This work is supported in part by MOE ARC (MOE2017-T2-1-149); NUHS Innovation Seed Grant 2017 (NUHSRO/2017/051/InnovSeed/02); Mechanobiology Institute of Singapore (R-714-106-004-135); and Institute of Bioengineering and Nanotechnology, Biomedical Research Council, Agency for Science, Technology and Research (A*STAR) (Project Numbers IAF-PP H18/01/a0/014, IAF-PP H18/01/a0/K14 and MedCaP-LOA-18-02) funding to Hanry Yu. Ng Chan Way is a research scholar of the National University of Singapore. We would like to thank Confocal Microscopy Unit &#38; Flow Cytometry Unit of the National University of Singapore for help and advice in hepatocyte purity analysis.

<ol>
	<li>Shen, L., Hillebrand, A., Wang, D. Q. H., Liu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+rat+hepatic+cells.">Isolation and primary culture of rat hepatic cells.</a> Journal of Visualized Experiments: JoVE. (64), e3917 (2012).</li><li>Cabral, F., 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=Purification+of+hepatocytes+and+sinusoidal+endothelial+cells+from+mouse+liver+perfusion.">Purification of hepatocytes and sinusoidal endothelial cells from mouse liver perfusion.</a> Journal of Visualized Experiments: JoVE. (132), e56993 (2018).</li><li>Green, C. J., 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=The+isolation+of+primary+hepatocytes+from+human+tissue:+optimising+the+use+of+small+non-encapsulated+liver+resection+surplus.">The isolation of primary hepatocytes from human tissue: optimising the use of small non-encapsulated liver resection surplus.</a> Cell and Tissue Banking. 18 (4), 597-604 (2017).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods in Cell Biology. 13, 29-83 (1976).</li><li>Gopalakrishnan, S., Harris, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+liver+endocytosis+followed+by+purification+of+liver+cells+by+liver+perfusion.">In vivo liver endocytosis followed by purification of liver cells by liver perfusion.</a> Journal of Visualized Experiments: JoVE. (57), e3138 (2011).</li><li>Wen, J. W., Olsen, A. L., Perepelyuk, M., Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+rat+portal+fibroblasts+by+in+situ+liver+perfusion.">Isolation of rat portal fibroblasts by in situ liver perfusion.</a> Journal of Visualized Experiments: JoVE. (64), e3669 (2012).</li><li>Korelova, K., Jirouskova, M., Sarnova, L., Gregor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+3D+collagen+sandwich+culture+of+primary+mouse+hepatocytes+to+study+the+role+of+cytoskeleton+in+bile+canalicular+formation+in+vitro.">Isolation and 3D collagen sandwich culture of primary mouse hepatocytes to study the role of cytoskeleton in bile canalicular formation in vitro.</a> Journal of Visualized Experiments: JoVE. (154), e60501 (2019).</li><li>Shi, W., 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=Isolation+and+purification+of+immune+cells+from+the+liver.">Isolation and purification of immune cells from the liver.</a> International Immunopharmacology. 85 (95), 106632 (2020).</li><li>Salem, E. S. B., 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=Isolation+of+primary+mouse+hepatocytes+for+nascent+protein+synthesis+analysis+by+non-radioactive+L-azidohomoalanine+labeling+method.">Isolation of primary mouse hepatocytes for nascent protein synthesis analysis by non-radioactive L-azidohomoalanine labeling method.</a> Journal of Visualized Experiments: JoVE. (140), e58323 (2018).</li><li>Xia, L., 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=Tethered+spheroids+as+an+in+vitro+hepatocyte+model+for+drug+safety+screening.">Tethered spheroids as an in vitro hepatocyte model for drug safety screening.</a> Biomaterials. 33 (7), 2165-2176 (2012).</li><li>Kegel, V., 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=Protocol+for+isolation+of+primary+human+hepatocytes+and+corresponding+major+populations+of+non-parenchymal+liver+cells.">Protocol for isolation of primary human hepatocytes and corresponding major populations of non-parenchymal liver cells.</a> Journal of Visualized Experiments: JoVE. (109), e53069 (2016).</li><li>Zhu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vertical-flow+bioreactor+array+compacts+hepatocytes+for+enhanced+polarity+and+functions.">A vertical-flow bioreactor array compacts hepatocytes for enhanced polarity and functions.</a> Lab on a Chip. 16 (20), 3898-3908 (2016).</li><li>Du, Y., 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=Synthetic+sandwich+culture+of+3D+hepatocyte+monolayer.">Synthetic sandwich culture of 3D hepatocyte monolayer.</a> Biomaterials. 29 (3), 290-301 (2008).</li><li>Tong, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constrained+spheroids+for+prolonged+hepatocyte+culture.">Constrained spheroids for prolonged hepatocyte culture.</a> Biomaterials. 80, 106-120 (2016).</li><li>Xia, L., 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=Laminar-flow+immediate-overlay+hepatocyte+sandwich+perfusion+system+for+drug+hepatotoxicity+testing.">Laminar-flow immediate-overlay hepatocyte sandwich perfusion system for drug hepatotoxicity testing.</a> Biomaterials. 30 (30), 5927-5936 (2009).</li><li>Gupta, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+canaliculi+contract+autonomously+by+releasing+calcium+into+hepatocytes+via+mechanosensitive+calcium+channel.">Bile canaliculi contract autonomously by releasing calcium into hepatocytes via mechanosensitive calcium channel.</a> Biomaterials. 259, 120283 (2020).</li><li>Horner, R., 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=Impact+of+Percoll+purification+on+isolation+of+primary+human+hepatocytes.">Impact of Percoll purification on isolation of primary human hepatocytes.</a> Scientific Reports. 9 (1), 6542 (2019).</li><li>Osypiw, J. C., 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=Subpopulations+of+rat+hepatocytes+separated+by+Percoll+density-gradient+centrifugation+show+characteristics+consistent+with+different+acinar+locations.">Subpopulations of rat hepatocytes separated by Percoll density-gradient centrifugation show characteristics consistent with different acinar locations.</a> The Biochemical Journal. 304, 617-624 (1994).</li><li>Beal, E. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+animal+model+of+ex+vivo+normothermic+liver+perfusion.">A small animal model of ex vivo normothermic liver perfusion.</a> Journal of Visualized Experiments: JoVE. (136), e57541 (2018).</li><li>Hillebrandt, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedure+for+decellularization+of+rat+livers+in+an+oscillating-pressure+perfusion+device.">Procedure for decellularization of rat livers in an oscillating-pressure perfusion device.</a> Journal of Visualized Experiments: JoVE. (102), e53029 (2015).</li></ol>

