Exposure to tissue culture conditions can adversely affect myoblast behavior in vivo in whole muscle grafts: Implications for myoblast transfer therapy

The effects of tissue culture conditions on the viability of myoblasts in w hole muscles transplanted in vivo were investigated. Whole male (SJL/J) don or muscles were exposed to various tissue culture reagents and proteolytic enzymes, and allografted into female (SJL/J) host mice. Desmin immunohistoc hemistry was used to assess the numbers of myogenic cells (as an index of m yoblast viability and the extent of regeneration) in tissue sections of who le-muscle grafts sampled on days 7 and 14. DNA quantitation with a Y-chromo some-specific probe was used to determine the total Y-1 sequence DNA (as an index of myoblast survival and proliferation) in whole-muscle grafts sampl ed on days 1, 3, and 7. In grafts exposed to serum-foe medium, there was a delay in myoblast fusion at 7 days that was recovered by 14 days, but expos ure to serum (10% or 20%) had a prolonged adverse effect on myotube formati on at 14 days. DNA quantitation demonstrated that either serum-free culture medium or 10% serum enhanced the number of male cells within whole-muscle grafts at 7 days. Proteolytic digestion (even for 5 min) of whole muscles p rior to grafting was extremely detrimental to myoblast survival and viabili ty at 7 and 14 days. The unexpected finding of adverse effects of tissue cu lture conditions on the regeneration of whole-muscle grafts in vivo appears to parallel the major problem of the rapid death of isolated cultured dono r myoblasts after injection in myoblast transfer therapy. The use of whole- muscle grafts provides an alternative and sensitive model to analyze the cr ucial effects of various tissue culture components on the subsequent surviv al and proliferation of myogenic cells in vivo.