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

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+functional+analysis+of+hepatocytes+from+mice+with+fatty+liver+disease.">Isolation, culture, and functional analysis of hepatocytes from mice with fatty liver disease.</a> STAR Protocols. 1 (3), 100222 (2020).</li><li>Dold, 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=Portal+hyperperfusion+after+extended+hepatectomy+does+not+induce+a+hepatic+arterial+buffer+response+(HABR)+but+impairs+mitochondrial+redox+state+and+hepatocellular+oxygenation.">Portal hyperperfusion after extended hepatectomy does not induce a hepatic arterial buffer response (HABR) but impairs mitochondrial redox state and hepatocellular oxygenation.</a> PLoS One. 10 (11), 0141877 (2015).</li><li>Boyce, S., Harrison, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+detailed+methodology+of+partial+hepatectomy+in+the+mouse.">A detailed methodology of partial hepatectomy in the mouse.</a> Laboratory Animals. 37 (11), 529-532 (2008).</li><li>Mitchell, C., Willenbring, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reproducible+and+well-tolerated+method+for+2/3+partial+hepatectomy+in+mice.">A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice.</a> Nature Protocols. 3 (7), 1167-1170 (2008).</li><li>Chen, T., Oh, S., Gregory, S., Shen, X., Diehl, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+omics+analysis+reveals+functional+diversification+of+hepatocytes+during+liver+regeneration.">Single-cell omics analysis reveals functional diversification of hepatocytes during liver regeneration.</a> JCI Insight. 5 (22), (2020).</li><li>Chembazhi, U. V., Bangru, S., Hernaez, M., Kalsotra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+plasticity+balances+the+metabolic+and+proliferation+dynamics+of+a+regenerating+liver.">Cellular plasticity balances the metabolic and proliferation dynamics of a regenerating liver.</a> Genome Research. 31 (4), 576-591 (2021).</li><li>Fiegel, H. C., Kaufmann, P. M., Kneser, U., Kluth, D., Rogiers, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priming+of+hepatocytes+for+cell+culture+by+partial+hepatectomy+prior+to+cell+isolation.">Priming of hepatocytes for cell culture by partial hepatectomy prior to cell isolation.</a> Journal of Tissue Engineering. 6 (6), 619-626 (2000).</li><li>Roche, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Liberase TM Research Grade. , (2020).</li><li>Giugliano, 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=Hepatitis+C+virus+infection+induces+autocrine+interferon+signaling+by+human+liver+endothelial+cells+and+release+of+exosomes,+which+inhibits+viral+replication.">Hepatitis C virus infection induces autocrine interferon signaling by human liver endothelial cells and release of exosomes, which inhibits viral replication.</a> Gastroenterology. 148 (2), 392-402 (2015).</li><li>Shetty, 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=Common+lymphatic+endothelial+and+vascular+endothelial+receptor-1+mediates+the+transmigration+of+regulatory+T+cells+across+human+hepatic+sinusoidal+endothelium.">Common lymphatic endothelial and vascular endothelial receptor-1 mediates the transmigration of regulatory T cells across human hepatic sinusoidal endothelium.</a> The Journal of Immunology. 186 (7), 4147-4155 (2011).</li><li>Edwards, S., Lalor, P. 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The authors declare that they have no competing interests.

