Name: An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation (Video) | JoVE Journal
Uploaded: 2021-10-25T13:00:00.000+00:00
Duration: 5 min 42 s
Description: Learn an improved time- and labor-efficient protocol for isolating mouse primary hepatocytes using C57BL/6J mice. This method enhances yield and reduces non-parenchymal cell interference for liver metabolism and drug testing research.

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5 minutes

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>

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</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

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...

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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JoVE Journal

Biology

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.

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

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.

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

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.

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

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.

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

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.

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Fatty acid β-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 β-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

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

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

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.

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

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.

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

This protocol presents an optimized two-step collagenase liver perfusion technique in a rat model and shows the use of isolated hepatocytes for in vitro long-term culture of 3D organoids.

Rat Liver Perfusion and Primary Hepatocytes Isolation: An Old Procedure Crucial for Cutting-Edge 3D Organoids Culture

Learn an improved time- and labor-efficient protocol for isolating mouse primary hepatocytes using C57BL/6J mice. This method enhances yield and reduces non-parenchymal cell interference for liver metabolism and drug testing research.

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

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