We have generated human hepatocyte spheroids with uniform size and shape by coculturing 1. In control hepatocyte preparations, there was typically a 3h delay prior to the onset of nitrite production. It is plausible that larger tissue weights would result in a greater hepatocyte cell yield. Isolation and primary culture of rat hepatic cells protocol. Review article potential for isolation of immortalized hepatocyte cell lines by liverdirected in vivo gene delivery of transposons in mice masahirosato,1 isseisaitoh,2 emiinada,3 shingonakamura,4 andsatoshiwatanabe5. However, these studies are often difficult to perform because of the inability of hepatocytes to proliferate in vitro. The hepatocyte isolation method by liver perfusion with the combination of collagenasedispase is feasible for obtaining a large amount of. A rapid twostep method for isolation of functional. We assessed the association between the total liver tissue weight and total viable hepatocyte cell yield following isolation and found a positive correlation between tissue weight and hepatocyte. After isolation were obtained 324, 48 1, 25 x 106 hepatocytes, with a viability of 94. Wen zhang primary hepatocyte isolation 20101121 purpose.
A simple method for the simultaneous isolation of stellate cells and hepatocytes from rat liver tissue article pdf available in molecular and cellular biochemistry 24812. A second way is to remove the required aliquot of cell suspension after filtration through the nylon mesh for hepatocyte isolation step 4. Exploring donor demographics effects on hepatocyte yield and. A technique to isolate human hepatocytes and nonparenchymal liver cells from the same donor is described.
Recent advances in the isolation of liver cells wiley. Potential for isolation of immortalized hepatocyte cell. Jun 01, 2016 hepatocyte media sekisui xenotechs optiplate media is used when plating attachable and inducible cryopreserved human, beagle dog, cynomolgus monkey, cd1 mouse, spraguedawley rat hepatocytes, etc. Hepatocyte proliferation an overview sciencedirect topics. Isolation and culture of adult mouse hepatocytes springerlink. Within the cell culture hood, use a cell scraper to gently disperse the cells into williams complete medium within a sterile petri dish. Protocols for isolation of human hepatocytes have been developed. The lowest viability result was for donor tissue number 2 1%, for which the cold ischemia time was 30 hours. We have partnered with lifenet health to provide highquality primary human hepatocytes products. Nitrite represents a stable end product of no production and is commonly used to assess no formation. Liver cells, or hepatocytes, have direct access to the livers blood supply through small capillaries called sinusoids. The mapk mek12erk12 pathway and its implication in hepatocyte cell cycle control provide an exhaustive overview of their own work and the literature regarding the role of this pathway in the hepatocyte cell cycle control. This method describes the steps from a living rat to a single cell solution of primary hepatocytes. Jan 01, 2000 following isolation, nitrite levels were measured in the extracellular media as a measure of no production fig.
Our laboratories have been involved in various applications of human liver cells and we have longlasting experiences in human liver cell isolation. Hepatocyte isolation system tissue dissociationcell isolation. Moreover, gene expression levels of cholangiocyte markers in hlcs were also not altered by culturing with hepatocytecm hepg2cm. Dec 20, 2007 primary hepatocyte cultures are a valuable tool for the understanding of cellular and molecular phenomena occurring during malaria liver stage. This chapter describes experimental protocols for the isolation of hepatocytes from human liver tissue and for the preparation of short and longterm cultures in which cells retain a. The aim of this chapter is to describe the authors experience in the isolation of hepatocytes from human liver tissue and the preparation of short and longterm cultures. These include hepatocytes, total liver cell population tlc, stellates, kupffers and intrahepatic biliary epithelial cells ihbec. However, the levels observed after 24 days of culture were 50 times lower than in hhpc. The establishment of a liver cell isolation method covering the different liver cell populations revealed that combining the parenchymal and nonparenchymal cell isolation is a critical step due to the difference in digestion time required for optimized results. Either of these separation procedures give a cell yield of about 2. Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. Isolation, culture, and quality procedures beside transport and logistics, there are additional reasons why cell isolation may be restricted or the cell isolation is. Isolation and coculture of rat parenchymal and nonparenchymal liver cells to evaluate cellular interactions and response the harvard community has made this article openly available.
