Name: isolation of regenerating hepatocytes after partial hepatectomy in mice
Uploaded: 2022-12-02T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice at JoVE.com

This study was supported by the Swiss National Fond (project grant 310030_189262).

All animal experiments were in accordance with Swiss Federal Animal Regulations and approved by the Veterinary Office of Zurich (n&#176; 007/2017, 156/2019) assuring human care. Male C57BL/6 mice aged 10-12 weeks were kept on a 12 h day/night cycle with free access to food and water. Each experimental group consisted of six to eight animals. See the Table of Materials for details related to all materials, equipment, and reagents used in this protocol.
1. Partial hepatect...

<ol>
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A., Tolba, R., Trautwein, C., Liedtke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+hepatectomy+in+mice.">Partial hepatectomy in mice.</a> Lab Animal. 49, 81-88 (2015).</li><li>Lehmann, K., 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+failure+after+extended+hepatectomy+in+mice+is+mediated+by+a+p21-dependent+barrier+to+liver+regeneration.">Liver failure after extended hepatectomy in mice is mediated by a p21-dependent barrier to liver regeneration.</a> Gastroenterology. 143 (6), 1609-1619 (2012).</li><li>Makino, 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+good+model+of+hepatic+failure+after+excessive+hepatectomy+in+mice.">A good model of hepatic failure after excessive hepatectomy in mice.</a> Journal of Surgical Research. 127 (2), 171-176 (2005).</li><li>Lizardo Thiebaud, M. 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The published protocol provides a reliable and straightforward method to isolate a high yield of normal and steatotic murine hepatocytes for single-cell downstream analyses or bulk analysis of cells following FACS sorting. The distinct advantage over density-gradient purification is that the cellular lipid content has essentially no impact on the effective yield of hepatocytes. Thus, the fraction of steatotic hepatocytes will be retained and included in downstream analyses. This is not only crucial for the study of steat...

