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 whole muscles transplanted in vivo were investigated. Whole male (SJL/J) donor muscles were exposed to various tissue culture reagents and proteolytic enzymes, and allografted into female (SJL/J) host mice. Desmin immunohistochemistry was used to assess the numbers of myogenic cells (as an index of myoblast viability and the extent of regeneration) in tissue sections of whole-muscle grafts sampled on days 7 and 14. DNA quantitation with a Y-chromosome-specific probe was used to determine the total Y-1 sequence DNA (as an index of myoblast survival and proliferation) in whole-muscle grafts sampled on days 1, 3, and 7. In grafts exposed to serum-free medium, there was a delay in myoblast fusion at 7 days that was recovered by 14 days, but exposure to serum (10% or 20%) had a prolonged adverse effect on myotube formation 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 prior to grafting was extremely detrimental to myoblast survival and viability at 7 and 14 days. The unexpected finding of adverse effects of tissue culture conditions on the regeneration of whole-muscle grafts in vivo appears to parallel the major problem of the rapid death of isolated cultured donor myoblasts after injection in myoblast transfer therapy. The use of whole-muscle grafts provides an alternative and sensitive model to analyze the crucial effects of various tissue culture components on the subsequent survival and proliferation of myogenic cells in vivo.
Smythe
G M
GM
Department of Anatomy and Human Biology, The University of Western Australia, Nedlands, Australia. gsmythe@anhb.uwa.edu.au
Grounds
M D
MD
D000818:Animals; D002470:Cell Survival; D017690:Cell Transplantation; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry; D016895:Culture Media, Serum-Free; D046508:Culture Techniques / Q000379:methods*; D004247:DNA / Q000032:analysis; D003893:Desmin / Q000032:analysis / Q000276:immunology; D010450:Endopeptidases / Q000378:metabolism; D005260:Female; D008297:Male; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000502:physiology; D018482:Muscle, Skeletal / Q000033:anatomy & histology / Q000166:cytology / Q000502:physiology* / Q000637:transplantation*; D012038:Regeneration; D013997:Time Factors; D019737:Transplants
D003470:Culture Media; D016895:Culture Media, Serum-Free; D003893:Desmin; D004247:DNA; D010450:Endopeptidases