incubator to contact incubated pluripotent cells with a protease inhibitor. 
     
     
         45 . The apparatus of  claim 44 , wherein the controller further directs at least one of: the liquid handler unit, the one or more reservoirs, and the incubator to contact incubated pluripotent cells with a Rho-associated kinase (ROCK) inhibitor. 
     
     
         46 . The apparatus of  claim 40 , wherein the liquid handler unit comprises at least a first reservoir and a second reservoir, wherein the first reservoir comprises a TeSR media, and wherein the second reservoir comprises a TeSR media, a Rho-associated kinase (ROCK) inhibitor, and a protease inhibitor. 
     
     
         47 . The apparatus of  claim 40 , wherein the Rho-associated kinase (ROCK) inhibitor is H-1152 or H-100. 
     
     
         48 . The apparatus of  claim 40 , wherein the pluripotent cells are ES cells or induced pluripotent cells (iPS). 
     
     
         49 . The apparatus of  claim 40 , wherein the liquid handler comprises a gripper tool and a liquid handling tool. 
     
     
         50 . The apparatus of  claim 40 , wherein the pluripotent cells are human ES cells. 
     
     
         51 . The apparatus of  claim 40 , further comprising a robotic device configured to facilitate fluid communication between the liquid handler unit and the incubator. 
     
     
         52 . The apparatus of  claim 40 , further comprising a second or more reservoirs in communication with the liquid handler unit. 
     
     
         53 . The apparatus of  claim 52 , wherein the second reservoir comprises cell culture plates, cell growth media, or a proteolytic enzyme solution. 
     
     
         54 . The apparatus of  claim 40 , wherein the liquid handler is in communication with at least a first reservoir and a second reservoir, wherein the first reservoir comprises a TeSR media, and wherein the second reservoir comprises a TeSR media which further comprises a Rho-associated kinase (ROCK) inhibitor and a protease inhibitor. 
     
     
         55 . The apparatus of  claim 40 , wherein the controller is comprised in a computer. 
     
     
         56 . The apparatus of  claim 40 , wherein said incubator comprises a portion of said pluripotent cells, and wherein the apparatus is configured to provide for expansion of the pluripotent cells with no or essentially no further differentiation of the cells in at least 97% of the expanded pluripotent cells. 
     
     
         57 . The apparatus of  claim 40 , wherein the incubator comprises a portion of said pluripotent cells, and wherein the pluripotent cells are human ES cells.