Epidermal stem cells are preserved during commercial-scale manufacture of a bilayered, living cellular construct (Apligraf®).
It is unknown if epidermal stem cells are maintained during the commercial-scale manufacture of Apligraf, a bilayered living cellular construct (BLCC). To answer this question, we genetically marked replicating keratinocytes, derived from production-scale expansion of working cell banks, in two-dimensional culture with a beta-galactosidase-expressing retrovirus and monitored their fate after incorporation into BLCC and subsequent in vivo transplantation to a nude mouse. Histological analysis of BLCCs showed distinct beta-galactosidase-positive clusters similar to clonal proliferation units visible 8-32 weeks after grafting. Keratinocytes recovered from grafts at week 32 were expanded in vitro in two-dimensional culture, and clonal growth of recovered cells was then compared to the original pregraft population of keratinocytes by colony-forming efficiency (CFE) assays. The CFE of the cells regrown from the grafts was similar to pregraft CFEs (45% and 40%, respectively). Cells regrown from the grafts were then used to produce a second BLCC and generated a well-differentiated epithelium that was histologically similar to pregraft BLCC. These findings provide clear evidence that epidermal stem cells were sustained during the process of large-scale tissue fabrication and that the process of isolation and expansion of cells in BLCC construction retains viable stem cells.
Carlson
Mark
M
Department of Oral and Maxillofacial Pathology, Tufts University, Boston, Massachusetts, USA.
Faria
Katie
K
Shamis
Yulia
Y
Leman
Jonathan
J
Ronfard
Vincent
V
Garlick
Jonathan
J
D000818:Animals; D002470:Cell Survival; D003094:Collagen / Q000737:chemistry*; D000078404:Epidermal Cells; D004817:Epidermis / Q000502:physiology; D006801:Humans; D008422:Materials Testing; D051379:Mice; D008819:Mice, Nude; D017768:Skin, Artificial; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology
C117898:Apligraf; D003094:Collagen