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

这项工作部分得到了MOE ARC（MOE2017-T2-1-149）的支持;NUHS创新种子拨款2017 （NUHSRO/2017/051/InnovSeed/02）;新加坡机械生物学研究所 （R-714-106-004-135）;和生物工程和纳米技术研究所，生物医学研究理事会，科学，技术和研究机构（A * STAR）（项目编号IAF-PP H18 / 01 / a0 / 014，IAF-PP H18 / 01 / a0 / K14和MedCaP-LOA-18-02）资助Hanry Yu。Ng Chan Way是新加坡国立大学的研究学者。我们要感谢新加坡国立大学的共聚焦显微镜单元和流式细胞术单元在肝细胞纯度分析方面的帮助和建议。

所有程序和动物收容均根据协议编号R15-0027和R19-0669根据新加坡国立大学机构动物护理和使用委员会（IACUC）的要求进行。
1. 溶液和手术器械的制备
<ol><li>使用超纯水制备 表1 中的缓冲液和细胞培养基。</li>
	<li>使用前在水浴中将无钙缓冲液和胶原酶缓冲液预热至37°C。</li>
	<li>高压灭菌器以下手术器械和实验室设备：一对锋利钝的手术剪刀，一对钝钝手术剪刀，?...

<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>

对于一般的两步胶原酶灌注手术，有几点特别重要。首先，切除肝脏时必须特别小心。确保胃肠道没有损坏，因为内容物的泄漏会导致细菌污染。此外，避免损坏Glisson的胶囊，该胶囊在动物手术过程中覆盖了肝脏表面。如果撕裂足够大，它可能允许分离的肝细胞过早释放到胶原酶缓冲液中。其次，胶原酶在确保良好的肝细胞活力的同时消化肝脏的能力可能因批号而异。使用的胶原酶量必须针对每...

原代肝细胞是肝脏相关基础研究、疾病治疗和药物检测等应用的重要工具。目前原发性肝细胞分离的金标准是Seglen在20世纪70年代引入的两步胶原酶灌注程序1，2，3 4。然而，这种手术在技术上具有挑战性，并且由新手外科医生进行时失败率很高。即使认为灌注成功，在分离之间也可能观察到肝细胞活力（通常为60%-95%）和产量（每200-300g大鼠0.5-5×108）的巨大差异。这会影响下游实验的质量和规模。除了技术程序之外，用于隔离的灌注装置（无论是市售的还是定制的）也是一个促成因素。必须注意灌注装置的组装、优化和维护。该方案的目的是通过技术程序的多参数灌注控制和两步胶原酶灌注程序的灌注设置来提高原代大鼠肝细胞分离之间的成功率和稳定性。
从技术方面来看，该程序中最困难的步骤是门静脉插管。至于其他步骤，如果遵守良好做法并采取一般预防措施，则可以提高隔离的稳定性。因此，了解每个步骤的推理非常重要，以便外科医生可以对手术过程中可能发生的各种变量做出反应。
已经发表了从大鼠和小鼠中分离肝细胞和肝脏非实质细胞的各种方案1，2，5，6，7，8，9。这些方案中使用的灌注装置有几个缺点，包括重复使用灌注管，温度控制问题，需要常规优化灌注参数，和/或使用不合适的静脉内（IV）导管进行门静脉插管。灌注管的重复使用会增加污染的机会，特别是如果管子没有得到适当的清洁和消毒。在没有常规更换的情况下重复使用管道也会使灌注装置暴露于诸如管道或连接器泄漏，气泡阱塞和管道收缩等问题，所有这些都将大大降低灌注液压力和流速，从而影响肝脏消化效率。在某些温度控制设置中没有恒定的热源，预热缓冲液会随着时间的推移而冷却，导致胶原酶活性和消化率低。虽然其他设置使用连接到水循环器的夹套玻璃冷凝器来加热缓冲器，但它们体积庞大，需要仔细清洁。在分离开始之前，必须测量和优化离开导管的缓冲液的温度、压力和流速，以确保稳定的灌注条件。即使在优化后，由于操作员的行为，参数在隔离期间仍可能中途变化，从而导致灌注和消化欠佳。大多数类型的静脉导管不适合门静脉插管，因为它们不允许在插管期间连续灌注。当插管成功时，他们无法立即通知外科医生。此外，将门静脉固定在软导管上而不使其变形具有挑战性。
在这里，我们使用标准化的一次性无菌管，用于精确稳定温度控制的硅胶加热器夹套，具有数据存储和管理的实时监控和报警系统以及使用特殊的静脉导管来解决这些问题，该导管允许在插管期间刺穿门静脉的同时进行连续灌注。据我们所知，我们是第一个将所有这些功能组合成紧凑的集成灌注系统（IPS）的团队，使其高度便携，能够安装到层流罩中以确保无菌运行。

