| Acute liver failure(ALF)is a serious disease that endangers human health and survival,bio-artificial liver(BAL)system has brought new hope for clinical treatment of ALF patients.Human induced hepatocyte(hiHep)is a type of hepatocyte-like cell that is directly transdifferentiated from human skin fibroblasts mediated by lentivirus.It has normal phenotypes and multiple functions of mature hepatocytes,which can be used as a potential source of seeding cells for the BAL system.In order to realize the clinical treatment application of the BAL system,at least 1010 or more functional hepatocytes are required,and these cells are expanded and obtained in a serum free medium(SFM)with a clear composition.However,the currently used serum-free media containing commercial serum substitutes are expensive and have unknown ingredients,making it difficult to precisely control the proliferation and function of hiHeps.Expansion of cells based on multi-layer plates is costly and difficult to scale up.In addition,the specific functions and activities of hiHeps cultured in vitro still need to be improved.These problems limit the clinical research and therapeutic application of the BAL system based on hiHep cells.Therefore,in this paper,by understanding the influence of the in vitro culture environment on the proliferation,activity and function of transdifferentiated hiHeps and related regulatory mechanisms,to establish a SFM with clear ingredients and no animal-derived components suitable for in vitro culture of hiHep cells and a serum-free large-scale expansion technology,and regulate the proliferation,activity and function of hiHep cells.The results of this study lay the foundation for the construction of a BAL system based on hiHeps and its future clinical treatment applications.Plackett-Burman(PB)experiment design was used to screen the initial serum-free medium OM14 that supports hiHeps growth in vitro.Then through factor effect analysis,it was found that the proliferation of hiHeps were significantly affected by the three effectors of vitamin C(VC),vitamin H(VH)and insulin.On the basis of optimizing the working concentration of the key effector insulin,and further explore the molecular mechanism that insulin affects hiHeps proliferation and function.The results show that the concentration of insulin at 1 mg/L was more conducive to the proliferation and functional-related gene expression of hiHeps,and insulin regulates hiHeps proliferation through the expression of Cyclin D1 protein mediated by PI3K-AKT1-mTOR-p70S6K signaling pathway and PI3K-AKT1-p53 signaling pathway.In addition,it also enhances the expression of albumin(ALB)and uridine diphosphate glucuronyl transferase(UGT)in hiHeps by promoting the expression of transcription factor HNF1A.Further studies showed that insulin treatment could maintain the normal morphology of hiHeps,promote cell proliferation and stable expression of liver function genes during long-term passage.By adding 1.5 mg/L VH and 30 mg/L VC,reducing the growth factor TGF-α concentration to 10 ng/mL,and replacing animal-derived bovine serum albumin(BSA)with plant-derived human serum albumin(Human Serum Albumin,HSA),the serum-free medium was further optimized.As a result,a serum-free medium with clear components and no animal-derived components was established.hiHeps cultured in the serum-free medium are comparable to the results in the serum-containing medium in terms of morphology,growth and function,and the degree of cell damage is lower.In order to establish the serum-free microcarrier culture system based on hiHeps,the effects of a series of key parameters on the attachment and growth of hiHep cells on microcarriers during serum-free culture were investigated,including microcarrier type,inoculation mode at adherent stage,microcarrier concentration,cell inoculation density,and stirring speed.The results showed that hiHeps exhibited better adhesion and proliferation capabilities on Cytodex 3 solid microcarriers,when compared with Cytopore 2 and Cultispher S macroporous microcarriers.In addition,50%of the final culture volume was used for inoculation during the adherence period,the microcarrier concentration is 3 mg/mL,the cell inoculation density is 2.0×105 cells/mL,and the stirring speed is 45 rpm,which was more conducive to the uniform attachment,growth and activity of hiHeps on the microcarriers.After 12 days of culture,the final cell density of hiHeps can reach 25.22×105 cells/mL,and the corresponding expansion fold is 12.61.Further,the study also found that addition of fibronectin(FN)significantly promotes hiHeps attachment and proliferation on Cytodex 3 microcarriers,and this effect is mediated by the integrin-β1/FAK/ERK/CyclinDl signaling pathway.Therefore,a serum-free microcarrier culture technique for hiHeps was established based on the flask culture system.hiHeps cultured with this technique were comparable to those cultured with serum-containing microcarriers in terms of cell proliferation and functional gene expression,and the cell activity was better.The harvested cells have been identified to maintain normal hepatocyte polygonal morphology and functional expression.Human umbilical cord derived mesenchymal stem cells(hUMSCs)were used to establish a serum-free co-culture system of hUMSCs and hiHeps,to explore the effects of hUMSCs on the function and activity of hiHep cells and the interaction mechanism.By investigating the effects of the inoculation sequences and inoculation ratios of hUMSCs and hiHeps on the function and activity of hiHeps,the co-culture conditions were determined.The results showed that the first inoculation of hUMSCs was followed by the inoculation of hiHeps in co-culture,and the co-culture ratio of hiHeps and hUMSCs was 1:1/2,which was more beneficial to hiHeps urea synthesis and cell viability.Based on different co-culture models,the main factors affecting the growth and function of hiHeps in direct co-culture were analyzed.It was found that co-culture conditioned medium(CO-CM)played a major role in promoting hiHeps ammonia removal and reducing apoptosis in the process of mixed co-culture.By detecting the extracellular matrix and cytokine content in Co-CM,it was found that the level of interleukin-6(IL-6)secretion increased significantly,and it was confirmed that TNF-α secreted by hiHeps during the co-culture process promoted the secretion of IL-6 by hUMSCs.Further studies showed that IL-6 is the main factor regulating hiHeps ammonia removal and apoptosis during co-culture.IL-6 reduces hiHeps apoptosis through the JAK-Stat3-Ref-1 pathway mediated ROS level reduction and the JAK-Stat3-Bcl-2/Bax-Caspase3 pathway,and IL-6 enhances the ammonia removal ability of hiHeps through JAK-PI3K/Akt-P53-ARG1 signaling pathway.hUMSCs and hiHeps were inoculated on polyethylene terephthalate(PET)fiber scaffolds for three-dimensional co-culture to investigate the functional expression of hiHeps during 3D co-culture under serum-free conditions.The results showed that compared with the 2D monoculture group,the liver-specific function,phase Ⅰ and phase Ⅱ metabolic enzyme activities of hiHeps were significantly improved in the 3D culture environment.Moreover,the albumin expression,urea synthesis and UGT enzyme activity of hiHeps were further enhanced during 3D co-culture.Subsequently,a 3D perfusion dynamic co-culture system based on PET fiber scaffolds was constructed,and the liver function and cell activity of hiHeps during 3D static co-culture,3D perfusion single culture and 3D perfusion co-culture were compared.Studies showed that the levels of albumin secretion and urea synthesis of hiHeps were higher,and the cell activity is better under 3D perfusion co-culture conditions.Therefore,the serum-free co-culture strategy can improve the function and activity of hiHeps.In summary,a serum-free medium with clear ingredients and no animal-derived components suitable for in vitro hiHeps culture and expansion is obtained through design and optimization in this paper,and a serum-free microcarrier cell expansion technology and serum-free co-culture technology based on hiHeps were established.After understanding the molecular mechanism of the interaction between hUMSCs and hiHeps,an in vitro 3D perfusion dynamic co-culture system to improve the function and activity of hiHeps were further constructed.These studies have laid a solid theoretical foundation for in vitro regulation of hiHeps serum-free large-scale expansion and functional maintenance,as well as the establishment of hiHeps-based BAL system and clinical treatment applications,and have important strategic guiding significance. |
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