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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 × 10 7  hAMSCs whereas CCMs and CMs achieved 7.86 ± 0.11 × 10 6  and 1.98 ± 0.86 × 10 6  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.

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