Effects Of Three Dimension Culture On The Function And Differentiation Of Hepatocytes

Part 1 Effects of 3D Culture on the Function of Hep G2 and Sk-hep1Objective: To compare the relative function of Hep G2 and Sk-hep1 hepatocellular carcinoma cells in 2D and 3D culture.Methods: Hep G2 and Sk-hep1 were seeded into 96-well plates by agar-covered with 1 ×104 cells/m L to construct spheroid.The changes of spheroids shape and diameter were observed by inverted microscope.Cell Titer-Glo(?) 3D Cell Viability was used to assess the viability of cells cultured under 2D and 3D environment.HE staining and immunohistochemistry were performed to observe the morphology of spheroids.Western blot was selected to detect the expression of E-Cadherin.In 2D and 3D culture,RT-q PCR was used to detect the expression of Albumin and related to drug metabolizing enzymes CYPs gene.The level of albumin production was assessed by ELISA,the synthesis of urea was measured by Jung Assay,and acetaminophen toxicity to hepatocytes under 2D and 3D culture was detected by Cell Titer-Glo(?) 3D Cell Viability.Results: Sk-hep1 could form compact spheroids gradually,while Hep G2 spheroids were loose in 96-well agar-coated plates observed by inverted microscope.HE staining and immunohistochemical showed that the peripheral cells of Sk-hep1 spheroids were closely arranged,while the internal cells were few.Hep G2 spheroids were loose and could not be sectioned for further histomorphological analysis.The results of immunohistochemical demonstrated that there were massive of collagen I deposit in spheroids.Western blot showed that there was no significant difference in the expression of E-Cadherin between 3D and 2D,the expression no change of E-Cadherin with the increase of culture days.However,Sk-hep1 had no expression of E-Cadherin both 2D with 3D.RT-q PCR results revealed that Albumin and CYPs expressed differently by 2D and 3D culture and varied by days under cultivation.Albumin and urea indicated that liver function of Hep G2 and Sk-hep1 in 3D culture were higher than those in 2D culture(P<0.01).As for APAP cytotoxicity,both of Hep G2 and Sk-hep1 3D IC50 were higher than 2D culture after 48 hours of culture.The IC50 were followed respectively: Hep G2(8.92 m M vs.11.31 m M),Sk-Hep1(9.22 m M vs.11.87 m M).Conclusion: Sk-hep1 were more inclined to form compact spheroids than Hep G2 under the same 3D culture conditions,and E-Cadherin had no obvious effect on the formation of spheroids.Compared to 2D culture,Sk-hep1 and Hep G2 cells delayed the proliferation,enhanced hepatocyte secretory factor and increased tolerance to APAP cytotoxicity.Part 2 Effects of 3D Culture on Differentiation of Adipose-derived Stem Cells into Hepatocyte-like CellsObjective: To compare the differentiation of adipose-derived stem cells into hepatocyte-like cells in 2D and 3D cultures.Methods: Adipose-derived stem cells(ASCs)were isolated and cultured by collagenase type I digestion.The expression of CD29,CD44,CD34 and CD45 were identified by flow cytometry.Their differentiation potentials were confirmed by the following ways: lipid induction was identified by oil red O staining,and osteogenesis was demonstrated by alizarin red S staining.ASCs were cultured directly in 2D and 3D after removal.In addition,after 6 days of culture,some of the3 D cells were digested and transformed into 2D culture,and some of the 2D cells were digested and then transformed into 3D cells.The remaining 2D and 3D ASCs were cultured continuously.All the four groups continued to culture for 6 days(12days containing two stages).ASCs were divided into three groups: 2D induction group,3D to 2D induction group,2D to 3D induction group and 3D induction group.The induction time was 16 days.Immunofluorescence was used to detect the expression of hepatocyte markers Albumin and AFP,RT-q PCR was qualified to detect the expression of CK18,Albumin and AFP,ELISA was performed to detect the secretion of albumin and Jung method was used to detect urea synthesis.To further explore its mechanism,laser confocal and RT-q PCR were used to measure the expression of multipotent markers Oct4,Nanog and Sox2.Results: ASCs showed fibroblast morphology.Flow cytometry indicated that ASCs were positive expression of CD29,CD44,and negative expression of CD34,CD45.Adipogenesis could be dyed red lipid droplets by oil red O,and alizarin red S staining results showed that calcium nodules can be dyed red after osteogenesis induction.Under the conditions of 2D,3D to 2D,2D to 3D and 3D,except for the expression of CK18 m RNA had no significance between 2D to 3D with 3D to2D(P>0.05).The expression of hepatocyte marker albumin,alpha-fetoprotein and their m RNA,albumin secretion and urea synthesis were higher in 3D group than in2 D to 3D group,higher in 2D to 3D group than in 3D to 2D group,and higher in 3D to 2D group than in 2D group(P<0.05).Laser confocal and RT-q PCR results confirmed that Nanog was consistent with its hepatogenesis trend,while the expression of Sox 2 and Oct 4 in 3D was significantly higher than in 2D(P < 0.01),there was no significant difference between 3D and 2D groups,and between 2D and3 D to 2D groups(P > 0.05).Conclusion: On the conditions of 2D,3D to 2D,2D to 3D and 3D,3D culture could increase the differentiation of ASCs into hepatocyte-like cells,further promote their phenotypic expression and hepatocyte-like maturation.3D culture were able to enhance the expression of Nanog,Sox 2 and Oct4 in ASCs.After 2D cells were transferred into 3D,the pluripotency of ASCs could be increased,but it could not reach the effect of 3D culture.The pluripotency of ASCs decreased when 3D cells were transferred into 2D,but it was higher than that of 2D cells.ASCs may affect the hepatogenic differentiation of adipose-derived stem cells through Nanog.