Name: an improved time- and labor- efficient protocol for mouse primary hepatocyte isolation
Uploaded: 2021-10-25T13:00:00.000+00:00
Duration: 5 min 42 s
Description: Watch this Scientific Journal Video about An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation at JoVE.com

This work was supported by the National Institutes of Health (Grant 5R01HD095512-02 to S.W.).

All procedures were approved by the Johns Hopkins Animal Care and Use Committee. C57BL/6 female mice (8-week old) were used in this study.
1. Preparation:
<ol>
	<li>Mix William&#39;s E Medium (GlutaMAX Supplement) with 10% FBS and 1% antibiotic-antimycotic solution to make the culture medium.</li>
	<li>Filter 25 mL of collagenase-dispase medium (e.g., Liver Digest Medium) through a 0.45 &#181;m syringe filter to remove particle debris.</li>
	<li>Warm 50 mL of double-distilled H2O (ddH2O), 35 mL of perfusion Medium (e.g., Liver Perfusion Medium) (or 50 mL at the first time using this protocol), and 25 mL of filtered collagenase-dispase medium in a 45 &#176;C water bath for 30 min.</li>
	<li>Within a sterile tissue culture hood, mix 2 mL of 10x HBSS and 18 mL of Percoll in a 50 mL tube to make 20 mL 1x Percoll-HBSS and keep on ice or 4 &#176;C. 
	NOTE: 1x Percoll-HBSS can be kept at 4 &#176;C for at least 6 months.</li>
	<li>Within a sterile tissue culture hood, pour 30 mL of wash medium (e.g., Hepatocyte Wash Medium) into a clean Petri dish, and keep on ice.</li>
	<li>Submerge the pumping tube in the water of a 45 &#176;C water bath. Results are most reliable if the room temperature is at 25 &#176;C.</li>
	<li>Prepare 2 mL of 1x anesthetics by mixing 225 &#181;L of Ketamine HCL, 93.75 &#181;L of Xylazine, and 1681 &#181;L of 1x PBS.</li>
	<li>Anesthetize one mouse using an approved method. Here the mouse was intraperitoneally injected with 150 &#181;L of 1x anesthetics. Perform the tests for loss of reflexes such as reaction to toe pinching to ensure full anesthesia.</li>
	<li>Secure the mouse on its back onto the dissection pad by four limbs, by either pining or using water-proof tape or other methods approved by the institution&#39;s Animal Care and Use Committee (or equivalents).</li>
	<li>Prepare sterilized forceps and scissors for dissection. To avoid possible contamination, conduct all steps within a sterile hood.</li>
</ol>
2. Procedure:
<ol>
	<li>Using a peristaltic pump, start pumping warmed-up ddH2O at a speed of 4 mL/min for 5 min. Change the pumping tube from water to a warmed-up perfusion medium.</li>
	<li>Disinfect the anesthetized mouse&#39;s abdomen with 70% EtOH and cut open with a scissor to expose the liver, portal vein, and inferior vena cava (IVC).</li>
	<li>Stop the peristaltic pump. Insert a 24 G catheter (e.g., Closed IV Catheter, 24 G, 0.75 IN) into IVC. Start pumping and cut the portal vein open.</li>
	<li>Continue pumping until the flushed-out liquid is clear (around 3-5 min). Press the portal vein every minute to let liquid reach every corner of the liver. Be careful not to let air bubbles enter the IVC. 
	NOTE: This step is to flush out as much blood as possible from the liver.</li>
	<li>Change the pumping tube from the perfusion medium to the collagenase-dispase medium. Continue pumping until all 25 mL of the collagenase-dispase medium is depleted while doing step 2.6.</li>
	<li>Press the portal vein every minute to let liquid reach every corner of the liver. 
	NOTE: At this stage, the complete loss of blood is fatal to the mouse. The death of the mouse can be confirmed by a lack of heartbeat after the experiment. Dispose of the carcass as per facility policies.</li>
	<li>Isolate the whole liver out without gallbladder to the 30 mL wash medium in the Petri dish on ice.</li>
	<li>Tear it up into pieces with forceps to release primary hepatocytes into solution. This step would turn the wash medium into a cloudy solution full of released primary hepatocytes and small liver pieces.</li>
	<li>Filter the cloudy solution in step 2.8 through a 70 &#181;m cell strainer into the 20 mL 1x Percoll-HBSS in a 50 mL tube on ice. Mix by inverting the tube 20 times.</li>
	<li>Centrifuge at 300 x g for 10 min at 4 &#176;C.</li>
	<li>Within the tissue culture hood, aspirate the supernatant. Wash the pellet with cold 30 mL of wash medium.</li>
	<li>Centrifuge at 50 x g for 5 min at 4 &#176;C.</li>
	<li>Remove the supernatant and resuspend the pellet in 25 mL of the culture medium (or appropriate other volumes) within the tissue culture hood.</li>
	<li>Count the cell number and plate the cells on desired culture plates according to the experimental design. 
	NOTE: Primary hepatocytes properly isolated from one 8-week-old mouse are usually sufficient to be plated on four 6-well plates or four 12-well plates.</li>
</ol>

