Name: an improved time- and labor- efficient protocol for mouse primary hepatocyte isolation
Uploaded: 2021-10-25T13:00:00
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-distille...

<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;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 MA2:D30edium</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 &#176;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 &#176;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 &#181;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 &#176;C</td>
		</tr>
		<tr>
			<td>William&#8217;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...

Watch this Scientific Journal Video about An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation at JoVE.com

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 ...

Mouse primary hepatocytes are important in liver studies. Especially for glucose metabolism, and hepatic drug testing. Time wise the isolation process can be time consuming and the energy costly.This protocol can provide an efficient way to isolate mouse primary hepatocytes. It is time and labor friendly. Requiring very few steps with all regions commercially available.Begin by using the peristaltic pump to start pumping warmed up double distilled water at a speed of 4 mL per minute, for five minutes. Then change the pumping tube from water to warmed up perfusion medium. Monitoring the air bubble between the water and perfusion medium.Will help track the flow of the perfusion medium through the system. Next disinfect the abdomen of an anesthetized eight week old C57 black 6J female mouse with 70%ethanol. And using a scissor, cut open the abdomen to expose the liver, portal vein and inferior vena cava.Then stop the peristaltic pump. After ensuring that the pumping tube contains perfusion medium and not water. Insert a 24 gauge catheter into the inferior vena cava.Switch the pump back on and cut the portal vein open. While the perfusion medium is being pumped, press the portal vein every minute to let the liquid reach every corner of the liver. Continue pumping for approximately three to five minutes or until the flushed out liquid is clear.Next change the pumping tube from the perfusion medium to the collagenase dispase medium. And press the portal vein every minute to let the liquid reach every corner of the liver. Continue pumping until all 25 mL of the collagenase dispase medium are depleted.Next isolate the whole liver without the gallbladder. And placed it in 30 mL of wash medium in a Petri dish on ice. Using forceps tear the liver into pieces to release primary hepatocytes into the solution.At this stage, the wash medium turns into a cloudy solution containing released primary hepatocytes and small liver pieces. Filter this cloudy solution through a 70 micron cell strainer, into 20 mL of 1X per call HBSS in a 50 mL tube on ice. And mix the solution by inverting the tube 20 times.Next centrifuge the solution. Then in the tissue culture hood, aspirate the supernatant. And wash the pellet with 30 mL of cold wash medium.Pellet the cells once again with centrifugation. Remove the supernatant, and re-suspend the pellet in 25 mL of culture medium. Count the cells and plate them on desired culture plates.According to the experimental design. After plating, isolated primary hepatocytes were firmly attached by one hour and fully expanded after 12 hours. In the isolated hepatocytes, mRNA levels of hepatocyte markers, such as transthyretin, CD95, ASGR1, and ASGR2 were significantly increased compared to the whole liver.In contrast the presence of immune cells, stellate cells, and endothelial cells was lower. As evidenced by a sharp decrease in CD45, collagen type 1 alpha 1, and endothelial cell 2 kinase 2 mRNA levels. Suggesting that this protocol can greatly reduce interference from other hepatic cells.After plating ASGR1 and ASGR2 levels decreased with time, but remain comparable to the whole liver until the 12 hour time point. Especially for ASGR1. The expression level of membrane bound CD81 was consistent for up to 48 hours.In comparison, toll-like receptor 4 expression generally increased and became higher than in vivo levels after 48 hours. Insulin sensitivity assays demonstrated that insulin significantly promoted phosphorylation of both AKT at serine 473. And forkhead box 01 at serine 256, in the hepatocytes.Indicating the sensitivity of primary hepatocytes to insulin. Phosphoenolpyruvate carboxykinase protein levels significantly increased after glucagon treatment. Suggesting that the glucose production pathway was activated.This activation was further confirmed by increased glucose production. And with other glucose production stimulators like Forskolin+IBMX. This protocol can save both time and energy for studies using mouse primary hepatocytes.Following this protocol, related experiments such as the hypothetical glucose metabolism and pharmaceutical biomarker assays can be carried out.

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