A New Multi-point Puncture Perfusion Technique For Adult Human Hepatocytes Isolation And Detection Of Cell Function In Vitro

Background:Artificial liver support system is recognized as the most effective treatment for a variety of acute liver failures and bioartificial liver(Bioartificial Liver,BAL) based liver cell may be a great hope for various acute and chronic liver diseases in the future. The bioartificial liver is mainly made up of the cultivated liver cells with activity in vitro,so bioartificial liver has the same functions of detoxification,secretion, synthesis and transformation as liver and liver cells function as its core part.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 failu and the target cell in the drug screening.Thus,the preparation and culture of high-active hepatocytes,in particular the preparation of human liver cells,have become the most basic link among all these studies.There are many kinds of hepatocyte isolation methods,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 drug detoxification trials in vivo were performed.Gradually,this method was abandoned because of a small number of hepatocytes with poor motility.In 1967,Howard established collagenase perfusion method,and In 1972,Seglen further developed the method into two-step perfusion,during which the preheated balanced saline without calcium was first infused to remove blood cells within hepatic sinusoid and some nonparenchymal cells,and then balanced saline containing was infused to perform purified in vitro digestion.Following these procedures,collagenase hepatocyte was separated,and obtained.With 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 a common method used in the laboratory.Two-step perfusion method is appropriate in liver separation of small animals such as mice,rats,rabbits and dogs.At present,the main application of two-step perfusion method for human liver which is discarded in transplantation for non-matching.The biggest flaw of this method is the lack of contributive liver in transplant,and the discarded liver for not matching is particularly rare.In this condition human liver cells are completely unable to meet the needs of the basic research,so that many basic researches have to choose animal's liver cells to replace human's.A lot of scholars have applied small pieces of liver tissue resected from hepatic lobes,through its remnants of the small blood vessels to separate liver cells by two-step perfusion method.This approach requires a larger block of liver tissue, and 3～4 blood vessels but in fact the liver tissue obtained in surgery is usually of irregular shape.So it is difficult to find blood vessels,two-step perfusion can not be implemented.Even if the blood vessels could be found,perfusion can only be done through the remnant irregular-shaped blood vessels,which would lead to insufficient perfusion,and unsatisfying separation result.So it is necessary to set up a practical cell separate technology to separate liver cells from human liver.On the basis of two-step perfusion,this study set up a new method of multi-point puncture perfusion to separate adult liver cells from small pieces of irregular-shaped liver tissue obtained in surgery.In this way,human liver cells will be obtained more simply and easily in many basic researches.This method will provide technical support to the application of human hepatocytes for bioartificial liver,cell transplantation research.This experiment further cultivated human liver parenchymal cells which were isolated by multi-point puncture perfusion in a short-term primary and external way and examined supernatant of the cultured cells for albumin,urea nitrogen,the level of intracellular enzyme,and cell metabolic function.Thus,on one hand,we identified the possibility of separate liver cells with functional activity by multi-point puncture perfusion;on the other hand,we analyzed primary hepatic parenchymal cell function in the course of adult primary liver cells cultured in vitro.Methods1,Human liver tissue samples were obtained from patients with liver cancer or hepatic hemangioma in the department of Hepatobiliary Surgery of Nanfang Hospital. Normal tissues around the lesions were sampled during the surgeries,and each of them weighed about 0.5～5g.2,Multi-point puncture perfusion was adopted to separate adult liver cells.Specific steps include:①multi-point puncture and perfuse with 38℃preheating perfusion fluid by pinhead injector until liver tissues change color from dark red to gray and the outflow of perfusion fluid get clean and bright.The pressure and flow rate during perfusion must be constant and produce no bubbles.This process lasts about 10-15 minutes.②multi-point puncture and perfuse with 38℃preheating collagenase typeⅣsolution with injector until the tissues get loose,lose elasticity, and the surface appears back of turtles.