Individual liver cell populations were isolated from the enriched npc. When the cell yield is low or selective removal of the nonparenchymal cells is needed, percoll. Primary hepatocyte cultures better reflect the properties of the liver in vivo than do cell lines derived from the liver. It is necessary to resuspend the cell pellet in optiplate hepatocyte media before seeding the cells onto culture dishes. Recently, a method of isolation of zonal hepatocytes.
Human hepatocyte transplantation for liver disease. The isolation of functionally heterogeneous hepatocytes of. Pdf primary hepatocyte cultures better reflect the properties of the liver in vivo than do cell lines derived from the liver. Harvardmit division of health sciences and technology hst.
Isolation, culture, and quality procedures beside transport and logistics, there are additional reasons why cell isolation may be restricted or the cell isolation. Isolation and primary culture of rat hepatic cells. Immortalized hepatocyte cell lines, such as hepg2 human hepatoblastoma kelly and darlington, 1989, the hepg2derived line c3a ellis et al. Hepatocyte transplantation has been employed in clinics in three types of procedures box 71. The isolated cells can be used not only in research but also for transplantation in patients with liver disease. Mouse hepatocyte isolation general surgery lab sop last updated 32315 by alicia frank 5 determining cell count add 190. Primary culture of hepatocytes is an in vitro model widely used to study various aspects of liver physiology and pathology. Primary mouse hepatocytes are an important tool in the biomedical research field for the assessment of hepatocyte function. Hepatocyte s carry out many metabolic functions, including the production of bile.
Also, modifications in the protocol of liver cell isolation, aimed at. Here we describe a method for the isolation and culture of mouse primary. When nonparenchymal cells were cultivated on collagen type icoated or uncoated dishes, clusters of small hepatocytelike cells appeared in 12 weeks. Density gradient centrifugation steps were used for removal of dead cells.
Coordinated interactions with hepatocytes induced id3 expression, whereas endothelial cells and stellate cells induced lxr. Potential for isolation of immortalized hepatocyte cell lines. This is accomplished by perfusing the liver with a type iv collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells. Isolation of primary human hepatocytes from normal and. The median hepatocyte viability by trypan blue exclusion was 52%, range, 1. Improved isolation of murine hepatocytes for in vitro. Protocol for isolation of primary human hepatocytes and. Xu ying xin institute of surgical research chinese pla general hospital, beijing harvardmit division of health sciences and technology hst. Pdf the use of isolated human liver cells in research and development. Hepatocyte isolation, culture, and its clinical applications synapse. Hepatocyte isolation system tissue dissociationcell. The morphology of the isolated primary human hepatocytes was assessed using tmrm mitochondrial stain 16 h post tissue isolation fig. Moreover, several publications report a percoll purification step in their protocol to recover liver cells after cryopreservation.
Survival of hepatocytes in suspension culture was dependent on fetal bovine serum concentration and temperature of incubation. Isolation of hepatocytes and their culture in vitro represent important avenues to explore the function of such cells. Primary hepatocytes provide scientists with a valuable tool for evaluating metabolic, biochemical, and molecular functions. An extensive selection of lots and multiple large lots are available ideal for reproducible longterm and multisite studies. Hepatocyte isolation system worthington enzyme manual. This study shows that the kupffer cell niche is composed of stellate cells, hepatocytes, and endothelial cells. Functional 3d human primary hepatocyte spheroids made by co. Rat hepatocyte and hepatic macrophage isolation simultaneous isolation of rat hepatocytes and hepatic macrophage hm is carried out using modified protocol of. Although an agent tends to cause initial damage in only one of these areas, the resulting disease may in time also involve other components. Human hepatocytes cell culture collagen fibronectin ecod ureogenesis. Isolation of human hepatocytes by a twostep collagenase. Arrival of the cryopreserved cells in your facilities place the cryogenic hepatocyte. If plating hepatocytes with an overlay, refer to the specification section for matrigel coating which will provide the protocol and technical tips. Methods and protocols guides readers through laboratory protocols for the generation of humanized livers for the assessment of biological actions in vivo and techniques to monitor cell engraftment after cell.