Zhou Yan and Hanry Yu declare competing interests as they hold equity in Vasinfuse, which manufactures and markets the Integrated Perfusion System. Hanry Yu holds equity in Histoindex, Invitrocue, Osteopore, Pishon Biomedical, Ants Innovate, and Synally Futuristech that have no competing interests with the information reported here.

There are a few points that are particularly important to observe for two-step collagenase perfusion procedure in general. Firstly, special care must be given when resecting the liver. Ensure that the gastrointestinal tract is not damaged as leakage of the contents will result in bacterial contamination. In addition, avoid damaging the Glisson&#39;s capsule, which covers the surface of the liver during the animal procedure. If the tear is large enough, it might allow premature release of disassociated hepatocytes into the collagenase buffer. Secondly, the ability of collagenase to digest the liver while ensuring good hepatocyte viability may vary by lot number. The amount of collagenase used must be optimized for every new lot of collagenase5. It is advisable to test a few lots of collagenase and select the best lot before buying in bulk. Ensure that the water is ultrapure, and the pH is correct. Impurities in the water such as ferrous and ferric ions and the wrong pH could affect collagenase activity, which will then drastically affect the quality of the isolated cells. Although collagenase buffer recirculation in this protocol can be optional, it is not advisable because the volume of collagenase buffer needed will increase to &#62;400 mL. Lastly, perfusion pressure and flow rate should be in an appropriate range. High pressure and flow rate will lead to liver cell death while low pressure and flow rate will result in insufficient buffer flow through the smaller vessels10. During calcium-free buffer perfusion, the pressure will initially rise. However, upon collagenase buffer perfusion, the pressure will then slowly drop over time since digestion by collagenase reduces flow resistance. If the pressure drop was not observed, the digestion time can be extended as required. Since the&#160;IPS has the optional pressure sensor, it is possible to vary the flow rate of the perfusate to compensate for pressure variations. On another note, although flushing the circuit with an oxygenator was used in some protocols2,5, we found that it was not critical for hepatocyte viability and yield. Thus, our device does not include an oxygenator.
In contrast to the common practice of liver digestion before resection, this protocol involves resection of the liver prior to digestion. The approach in this protocol holds several advantages compared to the common practice. Firstly, over-digestion of the liver can be avoided. Depending on the surgeon&#39;s skill, liver resection may take anywhere from 5-10 min. Following the common practice, even though the perfusion has been stopped, this still equates to an additional 5-10 min where the liver is exposed to collagenase. Over-digestion may decrease cell viability and reduce hepatocyte attachment and function. Secondly, this approach could potentially reduce hepatocyte loss. The Glisson&#39;s capsule is less likely to tear when the liver is still springy before digestion, compared to when it is soft and mushy after digestion. Finally, resection of the liver prior to digestion enables the possibility of collagenase recirculation. Collagenase recirculation eliminates the occurrence of failed isolations due to insufficient volume of collagenase being prepared. Our approach allows digestion time to be extended, as necessary. It also has a minor advantage of reducing the amount of collagenase needed per isolation. The main disadvantage of this approach is that it introduces a new problem of catheter detachment halfway during perfusion, but which could be addressed with a few technical precautions. Another difference is that this protocol does not involve the use of density gradient such as Percoll. By removing dead cells, Percoll has been reported to increase the final viability of cells with some loss of yield, from 20%-50%16. Our cell viability is high because we test and select collagenase lots. From our experience, some collagenase lots consistently give lower viability. Without density centrifugation, it will be necessary to avoid bad collagenase lots through prior testing. For those who do not have such liberty and must consider density gradient, care should be given during Percoll dilution to avoid selection of hepatocyte subpopulations with a slightly different metabolic profile17,18.