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 steatogenic liver diseases but also paramount for any analysis of processes following major hepatectomies, where hepatocytes display a temporally and spatially dynamic steatosis within the first 2-3 days of regeneration. Even though (single-cell) analyses of isolated hepatocytes have already been performed after hepatectomies19,20,21, it must be assumed that the cell population analyzed was, at least partially, deprived of lipid-laden hepatocytes and the results, therefore, were not fully representative and skewed toward lean hepatocytes.
Currently, the function of TRAS is not fully clarified. For example, the spatial differences in lipid content within liver lobules remain ill-understood. Therefore, a method is needed that allows quantitative and qualitative detection without precisely excluding these cells from analyses (e.g., for single-cell transcriptomics, flow cytometry, and metabolomics).
Overall, hepatocyte yield with this improved protocol is markedly superior over other methods for the isolation of fat-containing hepatocytes (e.g., from mice with nonalcoholic steatohepatitis, NASH) using density-gradient purification15. However, cell yield, viability, and fat content can vary between preparations. Several factors seem to be responsible.
First, different batches of collagenase or collagenase blends will vary in their enzymatic activity; however, the lot-to-lot differences are not as critical as with traditional collagenases. Nevertheless, adjusting the working concentration may be necessary. The differences can be further minimized by proper storage until use (dry lyophilizates and rehydrated stock solutions at -15 to -25 &#176;C) and by avoiding repetitive freeze-thaw cycles, but cannot be entirely eliminated. Therefore, it is recommended to use only collagenases of a specific batch for a given experimental series. In addition, the enzymatic activity depends on the temperature at which the digestion buffer reaches the liver, with an optimal temperature between 35 &#176;C and 37 &#176;C for a blend of collagenases I and II22. Lower temperatures will reduce the enzymatic activity, while temperatures above 50 &#176;C can lead to irreversible denaturation of the protein. Test this in advance and optimize the experimental conditions by adjusting the initial temperature of the digestion buffer, the length and diameter of the plastic tube, and the flow velocity. For example, a drop in buffer temperature of up to 10 &#176;C within a tube length of 60 cm is to be expected. Correct the temperature by using warming pads and infrared lamps, if needed. Collagenases display their optimum digestion capacity at a pH of 7.4; therefore, it is advised to adjust the pH of the buffer solutions already at a working temperature of approximately 37 &#176;C before use.
Second, it is paramount to flush the liver (or the whole mouse) sufficiently with perfusion buffer before starting the digestion process to eliminate potential inhibitors such as serum or albumin. Ideally, perfusion is started only after the systemic circulation has stopped, to ensure the digestion buffer moves only from the vena cava through the liver to the outflow via the opened portal vein. The superior vena cava can be closed with a small vascular clamp. This prevents contact with residual blood components/inhibitors. Another method of optimally flushing the liver is intermittent clamping of the portal vein. This should be done carefully and - once the digestion buffer has reached the liver - performed only once to avoid damaging the already digested cells.
Third, regenerating hepatocytes are sensitive, and experience has shown that they require shorter digestion times and more careful handling to avoid over-digestion and harm to the cells. To harvest the liver as gently as possible, it is recommended to grasp the hepatic hilus with a clamp and then, first cut the superior vena cava and free the liver from the connections to the vein and the diaphragm. Subsequently, the inferior vena cava and the portal vein can be cut, which enables mobilization of the liver and easily frees it from the gastrointestinal ligaments, the esophagus on one side, and the right kidney on the other side. In hepatectomized mice, it is possible to carefully grasp one of the ligatures from the median lobes to remove the liver. If an attempt is made to pull the liver out by force, the Glisson&#39;s capsule may rupture, and the isolated cells could get lost.
Finally, it is essential that the perfusion procedure and further processing of the hepatocytes are not interrupted or delayed, as this will immediately and inevitably affect viability. Ideally, the perfusion is performed in a team of at least two people and on one animal at a time. These requirements in time and manpower, however, are one of the limitations of this method, although this applies to alternative methods as well.
Another limitation is the final purity of the resulting cell suspension, as the advantage of not losing steatotic hepatocytes to a density gradient results in a small proportion of remaining non-parenchymal and/or immune cells. In the shown sample, the fraction of CD45+ (immune cells) and CD31+ (endothelial cells) is below 5%, and when selecting for cell size first during flow-cytometry, the percentages of CD45+ and CD31+ are 1.2% and 2.2%, respectively (Figure 5). For most applications, this level of purity is sufficient, but highly sensitive methods or the further use in primary cell cultures probably requires a higher degree. Numerous studies have used CD31 and CD45 as systemic markers to sort hepatic cell populations by MACS23,24 or FACS25.
This improved isolation method is ideally suited for the study of liver regeneration, particularly the early period that presents with significant amounts of steatotic hepatocytes. PHLF, featuring persisting steatosis, is a particularly relevant subject requiring research. Following extended hepatectomies leaving behind marginal remnants, PHLF can develop and indeed represents the most common cause of death due to liver surgery. Its pathobiology is only partially understood, and currently no treatment is available26. Given that PHLF significantly limits the application of liver surgery (such as for the cure of hepatic malignancies), exploring its causes and identifying potential measures is a clear medical need.
Need also exists for the study of diseases that share hepatic steatosis as a starting point. A prominent example is nonalcoholic fatty liver disease (NAFLD), which may progress to NASH and eventually to hepatocellular carcinoma (HCC)27. The spread of sedentary lifestyles in conjunction with the Western diet has led to a worldwide epidemic of NAFLD, and accordingly, HCC incidence is projected to rise28,29. Steatosis, in turn, is also tightly linked to obesity, the metabolic syndrome, and type-2-diabetes, all diseases with a rising incidence30. Therefore, steatosis represents an increasing burden for the current healthcare system, calling for effective means for its management. This improved two-step collagenase perfusion method enables the study of steatotic hepatocytes at the single-cell level, and hence should aid in exploring the causes and consequences of hepatocellular lipid accumulation.
This protocol presents an easy, fast, and reliable technique for the in vivo isolation of regenerating hepatocytes from mice after partial (70%) and extended (86%) hepatectomy. This approach does not rely on gradient-purification and can be used to investigate liver regeneration processes with the inclusion of lipid-laden hepatocytes that are usually lost during traditional isolation protocols. Moreover, this method can also be applied for the research of various liver diseases associated with steatotic changes and is not limited to the mouse species.

