Name: electroporation-mediated delivery of cas9 ribonucleoproteins and mrna into freshly isolated primary mouse hepatocytes
Uploaded: 2022-06-02T13:10:00
Description: Watch this Scientific Journal Video about Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes at JoVE.com

RNC获得了南卡罗来纳州生物工程再生和组织形成试点基金的资助，该基金由美国国立卫生研究院国家普通医学科学研究所（NIGMS），美国肝病研究基金会和美国基因&细胞治疗学会资助，拨款号为P30 GM131959，2021000920， 和2022000099分别。内容完全由作者负责，并不一定代表美国基因与细胞治疗学会或美国肝病研究协会基金会的官方观点。 图1 的原理图是用 BioRender.com 创建的。

动物实验都是按照机构动物护理和使用委员会指南和克莱姆森大学批准的协议进行的。在8至10周龄的麻醉野生型C57BL / 6J小鼠中进行外科手术。
1. 动物手术
<ol><li>溶液和仪器的制备 注意：灌注溶液和麻醉鸡尾酒配方显示在 材料表中。<ol><li>制备灌注液 1（肝素、EGTA 和 EBSS）、灌注液 2（聚乙二醇、乙二醇、高分子灌注酶、氯化钙2 和镁质<su...

<ol>
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(132), e56993 (2018).</li><li>Huang, P., 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=Induction+of+functional+hepatocyte-like+cells+from+mouse+fibroblasts+by+defined+factors.">Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors.</a> Nature. 475 (7356), 386-389 (2011).</li><li>Severgnini, M., 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+rapid+two-step+method+for+isolation+of+functional+primary+mouse+hepatocytes:+cell+characterization+and+asialoglycoprotein+receptor+based+assay+development.">A rapid two-step method for isolation of functional primary mouse hepatocytes: cell characterization and asialoglycoprotein receptor based assay development.</a> Cytotechnology. 64 (2), 187-195 (2012).</li><li>Jung, Y., Zhao, M., Svensson, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+functional+analysis+of+hepatocytes+from+mice+with+fatty+liver+disease.">Isolation, culture, and functional analysis of hepatocytes from mice with fatty liver disease.</a> STAR Protocols. 1 (3), 100222 (2020).</li><li>Kim, 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=Three-dimensional+(3D)+printing+of+mouse+primary+hepatocytes+to+generate+3D+hepatic+structure.">Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure.</a> Annals of Surgical Treatment and Research. 92 (2), 67-72 (2017).</li><li>Li, W. C., Ralphs, K. 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肝细胞分离方案中概述的步骤具有挑战性，需要熟练练习。从肝脏中成功分离肝细胞有几个关键步骤。首先，下腔静脉的适当插管对于完全肝脏灌注至关重要。灌注后肝脏无变白表明导管移位（表1）。下腔静脉（逆行灌注）在手术中管，因为它比门静脉（顺行灌注）更简单，更容易获得17，18。一些方案使用缝合线来固定导管1...

