Name: isolation of primary rat hepatocytes with multiparameter perfusion control
Uploaded: 2021-04-05T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control at JoVE.com

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.

All procedures and animal housing were carried out under protocol numbers R15-0027 and R19-0669 in accordance with the requirements of the Institutional Animal Care and Use Committee (IACUC) of the National University of Singapore.
1. Preparation of solutions and surgical instruments
<ol>
	<li>Prepare buffers and cell culture media in Table 1 using ultrapure water.</li>
	<li>Pre-warm the calcium-free buffer and collagenase buffer to 37 &#176;C in a water bath before use.</li...

<ol>
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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 th...

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 correc...

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 ...

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