METHOD FOR EFFICIENT TRANSFER OF HUMAN BLASTOCYST-DERIVED STEM CELLS (hBS CELLS) FROM A FEEDER-SUPPORTED TO A FEEDER-FREE CULTURE SYSTEM

Abstract

Technology is provided for the transfer of human blastocyst-derived stem cells (hBS cells) to a feeder-free culture system and propagation of the cells in such a feeder-free culture system, the method comprising the following steps of (a) transferring the blastocyst-derived stem cells from feeder to feeder free culture by mechanical treatment, (b) culturing the blastocyst-derived stem cells under feeder cell free growth conditions in a suitable growth medium and/or on a suitable support substrate, and (c) optionally passaging the blastocyst derived stem cell line every 3-10 days by enzymatic and/or mechanical treatment. The application of hBS cells cultured under a feeder-free condition in medicine (e.g., myocardial regeneration) and screening and toxicity testing also is provided.

1 - 39 . (canceled) 
     
     
         40 . A medicament for transplantation of hBS cells into a mammal for the prevention or treatment of a disease comprising an effective concentration of hBS cells and one or more agents selected from the group consisting of therapeutically active substances, prophylactically active substances, engraftment improving agents, viability improving agents, differentiation improving agents, and immunosuppressive agents, wherein said hBS cells are formed by the method comprising:
 (a) transferring the hBS cells from feeder to feeder-free culture without enzymatic treatment but solely by mechanical treatment; and   (b) following step (a), culturing the hBS cells under feeder-free growth conditions in a suitable growth medium and on a suitable support substrate, wherein said suitable growth medium is a cell-free medium conditioned by previous exposure to feeder cells, and said suitable support substrate comprises a cell-free matrix comprising extracellular matrix proteins.   
     
     
         41 . A method for treating a cardio-related disease in a mammal comprising transplanting hBS cells obtained by the method comprising steps (a) and (b) of  claim 40  into cardio-like cells in an effective amount to the mammal in need thereof. 
     
     
         42 . A kit comprising at least two of the following components in separate compartments, an agent that improves the engraftment and viability of hBS cells, the hBS cells obtainable by the method comprising steps (a) and (b) of  claim 40 , one or more agents that improve differentiation of hBS cell and one or more pharmaceutical and/or immunosuppressive agents. 
     
     
         43 . The kit of  claim 42 , further comprising a second cell-type that improves engraftment and survival of the hBS cells. 
     
     
         44 . The kit of  claim 42 , further comprising undissociated or dissociated differentiated hBS-cell colonies. 
     
     
         45 . A method for the production of monoclonal antibodies comprising utilizing the hBS cells obtained by the method comprising steps (a) and (b) of  claim 40 . 
     
     
         46 . A method for in vitro toxicity testing comprising utilizing the hBS cells obtained by the method comprising steps (a) and (b) of  claim 40 . 
     
     
         47 . A method for in vitro screening of potential drug substances comprising utilizing the hBS cells obtained by the method comprising steps (a) and (b) of  claim 40 . 
     
     
         48 . A method for identification of potential drug substances comprising utilizing the hBS cells obtained by the method comprising steps (a) and (b) of  claim 40 .