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【Background】 Acute liver failure(ALF) is a rapidly progressive liver disease with highmortality. Liver transplantation is still the only ultimate solution for end-stage liver failure,but scarcity of donor organs hampered its clinical application. Under such circumstance,extracorporeal liver support systems provide ALF patients with temporary liver support toget through the crisis period and prolong the waiting time for liver transplantation.Recently, several bioartificial liver (BAL) has been developed and entered clinical trials.BAL has great potentials in clinical application because of the compensation of entirefunction of the liver. However, the current BAL could not fully simulate themicroenvironment of liver. The short lifespan and rapid lose-of-function of the culturedhepatocytes block BAL clinical translation. Therefore, to maintain viability and prolongfunction of hepatocyte are critical issues in BAL development. Collagen hydrogels havebeen used extensively as an effective matrix platform to encapsulate cells in3D cellculture due to high water contents, easy transport of nutrients and waste, goodbiocompatibility, as well as simple manufacture and process. Moreover, our previousresults showed that the levels of albumin and urea were significantly increased when thehepatocytes were entrapped into collagen hydrogel. In addition, other studies have provedthat co-culture of hepatocyte with other stromal cells could improve the maintenance of hepatocyte function. In this study, we combine hydrogel3D culture and co-culture withaddition of growth factors to improve the hepatocyte functional maintenance and optimizethe culture system aiming to the development of efficient BAL system.【Objectives】To establishment a3D co-culture system by combining collagen hydrogel,HGF and NIH/3T3fibroblast to extend the life of the functional hepatocyte.【Methods】(1) Primary rat hepatocytes and3T3fibroblasts were co-encapsulated intocollagen I solution which pre-combined with HGF and DMEM to establish the3Dco-culture system. The morphology, phenotype and functions of glycogen and albuminsynthesis were determined by analysis of phase contrast microscopy, HE staining, PASstaining and immunofluorescence staining.(2) To optimize the culture system, fivedifferent hepatocyte to fibroblast (H:F) seeding ratio (1:0.5,1:1,1:2,1:4,1:8), fourvarious cell inoculum concentration (2×104/mL,2×105/mL,2×106/mL and2×107/ml) wereentrapped into collagen hydrogel and three ways of cultivation (single-culture,insert-culture, co-culture) were performed up to25days. Culture medium were collectedafter24h after changing the media and tested for albumin secretion and urea synthesis.The albumin and urea levels were compared to optimize the most appropriate cultureparameters and ways of cultivation.(3) Hepatocytes were cultured with optimum cultureconditions for25days. Cell viability was analyzed by LIVE/DEAD staining and WST-1assay and liver-cell-specific functions (LDL uptake, albumin secretion, urea synthesis)were evaluated. In addition, the expression levels of hepatocyte-specific genes were testedby Real-time PCR.【Results】(1) Hepatocyte/collagen gel construct were suspended in the medium andphase contrast microscopy revealed that hepatocytes were presented round shape anddistributed uniformly in collagen hydrogel. After7days of culture, HE staining showedclustery growing of hepatocytes with3T3fibroblasts scattering around. Within our culturesystem, hepatocytes remained PAS staining, CK18and albumin positive after7-dayculture, which provided the convincing evidence of highly differentiated primaryhepatocytes with functions of glycogen and albumin synthesis.(2) The albumin and urea productions of all groups (H:F1:0.5,1:1,1:2,1:4,1:8) were achieved peak at day7or day5and progressively decreased during the culture period of the experiment. But thealbumin and urea secreted by H:F ratio of1:0.5were apparently higher than other groupsat every time point of culture after day7(P<0.05) and still maintained high levels of30-50μg/24h,40-110μg/24h respectively; Cell density of2×106cells/mL did exhibitdifferences in albumin and urea production with other cell density and we did not observeany positive effects on liver-specific functions by further increase or decrease the cellseeding density; On day3, the albumin and urea productions made almost no difference inthree groups (single-culture, insert-culture, co-culture), while on day7and day20,improved albumin levels were observed in co-culture system and compared with singleculture and insert culture, the albumin levels of co-culture raised2.10and1.46times onday3,4.58and1.75times on day20.(3) Compared with single culture, our3D co-culturesystem exhibited high viability (52.25±4.62%vs.32.92±5.32%, P<0.01, day20), highlypreserved albumin secretion and urea synthesis as well as stronger LDL uptake. Inaddition, Real-time PCR revealed higher hepatocyte-specific gene expression (Albumin,HNF-4α, Claudin-3, and G6P) in3D co-culture than single culture model (P<0.05). Aftertreatment with liver enzyme inducer, induction of CYP isoenzyme expression was moresuccessful in3D co-culture group, where the compounds caused increasing (CYP2E1),decreasing (CYP1A1) or more stable (CYP1A2, CYP2B2, CYP3A1) enzyme expressionthan in single culture.【Conclusion】 We successfully established the hepatocyte3D co-culture system usingcollagen hydrogel as scaffolds. In this system, H:F of2:1, seeding density of2×106/ml anddirect contact of the co-culture cells yielded maximal function which could providereference to the preparation of BAL. The novel3D co-culture system which is thecombination of collagen hydrogel carrying groth factor and co-cultured cells provides avaluable way to prolong the viability and function of the encapsulated hepatocytes, whichwould have great application potentials in the BAL support therapy.
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