This work is supported in part by MOE ARC (MOE2017-T2-1-149); NUHS Innovation Seed Grant 2017 (NUHSRO/2017/051/InnovSeed/02); Mechanobiology Institute of Singapore (R-714-106-004-135); and Institute of Bioengineering and Nanotechnology, Biomedical Research Council, Agency for Science, Technology and Research (A*STAR) (Project Numbers IAF-PP H18/01/a0/014, IAF-PP H18/01/a0/K14 and MedCaP-LOA-18-02) funding to Hanry Yu. Ng Chan Way is a research scholar of the National University of Singapore. We would like to thank Confocal Microscopy Unit &#38; Flow Cytometry Unit of the National University of Singapore for help and advice in hepatocyte purity analysis.

<ol>
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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subpopulations+of+rat+hepatocytes+separated+by+Percoll+density-gradient+centrifugation+show+characteristics+consistent+with+different+acinar+locations.">Subpopulations of rat hepatocytes separated by Percoll density-gradient centrifugation show characteristics consistent with different acinar locations.</a> The Biochemical Journal. 304, 617-624 (1994).</li><li>Beal, E. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+animal+model+of+ex+vivo+normothermic+liver+perfusion.">A small animal model of ex vivo normothermic liver perfusion.</a> Journal of Visualized Experiments: JoVE. 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Zhou Yan and Hanry Yu declare competing interests as they hold equity in Vasinfuse, which manufactures and markets the Integrated Perfusion System. Hanry Yu holds equity in Histoindex, Invitrocue, Osteopore, Pishon Biomedical, Ants Innovate, and Synally Futuristech that have no competing interests with the information reported here.

There are a few points that are particularly important to observe for two-step collagenase perfusion procedure in general. Firstly, special care must be given when resecting the liver. Ensure that the gastrointestinal tract is not damaged as leakage of the contents will result in bacterial contamination. In addition, avoid damaging the Glisson&#39;s capsule, which covers the surface of the liver during the animal procedure. If the tear is large enough, it might allow premature release of disassociated hepatocytes into th...

Primary hepatocytes are important tools for liver-related basic research, disease treatment, and application such as drug testing. The current gold standard for primary hepatocyte isolation is the two-step collagenase perfusion procedure1,2,3 introduced by Seglen in the 1970s4. However, this procedure is technically challenging and has a high failure rate when performed by novice surgeons. Even when a perfusion is considered successful, drastic differences in hepatocyte viability (typically 60%-95%) and yield (0.5-5 x 108 per 200-300 g rat) may be observed between isolations. This influences the quality and scale of downstream experiments. Apart from the technical procedure, the perfusion setup used for the isolation, either commercially available or custom built, is a contributing factor. Attention must be given to the assembly, optimization, and maintenance of the perfusion setup. The purpose of this protocol is to improve the success rate and stability between isolations of primary rat hepatocytes through multiparameter perfusion control of the technical procedure and perfusion setup of the two-step collagenase perfusion procedure.
From the technical aspect, the most difficult step in the procedure is the portal vein cannulation. As for the other steps, if good practice is observed and general precautions are taken, the stability of the isolation can be improved. Therefore, understanding of the reasoning for each step is important so that the surgeon could respond to various variables that may occur during the procedure.
Various protocols for the isolation of hepatocytes and liver non-parenchymal cells from rat and mouse have been published1,2,5,6,7,8,9. The perfusion setups used in these protocols had several disadvantages, which include the reuse of perfusion tubing, problems with temperature control, need for routine optimization of perfusion parameters, and/or usage of unsuitable type of intravenous (IV) catheter for portal vein cannulation. The reuse of perfusion tubing will increase the chances of contamination, especially if the tubing was not cleaned and disinfected properly. Reuse of tubing without routine replacement will also expose the perfusion setup to problems such as leaky tubing or connectors, clogged bubble trap and constricted tubing, all of which will substantially reduce the perfusate pressure and flow rate, thus, affecting liver digestion efficiency. Without a constant heat source in some setups for temperature control, pre-warmed buffers will cool down over time, leading to low collagenase activity and digestion. Although other setups utilize a jacketed glass condenser connected to a water circulator to warm the buffer, they are bulky and require careful cleaning. Temperature, pressure, and flow rate of buffer exiting the catheter must be measured and optimized before the start of isolation toensure stable perfusion condition. Even after optimization, the parameters could still change halfway during isolation due to the actions of the operator, thereby leading to suboptimal perfusion and digestion. Most types of IV catheter are not suitable for portal vein cannulation because they do not allow continuous perfusion during cannulation. They are unable to immediately inform the surgeon when the cannulation is successful. Furthermore, it is challenging to secure the portal vein on the soft catheter without deforming it.
Here, we address these problems using standardized disposable sterile tubing, a silicone heater jacket for precise and stable temperature control, real-time monitoring and alarm system with data storage and management and use of a special IV catheter, which allows continuous perfusion while puncturing portal vein during cannulation. To the best of our knowledge, we are the first group to combine all these features into an integrated perfusion system (IPS) that is compact, making it highly portable and able to be fit into a laminar flow hood to ensure aseptic operation.

