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Background: Liver stem cells are multipotent differentiation potential of cells, not only from the original foregut endoderm and inner cell mass of blastocysts obtained, but also from fetal, neonatal and adult liver obtained. Can be formed in vitro cell clones expressing EpCAM, NCAM, ALB (albumin) / -, CK (cytokeratins) 8,18, 19, did not express AFP (α-fetoprotein). In certain conditions, can differentiate into liver cells, hepatocytes and bile duct cells. Because of the unique properties of stem cells, liver stem cells are considered cell therapy and bio-artificial liver ideal seed cells. If a large number of in vitro amplification to obtain a sufficient number of cells, then the liver stem cells will have broad clinical application. Currently, most of liver stem cell culture plate culture using traditional methods, even if cell adhesion at the bottom of petri dishes or flasks, in a static environment for fluids regularly passaged. However, this can lead to cell culture method some key phenotypes and biological functions vanish, the biological characteristics of the cells changed, obviously does not meet the requirements of hepatic stem cell expansion. Newly emerging three-dimensional culture method is considered better mimic in vivo cellular environment, gene expression and cell biology closer to the body changes; wherein the reactor is simulated microgravity Rotary bioreactor (rotating cell culture system, RCCS) because you can create a relatively weightless, low shear, low turbulent three-dimensional culture environment used cell research in recent years become a new means. Some scholars liver cells were placed in simulated microgravity reactor porcine hepatocytes cultured for 21 days showed maintained cell morphology and functional activity; mouse fetal liver cells in simulated microgravity reactor can form a similar structure of the liver tissue samples, which includes mature hepatocytes, blood samples and bile duct-like structures. Some academics have applied specific biological stent or three-dimensional perfusion culture systems cultured hepatic stem cells, found to induce liver stem cells to mature functional liver cells. The new cell culture applications, for our understanding of the growth characteristics of liver stem cells to create the conditions for the establishment and optimization of our liver stem cell expansion technology has brought new hope. Objective: This study aimed to previous hepatic stem cell research, based on the use of rotary bioreactor can create weightlessness characteristics simulated microgravity microcarrier-dimensional culture liver stem cells, liver stem cells flat culture and microcarrier culture as control, investigate microgravity environment on liver stem cell growth and proliferation and differentiation for future expansion of liver stem cells provide the basis for the system. Methods: from human fetal liver tissue were isolated and cultured liver stem cells for liver stem cells were identified; respectively liver stem cells conventional planar culture, microcarrier culture, simulated microgravity microcarrier three-dimensional culture, the use of phase contrast microscopy, scanning electron microscopy of liver stem cell morphological changes; culture supernatant by glucose consumption and lactate dehydrogenase levels of detection, compare three methods of cell culture and cell proliferation activity, etc., three-dimensional analysis of simulated microgravity environment on the growth and proliferation of hepatic stem cells effects; fluorescence quantitative PCR under different culture conditions liver stem cells ALB, EpCAM, CK19, AFP, HNF6, CYP3A4, CYP3A7mRNA expression, and to explore three-dimensional culture under simulated microgravity on liver stem cell differentiation. Results: 1. Microcarrier culture conditions liver stem cell proliferation was multidirectional direction to wrap the form of micro-carrier complex formation growth. Simulated microgravity conditions of three-dimensional culture liver stem cells to microcarriers sphere as the base, multi-directional growth and proliferation in a three-dimensional structure can be observed under the inverted microscope sizes closely connected with microcarrier cell mass, scanning electron microscope cell mass surface is rich in extracellular matrix and microvilli, form the largest diameter of 100um cell mass or more. 2 supernatant glucose consumption and LDH test results showed: under conditions of simulated microgravity liver stem cells from the first nine days beginning glucose consumption increased significantly, to 17 days reached a peak 21 days decreased significantly; while LDH levels until day 17 remains low, but the first 21 days are significantly increased, and the plane microcarrier culture culture and compared to the results, a significant difference; prompts liver stem cells in simulated microgravity environment of a longer period of cell growth starting to enter the logarithmic growth Subsequent cell growth and proliferation capacity is far stronger than the flat culture and microcarrier culture liver stem cells. When the three-dimensional growth of a certain volume of cell mass, due to the exchange of nutrients within the cell mass disorders, further affecting cell proliferation. 3 fluorescence quantitative RT-PCR test results: After 21 days training, simulated microgravity three-dimensional cell culture expression of EpCAM, ALB, CK19, HNF6, CYP3A7, did not express AFP, CYP3A4; microcarrier culture results were compared with analog micro- gravity environment liver stem cells EpCAM, ALB, CK19, HNF6, CYP3A7 expression microcarrier culture were 29 times, 69 times, 27 times, nine times, and 38 times, both compared to P values ??were less than 0.05, a statistically Learn the difference. Tip simulated microgravity environment of three-dimensional culture did not happen hepatic stem cell differentiation; compared with microcarrier culture, simulated microgravity environment more conducive to liver stem cells maintain cell phenotype, which can maintain good stem cell properties. Conclusions: 1. Simulated microgravity environment can promote the proliferation of hepatic stem cells was more than three-dimensional structure to grow; 2 with train and plane microcarrier culture compared to liver stem cells in simulated microgravity environment can get a stronger ability to cell growth and proliferation and cell activity; When the liver stem cell growth after the formation of larger cell clusters, nutrient exchange impairment can affect cell proliferation; 3. simulated microgravity environment more conducive to liver stem cells maintain cell phenotype, thereby maintaining the stem cell properties. 4 simulated microgravity three-dimensional culture technique is superior to microcarrier culture and flat culture, can be used for liver stem cell expansion, especially for short-term expansion of liver stem cells.
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