<ol>
	<li>Han, H. S., Kang, G., Kim, J. S., Choi, B. H., Koo, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+glucose+metabolism+from+a+liver-centric+perspective.">Regulation of glucose metabolism from a liver-centric perspective.</a> Experimental and Molecular Medicine. 48, 218 (2016).</li><li>Severgnini, M., 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+rapid+two-step+method+for+isolation+of+functional+primary+mouse+hepatocytes:+cell+characterization+and+asialoglycoprotein+receptor+based+assay+development.">A rapid two-step method for isolation of functional primary mouse hepatocytes: cell characterization and asialoglycoprotein receptor based assay development.</a> Cytotechnology. 64 (2), 187-195 (2012).</li><li>Guidotti, J. E., 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=Liver+cell+polyploidization:+a+pivotal+role+for+binuclear+hepatocytes.">Liver cell polyploidization: a pivotal role for binuclear hepatocytes.</a> Journal of Biological Chemistry. 278 (21), 19095-19101 (2003).</li><li>Morales-Navarrete, H., 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+versatile+pipeline+for+the+multi-scale+digital+reconstruction+and+quantitative+analysis+of+3D+tissue+architecture.">A versatile pipeline for the multi-scale digital reconstruction and quantitative analysis of 3D tissue architecture.</a> Elife. 4, 11214 (2015).</li><li>Bruening, J., 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=Hepatitis+C+virus+enters+liver+cells+using+the+CD81+receptor+complex+proteins+calpain-5+and+CBLB.">Hepatitis C virus enters liver cells using the CD81 receptor complex proteins calpain-5 and CBLB.</a> PLoS Pathogens. 14 (7), 1007111 (2018).</li><li>Silvie, O., 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=Hepatocyte+CD81+is+required+for+Plasmodium+falciparum+and+Plasmodium+yoelii+sporozoite+infectivity.">Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity.</a> Nature Medicine. 9 (1), 93-96 (2003).</li><li>Crispe, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocytes+as+immunological+agents.">Hepatocytes as immunological agents.</a> Journal of Immunology. 196 (1), 17-21 (2016).</li><li>Liu, N. 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=Loss+of+TR4+orphan+nuclear+receptor+reduces+phosphoenolpyruvate+carboxykinase-mediated+gluconeogenesis.">Loss of TR4 orphan nuclear receptor reduces phosphoenolpyruvate carboxykinase-mediated gluconeogenesis.</a> Diabetes. 56 (12), 2901-2909 (2007).</li><li>Wang, Z., 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=Gonadotrope+androgen+receptor+mediates+pituitary+responsiveness+to+hormones+and+androgen-induced+subfertility.">Gonadotrope androgen receptor mediates pituitary responsiveness to hormones and androgen-induced subfertility.</a> JCI Insight. 5 (17), 127817 (2019).</li><li>Santos, S. D., 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=CSF+transthyretin+neuroprotection+in+a+mouse+model+of+brain+ischemia.">CSF transthyretin neuroprotection in a mouse model of brain ischemia.</a> Journal of Neurochemistry. 115 (6), 1434-1444 (2010).</li><li>Pinhu, L., 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=Overexpression+of+Fas+and+FasL+is+associated+with+infectious+complications+and+severity+of+experimental+severe+acute+pancreatitis+by+promoting+apoptosis+of+lymphocytes.">Overexpression of Fas and FasL is associated with infectious complications and severity of experimental severe acute pancreatitis by promoting apoptosis of lymphocytes.</a> Inflammation. 37 (4), 1202-1212 (2014).</li><li>Mi, Y., Lin, A., Fiete, D., Steirer, L., Baenziger, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+mannose+and+asialoglycoprotein+receptor+expression+determines+glycoprotein+hormone+half-life+at+critical+points+in+the+reproductive+cycle.">Modulation of mannose and asialoglycoprotein receptor expression determines glycoprotein hormone half-life at critical points in the reproductive cycle.