<table><tbody><tr><td>&lt;strong&gt;Material/Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1 mL syringe</td><td>Nipro</td><td></td><td></td></tr><tr><td>27G needle</td><td>Nipro</td><td></td><td></td></tr><tr><td>Black braided silk non-absorbable, non-sterile surgical suture</td><td>Look</td><td>SP117</td><td></td></tr><tr><td>Bochem 18/10 stainless steel forceps, sharp tip contain bent round tip</td><td>Bochem</td><td>10333511</td><td></td></tr><tr><td>Disposable Perfusion Set</td><td>Vasinfuse</td><td>BPF-112</td><td></td></tr><tr><td>Floating circular 1.5 mL microcentrifuge tube rack</td><td>Sigma-Aldrich</td><td>R3133</td><td></td></tr><tr><td>German Standard Tissue Forceps, Serrated / 1&amp;times;2 teeth , 14.5cm</td><td>Walentech</td><td></td><td></td></tr><tr><td>Greiner Cellstar aspirating pipette</td><td>Merck</td><td>GN710183</td><td></td></tr><tr><td>Haemocytometer</td><td></td><td></td><td></td></tr><tr><td>Integrated Perfusion System</td><td>Vasinfuse</td><td>IPS-001</td><td></td></tr><tr><td>Iris Scissors curved, stainless, 11cm</td><td>Optimal Medical Products Pte Ltd</td><td>CVD</td><td></td></tr><tr><td>Light microscope with 10X lens</td><td>Olympus</td><td></td><td></td></tr><tr><td>Mesh Sheet 100&amp;micro;M Nylon</td><td>Spectra-Teknic(s) Pte Ltd</td><td>06630-75</td><td></td></tr><tr><td>Operating Scissors, BL/BL, 13cm</td><td>Optimal Medical Products Pte Ltd</td><td>STR &amp;ndash; BL/BL</td><td></td></tr><tr><td>Operating Scissors, SH/BL, 13cm</td><td>Optimal Medical Products Pte Ltd</td><td>STR &amp;ndash; SH/BL</td><td></td></tr><tr><td>Reverse force hemostatic clip</td><td>Shanghai Jin Zhong Pte Ltd</td><td>XEC230</td><td></td></tr><tr><td>Water bath</td><td>Grant</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Reagents/Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10X Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A9056</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck</td><td>137101</td><td></td></tr><tr><td>Collagenase Type IV</td><td>Gibco</td><td>17104019</td><td></td></tr><tr><td>Dexamethasone</td><td>TCI</td><td>D1961</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>31600-034</td><td></td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050061</td><td></td></tr><tr><td>HEPES</td><td>Invitrogen</td><td>11344-041</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>1-9278</td><td></td></tr><tr><td>KCl</td><td>VWR</td><td>VWRC26764.298</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5379</td><td></td></tr><tr><td>Linoleic acid</td><td>Sigma-Aldrich</td><td>L9530</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S5886</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S8875</td><td></td></tr><tr><td>NaOH</td><td>Merck</td><td>106462</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>Type I bovine collagen</td><td>Advanced BioMatrix</td><td>5005-100ml</td><td></td></tr><tr><td>William&amp;rsquo;s E Media</td><td>Sigma-Aldrich</td><td>W1878</td><td></td></tr></tbody></table>

isolation of primary rat hepatocytes with multiparameter perfusion control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

A surgeon could tell whether liver perfusion is going on smoothly by observing the outcome after certain steps. The first outcome can be observed upon cannulation, cutting of the infrahepatic IVC, and restoring the perfusion flow rate. The liver should have completely changed color from dark red to brown, while maintaining its volume. If the liver looks slightly deflated and has a reddish tint or blotches of red, it means that the perfusion flow rate was set wrongly (too low), or the portal vein was not cannulated correc...

Watch this Scientific Journal Video about Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control at JoVE.com

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

isolation-primary-rat-hepatocytes-with-multiparameter-perfusion

Research

JoVE Journal

Biology

4.6K Views.  National University of Singapore. Dit isolatieprotocol voor rattenhepatocyten is gebaseerd op de leverresectie voorafgaand aan de verteringsbenadering. Het protocol introduceert een compact en draagbaar geïntegreerd perfusiesysteem voor celisolatie. Deze techniek omvat collagenase-recirculatie die het volume van collagenasegebruik vermindert en meer flexibiliteit biedt bij het verlengen van de verteringstijd, terwijl een hoge zwaartekrachtopbrengst en hepatocytenspecifieke functie worden gegarandeerd.Zet de rat in de ...

Video: Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

Biochemistry

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Immunology and Infection

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Isolation and Primary Culture of Rat Hepatic Cells

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. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Medicine

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

In Situ Mouse Liver Perfusion Model to Study Metabolic Responses to Substrates

Hepatic Glucose Production, Ureagenesis, and Lipolysis Quantified using the Perfused Mouse Liver Model

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused liver allows the study of liver biology and metabolism in the whole liver with an intact hepatic architecture, separated from the influence of extra-hepatic factors. Liver perfusions were originally developed for rats, but the method has been adapted to mice as well. Here we describe a protocol for in situ perfusion of the mouse liver. The liver is perfused antegradely through the portal vein with oxygenated Krebs-Henseleit bicarbonate buffer, and the output is collected from the suprahepatic inferior vena cava with clamping of the infrahepatic inferior vena cava to close the circuit. Using this method, the direct hepatic effects of a test compound can be evaluated with a detailed time resolution. Liver function and viability are stable for at least 3 h, allowing the inclusion of internal controls in the same experiment. The experimental possibilities using this model are numerous and may infer insight into liver physiology and liver diseases.

Author Spotlight: Investigating Hepatic Adaptations and Prediabetic Progression in Liver Diseases 

Here, we present a robust method for in situ perfusion of the mouse liver to study the acute and direct regulation of liver metabolism without disturbing the hepatic architecture but in the absence of extra-hepatic factors.

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

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