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

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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><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</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&#039; Balanced Salt Solution (HBSS), Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;, phenol red</td><td>Sigma-Aldrich</td><td>H9269-6x600ML</td><td>For digestion/preservation</td></tr><tr><td>Hanks&#039; Balanced Salt solution (HBSS), w/o Ca&lt;sup&gt;2+&lt;/sup&gt;, w/o Mg&lt;sup&gt;2+&lt;/sup&gt;, 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 &amp;ndash; 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&amp;rsquo; Medium E</td><td>Sigma-Aldrich</td><td>W4128-500ML</td><td>-</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</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 &amp;micro;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>&lt;strong&gt;Equipment&lt;/strong&gt;</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 &amp;ndash; 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 &amp;ndash; EverFlo</td><td>Philips&amp;nbsp;</td><td>1020007</td><td>0 &amp;ndash; 5 L/min</td></tr><tr><td>Pipetboy &amp;ndash; Pipettor Turbo-Fix</td><td>TPP</td><td>94700</td><td>-</td></tr><tr><td>Shenchen perfusion pump &amp;ndash; YZ1515x</td><td>Shenchen</td><td>YZ1515x</td><td>-</td></tr><tr><td>Surgical microscope &amp;ndash; 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 &amp;ndash; Precision GP 02</td><td>Thermo scientific</td><td>-</td><td>Adjust to 42 &amp;deg;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 × 106 hepatocytes after 70%-hepatectomy and 4-9 × 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 × 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="Cell count, volume, and hepatocyte viability bar charts; pre/post-purification analysis." class="xfigimg" src="/files/ftp_upload/64493/64493fig06.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
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="Flow cytometry analysis; dot plots and histogram showing cell granularity; statistical bar chart." class="xfigimg" src="/files/ftp_upload/64493/64493fig07.jpg" data-altupdatedat="1747911167000" data-alt="Figure 7"> 
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="Sudan IV staining; bar chart, microscope images compare steatotic hepatocytes: control, 70% sHx, 86% eHx." class="xfigimg" src="/files/ftp_upload/64493/64493fig08.jpg" data-altupdatedat="1747911167000" data-alt="Figure 8"> 
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 µm (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="Flow cytometry analysis; scatter plots of SSC-A vs. FSC-A (A), SSC-H (B), CD31+ vs. CD45+ (C)." class="xfigimg" src="/files/ftp_upload/64493/64493fig05.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
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="Centrifugation diagram showing hepatocyte isolation using density gradient solution, cellular layers visible." class="xfigimg" src="/files/ftp_upload/64493/64493fig09.jpg" data-altupdatedat="1747911167000" data-alt="Figure 9"> 
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) The pellet obtained following density-gradient centrifugation contains lean hepatocytes of smaller size and is mostly devoid of steatotic cells. This is the "pure" 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 µm. <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%20File_R3_revised.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure S1: Overview on the perfusion setup. (1) 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's capsule. (A) The Glisson'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'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

4.2K Views.  University Hospital Zurich (USZ). Het algemene doel van dit protocol is om een geoptimaliseerde methode te presenteren voor hepatocytenisolatie bij muizen na gedeeltelijke hepatectomie zonder de noodzaak van dichtheidsgradiëntcentrifugatie. We zien meestal een groot percentage lipide-beladen hepatocyten tijdens vroege regeneratie. Die steatotische hepatocyten die meestal verloren gaan bij dichtheidsgradiëntcentrifugatie vanwege de lage dichtheid, worden met dit protocol behouden.Om de perfusieapparatuur voor te berei...

Video: Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

Developmental Biology

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult liver regeneration after 70% partial hepatectomy results in hepatocyte hypertrophy with some replication in remaining lobes with restoration of metabolic activity, but with permanent loss of the injured lobe&#39;s morphology and architecture. Here, we detail a new surgical method in the neonate that leaves a physiologic environment conducive to regeneration. This model involves amputation of the left lobe apex and a subsequent conservative management regimen, and lacks the necessity for ligation of major liver vessels or chemical injury, leaving a physiologic environment where regeneration may occur. We extend this protocol to amputations on juvenile (P7-14) mice, during which the injured liver transitions from organ regeneration to compensatory growth by hypertrophy. The presented, brief 30 min protocol provides a framework to study the mechanisms of regeneration, its age-associated decline in mammals, and the characterization of putative hepatic stem or progenitors.

Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting.

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Immunology and Infection

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 ...

The Influence of Liver Resection on Intrahepatic Tumor Growth

The high incidence of tumor recurrence after resection of metastatic liver lesions remains an unsolved problem. Small tumor cell deposits, which are not detectable by routine clinical imaging, may be stimulated by hepatic regeneration factors after liver resection. It is not entirely clear, however, which factors are crucial for tumor recurrence.
The presented mouse model may be useful to explore the mechanisms that play a role in the development of recurrent malignant lesions after liver resection. The model combines the easy-to-perform and reproducible techniques of defined amounts of liver tissue removal and tumor induction (by injection) in mice. The animals were treated with either a single laparotomy, a 30% liver resection, or a 70% liver resection. All animals subsequently received a tumor cell injection into the remaining liver tissue. After two weeks of observation, the livers and tumors were evaluated for size and weight and examined by immunohistochemistry.
After a 70% liver resection, the tumor volume and weight were significantly increased compared to a laparotomy alone (p &#60;0.05). In addition, immunohistochemistry (Ki67) showed an increased tumor proliferation rate in the resection group (p &#60;0.05).
These findings demonstrate the influence of hepatic regeneration mechanisms on intrahepatic tumor growth. Combined with methods like histological workup or RNA analysis, the described mouse model could serve as foundation for a close examination of different factors involved in tumor growth and metastatic disease recurrence within the liver. A considerable number of variables like the length of postoperative observation, the cell line used for injection or the timing of injection and liver resection offer multiple angles when exploring a specific question in the context of post-hepatectomy metastases. The limitations of this procedure are the authorization to perform the procedure on animals, access to an appropriate animal testing facility and acquisition of certain equipment.

A high incidence of tumor recurrence after resection of liver metastases remains an unsolved problem. The illustrated mouse model may be useful to investigate the reasons for such recurrences. It combines a liver resection model with intrahepatic tumor cell injection for the first time.

The high incidence of tumor recurrence after resection of metastatic liver lesions remains an unsolved problem. Small tumor cell deposits, which are not ...

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 ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Biology

Studying Liver Regeneration of ALLPS in Animal Models for Staged Hepatectomy

A Mouse Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy Procedure Aided by Microscopy

Hepatectomy is widely regarded as the primary treatment for hepatic malignancies; yet, postoperative liver failure remains a major cause of perioperative mortality, severely impacting patient outcomes. In a robust hepatic environment, the future liver remnant (FLR) must exceed 25%, and in cases of cirrhosis, this requirement increases to over 40%. The inadequacy of FLR is currently a major obstacle in the progression of hepatic surgery.
Traditional methods to enhance FLR hypertrophy mainly focus on portal vein embolization (PVE), but its effectiveness is considerably limited. In recent years, there have been numerous reports on a novel biphasic hepatectomy method involving hepatic partitioning and portal vein ligation, known as associating liver partition and portal vein ligation for staged hepatectomy (ALPPS). ALPPS surpasses PVE in efficiently and considerably inducing FLR hypertrophy. However, the detailed mechanisms driving ALPPS-facilitated hepatic regeneration are not fully understood. Thus, replicating ALPPS in animal models is crucial to thoroughly investigate the molecular mechanisms of hepatic regeneration, offering valuable theoretical and practical insights.

Author Spotlight: Advancing Liver Regeneration Research through ALPPS Mouse Model

Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) has been shown to result in remarkable liver regeneration after several weeks post first-stage surgery. Animal models that have undergone ALPPS have been developed to explore potential liver regeneration ability and achieve favorable clinical outcomes.

Hepatectomy is widely regarded as the primary treatment for hepatic malignancies; yet, postoperative liver failure remains a major cause of perioperative ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

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