肝脏IMD是遗传性疾病，其特征在于缺乏参与代谢的关键肝酶，导致有毒代谢物的积累。如果不进行治疗，肝脏IMD会导致器官衰竭或过早死亡1，2。肝脏IMD患者的唯一治疗选择是原位肝移植，由于供体器官的可用性低以及手术后免疫抑制治疗的并发症，这是有限的3，4。根据器官采购和移植网络最近收集的数据，肝移植等待名单上只有40%-46%的成年患者接受器官，而这些患者中有12.3%在等待名单上死亡5。此外，只有5%的罕见肝病有FDA批准的治疗方法6。很明显，迫切需要对肝脏IMDs进行新的治疗。然而，需要适当的疾病模型来开发新的治疗方案。
使用 体外 和 体内 系统对人类疾病进行建模仍然是开发有效疗法和研究肝脏IMD病理学的障碍。来自罕见肝病患者的肝细胞很难获得7。动物模型对于发展对疾病病理学的理解和测试治疗策略至关重要。然而，一个障碍是从携带致命突变的胚胎中生成模型。例如，试图创建Alagille综合征（ALGS）的小鼠模型，其中胚胎含有 Jag1 基因5′端附近5 kb序列的纯合子缺失，导致胚胎8的早期死亡。此外，通过在胚胎干细胞中进行基因编辑来生成小鼠模型可能是时间和资源密集型的9。最后，突变将出现在目标组织之外，导致混淆变量，可能阻碍对疾病的研究9。体细胞基因编辑将允许在肝脏组织中更容易地进行编辑，并绕过与使用胚胎干细胞生成模型相关的挑战。
电穿孔使CRISPR-Cas9能够通过施加高压电流使细胞膜透化而直接输送到细胞核中，并且与许多细胞类型兼容，包括那些对转染技术不妥协的细胞类型，例如人胚胎干细胞，多能干细胞和神经元10，11，12.然而，低生存能力是电穿孔的潜在缺点;优化程序可以产生高水平的递送，同时限制毒性13。最近的一项研究表明，将CRISPR-Cas9组分电穿孔到初级小鼠和人肝细胞中作为一种高效的方法的可行性14.肝细胞中的 离体 电穿孔有可能应用于为肝脏的人类IMD生成新的小鼠模型。
该协议提供了详细的分步程序，用于从肝脏中分离小鼠肝细胞，随后电穿孔CRISPR-Cas9作为RNP复合物，由Cas9蛋白和合成单向导RNA（sgRNA）组成，或Cas9 mRNA与sgRNA结合以获得高水平的靶向基因编辑。此外，该协议还提供了在CRISPR-Cas9电穿孔到新分离的小鼠肝细胞后量化基因编辑效率，活力和功能的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 mL PCR 8-tube FLEX-FREE strip, attached clear flat caps, natural</td>
			<td>USA Scientific</td>
			<td>1402-4700</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Collagen Plates</td>
			<td>Advanced Biomatrix</td>
			<td>5073</td>
			<td></td>
		</tr>
		<tr>
			<td>accuSpin Micro 17R</td>
			<td>Fisher Scientific</td>
			<td>13-100-675</td>
			<td></td>
		</tr>
		<tr>
			<td>All-in-one Fluorescent Microscope</td>
			<td>Keyence</td>
			<td>BZ-X810</td>
			<td></td>
		</tr>
		<tr>
			<td>Analog Vortex Mixer</td>
			<td>VWR</td>
			<td>97043-562</td>
			<td></td>
		</tr>
		<tr>
			<td>ART Wide BORE filtered tips 1,000 &#181;L</td>
			<td>ThermoFisher Scientific</td>
			<td>2079GPK</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>Bio-Rad Laboratories</td>
			<td>TC20</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue Wax Dissection Tray</td>
			<td>Braintree Scientific Inc.</td>
			<td>DTW1</td>
			<td>9&#34; x 6.5&#34; x 1/2&#34;+F21</td>
		</tr>
		<tr>
			<td>Cell scraper/lifter</td>
			<td>Argos Technologies</td>
			<td>UX-04396-53</td>
			<td>Non-pyrogenic, sterile</td>
		</tr>
		<tr>
			<td>Conical tubes (15 mL)</td>
			<td>Fisher Scientific</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes (50 mL)</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton applicators</td>
			<td>Fisher Scientific</td>
			<td>22-363-170</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved scissors</td>
			<td>Cooper Surgical</td>
			<td>62131</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes</td>
			<td>Falcon</td>
			<td>351029</td>
			<td>100mm,sterile</td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes</td>
			<td>VWR</td>
			<td>25373-100</td>
			<td>35mm, sterile</td>
		</tr>
		<tr>
			<td>Epoch Microplate Spectrophotometer</td>
			<td>BioTek Instruments</td>
			<td>250082</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Cell strainer (70 &#181;m)</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Cooper Surgical</td>
			<td>61864</td>
			<td>Euro-Med Adson Tissue Forceps</td>
		</tr>
		<tr>
			<td>IV catheters&#160;</td>
			<td>BD</td>
			<td>382612</td>
			<td>24 G x 0.75 in</td>
		</tr>
		<tr>
			<td>IV infusion set</td>
			<td>Baxter</td>
			<td>2C6401</td>
			<td></td>
		</tr>
		<tr>
			<td>MyFuge 12 Mini Centrifuge</td>
