Abstract: Objective To establish a three-dimensional cultivation platform for human adipose derived stem cells (hADSCs) by utilizing micro-scale technology, and to explore the influence of three-dimensional stacking condition to cell proliferation and apoptosis. Methods Micro-columnar structure were generated by micro-scale technology on silicon master with diameter of 60 μm, 80 μm, 100 μm and 150 μm respectively. The same specification micro-well arrays were acquired by reversing mould aperture using polydimethylsiloxane (PDMS), which equipped with PDMS ring on the outer wall to constitute micro-reactor. The error rate of this platform was analyzed. Then the third generation of human adipose-derived stem cells (hADSCs) were selected and mixed into single cell suspension mixture with three kinds of concentration, including 3×104 cells/mL, 6×104 cells/mL and 1×105 cells/mL. And 200μL cell suspension was injected into micro-reactor to establish three-dimensional culture models with different stacked layers. Then 24 hours, 72 hours, 120 hours and 168 hours were choosed as sample collection time point. The morphology description of cell distribution in each group was analyzed with scanning electron microscopy, the arrangement inside the microwells and the cell distribution outside the microwells were described, and the live/dead cells were counted and tested in each aperture and micro hole by using laser scanning confocal microscope. Results The error rate of diameter and pore diameter were no more than 0.2%. When hADSCs were planted by the density of 6 ×104 cells/mL, the stacking layer condition can be acquired. The number of cell within microwells was at static condition no more than 7 days. The 60 μm group had the highest number of out-of-well cell (P<0.05) and lowest 05). The highest apoptosis ratio was also observed in the 60 μm group (P<0.05). Conclusion The application of microscale technique can acquire extremely low error rate, which can satisfy the requirements of high precision design. The static proliferation condition of planted hADSCs can be maintained within microwells, which can stabilize the condition of cells and be designed as a perfect three dimention model for cell research at early stage of plant. The stackable condition of hADSCs can be acquired by planting into microwells with different diameters, which can significant affect the rate of apoptosis under this three dimentional cultivate condition. Thinner layer of stack may reduce the rate of apoptosis.

Key words: Micro-well arrays, Micro-scale technology, Polydimethylsiloxane, Human adipose tissue derived stem cells