Ⅳcollagenase solution can be recycled.This process takes around 15-20 minutes.③Tear the liver tissues into pieces bluntedly with sterile forceps,remove the residual amicula and fibrous connective tissue, collect the liver cell suspension into sterile bottle,and then continue to digest with shake in collagenase solution for 15 minutes at 37℃.④Make the digest into cell suspension with thick-mouth straw.Filter with 100-mesh stainless steel screen,and collect the liver cell suspension with a centrifuge tube on the ice bath.⑤Centrifuge 5min at 4℃with a speed of 50g,remove the supernatant,add some red cell disruption to the sediments at the bottom,allow it to stand for 3 minutes at room temperature,and then centrifuge at the same speed for 5min again.⑥Collect the bottom sediment,re-suspend the cells with liver cell washing buffer solution containing DNase I,and then filter with 200-mesh stainless steel screen.⑦Centrifuge 5min with 50g at 4℃,remove the supernatant,re-suspend the cells with serum-free DMEM medium,centrifuge again,discard the supernatant,and put the sediment cells on the ice bath for use.3,Collect the liver cells,stain them with trypan blue and then calculate the motility rate and the yield.Culture the liver cells with RPMI-1640(contained 15% fetal bovine serum),and calculate the adherence rate after 24h.4,Observe the liver cells' shape and structure of the adult primary liver cells when they were freshly isolated and cultured with the inverted phase contrast microscope and the fully automatic biology photomicrographic device.5,Draw a growth curve of adult primary liver cells isolated by multi-point puncture perfusion. 6,Collect cell supernatant of the primary human liver cells that were isolated by multi-point puncture perfusion respectively after cultures for 1 day,3 days,5 days,7 days,9 days.Analyze the concentrations of urea(BUN),alkaline phosphatase(ALP), alanine aminotransferase(ALT),aspartate aminotransferase(AST),alpha-fetoprotein (AFP) etc in cell culture supernatant with fully automatic biochemical analyzer.7,Detect the levels of albumin of adult primary liver cell culture supernatant separated by multi-point separation of puncture perfusion by ELISA.8,Detect AlbmRNA and the expression of cytochrome P450 2E1 mRNA of primary cultured human liver cells by RT-PCR.9,Detect the metabolic function of the primary cultured human hepatocyte by the diazepam transformation test.Results1,The method of multi-point puncture perfusion set up by us was used to separate liver cells from adult small irregular liver tissue resectted in surgery.Survival Rate of liver cells were 87%±7%,a total of(2.2±0.7)×10~7 liver cells were isolated from one gram liver tissue,liver cell adhesion rate was 75%,and there were rare cell debris and other non-parenchymal cell pollution.In addition,the methods are good for isolation of primary liver tumor cells,and the success rate is almost 100%.2,Automatic biochemical analyzer was used to detect the level of biochemical indicators in primary adult liver cell culture supernatant.As a result,the levels of ALT and AST rose with the increased culture days,ALP showed no significant change trend,and AFP was not detected all the time.Although the content of BUN demonstrated a slight downward trend,it maintained at a relatively high level in the short-term culture in vitro.3,ELISA was used to detect albumin secretion of adult liver cells in vitro.The secretion amount reached a peak on the third day and then went down.The cell supernatant concentrations of human albumin excreted by human primary liver cells cultured from 1 day to 7 days were respectively 48.2±4.5 ng/ml,55.2±6.9 ng/ml,63.3±6.4 ng/ml,37.0±10.6 ng/ml,22.5±4.9 ng/ml,14.9±5.3 ng/m,10.0±2.9ng/ml.4,RT-PCR can detect the expression of AlbmRNA and cytochrome P450 2E1 mRNA in the adult primary liver cells obtained from multi-point puncture perfusion. 374bp and 338bp bright bands separately appeared.5,Adult liver cells can be effective in playing its role in drug metabolism in vitro within 7 days via the diazepam transformation test.In vitro on the third day it had the greatest transforming capacity,and then there was a gradual downward trend.Conclusions1,The laboratory operating procedures of multi-point puncture perfusion were established to separate human liver cells,which can reduce the requiment for liver tissue samples.So it is no longer difficult to get primary adult liver cells.The new method was easy,economical and practical.2,Through the functional detection of human primary liver cells obtained from multi-point puncture perfusion,we know that this method can provide liver cells with the biological activity and metabolic function that can be used to do researches on source of human liver cells.3,The dynamic change of functional activity of adult primary liver cells was studied in the process of culture in vitro.