<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

原代肝细胞广泛用于肝病的基础研究和 体外毒性试验。用于原发性肝细胞分离的两步胶原酶灌注手术在技术上具有挑战性，特别是在门静脉插管术中。由于灌注装置的组装、优化或维护困难，该程序也容易偶尔受到污染和灌注条件的变化。在这里，提出了一个改进的两步胶原酶灌注程序与多参数灌注控制的详细方案。通过在手术的关键步骤采取必要的技术预防措施，并通过采用特殊的静脉导管，标准化的无菌一次性导管，温度控制和实时监测和报警系统，降低操作难度和减轻灌注参数的可变性，成功可靠地分离了原代大鼠肝细胞。分离的原代大鼠肝细胞始终表现出高细胞活力（85%-95%），产量（每200-300g大鼠2-5×108 个细胞）和功能（白蛋白，尿素和CYP活性）。该程序由一个集成的灌注系统补充，该系统足够紧凑，可以安装在层流罩中以确保无菌操作。

外科医生可以通过观察某些步骤后的结果来判断肝脏灌注是否顺利进行。第一个结果可以在插管，切割静脉IVC和恢复灌注流速时观察到。肝脏应该已经完全改变颜色从深红色到棕色，同时保持其体积。如果肝脏看起来略微放气，并有红色或红色斑点，则意味着灌注流速设置不正确（太低），或者门静脉没有正确插管。如果肝脏不仅变成棕色，而且变得臃肿和僵硬，则意味着流速设置不正确（太高?...

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Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

该协议详细说明了使用特殊的静脉内导管，标准化的无菌一次性导管，通过实时监测补充的温度控制，以及用于两步胶原酶灌注手术的报警系统，以提高分离的原代大鼠肝细胞的活力，产量和功能的一致性。