Human hepatocytes, cell isolation, hepatocyte culture. For this purpose, the sample must be encapsulated in glissons capsula on all areas except, obviously, the edge left by the surgeon. Cell transplants have been used to provide shortterm liver support to patients who were dying of their disease before a suitable organ could be found. In brief, the rat liver is perfused through portal. Asialoglycoprotein receptor 1 is a specific cellsurface. This paper describes an improved perfusiondissociation procedure to isolate hepatocytes from mouse liver that is suitable for malaria studies and allows reproducible preparation of primary hepatocytes with consistent cell yields and controlled purity. F experimental design for the isolation, expansion, and characterization of single cell hepatocytederived organoids.
Hepatocyte isolation system worthington biochemical. The protocol for mouse hepatocyte isolation may be applied to both normal and transgenic mice. Impact of percoll purification on isolation of primary human. Hepatocytes are the primary functional cells of the. The hadscs in spheroids could compensate for the low viability and improve the functional maintenance of hheps. Isolation and coculture of rat parenchymal and non. Rat hepatic stellate cells and hepatic macrophage isolation pronase and collagenase.
To evaluate the hepatocyte enrichment of these preparations we used facs analysis figure 2 and stained cells at different steps of the hepatocyte isolation procedure with anticd95 pe, cd45rbbiotin and streptavidinapc from bdpharmingen, the hepatocyte morphological gate was defined on basis of size and confirmed by the presence of high frequency of cd95 low cells. Although the clinical efficacy of hepatocyte transplantation has been demonstrated, the shortage of good quality donor livers for hepatocyte isolation and the lack of dependable methods of cryopreservation will limit widespread clinical application of this method until further research overcomes these problems. They have been validated with our hepatocyte products by our scientific team to deliver the highest. Hepatocyte isolation, culture, and its clinical applications. A modified twostep collagenase perfusion procedure for isolation of human hepatocytes is described. The choice of cell type is a critical parameter for any cell based therapy. Moderate increases in h2o2 production induced by hepatocyte isolation mediate stress adaptation and enhanced survival izak patrik miller 1, ivan pavlovic. A experimental scheme for the induction of recombination in ck19creertr26 yfp mice and isolation of ck19negative hepatocytes for in vitro culture. These technologies may benefit from the use of cloned and immortalized hepatocytes. Pdf a simple method for the simultaneous isolation of. Indeed, key factors that compromise the quality of the isolated hepatocytes include high liver fat content, prolonged warm ischemia andor storage of the organ.
Liver cell isolation is mainly performed using the twostep collagenase perfusion protocol. Isolation and identification of bovine primary hepatocytes. Although limited in number of data points, which should be taken into consideration, the conclusions of this study could be utilized as indications, allowing for expansion of liver selection criteria for hepatocyte isolation and provide the necessary quality hepatocytes in large quantities for the growing pharmaceutical, biomedical, and. Here, we proposed that a combination of transposon and liverdirected introduction of nucleic acids may confer acquisition of unlimited cellular proliferative potential on hepatocytes, enabling the possible isolation of immortalized hepatocyte cell lines, which has often failed using more traditional immortalization methods. Isolation of human hepatocytes from livers rejected for liver. Since it has been established that expression of hnf4. These methods for hepatocyte isolation have been modified and continuously improved and are now standard approaches for isolation of hepatocytes in cultures, in cell suspensions, and from tissues. By using the preoptimized combination of enzymes contained in this kit, it is possible to minimize the lottolot variation and improve the quality of the isolated hepatocytes.