Our approach in using an IPS for primary rat hepatocyte isolation resolves several issues with existing perfusion setups1,2,5,6,7,8,9. Usage of disposable sterile tubing saves time by removing the need to clean and disinfect perfusion tubing before and after each isolation. It reduces fire hazard since the perfusion tubing need not to be filled with flammable 70% ethanol when not in use to maintain sterility. The risk of contamination due to improper disinfection and storage can also be eliminated. Most importantly, the need for routine replacement of old tubing can be circumvented; this process is essential but is often complicated and time consuming. The addition of a bubble filter to the tubing near the catheter allows bubbles to escape before reaching the catheter and prevents air from passing through when the perfusion buffer runs out. Usage of a silicone heater jacket as the temperature control system allows precise and stable maintenance of perfusate temperature during digestion, in contrast with pre-warmed buffers which will significantly cool down over time. In addition, the silicone heater jacket is compact and can be easily maintained, unlike a glass jacket coupled to a water bath circulator. Usage of monitoring sensors for temperature and pressure replaces the optimization step before isolation. Temperature variation may occur during or between experiments. Pressure (and thus flow rate) may also change due to issues with perfusion tubing. Placement of sensors near the cannula allows more accurate reading of the temperature of buffer perfusing into the liver. Sensor alarms can remind the user to take corrective action. The recorded log also allows for troubleshooting. Usage of a special IV catheter for cannulation simplifies cannulation of the portal vein. Unlike other types of IV catheter, the catheter described here allows fluid communication between the cannula and the tubing in both its puncturing and retracted needle position due to a hole at the side of the needle. Thus, surgeons could use color change of the liver as a sign of successful cannulation. Upon successful cannulation, the needle tip can be retracted behind the catheter tip to prevent the re-piercing of the portal vein. The retracted needle could also act as a foundation for the ligature to secure the portal vein onto the soft cannula and prevent it from being deformed. This catheter can be operated with one hand; therefore, the non-dominant hand can be used to support the portal vein with forceps. The compact integrated design allows the whole perfusion device to be put into the laminar hood, reducing the risk of contamination, and saving space. This is especially important if the animal procedure is to be performed in a dedicated animal facility where space availability can be a challenge.
In comparison to previously reported rat hepatocyte isolation protocols using the two-step collagenase perfusion procedure1, the conditions described consistently generate higher cell yield of up to 5 x 108 hepatocytes per isolation, cell viability between 88%-94.8% and good hepatocyte-specific function.
This protocol can be concurrently used to isolate other non-parenchymal liver cells such as Kupffer cells, liver endothelial cells and hepatic stellate cells from the same rat. This protocol can also be modified for the isolation of liver cells from mice by using a smaller gauge IV catheter for cannulation and decreasing the perfusion flow rate to a range suitable for mouse species6. The perfusion procedure for mice can be simplified by resecting the liver after digestion.
The IPS device used in this experiment can be also used for ex vivo liver perfusion application such as experiments on liver transplantation as well as decellularization of rodent and discarded human livers19,20. The IPS has an advantage where the liver must be perfused for long periods because the IPS can monitor perfusion conditions in real-time and allow discontinuation of the perfusion upon completion either automatically or manually by remote control.