</a> Journal of Biological Chemistry. 289 (17), 12157-12167 (2014).</li><li>Tanowitz, M., 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=Asialoglycoprotein+receptor+1+mediates+productive+uptake+of+N-acetylgalactosamine-conjugated+and+unconjugated+phosphorothioate+antisense+oligonucleotides+into+liver+hepatocytes.">Asialoglycoprotein receptor 1 mediates productive uptake of N-acetylgalactosamine-conjugated and unconjugated phosphorothioate antisense oligonucleotides into liver hepatocytes.</a> Nucleic Acids Research. 45 (21), 12388-12400 (2017).</li><li>Manczak, M., 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=Neutralization+of+granulocyte+macrophage+colony-stimulating+factor+decreases+amyloid+beta+1-42+and+suppresses+microglial+activity+in+a+transgenic+mouse+model+of+Alzheimer's+disease.">Neutralization of granulocyte macrophage colony-stimulating factor decreases amyloid beta 1-42 and suppresses microglial activity in a transgenic mouse model of Alzheimer's disease.</a> Human Molecular Genetics. 18 (20), 3876-3893 (2009).</li><li>Zhang, Y., 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=JAK1-dependent+transphosphorylation+of+JAK2+limits+the+antifibrotic+effects+of+selective+JAK2+inhibitors+on+long-term+treatment.">JAK1-dependent transphosphorylation of JAK2 limits the antifibrotic effects of selective JAK2 inhibitors on long-term treatment.</a> Annals of the Rheumatic Diseases. 76 (8), 1467-1475 (2017).</li><li>Shih, S. 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=Molecular+profiling+of+angiogenesis+markers.">Molecular profiling of angiogenesis markers.</a> American Journal of Pathology. 161 (1), 35-41 (2002).</li><li>Liu, G., 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=miR-147,+a+microRNA+that+is+induced+upon+Toll-like+receptor+stimulation,+regulates+murine+macrophage+inflammatory+responses.">miR-147, a microRNA that is induced upon Toll-like receptor stimulation, regulates murine macrophage inflammatory responses.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (37), 15819-15824 (2009).</li><li>Ding, Q., 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=Mice+expressing+minimally+humanized+CD81+and+occludin+genes+support+Hepatitis+C+virus+uptake+in+vivo.">Mice expressing minimally humanized CD81 and occludin genes support Hepatitis C virus uptake in vivo.</a> Journal of Virology. 91 (4), 01799 (2017).</li><li>Renshaw, M., 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=Cutting+edge:+impaired+Toll-like+receptor+expression+and+function+in+aging.">Cutting edge: impaired Toll-like receptor expression and function in aging.</a> Journal of Immunology. 169 (9), 4697-4701 (2002).</li><li>Li, W. C., Ralphs, K. L., Tosh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+mouse+hepatocytes.">Isolation and culture of adult mouse hepatocytes.</a> Methods in Molecular Biology. 633, 185-196 (2010).</li><li>Salem, E. S. B., 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=Isolation+of+primary+mouse+hepatocytes+for+nascent+protein+synthesis+analysis+by+non-radioactive+l-azidohomoalanine+labeling+method.">Isolation of primary mouse hepatocytes for nascent protein synthesis analysis by non-radioactive l-azidohomoalanine labeling method.</a> Journal of Visualized Experiments: JoVE. (140), e58323 (2018).</li><li>Cabral, F., 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=Purification+of+Hepatocytes+and+Sinusoidal+Endothelial+Cells+from+Mouse+Liver+Perfusion.">Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion.</a> Journal of Visualized Experiments: JoVE. (132), e56993 (2018).</li><li>Korelova, K., Jirouskova, M., Sarnova, L., Gregor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+3D+collagen+sandwich+culture+of+primary+mouse+hepatocytes+to+study+the+role+of+cytoskeleton+in+bile+canalicular+formation+in+vitro.">Isolation and 3D collagen sandwich culture of primary mouse hepatocytes to study the role of cytoskeleton in bile canalicular formation in vitro.