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%-hepatectomy in mice, with a mean final viability of 78% and 65%, respectively. This corresponds to the expected percentage compared to a total yield of 50-70 &#215; 106 from a whole mouse liver (Figure 6A,C). After partial hepatectomies, hepatocytes show an increased cell size compared to normal livers, corresponding to their increased lipid-content (Figure 6B). The gating strategy is shown in Supplementary Figure S4.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64493/64493fig06.jpg" /> 
Figure 6: Hepatocyte yield, cell size, and viability. (A) Cell count of one whole mouse liver and of remnants 24 h post 70%- and 86%-hepatectomy. (B) Calculated cell volume of isolated hepatocytes. Note the marked increase in cell size 24 h post hepatectomy, for both 70% and 86%. (C) Cell viability of isolated hepatocytes after each step of the purification process. Viability was measured by flow cytometry using Alexa Fluor 488 Zombie green viability dye. Percentages are taken from all hepatocyte singlets. For the gating strategy, see Supplementary Figure S6 and Supplementary Figure S4. Error bars refer to standard deviations with n = 6-8/group. Abbreviations: sHx = standard hepatectomy; eHx = extended hepatectomy; w/o resection = sham surgery. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64493/64493fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
An increased lipid content in murine hepatocytes 24 h after partial hepatectomy can be observed by an increase in granularity (Figure 7; for gating strategy, see Supplementary Figure S5) or directly observed by the presence of lipid vesicles inside enlarged hepatocytes (Figure 8 and Supplementary File 1).
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64493/64493fig07.jpg" /> 
Figure 7: Increased granularity and cell size measured by flow cytometry. (A) Increased granularity among isolated hepatocytes 24 h post hepatectomy as an indirect marker for the presence of lipid vesicles. (B) Histogram showing SSC. (C) The percentages represent the fraction of cells with high cytoplasmic granular intensity, measured by the SSC signal. Error bars refer to standard deviations with n = 6-8/group. Abbreviations: sHx = standard hepatectomy; eHx = extended hepatectomy; w/o resection = sham surgery; FSC = forward scatter signal; SSC = side scatter. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64493/64493fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/64493/64493fig08.jpg" /> 
Figure 8: Sudan IV staining as a marker of fat content of isolated fresh hepatocytes. (A) Hepatocytes from a fresh whole mouse liver without prior surgery. Increased size and fat content of hepatocytes 24 h (B) post 70%-hepatectomy and (C) 86%-hepatectomy. Note the concomitant presence of both larger, steatotic hepatocytes and smaller, non-steatotic hepatocytes. Scale bars = 30 &#181;m (B-D). Error bars refer to standard deviations with n = 6-8/group. Abbreviations: sHx = standard hepatectomy; eHx = extended hepatectomy; w/o resection = sham surgery. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64493/64493fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A very low percentage of immune and non-parenchymal cells remain in the final suspension. Further purification is achieved by FACS or MACS, if desired. Negative selection of CD31+ and CD45+ cells by flow cytometry is used to demonstrate and quantify the non-parenchymal cells (Figure 5).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64493/64493fig05.jpg" /> 
Figure 5: Flow cytometry gating strategy and selection of CD31- and CD45- parenchymal cells. (A) Gating chart with all events recorded; consider the relatively large size of hepatocytes and use adjusted voltages. Do not go higher than 350 V for FSC and 220 V for SSC. (B) Singlet gating. (C) Hepatocytes are selected by gating CD31- (endothelial marker) and CD45- (immune marker) cells. This selection can be performed in a fluorescence-activated cell sorter to select parenchymal cells for further downstream analyses, if needed; n = 4-5/group. Abbreviations: FSC = forward scatter; SSC = side scatter. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64493/64493fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To demonstrate the efficacy of the modified protocol in retaining steatotic cells, hepatocytes were isolated using the classic density-gradient approach7 (Figure 9A). Unlike with the modified procedure (Figure 9B), a fatty cell layer was clearly visible on top of the density gradient. Analysis of cells confirmed a gross absence of lipid-laden hepatocytes in the pellet following density-gradient centrifugation. In contrast, cells from the fatty layer were enriched with steatotic hepatocytes and a substantial fraction of non-parenchymal and dead cells (Figure 9A). Thus, classic isolation deprives the harvest of steatotic cells, while this modified protocol omitting the density gradient retains lipid-laden subpopulations, enabling the unbiased exploration of liver regeneration without skewing toward lean hepatocytes.
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/64493/64493fig09.jpg" /> 
Figure 9: Yields of steatotic hepatocytes following the classic density-gradient isolation protocol compared to the improved protocol. (A) With the classic density-gradient purification method (final concentration of 90% density-gradient solution in phosphate-buffered saline), dead hepatocytes are collected in a cell layer on top of the density-gradient solution. (B) This layer does not only consist of non-parenchymal cells and non-viable hepatocytes but also of viable, large, fatty hepatocytes. (C)&#160;The pellet obtained following density-gradient centrifugation contains lean hepatocytes of smaller size and is mostly devoid of steatotic cells. This is the &#34;pure&#34; fraction that is isolated with the classic protocol. (D) With the improved protocol, the lipid-filled hepatocytes are not lost and all hepatocytes are pelleted. The supernatant (E) consists mostly of dead and fragmented hepatocytes and non-parenchymal cells, while the pellet (F) is a mixture of hepatocytes of variable size and lipid-content. Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64493/64493fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Supplementary protocols. (A) Lipid staining with Sudan IV; (B) standard and extended hepatectomy in mice. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64493/64493_Supplementary File_R3_revised.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure S1: Overview on the perfusion setup. (1)&#160;Cannulate the inferior vena cava. (2) Perfuse the liver with warm perfusion buffer to chelate calcium. (3) Warm the digestion buffer with collagenases and Ca2+ to dissociate cells in vivo. Remove the liver and (4) store in ice-cold preservation buffer (maximum 30 min). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64493/Supp_FigureS1_R2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure S2: Perfusion setup. (A) Perfusion table with an isoflurane nozzle, red light heating lamp, and perfusion tube. A heavy object can be placed underneath the tube to stabilize it and reduce the risk of displacement. (B) During the first few minutes of the perfusion, some blood will outflow through the portal vein and pool in the open abdominal cavity. (C) The abdominal wall is incised on one side to drain the blood. A cotton swab facilitates the drainage. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64493/Supp_FigureS2_R2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure S3: Glisson&#39;s capsule. (A) The Glisson&#39;s capsule is ruptured with fine-tip tweezers in a few locations along the liver surface, and the liver cells are released by gently shaking the capsule. (B) The liver should tear apart easily. (C) The empty liver sac (Glisson&#39;s capsule) with released hepatocytes in suspension. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64493/Supp_FigureS3_R2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure S4: Flow cytometry gating strategy for viability screening. (A) All events were recorded. (B) Singlet gating. (C) Live/dead staining with Alexa Fluor 488 Zombie green viability dye. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64493/Supp_FigureS4_R2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure S5: Flow cytometry gating strategy for granularity measurement. (A) All events were recorded. (B) Singlet gating. (C) Dot plot of side scatter (SSC; x axis) versus forward scatter (FSC; y axis) to analyze the levels of granularity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64493/Supp_FigureS5_R2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure S6: Hepatocyte purification. (A) Cell pellet after the first centrifugation step; note the fatty layer on top of the medium. (B) Hepatocyte pellet after the second round of centrifugation. (C) Purified hepatocytes at the end of the purification process. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64493/Supp_FigureS6_R2.pdf" target="_blank">Please click here to download this File.</a>