Primary hepatocytes are important tools for liver-related basic research, disease treatment, and application such as drug testing. The current gold standard for primary hepatocyte isolation is the two-step collagenase perfusion procedure1,2,3 introduced by Seglen in the 1970s4. However, this procedure is technically challenging and has a high failure rate when performed by novice surgeons. Even when a perfusion is considered successful, drastic differences in hepatocyte viability (typically 60%-95%) and yield (0.5-5 x 108 per 200-300 g rat) may be observed between isolations. This influences the quality and scale of downstream experiments. Apart from the technical procedure, the perfusion setup used for the isolation, either commercially available or custom built, is a contributing factor. Attention must be given to the assembly, optimization, and maintenance of the perfusion setup. The purpose of this protocol is to improve the success rate and stability between isolations of primary rat hepatocytes through multiparameter perfusion control of the technical procedure and perfusion setup of the two-step collagenase perfusion procedure.
From the technical aspect, the most difficult step in the procedure is the portal vein cannulation. As for the other steps, if good practice is observed and general precautions are taken, the stability of the isolation can be improved. Therefore, understanding of the reasoning for each step is important so that the surgeon could respond to various variables that may occur during the procedure.
Various protocols for the isolation of hepatocytes and liver non-parenchymal cells from rat and mouse have been published1,2,5,6,7,8,9. The perfusion setups used in these protocols had several disadvantages, which include the reuse of perfusion tubing, problems with temperature control, need for routine optimization of perfusion parameters, and/or usage of unsuitable type of intravenous (IV) catheter for portal vein cannulation. The reuse of perfusion tubing will increase the chances of contamination, especially if the tubing was not cleaned and disinfected properly. Reuse of tubing without routine replacement will also expose the perfusion setup to problems such as leaky tubing or connectors, clogged bubble trap and constricted tubing, all of which will substantially reduce the perfusate pressure and flow rate, thus, affecting liver digestion efficiency. Without a constant heat source in some setups for temperature control, pre-warmed buffers will cool down over time, leading to low collagenase activity and digestion. Although other setups utilize a jacketed glass condenser connected to a water circulator to warm the buffer, they are bulky and require careful cleaning. Temperature, pressure, and flow rate of buffer exiting the catheter must be measured and optimized before the start of isolation toensure stable perfusion condition. Even after optimization, the parameters could still change halfway during isolation due to the actions of the operator, thereby leading to suboptimal perfusion and digestion. Most types of IV catheter are not suitable for portal vein cannulation because they do not allow continuous perfusion during cannulation. They are unable to immediately inform the surgeon when the cannulation is successful. Furthermore, it is challenging to secure the portal vein on the soft catheter without deforming it.
Here, we address these problems using standardized disposable sterile tubing, a silicone heater jacket for precise and stable temperature control, real-time monitoring and alarm system with data storage and management and use of a special IV catheter, which allows continuous perfusion while puncturing portal vein during cannulation. To the best of our knowledge, we are the first group to combine all these features into an integrated perfusion system (IPS) that is compact, making it highly portable and able to be fit into a laminar flow hood to ensure aseptic operation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Material/Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Nipro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>27G needle</td>
			<td>Nipro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Black braided silk non-absorbable, non-sterile surgical suture</td>
			<td>Look</td>
			<td>SP117</td>
			<td></td>
		</tr>
		<tr>
			<td>Bochem 18/10 stainless steel forceps, sharp tip contain bent round tip</td>
			<td>Bochem</td>
			<td>10333511</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Perfusion Set</td>
			<td>Vasinfuse</td>
			<td>BPF-112</td>
			<td></td>
		</tr>
		<tr>
			<td>Floating circular 1.5 mL microcentrifuge tube rack</td>
			<td>Sigma-Aldrich</td>
			<td>R3133</td>
			<td></td>
		</tr>
		<tr>
			<td>German Standard Tissue Forceps, Serrated / 1&#215;2 teeth , 14.5cm</td>