</a> Journal of Visualized Experiments: JoVE. (154), e60507 (2019).</li><li>Goncalves, L. A., Vigario, A. M., Penha-Goncalves, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+isolation+of+murine+hepatocytes+for+in+vitro+malaria+liver+stage+studies.">Improved isolation of murine hepatocytes for in vitro malaria liver stage studies.</a> Malar Journal. 6, 169 (2007).</li><li>Yin, Z., Ellis, E. C., Nowak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mouse+hepatocytes+for+transplantation:+a+comparison+between+antegrade+and+retrograde+liver+perfusion.">Isolation of mouse hepatocytes for transplantation: a comparison between antegrade and retrograde liver perfusion.</a> Cell Transplant. 16 (8), 859-865 (2007).</li><li>. Hepatocyte media, Thermofisher Available from: <a target="_blank" href="https://assets.thermofisher.com/TFS-Assets/LSG/manuals/Hepatocyte_Media_UG.pdf">https://assets.thermofisher.com/TFS-Assets/LSG/manuals/Hepatocyte_Media_UG.pdf</a> (2021)</li><li>Sia, D., Villanueva, A., Friedman, S. L., Llovet, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+cancer+cell+of+origin,+molecular+class,+and+effects+on+patient+prognosis.">Liver cancer cell of origin, molecular class, and effects on patient prognosis.</a> Gastroenterology. 152 (4), 745-761 (2017).</li><li>Freitas-Lopes, M. A., Mafra, K., David, B. A., Carvalho-Gontijo, R., Menezes, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+location+and+distribution+of+hepatic+immune+cells.">Differential location and distribution of hepatic immune cells.</a> Cells. 6 (4), 48 (2017).</li><li>Schachtrup, C., Le Moan, N., Passino, M. A., Akassoglou, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells+and+astrocytes:+Stars+of+scar+formation+and+tissue+repair.">Hepatic stellate cells and astrocytes: Stars of scar formation and tissue repair.</a> Cell Cycle. 10 (11), 1764-1771 (2011).</li><li>Geerts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History,+heterogeneity,+developmental+biology,+and+functions+of+quiescent+hepatic+stellate+cells.">History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells.</a> Seminars in Liver Disease. 21 (3), 311-335 (2001).</li><li>Poisson, J., 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=Liver+sinusoidal+endothelial+cells:+Physiology+and+role+in+liver+diseases.">Liver sinusoidal endothelial cells: Physiology and role in liver diseases.</a> Journal of Hepatology. 66 (1), 212-227 (2017).</li><li>Peter, M. E., 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=The+CD95+receptor:+apoptosis+revisited.">The CD95 receptor: apoptosis revisited.</a> Cell. 129 (3), 447-450 (2007).</li><li>Peters, D. T., 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=Asialoglycoprotein+receptor+1+is+a+specific+cell-surface+marker+for+isolating+hepatocytes+derived+from+human+pluripotent+stem+cells.">Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells.</a> Development. 143 (9), 1475-1481 (2016).</li><li>Willoughby, J. L. S., 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=Evaluation+of+GalNAc-siRNA+conjugate+activity+in+pre-clinical+animal+models+with+reduced+asialoglycoprotein+receptor+expression.">Evaluation of GalNAc-siRNA conjugate activity in pre-clinical animal models with reduced asialoglycoprotein receptor expression.</a> Molecular Therapy. 26 (1), 105-114 (2018).</li><li>Hutchins, N. A., Chung, C. S., Borgerding, J. N., Ayala, C. A., Ayala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kupffer+cells+protect+liver+sinusoidal+endothelial+cells+from+Fas-dependent+apoptosis+in+sepsis+by+down-regulating+gp130.">Kupffer cells protect liver sinusoidal endothelial cells from Fas-dependent apoptosis in sepsis by down-regulating gp130.</a> American Journal of Pathology. 182 (3), 742-754 (2013).</li></ol>