The liver can regenerate itself even after major tissue loss. This unique regenerative capacity is explicitly illustrated by the experimental model of partial (70%) hepatectomy, first described in rats by Higgins and Anderson in 19311. In this model, 70% of the liver is surgically removed from animals by clipping off larger liver lobes. The remaining lobes then grow through compensatory hypertrophy to restore the original liver mass within about 1 week after surgery, albeit without restoration of the original liver architecture2,3. Additional hepatectomies with varying amounts of tissue removal have been developed, such as 86%-extended hepatectomy where the liver remnant is too small to recover, eventually leading to posthepatectomy liver failure (PHLF) and subsequent death in 30%-50% of the animals4,5,6. These models enable the study of normal and failed liver regeneration, depending on the amount of resected tissue (Figure 1).
Although mouse models of hepatectomies have been used successfully for many years, only recently have more advanced analytical methods allowed for a deeper insight at the single-cell level. For most of these methods, however, the presence of individual hepatocytes is a basic prerequisite. Most protocols for the isolation of primary hepatocytes are based on a two-step collagenase perfusion technique and subsequent density-gradient purification to separate viable hepatocytes from debris and non-parenchymal, as well as dead cells7,8,9. This method was first described by Berry and Friend in 196910 and adapted by Seglen and colleagues in 197211,12. However, as gradient centrifugation relies on the density and size of cells, lipid-laden hepatocytes are often lost during standard purification. While such loss may be negligible for many research questions, it is a crucial aspect for early liver regeneration. During the first 2 days, hepatocytes within the regenerating mouse liver accumulate lipids, thereby growing in size and dipping in density. This transient regeneration-associated steatosis (TRAS) serves to provide regenerative fuel and is temporary, but partially overlaps the major proliferative phase and is unevenly distributed within the liver lobules - the functional units of the liver13,14. After extended 86%-hepatectomy, however, TRAS also occurs but persists, because regeneration is stalled and lipids are not being oxidized14. Therefore, gradient-purification of hepatocytes following 70%- or 86%-hepatectomies will yield non-representative fractions, as most lipid-laden hepatocytes are lost due to their low density15.
In this modified isolation protocol, hepatocytes from C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. Usually, cannulation and perfusion of the remnant for cell isolation are done via the portal vein. However, portovascular resistance in small remnants left after major resection is high16, and thus perfusion is delicate. Because the vena cava remains unaffected by hepatectomies, perfusion can be easily performed in the retrograde direction via cannulation of the vena cava. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the liver remnant, using retrograde perfusion with outflow through the portal vein (Supplementary Figure S1). Hepatocytes are dissociated by collagenases and released from the Glisson&#39;s capsule. After washing and careful processing of viable hepatocytes by stepwise isolation using a low-speed centrifugation approach, the hepatocytes can be used for any downstream analyses.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Zombie green</td>
			<td>BioLegend</td>
			<td>423111</td>
			<td>Amine-reactive viability dye</td>
		</tr>
		<tr>
			<td>Attane Isoflurane ad us. vet. 99.9%</td>
			<td>Provet AG</td>
			<td>QN01AB06</td>
			<td>CAUTION: needs ventilation</td>
		</tr>
		<tr>
			<td>EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>E8008-100ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>V001229</td>
			<td>Dilute with water to 70%</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FCS)</td>
			<td>Gibco</td>
			<td>A5256701</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS), Ca2+, Mg2+, phenol red</td>
			<td>Sigma-Aldrich</td>
			<td>H9269-6x600ML</td>
			<td>For digestion/preservation</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt solution (HBSS), w/o Ca2+, w/o Mg2+, no phenol red</td>
			<td>Sigma-Aldrich</td>
			<td>H6648-6x500ML</td>
			<td>For perfusion buffer</td>
		</tr>
		<tr>
			<td>HEPES solution, 1 M</td>
			<td>Sigma-Aldrich</td>
			<td>83264-100ML-F</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Histoacryl tissue adhesive (butyl-2-cyanoacrylate)</td>
			<td>B. Braun</td>
			<td>1050052</td>
			<td>For stabilization of cannulation site</td>
		</tr>
		<tr>
			<td>Hoechst 33258 Staining Dye Solution</td>
			<td>Abcam</td>
			<td>ab228550</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Liberase Research Grade</td>
			<td>Roche</td>
			<td>5401119001</td>
			<td>Lyophilized collagenases I/II</td>
		</tr>
		<tr>
			<td>NaCl 0.9% 500 mL Ecotainer</td>
			<td>B. Braun</td>
			<td>123</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Paralube Vet Ointment</td>
			<td>Dechra</td>
			<td>17033-211-38</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>A1286301</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sudan IV &#8211; Lipid staining</td>
			<td>Sigma-Aldrich</td>
			<td>V001423</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Temgesic (Buprenorphine hydrochloride), Solution for Injection 0.3 mg/mL</td>
			<td>Indivior Europe Ltd.</td>
			<td>345928</td>
			<td>Narcotics. Store securely.</td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4%, sterile-filtered</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td>For cell counting</td>
		</tr>
		<tr>
			<td>Williams&#8217; Medium E</td>
			<td>Sigma-Aldrich</td>
			<td>W4128-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL serological pipette, Greiner Cellstar</td>
			<td>Merck</td>
			<td>P7865</td>
			<td>-</td>
		</tr>
		<tr>
			<td>50 mL Falcon tubes</td>
			<td>TPP</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BD Neoflon, Pro IV Catheter 26 G</td>
			<td>BD Falcon</td>
			<td>391349</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cell scraper, rotating blade width 25 mm</td>
			<td>TPP</td>
			<td>99004</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Falcon Cell Strainer 100 &#181;m Nylon</td>
			<td>BD Falcon</td>
			<td>352360</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fenestrated sterile surgical drape</td>
			<td>-</td>
			<td>-</td>
			<td>Reusable cloth material</td>
		</tr>
		<tr>
			<td>Filling nozzle for size 16# tubing (ID 3.1 mm)</td>
			<td>Drifton</td>
			<td>FILLINGNOZZLE#16</td>
			<td>To go into the tubes</td>
		</tr>
		<tr>
			<td>Flow cytometry tubes, 5 mL</td>
			<td>BD Falcon</td>
			<td>352008</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Male Luer to Barb, Tubing ID 3.2 mm</td>
			<td>Drifton</td>
			<td>LM41</td>
			<td>Connection tube to syringe</td>
		</tr>
		<tr>
			<td>Petri dishes, 96 x 21 mm</td>
			<td>TPP</td>
			<td>93100</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Prolene 5-0</td>
			<td>Ethicon</td>
			<td>8614H</td>
			<td>To retract the sternum</td>
		</tr>
		<tr>
			<td>Prolene 6-0</td>
			<td>Ethicon</td>
			<td>8695H</td>
			<td>For skin suture</td>
		</tr>
		<tr>
			<td>Prolene 8-0</td>
			<td>Ethicon</td>
			<td>EH7470E</td>
			<td>Ligature gall bladder</td>
		</tr>
		<tr>
			<td>Tube 16#, WT 1.6 mm, ID 3.2 mm, OD 6.4 mm</td>
			<td>Drifton</td>
			<td>SC0374T</td>
			<td>Perfusion tube</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRFortessa Cell Analyzer Flow Cytometer</td>
			<td>BD</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Isis rodent shaver</td>
			<td>Aesculap</td>
			<td>GT421</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Isofluran station</td>
			<td>Provet</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Low-speed centrifuge &#8211; Scanspeed 416</td>
			<td>Labogene</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Neubauer-improved counting chamber</td>
			<td>Marienfeld</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Oxygen concentrator &#8211; EverFlo</td>
			<td>Philips&#160;</td>
			<td>1020007</td>
			<td>0 &#8211; 5 L/min</td>
		</tr>
		<tr>
			<td>Pipetboy &#8211; Pipettor Turbo-Fix</td>
			<td>TPP</td>
			<td>94700</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Shenchen perfusion pump &#8211; YZ1515x</td>
			<td>Shenchen</td>
			<td>YZ1515x</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Surgical microscope &#8211; SZX9</td>
			<td>Olympus</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>ThermoLux warming mat</td>
			<td>Thermo Lux</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Vortex Genie 2, 2700 UpM</td>
			<td>NeoLab</td>
			<td>7-0092</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Water bath &#8211; Precision GP 02</td>
			<td>Thermo scientific</td>
			<td>-</td>
			<td>Adjust to 42 &#176;C</td>
		</tr>
	</tbody>
</table>

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.

TRAS peaks at 16 h post hepatectomy and gradually vanishes 32-48 h after standard hepatectomy, but persists beyond 48 h after extended hepatectomy. Macroscopically, TRAS is readily visible as a pale complexion of the liver remnant (Figure 1F) and can be observed in hepatectomized mice between 16 h and 48 h after surgery.
The estimated final yield is 10-15 &#215; 106 hepatocytes after 70%-hepatectomy and 4-9 &#215; 106 after extended 86%-hepat...

Watch this Scientific Journal Video about Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice at JoVE.com

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

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 ...

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