Study On Culture Of Human Amniotic Epithelial Cells And Its Differentiation Into Hepatocyte-like Cells

Objective To establish the isolation, culture and cryopreservation process of human amniotic epithelial cells, to study the basic biological characteristics of hAECs, and the differentiation of human amniotic epithelial cells into hepatocyte like cells in vitro.Methods We collect cesarean section newborn amniotic membrane under aseptic conditions, using orthogonal method for different isolation, culture and cryopreservation condition design suitable factors levels, using the corresponding orthogonal table to test. SSEA-4 positive cells amount of the same quality of amniotic membrane for comparison, research the tripsin concentration, digestion time, the number of digestion and other influence factors on the separation effect; SSEA-4 positive cells amount/cell inoculation amount for comparison, research the cell inoculation density, concentration of serum and cell growth factor (EGF) concentration influence factors on the cell cluture effect; survival rate of resuscitation cells for comparison, research the frozen storage density, DMSO concentration and serum concentration of influence factors on the effect of cryopreservation. Put forward the optimization of human amniotic epithelial cells isolation, culture and cryopreservation process. cells cryopreserved experiment use 10% DMSO serum culture medium for freeze protection fluid. Take primary, subculture and cryopreservation resuscitation cell morphological observation, draw cell growth curve, related surface markers of immunofluorescence staining for the primary cells, and the primary and P4 cells for immune flow cytometry. Take P1 hAECs, 5% FBS DMEM/F12 culture medium added 20 ng/ml HGF, 10ng/ml FGF4, lOng/ml IGF-1 and 500nm dexamethasone(DEX) induction of the reagent combination in the induced group; 5% FBS DMEM/F12 medium in the control group. The induction period was 3 weeks. We contrast induced cells and control the change of cell morphology microscopically, changes of cell immunofluorescence staining in induced process, use transcription and fluorescence quantitative PCR to detect the change of liver cell related genes in AFP, ALB, HNF4 alpha and CYP1A2 expressionreverse in the induced process, and take a glycogen staining to the inducted hAECs of 3 weeks.Results We found through the data of the orthogonal method analysis, the optimized conditions of trypsin digestion amniotic epithelial cells is 0.25% trypsin concentration,4 times digestion,20 minutes digestion time each time, the effect of separation factors magnitude: digestion times>digestion time>trypsin concentration. The optimized culture conditions: 4*105/ml Cell inoculation density, 10ng/ml the concentration of EGF,5% the concentration of serum, the effect of culture factor:cell inoculation density>the concentration of serum>the concentration of EGF. The optimized conditions of cryopreservation:cryopreservation density is 1*107/ml, the concentration of DMSO was 10%, the serum concentration of 80%. The factors affecting the freezing storage were as follows:DMSO concentration> cell frozen storage density> serum concentration.The primary cells were inoculated in the sink adherent growth in 2-3days, adherence was flat and irregular polygon, with cobblestone growth. After passage, the cell wall and growth rate was accelerated, and the growth of P2 cells and the ability of the expansion of frozen storage were not significantly decreased. Immunofluorescence staining showed that the expression of CK19, SSEA-4, HLA-DR, CD45 and Vimentin were not expressed in the primary cells. Statistical primary and P4 cells flow cytometry data, P4 hAECs ssea-4 and CK19 immuno positive cells percentage than P0 generation has decreased significantly (P<0.0001); and vimentin and CD 105 is P0 generation have increased significantly (P<0.0001); P4 and P0 generation hAECs CD73 immune positive cells percentage were very high, and no significant difference P=0.162>0.05; HLA-DR in P0 or P4 generation of hAECs were not expressed. After 2 weeks induction, hAECs transformed gradually from ellipse to multi angle and flat shape, cell refraction increased. Immunofluorescence staining results showed that, AFP expression amount is large in cells of 1 week induction, but after 3 weeks AFP had decreased significantly; ALB and CK18 expression of 3 weeks induction of cells is markedly increased from 1 week; and SSEA-4 expression gradually weakened.2-△△CT method on each of the gene for relative quantification revealed by fluorescence quantitative PCR, after 1 week of induction of HAECs AFP relative expression was significantly higher than that before induction (P<0.05),3 weeks after induction, AFP expression decreased significantly (P<0.05); and for ALB, HNF4 alpha and CYP1A2, after 3 weeks of induction of hAECs each gene relative expression levels were significantly higher than those before induction (P value was less than 0.05).Conclusion we established a relatively suitable method for the isolation, culture and cryopreservation process of human amniotic epithelial cells, to achieve the expected purpose. Human amniotic epithelial cells are easy to obtain and proliferate in vitro, express embryonic stem cells’ maintain undifferentiated state surface markers.hAECs could be differentiate into hepatocyte like cells in vitro, express the specific surface markers and unique functional characteristics of hepatocytes, which is expected to become the source of stem cells treatment of end-stage liver disease.