The authors have nothing to disclose.

Various primary hepatocyte isolation protocols have been developed. They also have been kept optimized and adapted based on different needs (Table 1). Isolation protocols are generally composed of two parts: perfusion (including enzyme digestion) and purification.
The perfusion can be performed with the entire liver in vivo2,20,21,22,</...

The liver serves as one of the most important organs in the vertebrate body due to the vital role it plays in numerous life-supporting functions like food digestion, blood circulation, and detoxification. Usage of mouse primary hepatocyte in vitro culture is increasingly popular in studies of carbohydrate metabolism and hepatic carcinoma. Therefore, it is important to develop a convenient method for mouse primary hepatocyte isolation while maintaining its innate physiological function. Due to its function as a hub of glucose metabolism, the liver is also central to glucose production and storage1. Experiments with primary hepatocytes in vitro are a must to most glucose metabolism studies. Therefore, for years, various research groups have developed protocols for mouse primary hepatocyte isolation.
The general procedure of mouse hepatocyte isolation is to first flush out blood in the liver with an isosmotic liquid such as phosphate-buffered saline (PBS) or Hanks&#39; Balanced Salt Solution (HBSS) and then use collagenase-containing solution to dissociate hepatocytes. These protocols share a general procedure but differ in reagents and steps based on different needs. However, preparing required reagents and performing isolation steps take time. In developing the present protocol, efficiency was set as a priority, with all reagents ready-to-use and available from the market, and as few steps as possible. The overall goal of this protocol is to provide a fast and labor-efficient method to isolate primary hepatocytes from mouse, without jeopardizing the isolated primary hepatocyte purity and viability.

<table><tbody><tr><td>1x PBS</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>10x HBSS</td><td>Gibco</td><td>14065-056</td><td></td></tr><tr><td>12-well Plate</td><td>FALCON</td><td>353043</td><td>Coating not required</td></tr><tr><td>6-well Plate</td><td>FALCON</td><td>353046</td><td>Coating not required</td></tr><tr><td>anti-AKT</td><td>Cell Signaling</td><td>2920S</td><td></td></tr><tr><td>Antibiotic Antimycotic Solution (100x), Stabilized</td><td>Sigma-Aldrich</td><td>A5955</td><td></td></tr><tr><td>anti-FOXO1</td><td>Cell Signaling</td><td>97635S</td><td></td></tr><tr><td>anti-GAPDH</td><td>Cell Signaling</td><td>2118S</td><td></td></tr><tr><td>anti-p-AKT (S473)</td><td>Cell Signaling</td><td>9271L</td><td></td></tr><tr><td>anti-PEPCK</td><td>Santa Cruz</td><td>SC-166778</td><td></td></tr><tr><td>anti-p-FOXO1 (S256)</td><td>Cell Signaling</td><td>84192S</td><td></td></tr><tr><td>Cell Strainer, 70 &amp;micro;m</td><td>CELLTREAT</td><td>229483</td><td></td></tr><tr><td>Closed IV Catheter, 24 Gauge 0.75 IN</td><td>Becton Dickinson</td><td>383511</td><td></td></tr><tr><td>DMEM, no glucose, no glutamine, no phenol red</td><td>ThermoFisher Scientific</td><td>A1443001</td><td></td></tr><tr><td>EnzyChrom Glucose Assay Kit</td><td>BioAssay Systems</td><td>EBGL-100</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Hyclone</td><td>SH30071.03</td><td></td></tr><tr><td>Forskolin</td><td>MilliporeSigma</td><td>F3917-10MG</td><td></td></tr><tr><td>Glucagon</td><td>Sigma-Aldrich</td><td>G2044</td><td></td></tr><tr><td>Goat Anti-mouse IgG Secondary Antibody</td><td>LI-COR</td><td>926-68070</td><td></td></tr><tr><td>Goat Anti-rabbit IgG Secondary Antibody</td><td>LI-COR</td><td>926-32211</td><td></td></tr><tr><td>GraphPad Prism 8</td><td>GraphPad Software</td><td>NA</td><td></td></tr><tr><td>Hepatocyte Wash Medium</td><td>Gibco</td><td>17704-024</td><td></td></tr><tr><td>IBMX</td><td>Cell Signaling</td><td>13630S</td><td></td></tr><tr><td>Insulin</td><td>Lilly</td><td>NDC 0002-8215-01</td><td></td></tr><tr><td>Ketamine HCL (100 mg/mL)</td><td>Hospira Inc</td><td>NDC 0409-2051-05</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030081</td><td></td></tr><tr><td>Liver Digest Medium</td><td>Gibco</td><td>17703-034</td><td>Aliquot within tissue culture hood to 25 mL each in 50 mL tube, and keep in -20 &amp;deg;C freezer</td></tr><tr><td>Liver Perfusion Medium</td><td>Gibco</td><td>17701-038</td><td></td></tr><tr><td>Pen Strep</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-01</td><td></td></tr><tr><td>Peristaltic Pump</td><td>Gilson</td><td>Minipuls 2</td><td>Capable of pumping at 4 mL/min</td></tr><tr><td>Petri Dish</td><td>Fisherbrand</td><td>08-757-12</td><td></td></tr><tr><td>Refrigerated Centrifuge</td><td>Sorvall</td><td>Legend RT</td><td>Capable to centrifuge 50 mL tube at 4 &amp;deg;C</td></tr><tr><td>Sodium L-Lactate</td><td>Sigma-Aldrich</td><td>L7022</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Gibco</td><td>11360070</td><td></td></tr><tr><td>Syringe Filter, PVDF 0.45 &amp;micro;m 30mm diameter</td><td>CELLTREAT</td><td>229745</td><td></td></tr><tr><td>Syringe, 0.5 mL</td><td>Becton Dickinson</td><td>329461</td><td></td></tr><tr><td>Syringe, 60 mL</td><td>Becton Dickinson</td><td>309653</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Gibco</td><td>15250061</td><td></td></tr><tr><td>Tube, 15 mL</td><td>Corning</td><td>430052</td><td></td></tr><tr><td>Tube, 50 mL</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>Water Bath Tank</td><td>Corning</td><td>CLS6783</td><td>Or any water bath tank capable of heating up to 45 &amp;deg;C</td></tr><tr><td>William&amp;rsquo;s E Medium (GlutaMAX Supplement)</td><td>Gibco</td><td>32551020</td><td></td></tr><tr><td>Xylozine (100 mg/mL)</td><td>Vetone Anased LA</td><td>NDC13985-704-10</td><td></td></tr></tbody></table>

