Bioreactor system and method of enhancing functionality of muscle cultured in vitro

Abstract

A method of producing organized skeletal muscle tissue from precursor muscle cells in vitro comprises: (a) providing precursor muscle cells on a support in a tissue media; then (b) cyclically stretching and relaxing the support at least twice along a first axis during a first time period; and then (c) optionally but preferably maintaining the support in a substantially static position during a second time period; and then (d) repeating steps (b) and (c) for a number of times sufficient to enhance the functionality of the tissue formed on the support and/or produce organized skeletal muscle tissue on the solid support from the precursor muscle cells.

1 . A method of culturing organized skeletal muscle tissue from precursor muscle cells, comprising:
 (a) providing precursor muscle cells on a support in a tissue media; then   (b) cyclically stretching and relaxing said support at least twice along a first axis during a first time period; and then   (c) maintaining said support in a substantially static position during a second time period; and then   (d) repeating steps (b) and (c) for a number of times sufficient to enhance the functionality of the muscle tissue or produce organized skeletal muscle tissue on said solid support from said precursor muscle cells.   
     
     
         2 . The method of  claim 1 , wherein said cyclically stretching and relaxing is carried out at least three times during said first time period. 
     
     
         3 . The method of  claim 1 , wherein said stretching comprises extending said support to a dimension at least 5% greater in length than said static position. 
     
     
         4 . The method of  claim 1 , wherein said relaxing comprises retracting said support to a dimension not greater in length than said static position. 
     
     
         5 . The method of  claim 1 , wherein said relaxing comprises retracting said support to a dimension at least 5% lesser in length than said static position. 
     
     
         6 . The method of  claim 1 , wherein said first time period is from 2 to 30 minutes in duration. 
     
     
         7 . The method of  claim 1 , wherein said second time period is from 10 to 100 minutes in duration. 
     
     
         8 . The method of  claim 1 , wherein said repeating of steps (b) and (c) is carried out for a time of five days to three weeks. 
     
     
         9 . Cultured muscle tissue produced by the process of  claim 1 . 
     
     
         10 . The cultured muscle tissue of  claim 9 , wherein said tissue is characterized by a contractile response to KCl-induced depolarization in vitro. 
     
     
         11 . The cultured muscle tissue of  claim 10 , wherein said tissue is further characterized by:
 cells that exhibit unidirectional orientation on histological examination;   the presence of multinucleated myofibril cells;   cells that express muscle markers as confirmed by immunohistochemistry; and   extracellular matrices as confirmed by Mason's Trichrome.   
     
     
         12 .- 22 . (canceled) 
     
     
         23 . The cultured muscle tissue of  claim 9 , wherein said tissue is suturable and is 1 to 50 cm in length. 
     
     
         24 . The cultured muscle tissue of  claim 9 , wherein said tissue contracts in response to electrical stimulation four weeks after of in vivo implantation. 
     
     
         25 . A method of reconstructing a muscle in a subject in need thereof, comprising implanting muscle tissue of  claim 9  in said subject in an orientation effective to reconstruct said muscle. 
     
     
         26 . A method of building soft tissue in a subject in need thereof, comprising implanting muscle tissue of  claim 9  in said subject in an orientation effective to build soft tissue.