			<td>Walentech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Greiner Cellstar aspirating pipette</td>
			<td>Merck</td>
			<td>GN710183</td>
			<td></td>
		</tr>
		<tr>
			<td>Haemocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Integrated Perfusion System</td>
			<td>Vasinfuse</td>
			<td>IPS-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors curved, stainless, 11cm</td>
			<td>Optimal Medical Products Pte Ltd</td>
			<td>CVD</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope with 10X lens</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh Sheet 100&#181;M Nylon</td>
			<td>Spectra-Teknic(s) Pte Ltd</td>
			<td>06630-75</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissors, BL/BL, 13cm</td>
			<td>Optimal Medical Products Pte Ltd</td>
			<td>STR &#8211; BL/BL</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissors, SH/BL, 13cm</td>
			<td>Optimal Medical Products Pte Ltd</td>
			<td>STR &#8211; SH/BL</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse force hemostatic clip</td>
			<td>Shanghai Jin Zhong Pte Ltd</td>
			<td>XEC230</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Grant</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents/Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9056</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Merck</td>
			<td>137101</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Gibco</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>TCI</td>
			<td>D1961</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>31600-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Invitrogen</td>
			<td>11344-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>1-9278</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>VWR</td>
			<td>VWRC26764.298</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5379</td>
			<td></td>
		</tr>
		<tr>
			<td>Linoleic acid</td>
			<td>Sigma-Aldrich</td>
			<td>L9530</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S8875</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Merck</td>
			<td>106462</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Type I bovine collagen</td>
			<td>Advanced BioMatrix</td>
			<td>5005-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>William&#8217;s E Media</td>
			<td>Sigma-Aldrich</td>
			<td>W1878</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A surgeon could tell whether liver perfusion is going on smoothly by observing the outcome after certain steps. The first outcome can be observed upon cannulation, cutting of the infrahepatic IVC, and restoring the perfusion flow rate. The liver should have completely changed color from dark red to brown, while maintaining its volume. If the liver looks slightly deflated and has a reddish tint or blotches of red, it means that the perfusion flow rate was set wrongly (too low), or the portal vein was not cannulated correctly. If the liver not only turns brown but becomes bloated and stiff, it means that the flow rate was set wrongly (too high), or the infrahepatic IVC was not cut properly. If only a few lobes are not being perfused well, it means that the catheter was inserted too deep and is only perfusing one branch of the portal vein. The second outcome can be observed after resection and clipping of the infrahepatic IVC. If the outflow from the suprahepatic IVC is slow and weak, it might mean that the catheter had detached during resection, or that the infrahepatic IVC was not clipped properly. The third outcome can be observed during digestion. On the brown liver, translucent spots/network should appear and enlarge. The liver should lose its springy consistency and become soft and mushy. We optimize our collagenase concentration so that this state can be reached within 12 min. When trying out a new lot of collagenase, digestion time should be extended until this state is reached. The fourth outcome can be observed when cells are released from the liver. When the cells are released, the media should look cloudy. If a lot of grainy texture is observed, it could mean that the liver was insufficiently digested. If the digestion is complete, only white weblike vessels should be left. Even if the digestion is partial (Figure 6), it is still possible to acquire good cells.
Our protocol described here consistently generate higher cell yield of up to 5 &#215; 108 hepatocytes per isolation from rats weighing 200-300 g and cell viability between 88%-94.8% as determined by trypan blue counting12,13,14,15,16 (Table 2). Hepatocyte purity obtained as determined by immunostaining of albumin was 96.8 &#177; 2.0%11 (Figure 7). Hepatocytes in sandwich culture formed distinct bile canaliculi and had good cell-cell contact (Figure 8). The hepatocytes have high functionality as shown by albumin, urea, and CYP assays10 (Table 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62289/62289fig01.jpg" /> 