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.

In order to test the efficiency, the present primary hepatocyte isolation protocol was performed on 8-week old female C57BL/6 mice. The attachment and purity of isolated primary hepatocytes were tested. Primary hepatocyte isolation is used for a wide variety of experiments on liver physiology, such as hepatic drug effects and glucose metabolism, pharmaceutical biomarker activity2, insulin sensitivity, and glucose production. Therefore, the activities of the primary hepatocytes isolated with this p...

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

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 ...

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Biology

9.6K Views.  Johns Hopkins University School of Medicine. 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.

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

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. 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.

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Biochemistry

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 ...

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 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 ...

Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes

This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 as ribonucleoproteins (RNPs) and mRNA. Primary mouse hepatocytes were isolated using a three-step retrograde perfusion method resulting in high yields of up to 50 &#215; 106 cells per liver and cell viability of &#62;85%. This protocol provides detailed instructions for plating, staining, and culturing hepatocytes. The results indicate that electroporation provides a high transfection efficiency of 89%, as measured by the percentage of green fluorescent protein (GFP)-positive cells and modest cell viability of &#62;35% in mouse hepatocytes.
To demonstrate the utility of this approach, CRISPR-Cas9 targeting the hydroxyphenylpyruvate dioxygenase gene was electroporated into primary mouse hepatocytes as proof-of-principle gene editing to disrupt a therapeutic gene related to an inherited metabolic disease (IMD) of the liver. A higher on-target edit of 78% was observed for RNPs compared to 47% editing efficiency with mRNA. The functionality of hepatocytes was evaluated in vitro using an albumin assay that indicated that delivering CRISPR-Cas9 as RNPs and mRNA results in comparable cell viability in primary mouse hepatocytes. A promising application for this protocol is the generation of mouse models for human genetic diseases affecting the liver.

This protocol describes techniques for isolating primary mouse hepatocytes from the liver and electroporating CRISPR-Cas9 as ribonucleoproteins and mRNA to disrupt a therapeutic target gene associated with an inherited metabolic disease of the liver. The methods described result in high viability and high levels of gene modification after electroporation.

This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 ...

Bioengineering

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

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 ...

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Presented here is a protocol for the isolation of mouse hepatocytes from adult mouse livers using a modified collagenase perfusion technique. Also described is the long-term culture of hepatocytes in a 3D collagen sandwich setting as well as immunolabeling of the cytoskeletal components to study bile canalicular formation and its response to treatment.

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...

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.

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 ...