title: Epidermal stem cells are preserved during commercial-scale manufacture of a bilayered, living cellular construct (Apligraf®).
abstract: 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.
authors.affiliation: Department of Oral and Maxillofacial Pathology, Tufts University, Boston, Massachusetts, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Carlson
authors.forename: Katie
authors.initials: K
authors.lastname: Faria
authors.forename: Yulia
authors.initials: Y
authors.lastname: Shamis
authors.forename: Jonathan
authors.initials: J
authors.lastname: Leman
authors.forename: Vincent
authors.initials: V
authors.lastname: Ronfard
authors.forename: Jonathan
authors.initials: J
authors.lastname: Garlick
mesh_terms: 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
chemical_list: C117898:Apligraf; D003094:Collagen