Figure 1:&#160;Schematic diagram of the IPS. The perfusate starts in the perfusate bag or bottle (1, 2). Two flow regulators (3, 4) work together to control the passage of perfusate. Perfusate proceeds to a bubble trap (5). Subsequent processes are controlled by the LCD touch screen (6) of the main system. Perfusate is then pulled by a peristaltic pump (7) and passes through a silicone jacketed electronic heater (8), which heats up the perfusate to the desired temperature. The silicone heater jacket (8) is connected to the main system by a connector (9). When necessary, a disposable pressure sensor (10) can be connected to the main system by a connector (11), to allow measurement of the perfusion pressure. The perfusate then passes through a perfusion monitor (12), which measures the temperature of the perfusate and detects the presence of bubbles. The monitor can also determine whether the perfusate has run out. Monitor readings are transmitted to the main system via LoRa. The perfusate then proceeds to a bubble filter (13) and enters the liver through the portal vein cannula (14). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62289/62289fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62289/62289fig05.jpg" /> 
Figure 5:&#160;Schematic diagram of the two-step collagenase perfusion for primary rat hepatocyte isolation. (A) Perfusion of calcium-free buffer. The roller clamp is completely loosened for inlet 1 and completely tightened for inlet 2. (B) Liver digestion with collagenase buffer. Resected liver is transferred to the top of a beaker to allow buffer recirculation. The roller clamp is completely loosed for inlet 2 and completely tightened for inlet 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62289/62289fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62289/62289fig06.jpg" /> 
Figure 6: Liver after collagenase digestion and hepatocyte isolation. Shown is a partially digested liver lobe. Undigested parts of the liver remained brown. In digested parts of the liver, only white vessels were left behind. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62289/62289fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62289/62289fig07.jpg" /> 
Figure 7:&#160;Immunostaining of hepatocytic marker for quantification of hepatocyte purity. (A) Sandwich culture of unpurified liver cell suspension (before differential centrifugation; left) and purified hepatocyte suspension (after differential centrifugation; right) seeded at low cell density. (B) Representative images show cells stained with DAPI (nucleus; blue) and antibody against albumin (hepatocyte-specific marker; green). (C) Quantification of hepatocyte purity. The purity was determined by counting albumin positive fluorescent stained cells in relation to the total cell number visualized by DAPI nuclear staining. Images in false colors were shown. Albumin (green), nucleus of cells with albumin (blue), nucleus of cells without albumin (red). Scale bars 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62289/62289fig07large.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/62289/62289fig08.jpg" /> 
Figure 8:&#160;Phase contrast image of isolated primary rat hepatocytes from two-step collagenase perfusion in sandwich culture at day 3. Scale bar 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62289/62289fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name of Buffers/Media</td>
			<td>Final concentration</td>
			<td>Amount</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Hepatocyte culture media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>William&#8217;s E media</td>
			<td></td>
			<td>500 mL</td>
			<td rowspan="7">Add all reagents into William&#39;s E media. Filter through 0.22 &#181;m filter. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>1 mg/mL</td>
			<td>0.5 g</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100X)</td>
			<td>1X</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Insulin (20 mg/ml)</td>
			<td>0.5 &#181;g/mL</td>
			<td>12.5 &#181;L</td>
		</tr>
		<tr>
			<td>Dexamethasone (1 mM)</td>
			<td>100 nM</td>
			<td>50 &#181;L</td>
		</tr>
		<tr>
			<td>Linoleic acid (7.5 &#181;g/mL)</td>
			<td>50 ng/mL</td>
			<td>3.33 &#181;L</td>
		</tr>
		<tr>
			<td>Glutamax (100X)</td>
			<td>1X</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Collagenase buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td>0.100 g</td>
			<td rowspan="5">Dissolve in 380 mL of ultrapure water. Adjust to pH 7.49 - 7.51 with NaOH. Top up to 400 mL with ultrapure water. Filter through 0.22 &#181;m filter. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td></td>
