title: Large-scale expansion of human skin-derived precursor cells (hSKPs) in stirred suspension bioreactors.
abstract: Human skin-derived precursor cells (hSKPs) are multipotent adult stem cells found in the dermis of human skin. Incorporation of hSKPs into split-thickness skin grafts (STSGs), the current gold standard to treat severe burns or tissue resections, has been proposed as a treatment option to enhance skin wound healing and tissue function. For this approach to be clinically viable substantial quantities of hSKPs are required, which is the rate-limiting step, as only a few thousand hSKPs can be isolated from an autologous skin biopsy without causing donor site morbidity. In order to produce sufficient quantities of clinically viable cells, we have developed a bioprocess capable of expanding hSKPs as aggregates in stirred suspension bioreactors (SSBs). In this study, we found hSKPs from adult donors to expand significantly more (P < 0.05) at 60 rpm in SSBs than in static cultures. Furthermore, the utility of the SSBs, at 60 rpm is demonstrated by serial passaging of hSKPs from a small starting population, which can be isolated from an autologous skin biopsy without causing donor site morbidity. At 60 rpm, aggregates were markedly smaller and did not experience oxygen diffusional limitations, as seen in hSKPs cultured at 40 rpm. While hSKPs also grew at 80 rpm (0.74 Pa) and 100 rpm (1 Pa), they produced smaller aggregates due to high shear stress. The pH of the media in all the SSBs was closer to biological conditions and significantly different (P < 0.05) from static cultures, which recorded acidic pH conditions. The nutrient concentrations of the media in all the SSBs and static cultures did not drop below acceptable limits. Furthermore, there was no significant build-up of waste products to limit hSKP expansion in the SSBs. In addition, hSKP markers were maintained in the 60 rpm SSB as demonstrated by immunocytochemistry. This method of growing hSKPs in a batch culture at 60 rpm in a SSB represents an important first step in developing an automated bioprocess to produce substantial numbers of clinically viable hSKPs aimed at regenerating the dermis to improve healing of severe skin wounds. Biotechnol. Bioeng. 2016;113: 2725-2738. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Pharmaceutical Production Research Facility (PPRF), Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada.|Department of Chemical and Petroleum Engineering, Schulich School of Engineering, University of Calgary, 2500 University Drive NW, Calgary, Alberta, T2N 1N4, Canada.
authors.forename: Denver C
authors.initials: DC
authors.lastname: Surrao
authors.affiliation: Pharmaceutical Production Research Facility (PPRF), Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada.|Biomedical Engineering Graduate Program, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Boon
authors.affiliation: Pharmaceutical Production Research Facility (PPRF), Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada.|Department of Chemical and Petroleum Engineering, Schulich School of Engineering, University of Calgary, 2500 University Drive NW, Calgary, Alberta, T2N 1N4, Canada.
authors.forename: Breanna
authors.initials: B
authors.lastname: Borys
authors.affiliation: Faculty of Veterinary Medicine, Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Sarthak
authors.initials: S
authors.lastname: Sinha
authors.affiliation: Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Ranjan
authors.initials: R
authors.lastname: Kumar
authors.affiliation: Faculty of Veterinary Medicine, Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, Alberta, Canada.|Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.|Department of Surgery, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.|Alberta Children's Hospital Research Institute, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Jeff
authors.initials: J
authors.lastname: Biernaskie
authors.affiliation: Pharmaceutical Production Research Facility (PPRF), Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada. mskallos@ucalgary.ca.|Department of Chemical and Petroleum Engineering, Schulich School of Engineering, University of Calgary, 2500 University Drive NW, Calgary, Alberta, T2N 1N4, Canada. mskallos@ucalgary.ca.|Biomedical Engineering Graduate Program, University of Calgary, Calgary, Alberta, Canada. mskallos@ucalgary.ca.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Kallos
mesh_terms: D000328:Adult; D053687:Adult Stem Cells / Q000166:cytology* / Q000502:physiology; D000368:Aged; D061249:Batch Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D019149:Bioreactors; D049109:Cell Proliferation / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005260:Female; D006801:Humans; D008297:Male; D039902:Multipotent Stem Cells / Q000166:cytology* / Q000502:physiology; D012867:Skin / Q000166:cytology*
keywords: bioprocess engineering; human SKPs (hSKPs); stirred suspension bioreactors (SSBs)