Study On Co-culture Of Primary Hepatocytes With Kupffer Cells From Suckling Piglets In RCCS

Research backgroundLiver failure is very severe clinical syndromes with high mortality.To date,liver transplantation is the only effective treatment for patients with liver failure.However orthotopic liver transplantation is critically limited by the shortage of liver donors.An alternative approach as replacement for liver transplantation could be a cell-based bioartificial liver(BAL).The progress in the development of BAL has been immense over the past years,however,many important BAL characteristics that are necessary to meet clinical demands have not been sufficiently addressed,as lack of suitable hepatocytes source,perfect culture method,enough cells with normal liver function and appropriate bioreactors that meet the characteristics of liver bioengineering.Numerous studies have shown that liver non-parenchymal cells(NPC)play an important role in liver pathophysiology and maintenance of primary hepatocyte function.Thus focus has been given to incorporation of multiple cell types into the liver culture over the past few years,to find the most suitable NPC and the most appropriate cell ratio is one of the research hotspots.Kupffer cells(KCs)are the liver resident macrophages,have phagocytosis and clearance function,in addition can secrete the cytokines such as tumor necrosis factor-α(TNF-α),hepatocyte growth factor(HGF),interleukin-6(IL-6),which can effectively promote the proliferation of hepatocytes.So in theory,KCs could make the function of artificial liver more perfect.However,the study on co-culture of hepatocytes and KCs as the function cells source of BAL is less.Our research team successfully established a two-dimensional(2D)co-cultivation model of primary hepatocytes and Kupffer cells isolated from suckling piglets in the early stage and set up the suitable ratio of 12:1.In addition,many studies have found that three-dimensional(3D)culture can reconstruct a physiological environment and improve cell function.The Rotary Cell Culture System(RCCS)can simulate microgravity environment and has characteristics of promoting 3D cell proliferation and differentiation,low shear force,high-efficient material delivery and high density culture,which is a rare system for cell co-cultivation.This study combined with the advantages of co-culture and 3D culture use microcarriers to establish a co-culture model of direct contact between hepatocytes and KCs at 12:1 ratio and to explore the co-culture mechanism,in order to provide experimental basis for the construction of efficient,economical and practical seed cells of BAL.Objective:This study aims to establish a stable and efficient experimental method of isolating and purificating large quantity of high-quality and high-purity Kupffer cells from suckling piglets.Then set up a co-cultivation model of hepatocytes with KCs on microcarriers with 12:1 ratio in RCCS to evaluate the feasibility of co-cultivating hepatocytes and KCs on microcarriers in RCCS,and to provide experimental basis for constructing efficient,economical and practical seed cells for BALs.To investigate further effect of KCs and RCCS on hepatocytes in terms of hepatocyte function genes expression,and to investigate the role of soluble cytokines,such as HGF,TNF-α,IL-6 in co-culture.Method:This study was divided into three parts.1.Two-step collagenase perfusion in vitro and modified four-step semi in situ collagenase perfusion method were performed respectively on suckling piglets liver(n=5)to obtain liver cells suspension by using a self-designed circular perfusion system.Liver cells were separated into hepatocytes and NPC fraction by low-speed centrifugation,KCs were isolated by 25%Percoll and 50%Percoll density gradient centrifugation combined with 30 minutes or 15 minutes adherent purification.The yield,viability and purity of hepatocytes(HCs)and KCs were compared.2.The coculture models of direct contact between primary suckling pig hepatocytes and Kupffer cells in culture plate(2D)and RCCS(3D)were established.Morphological changes of cells were observed by microscope and cells activity observed by MTT staining and cell proliferation detected by CCK-8 and cell growth curve drew.The ALT,AST,LDH,GLU,BUN and ALB levels of coulture supernatant on day 1,3,5,7,9 and 11 were measured respectively in the hepatocyte culture group(2D、3D)and the co-culture group(2D、3D).The concentration of diazepam was detected by ultra-high performance