| BackgroundThe main pattern of tissue engineering in vitro was co-culturing seed cells with porous scaffolds (synthetic or natural), combined with specific culture conditions that induce differentiation and proliferation of the seed cells to specific directions. Thess protocals can meet the needs of directional tissue construction. Adipose-derived stem cells (ADSCs), which possess characteristics of convenient for harvesting, high proliferation activity, multiple differentiation property and stable phenotypic expression features, have increasingly respected as an perfect candidate of seed cells in tissue engineering.At present, more and more researches realized that internal special factors might have greatly influence on proliferation and differentiation of seed cells. Due to the opacity of commonly used materials, the observation of cell features is usually destructive to the cell microenvironment. If we can observe and detect the cells in a relatively stable environment without interference, it will provide greatly help for explaining the mechanisms of spatial effects on seed cells.With the development of micro-scale technologies, the designing and manufacture of micro-scale or even nano-scale structures have become possible. Among all these innovative technologies, micro-well arrays got more and more attentions for its advantages, including easy for processing, high biological affinity, versatility and low equipment requirements. We can observe cells almost without interference on this platform, which also provides opportunities for detecting the proliferation and differentiation of seed cells under versital physical and spatial influences.ObjectiveIn this study, a newly experimental platform was designed by manufacturing polydimethylsiloxane (PDMS) micro-well arrays, which produced by micro-scale technologies. Combined with different implanting density of hADSCs, this platform can make different three dimension stacking forms possible in these wells. For it can easily observing the internal behaviors of cell within micro-well arrays, the stack conditions might greatly affect the differentiation and proliferation of seed cells. For one, we can possibly test the safety and quality of this platform. For another, we can also explore the potential links between local stack condition and the bio-behaviors of seed cells under different pile-up conditions.Methods(1) The construction of universal PDMS micro-well arrays reactors. With the help of high precise photolithography technique, we can design and manufacture a high-precision PDMS micro-well arrays platform consist of thousands of wells with 50μm depth and a series of diameter of 60μm,80μm, 100μm and 150μm. The micro-well arrays consisted the floor of micro-reactor, and PDMS wells formed the out wall. The precision of these wells and the sealing proterty of reactors were tested in this part of experiment.(2) The qualification testing of proliferation and differentiation of hADSCs in vitro. The proliferation and differentiation characteristics of finished-product hADSCs(Cyagen(?)) were tested during this part of experiment. The proliferation curve, multiplication timing and the pecentage of live cells in the 1st,3rd and 6th generation groups of hADSCs were considered as assessing targets. The ability of differentiation of the 3rd generation of hADSCs was also assessed by specific dying (Oilred O for adipogenesis/alizarin red for osteogenesis) and markers (PPAR-γ, β-Actin and C/EBPα for adiposensis/OPN, OCN and AKP for osteogenesis) through RT-PCR testing.(3) Identifing the impacts of special stacking condition among proliferation and apoptosis of hADSCs by using PDMS micro-well arrays reactors. Three kinds of hADSCs seeding density(3×104/ml,6×104/ml and 1×105/ml) were designiated within the micro-well arrays reactors(60μm,80μm, 100μm and 150μm). The optical microscope, scanning electronic microscope and laser scanning confocal microscope were used to analyze the seeding condition, numbers, and apoptosis rate of hADSCs in these reactors.(4) Identifing the impacts of special stacking condition among adipogenesis and osteogenesis of hADSCs by using PDMS micro-well arrays reactors. Three diameters of micro-well arrays(80μm, 100μm and 150μm) were utilized for hADSCs seeding combined with a seeding density of 6×104/ml. The ability of differentiation in the 3rd generation of hADSCs was assessed by specific dying (Oilred O for adipogenesis/ alizarin red for osteogenesis) and RNA markers (PPAR-γ,β-Actin and C/EBPα for adiposensis/OPN, OCN and AKP for osteogenesis) through RT-PCR testing.Results(1) Universal PDMS micro-well arrays reactor with highly precision was successfully built in this experiment. The error rates of each kind of wells were lower than 0.2%. The outer walls of reactor were firmly stick to the floor and no leakage can be observed.(2) The finished-product hADSCs could be verified as competent resources of seed cells, which reflected by its energetic proliferation and stable condition of surface markers. It could successfully differentiate into adipose cells and osteon cells, which tested by specific dying and RNA markers.(3) The rate of proliferation and apoptosis had no significant difference between culturing on PDMS surface and Petri dish surface, which guaranteed the safety of this platform. A controllable stacking culture platform could successfully built by combining three diameters of micro-well arrays(80μm, 100μm and 150μm) and a planting density of 6×104/ml of cells. The cell numbers were stable at the first week after planting. The percentage of cell apoptosis was linked to the layers of stacking conditon.(4) Different stacking condition leads to different level of of adipogenesis and osteogenesis. Single layer planting of hADSCs tended to differentiate into adipose cells. Double layers planting tended to differentiate into osteon cells. The level of specific marker of differentiation in PDMS micro-well arrays was raised behind those cells which cultured on plat PDMS surface.ConclusionsThe universal micro-well arrays reactors were successfully built by consisting micro-well arrays as floor and PDMS shelt as walls. The error rates of each well were extremely low and the structure of reactor were reliable for cell culture.Finished-product hADSCs behaved booming activity of proliferation and stable surface markers, which guaranteed the persistence of cell culture on different platform. The ability of adipogenesis and osteogenesis was accorded with the characteristic of mesenchymal stem cells.The stable cells growth rate in PDMS micro-well arrays reactors could guarantee a experimental platform which acquired controllable stacking condition by coordinating the density of seeding cells and the diameter of micro-wells. The results of this experiment affirmed that the special factor could be considered as a essential factor guiding the proliferation, apoptosis and differentiation of hADSCs. |
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