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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.

An appropriate ultrasonication of collecting foreign recombinant protein (human-adiponectin) from yeast was established. Regarded the bioactivity of foreign recombinant protein tested by Western Blotting analysis as parameter, a L9(33) orthogonal array design based on a series of single-factor experiments was employed to optimize conditions for ultrasonic disruption. The results showed that the ultrasonic power of 450 W, duration time on 25 min and operation interval (work time: intermittent time) of 10:10 (s/s) were optimal process with the highest protein bioactivity, and meanwhile, the cell breaking rate was (67.8±2.1) %.

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