liquid chromatography tandem mass spectrometry(UHPLC-MS/MS)to evaluate the metabolic ability of hepatocytes to diazepam in different culture groups.3.The expression level of hepatocyte function genes was detected by RT-PCR on day 1,3 and 5,and the difference of hepatocyte function gene expression level between single culture group(2D,3D)and co-culture group(2D,3D)was compared.HGF,TNF-α and IL-6 levels of supernatant in hepatocyte culture group(2D,3D),Kupffer cell culture group(2D)and hepatocyte and KCs co-culture group(2D,3D)were measured and compared.Result:1.The average yield,viability and purity of hepatocytes and viability and purity of KCs obtained by the modified four-step semi in situ perfusion method were higher respectively than that obtained by the two-step perfusion method.The average yield of KCs obtained by two-step collagenase perfusion in vitro and modified four-step semi in situ collagenase perfusion combined with adherent purification for 15 minutes was lower than that of adherent purification for 30 minutes,but the purity of KCs by adherent purification for 15 minutes was significantly higher than that of adherent purification for 30 minutes.2.The hepatocytes in the 2D co-cultured group could maintain the morphology of hepatocytes for longer time;The cell-microcarrier aggregates formed in the 3D co-culture group were larger,the hepatocytes had stronger proliferation ability and grew for longer time compared to monoculture group.The AST,LDH level of the 3D culture group was lower than that of the corresponding 2D monoculture and co-culture group;The AST and LDH level of culture supernatants in 3D and 2D co-culture group was lower than that in monoculture group.The glucose level of culture supernatants was lower both in 2D and 3D co-culture group compared to relevant 2D and 3D monoculture group.The ALB and BUN level in the 2D and 3D co-cultured group was higher than that in the monoculture group;The ALB and BUN level in 3D culture group was higher than that in 2D culture group.The diazepam metabolic ability of hepatocytes in 2D co-culture group was better than that in monoculture group,there was no significant difference between the 3D co-culture group and monoculture group,and that both in 3D monoculture and co-culture group was better than that in 2D culture group on day 9 and 11.3.The expression level of functional genes,such as Albumin、G6P、CSP-1、HNF-4α、Claudin-3、CYP1A2、CYP3A4,etc.both in 3D monoculture and co-culture group was higher than that in corresponding 2D culture group on day3 or 5.The expression of Albumin、G6P、CSP-1、CYP1A2 and CYP3A4 genes in 2D and 3D co-culture group was higher than that in the corresponding individual culture group.HGF,IL-6 and TNF-α was not detected both in 2D and 3D hepatocyte monoculture group.In addition,the level of HGF and IL-6 both in 3D and 2D co-culture group was higher than that in KCs 2D monoculture group.The level of HGF and IL-6 in 3D co-culture group was higher than that of 2D co-culture group on day 5,7and 9.Conclusion:1.Viability and purity of the KCs isolated by improved four-step semi-in situ collagenase perfusion combined with 15 minutes adherence purification method was higher,and the yield,viability and purity of hepatocytes isolated by improved four-step was significantly increased,so which is a stable,economical method to isolate KCs with high Viability and purity,and to obtaine simultaneously hepatocytes with high yeild,viability and purity.2.The direct contact co-culture model of primary suckling pig hepatocytes and KCs at a ratio of 12:1 on microcarriers(Cytodex-3)in RCCS was successfully established for the first time,High-density and large-scale cultivation of hepatocytes was carried out under the simulated microgravity environment.Hepatocytes co-cultured in RCCS had better proliferation,synthesis and metabolism functions.The co-culture of hepatocytes with KCs in RCCS is expected to become one of the ideal cell source and culture methods for BAL.3.The simulated microgravity environment in RCCS could promote expression of hepatocytes function Genes,such as Albumin,G6P,CSP-1,HNF-4α,Claudin-3,CYP1A2,CYP3A4,etc.and promote hepatocytes proliferation and differentiation.Co-culture of hepatocytes with Kupffer cells at a ratio of 12:1 could promote the expression of hepatocytes function genes such as G6P,CSP-1,CYP1A2,CYP3A4,etc.The cytokines,HGF,IL-6 and TNF-α,secreted by KCs may be an important factor to promote hepatocyte proliferation and improve hepatocyte function.