The hepatocyte plates are one cell thick in mammals and two cells thick in the chicken. Serum concentrations of 5, 10, and 20% produced the highest survival during primary culture. The endothelial cells have no basement membrane and are separated from the hepatocytes by the space of disse, which drains lymph into the portal tract lymphatics. Several methods for hepatocyte isolation have been published. Jul 17, 2017 as a readout of hepatocyte function the amount of urea in the cell medium was measured and all hepatocyte isolations measured had urea in the media above background concentrations fig. Hepatic cells, human hepatocytes and nonhuman hepatic cells. The method based on twostep collagenase perfusion for isolation. Protocol for isolation of primary human hepatocytes and corresponding major populations of nonparenchymal liver cells. Hence, there is a significant effort to develop hepatocyte cell lines for this purpose. Following isolation, the cells can either be used for shortterm studies or, alternatively, maintained in culture for prolonged periods to study longterm changes in gene expression. Minimum leakage if any must occur during perfusion. These methods for hepatocyte isolation have been modified and continuously improved and are now standard approaches for isolation of hepatocytes in cultures, in cell suspensions, and. Review article potential for isolation of immortalized hepatocyte cell lines by liverdirected in vivo gene delivery of transposons in mice masahirosato,1 isseisaitoh,2 emiinada,3. Moderate increases in h2o2 production induced by hepatocyte isolation mediate stress adaptation and enhanced survival.
This method can also be applied to other mammalian livers. Isolation, characterization, and differentiation to. For example, primary culture is used to assess the expression and function of drugmetabolizing enzymes including cytochromes p450, drug metabolism, drugdrug interactions, and the mechanisms of cytotoxicity and genotoxicity 37. The isolated cells can be used not only in research but also for transplantation in patients with liver disease, especially acute liver failure and liverbased metabolicsynthetic conditions. Pdf isolation and culture of adult mouse hepatocytes. Thus, although viral hepatitis inflammation of the liver. I have watched your viedo about isolation and primary culture of rat hepatic cells. Rat hepatocyte and hepatic macrophage isolation simultaneous isolation of rat hepatocytes and hepatic macrophage hm is carried out using modified protocol of collagenase digestion 1, 2.
An initial high quality cell suspension after isolation remains essential prior to cryopreservation. Our experience highlights that the isolation of primary human hepatocytes from normal or diseased livers is a viable proposition given appropriate methods. I have watched your viedo about isolation and primary culture of rat hepatic cells which was perfectly made. Development, characterization and isolation of small. Our hepatic cell culture reagents will ensure the success of your metabolism studies. Characterization of nitric oxide production following.
Stellate cells, hepatocytes, and endothelial cells imprint. Primary hepatocytes provide scientists with a valuable tool for evaluating metabolic, biochemical, and molecular functions in a physiologically relevant, readily controlled in vitro experimental system. Here we describe a method for the isolation and culture of mouse. This requires surgery on the lab animal, a perfusion with buffer solution, a digestion with collagenase and a filtration step to obtain primary hepatocytes. The use of human liver samples for hepatocyte preparation for scientific. Primary hepatocyte isolation protocols ucla center for. Gibco hepatocytes offer high viabilities routinely great than 80%, in vivolike enzyme expression levels, and are characterized for phase i and phase ii drug metabolizing enzyme activities. Sinusoids display a discontinuous, fenestrated endothelial cell lining.
However, the isolation of human liver cells requires a welldeveloped logistic network among surgeons, biologists, and technicians to obtain a high quality of cells. The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. We assessed the association between the total liver tissue weight and total viable hepatocyte cell yield following isolation and found a positive correlation between tissue weight and hepatocyte yield r s 0. Mar 29, 2011 we know of no other studies which evaluate and compare the outcome of human hepatocytes isolation from all sources, including endstage liver disease, using one standard isolation protocol. Improved isolation of murine hepatocytes for in vitro malaria. Protocol for isolation of primary human hepatocytes. Purification of hepatocytes and sinusoidal endothelial. Primary hepatocyte isolation protocols ucla center. Dissolve collagenase for 3060min before digestion, temperature 37c not to go over 42 c, also not necessary to use higher than 37 c rinse pump with 70% alcohol, sterile water and perfusion buffer. The worthington hepatocyte isolation system has been developed to provide researchers with a reliable, convenient, and consistent hepatocyte cell isolation system. Rat hepatocyte isolation specialty media thermo fisher. The isolation of primary hepatocytes from human tissue. We differentiated multiple human embryonic stem cell hesc and human induced pluripotent stem cell hipsc lines representing a range of hlc differentiation propensities and characterized the expression of hepatocyte markers among cells positive for surface asgr1.