A New 4-step Perfusion Technique For Mass Hepatocyte Isolation

Background:Hepatocyte belongs to a sort of high-differentiated parenchymal cell with multiple functions. It may change gradually to be of hypofunction, dedifferentiation and even death rapidly under the inappropriate environment in vitro, for instance, absence of hepatocyte growth factor (HGF) and epidermal growth factor (EGF) can cause cell senescence and nonfunction, inappropriate pH value and lack of hormone can cause cell death, and gene activation can cause apoptosis.With the development of modern cell engineering and the latest achievements in cytobiology, tissue culture technique and biology, nowadays hepatocyte has become the hotspot of multiple studies because it can be used as the biological section in the construction of bioartificial liver, the seed cell in cellular transplantation to cure acute and chronic liver failure and the target cell in the drug screening. Thus, the preparation and culture of a large number of high-active hepatocytes have become the most basic link among all these studies.There are many kinds of hepatocyte isolation method, such as mechanical separation, chelation, enzyme digestion, and so on. The mechanical separation began more than half a century ago. In 1956, for the first time, Sorrentino prepared fresh hepatic tissue homogenate using mechanical method and many in vivo drug detoxification trials were performed. It was demonstrated that the homogenate could digest salicylic acid, barbital sodium and ketone body, and induce the urea synthesis of ammonia. Gradually, this method was abandoned because of a little number of hepatocytes with poor motility. In 1967, Howard established collagenase perfusion method that was then improved by Berry and Friend. Using the perfusion of digestive liquid that was prepared through balanced saline without calcium, collagenase and hyaluronidase, the method could yield a larger number of hepatocytes with better motility than the mechanical separation. In 1972, Seglen further developed the method into two-step perfusion, during which the preheated balanced saline without calciumwas first infused to remove blood cells within hepatic sinusoid and some nonparenchymal cells, and then balanced saline containing collagenase was infused to perform in vitro digestion. Following these procedures, hepatocyte was separated, purified and obtained. By this method, the number of hepatocyte and its motility were both improved and foreign matters in hepatocyte suspension were reduced. Nowadays, the two-step perfusion has become one common method used in the laboratory.However, two-step perfusion method is appropriate in liver separation of small animals such as mice, rats, rabbits and dogs, and the effectiveness of separation is poor in pigs because of the more collagens and fibrous tissue in livers. Nevertheless, a large number of high-quality hepatocytes are needed in bioartificial liver and cellular transplantation studies. Therefore, it is necessary to establish novel cell separation techniques from large animal organs (i.e. pig) and human livers.In the current study, with two-step perfusion as control, we established a novel four-step perfusion method in order to provide new approaches to obtaining high quality hepatocytes.Materials and Methods:The following materials were obtained commercially:Dispase, DNase and bovine serum albumin (BSA) were purchased from Roche Holding AG, USA; type â…£ collagenase and glutamine were from Sigma corporation of USA; Walliams' E was from Gibco corporation of USA; glucose, Hepes, NaHCO₃, NaCl, KCl, KH₂PO₃, Na₂HPO₄·H₂O, CaCl₂·2H₂O, Trypan Blue, MgCl₂·6H₂O, MgSO₄·7H₂O, Trypsin and EDTA·4Na were from Shanghai Bio-engineering Co., Ltd; fetal bovine serum (FBS) was from Hyclone corporation of USA and heparin was from Shanghai No.1 Biochemical & Pharmaceutical Co., Ltd. Culture flask and culture plate were from Nunc corporation of Denmark. Filtrator(0.22μm) was from Millipore ultrapure water system (Germany). Chinese Experimental Miniature Pigs were purchased from the Experimental Hogpen of China Agricultural University.Experimental instruments include Olympus inverted phase contrast microscope, H-600A transmission electron microscope (Hitachi, Japan), CO₂ incubator (FormaScientific, USA), liquid nitrogen tank (Thermo Scientific, USA), acidometer and electronic balance (Sartorius, Germany), Millipore ultrapure water system (Germany), ice machine (Scotsman, Germany), large capacity refrigerated centrifuge (Hitachi, Japan), vapor sterilization machine (Tomy, Japan), peristaltic pump (Eppendorf, Germany), electrothermal thermostatic waterbath box (Shanghai Jinghong Laboratory Instrument Co., Ltd) and heating magnetic stirrer (Hangzhou Instrument and Electrical Machinery Factory). Stainless steel wire mesh (#80 and #150) were purchased from Huadong Medicine Co., Ltd and the extracorporeal perfusion apparatus were self-designed.Porcine hepatocyte separation was performed using perfusate containing EDTA and collagenase according to the routine two-step perfusion method in vitro.Liver was obtained by the same method and put in stainless steel pot, and then was crushed gently. Blood outside the liver and residual blood in great vessels were washed as much as possible using 500 ml Perfusion Buffer (PB) precooled at 4℃. First, liver was consecutively infused with 300 ml Perfusion Buffer with EDTA(PBE) with normal temperature and 700 ml PBE liquid preheated at 39 °C through peristaltic pump. Second, 500 ml PB liquid preheated at 39 °C was infused into liver and then was removed. Then liver was transferred into a new sterile stainless steel pot. Third, 500 ml Perfusion Buffer with Dispase (PBD) preheated at 39 °C was infused. Fourth, about 200 ml perfusate was removed and then 500 ml Perfusion Buffer with Collagenase (PBC) preheated at 39℃ is infused. The 500 ml PBC liquid and the residual 300 ml PBD liquid were infused in the recycling way until liver capsule was separated. Cholecyst was removed and liver capsule was sharply torn. Fibrous connective tissue was removed and hepatocyte was obtained under the ice bath through #80 and #150 stainless steel wire mesh, and then hepatocyte suspension was collected.Hepatocyte was collected via the two different perfusion methods and was stained using trypan blue. Motility and recovery rates were calculated. Hepatocyte was cultured using William's Medium E and 24 h adherent rate of hepatocytes was calculated.Results:Using self-designed extracorporeal perfusion system, hepatocyte was separated from miniature pig via the routine two-step perfusion and the four-step perfusion method we had established, respectively. The hepatocyte motility rate was 88 %±5 % and 95%±4% in two- and four-step methods, respectively. The total number of separated hepatocytes was 3:1±0.7×10⁷/g hepatic tissue and 5.3±1.1×10⁷/g hepatic tissue, respectively. The adherent rate of hepatocyte was 80% and higher than 90%, respectively. There were significantly higher hepatocyte motility and recovery rates and 24 h adherent rate in the four-step method as compared with the two-step method (P<0.05 ), with extremely little cellular fractions and nonparenchymal cell contamination. Under the microscope, the contamination rate of nonparenchymal cells was lower.Conclusion:Four-step perfusion method can result in high hepatocyte motility and recovery rates, and significantly improve the adherence ability of cultured cells. Furthermore, there are extremely little cellular fractions and nonparenchymal cell contamination. This method may be extensively used during separating hepatocyte for bioartificial liver and in other large studies on hepatocyte separation. It can provide high quality hepatocytes in bioartificial liver and cellular transplantation studies.