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 ...

isolation-regenerating-hepatocytes-after-partial-hepatectomy

Nanyang Technological University

Research

JoVE Journal

Medicine

4.1K Views.  University Hospital Zurich (USZ). 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.

Video: Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological conditions. Partial hepatectomy also facilitates the increased engraftment and proliferation of transplanted cells by accelerating neovascularization and cell migration towards the liver. Here, we describe a simple protocol for performing 30% hepatectomy and transplantation of cells in the spleen of a non-obese diabetic/severe combined immunodeficient NOD.SCID (NOD.CB17-Prkdcscid/J) mouse. 
In this procedure, two small incisions are made. The first incision is to expose and resect the left lobe of the liver, and another small incision is made to expose the spleen for the intrasplenic transplantation of cells. This procedure does not require any specialized surgical skills, and it can be completed in 5-7 minutes with less stress and pain, faster recovery, and better survival. We have demonstrated the transplantation of hepatocytes isolated from a green fluorescent protein (GFP) expressing mouse (Transgenic C57BL/6-Tg (UBC-GFP) 30Scha/J), as well as hepatocyte like cells of human origin (NeoHep) in partially hepatectomized NOD.SCID mice.

We have described a protocol for performing partial hepatectomy (PHx) and cell transplantation via spleen in NOD.SCID (NOD.CB17-Prkdcscid/J) mice. In this protocol, an incision is made to expose and resect the left lobe of the liver followed by another incision for the intrasplenic transplantation of cells.

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

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 ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

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

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Isolation of Atrial Myocytes from Adult Mice