			<td>0.789 g</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>80 - 200 U/mL</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td></td>
			<td>0.140 g</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td></td>
			<td>4.800 g</td>
		</tr>
		<tr>
			<td>Calcium-free buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td></td>
			<td>0.0815 g</td>
			<td rowspan="4">Dissolve in 900 mL of ultrapure water. Adjust to pH 7.49-7.51 with HCl. Top up to 1 L with ultrapure water. Filter through 0.22 &#181;m filter.</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td></td>
			<td>1.0500 g</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td>3.4480 g</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td></td>
			<td>0.1750 g</td>
		</tr>
		<tr>
			<td>DMEM for cell isolation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td></td>
			<td>1 sachet</td>
			<td rowspan="3">Top up to 1 L with ultrapure water. Filter through 0.22 &#181;m filter. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td></td>
			<td>1.5 g</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100X)</td>
			<td>1X</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Collagen overlay (1 mL)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.1 M NaOH</td>
			<td>1 part</td>
			<td>20.8 &#181;L</td>
			<td rowspan="5">Prepare fresh. Add collagen last.</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>1 part</td>
			<td>20.8 &#181;L</td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>6 parts</td>
			<td>125 &#181;L</td>
		</tr>
		<tr>
			<td>Hepatocyte culture media</td>
			<td>32 parts</td>
			<td>667 &#181;L</td>
		</tr>
		<tr>
			<td>Type I bovine collagen</td>
			<td>8 parts</td>
			<td>167 &#181;L</td>
		</tr>
		<tr>
			<td colspan="3">Collagen coating solution (1 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>Type I bovine collagen</td>
			<td>1 part</td>
			<td>0.5 mL</td>
			<td rowspan="2">Prepare fresh.</td>
		</tr>
		<tr>
			<td>0.01 N HCl</td>
			<td>1 part</td>
			<td>0.5 mL</td>
		</tr>
	</tbody>
</table>
Table 1: Recipes for buffers and solutions.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hepatocyte viability (%)</td>
			<td>Hepatocyte yield (x 108 cells)</td>
			<td>Viable hepatocyte yield (x 108 cells)</td>
		</tr>
		<tr>
			<td>91.4 &#177; 3.4</td>
			<td>4.39 &#177; 1.58</td>
			<td>4.04 &#177; 1.48</td>
		</tr>
	</tbody>
</table>
Table 2: Cell viability and yield. Values &#177; SD from 18 different experiments are shown.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Urea (&#181;g/million cells/day)</td>
			<td>Albumin (&#181;g/million cells/day)</td>
			<td>CYP1A2 activity (&#181;g/million cells/day)</td>
			<td>CYP2B1/2 activity (&#181;g/million cells/day)</td>
			<td>CYP3B2 activity (&#181;g/million cells/day)</td>
		</tr>
		<tr>
			<td>Day 1&#160;</td>
			<td>124 &#177; 2.6</td>
			<td>39.0 &#177; 3.6</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Day 3&#160;</td>
			<td>65.7 &#177; 3.8</td>
			<td>516.0 &#177; 95.1</td>
			<td>2249 &#177; 56</td>
			<td>188 &#177; 49</td>
			<td>93.7 &#177; 0.6</td>
		</tr>
	</tbody>
</table>
Table 3:&#160;Functional levels of isolated primary rat hepatocyte in sandwich culture. Values &#177; SD of urea, albumin, CYP1A2, CYP2B1/2, and CYP3B2 assays from three different experiments are shown.

Watch this Scientific Journal Video about Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control at JoVE.com

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

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-primary-rat-hepatocytes-with-multiparameter-perfusion

Nanyang Technological University

Research

JoVE Journal

Biology

4.6K Views.  National University of Singapore. 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.

Video: Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

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. ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

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

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Presented here is a protocol for the isolation of mouse hepatocytes from adult mouse livers using a modified collagenase perfusion technique. Also described is the long-term culture of hepatocytes in a 3D collagen sandwich setting as well as immunolabeling of the cytoskeletal components to study bile canalicular formation and its response to treatment.

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Primary Culture of Adult Rat Heart Myocytes

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Isolation and Primary Culture of Rat Hepatic Cells

Isolation of Immune Cells from Primary Tumors

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

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

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