多参数灌注对照分离原代大鼠肝细胞

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

这种大鼠肝细胞分离方案是基于消化前的肝切除方法。该协议引入了用于细胞分离的紧凑便携式集成灌注系统。该技术涉及胶原酶再循环，其减少了胶原酶的使用量，并在延长消化时间方面提供了更大的灵活性，同时确保了高重力产量和肝细胞特异性功能。把老鼠放在引擎盖上。切开腹部肌肉后，用弯曲的镊子的后部将肠道向右推。用弯曲的镊子尖端打破胆管下方的膜，以将缝合线绕在门静脉和胆管周围。使用丝外科缝合线在靠近肝脏的门静脉周围形成一个非常松散的结扎，就在静脉分支左右进入不同的肝叶之前。在第一个结扎的上游约两到三厘米处，就在胃静脉从门静脉分支之前，用两对弯曲的镊子的尖端穿过门静脉下方的组织形成一个孔。然后在插管期间拉伸孔以支撑门静脉。通过将泵速降低到四 RPM，以大约每分钟三毫升的流速，防止肝脏中的压力积聚。并确保从静脉导管流出的无钙缓冲液减少到缓慢滴注。在用镊子轻轻支撑门静脉的同时，握住静脉导管，使针的斜面朝上。并将针以10至20度角插入门静脉。然后缓慢前进，直到整个斜面在静脉内。正确插入应导致肝脏颜色变白。将套管向前穿过针头，用食指向后拉动翅膀，同时用拇指和中指握住导管，将针头缩回套管后面。在门静脉下方用静脉夹子将门静脉固定在导管上，以避免灌注血流中断。接下来，切开下腔静脉以防止肝脏中的压力积聚，并观察血液喷出的冲动，以确保正确的切割。将泵速提高到 38 RPM，流量约为每分钟 33 毫升。用镊子关闭下腔静脉两到三秒钟，然后重新打开肝脏，冲洗肝脏三次。为了灌注所有肝叶，调整套管尖端的位置，并确保将其放置在门静脉分支进入不同肝叶之前。然后，收紧门静脉周围的松弛结扎，略微上游分支点，并固定门静脉中套管的位置，通过在斜面和侧孔之间的硬针支撑套管的点处打三个结来防止缓冲液的回流。肝切除术后，让肝脏用游离钙缓冲液灌注12分钟。然后，拧紧游离钙缓冲液的辊夹，拧松胶原酶缓冲液的辊夹以更换灌注缓冲液。胶原酶缓冲液到达肝脏后，通过关闭上下腔静脉两到三秒钟并重新打开它来冲洗肝脏三次。小心地将肝脏移动到烧杯顶部的阶段，通过在支撑导管的同时用镊子握住隔膜来再循环胶原酶缓冲液，注意不要拉导管以防止意外脱落。消化肝脏12分钟，当肝脏失去光滑的棕色质地并变得糊状时，停止灌注。如有必要，请延长消化时间。肝脏消化后切开门静脉，小心地取出套管。然后将肝脏转移到冷的DMEM中以分离肝细胞。使用弯曲的镊子的背面轻轻敲击肝脏，以打破并剥离闪光胶囊。并通过在DMEM中轻轻摇动肝脏来释放肝细胞，直到细胞在培养基中完全解离。肝细胞的存活率在88%至94.8%之间，由行程和蓝色计数确定。重达200至300克的大鼠的肝细胞产量高达每次分离10至8个细胞的五倍。代表性的图像显示未纯化的肝细胞悬浮液和纯化的肝细胞悬浮液。分离的肝细胞的纯度约为96.8%，通过计数白蛋白阳性荧光染色细胞相对于细胞核总数来量化。在以假彩色显示的代表性图像中，绿色代表白蛋白，蓝色表示具有白蛋白的细胞核。没有白蛋白的细胞核呈红色突出显示。三明治培养物中的肝细胞形成明显的胆汁小管，并具有良好的细胞接触。如白蛋白，尿素和细胞色素P450测定所示，肝细胞表现出高尿素和白蛋白分泌。第3天，肝细胞对CYP1A2、CYP2B1/2和CYP3B2表现出较高的酶活性。在将针头插入门静脉之前，要做好心理准备。插入后，外科医生将需要在没有脉搏的情况下继续进行，直到切割IVC后泵速增加。

Research

JoVE Journal

Biology

Primary Culture of Adult Rat Heart Myocytes

成年大鼠心脏心肌细胞的原代培养

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

科研

生物学

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

大鼠门户成纤维细胞的分离<em&gt;原位</em&gt;肝脏灌注

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

大鼠肝细胞的分离和小学文化

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

从原发肿瘤的免疫细胞的分离

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

Culturing Primary Rat Inner Medullary Collecting Duct Cells

原代培养大鼠内髓集合管细胞

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

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

人肝细胞通过一个两步胶原酶灌注过程隔离

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

隔离和鼠标主动脉内皮细胞原代培养

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

在小鼠原代肝细胞测定脂肪酸氧化和脂肪生成的

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

协议主要人肝细胞的分离和相应的非实质肝细胞的主要种群

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