Enhanced efficiency in isolation and expansion of hAMSCs via dual enzyme digestion and micro-carrier

A two-stage method of obtaining viable human amniotic stem cells (hAMSCs) in large-scale is described. First, human amniotic stem cells are isolated via dual enzyme (collagenase II and DNAase I) digestion. Next, relying on a culture of the cells from porous chitosan-based microspheres in vitro, high purity hAMSCs are obtained in large-scale. Dual enzymatic (collagenase II and DNase I) digestion provides a primary cell culture and first subculture with a lower contamination rate, higher purity and a larger number of isolated cells. The obtained hAMSCs were seeded onto chitosan microspheres (CM), gelatin-chitosan microspheres (GCM) and collagen-chitosan microspheres (CCM) to produce large numbers of hAMSCs for clinical trials. Growth activity measurement and differentiation essays of hAMSCs were realized. Within 2 weeks of culturing, GCMs achieved over 1.28 ± 0.06 × 10⁷ hAMSCs whereas CCMs and CMs achieved 7.86 ± 0.11 × 10⁶ and 1.98 ± 0.86 × 10⁶ respectively within this time. In conclusion, hAMSCs showed excellent attachment and viability on GCM-chitosan microspheres, matching the hAMSCs' normal culture medium. Therefore, dual enzyme (collagenase II and DNAase I) digestion may be a more useful isolation process and culture of hAMSCs on porous GCM in vitro as an ideal environment for the large-scale expansion of highly functional hAMSCs for eventual use in stem cell-based therapy.