			<td>Benchmark Scientific</td>
			<td>1220P38</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles</td>
			<td>Fisher Scientific</td>
			<td>05-561-20</td>
			<td>25 G</td>
		</tr>
		<tr>
			<td>Nucleofector 2b Device</td>
			<td>Lonza</td>
			<td>AAB-1001</td>
			<td>Program T-028 was used for electroporation in mouse hepatocytes</td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Masterflex</td>
			<td>&#160;HV-77120-42</td>
			<td>10 to 60 rpm; 90 to 260 VAC</td>
		</tr>
		<tr>
			<td>Precision pump tubing</td>
			<td>Masterflex</td>
			<td>HV-96410-14</td>
			<td>25 ft, silicone</td>
		</tr>
		<tr>
			<td>Primaria Culture Plates</td>
			<td>Corning Life Sciences</td>
			<td>353846</td>
			<td>Nitrogen-containing tissue culture plates</td>
		</tr>
		<tr>
			<td>Serological Pipets (25 mL)</td>
			<td>Fisher Scientific</td>
			<td>12-567-604</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>BD</td>
			<td>329464</td>
			<td>1 mL, sterile</td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>Bio-Rad Laboratories</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>ALT</td>
			<td>27577</td>
			<td>Thermo Scientific Precision Microprocessor Controlled 280 Series, 2.5 L</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R S.p. Cas9 Nuclease V3</td>
			<td>IDT</td>
			<td>1081058</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Coulter AMPure XP, 5 mL</td>
			<td>Fisher Scientific</td>
			<td>NC9959336</td>
			<td></td>
		</tr>
		<tr>
			<td>CleanCap Cas9 mRNA</td>
			<td>Trilink Biotechnologies</td>
			<td>L-7606-100</td>
			<td></td>
		</tr>
		<tr>
			<td>CleanCap&#160;EGFP mRNA</td>
			<td>Trilink Biotechnologies</td>
			<td>L-7201-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Matrigel Matrix</td>
			<td>Corning Life Sciences</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher Scientific</td>
			<td>11885076</td>
			<td>Low glucose, pyruvate</td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>VWR</td>
			<td>71001-652</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermoscientific</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepatocyte Maintenance Medium (MM)</td>
			<td>Lonza</td>
			<td>MM250</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepatocyte Plating Medium (PM)</td>
			<td>Lonza</td>
			<td>MP100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Albumin ELISA Kit</td>
			<td>Fisher Scientific</td>
			<td>NC0010653</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse/Rat Hepatocyte Nucleofector Kit</td>
			<td>Lonza</td>
			<td>VPL-1004</td>
			<td></td>
		</tr>
		<tr>
			<td>OneTaq HotStart DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0481L</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 10x pH 7.4&#160;</td>
			<td>Thermoscientific</td>
			<td>70011-044</td>
			<td>No calcium or magnesium chloride</td>
		</tr>
		<tr>
			<td>Percoll (PVP solution)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-500790A</td>
			<td></td>
		</tr>
		<tr>
			<td>Periodic acid</td>
			<td>Sigma-Aldrich</td>
			<td>P7875-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Permount Mounting Medium</td>
			<td>VWR</td>
			<td>100496-550</td>
			<td></td>
		</tr>
		<tr>
			<td>QuickExtract DNA Extraction Solution</td>
			<td>Lucigen Corporation</td>
			<td>QE05090</td>
			<td></td>
		</tr>
		<tr>
			<td>Schiff&#8217;s fuchsin-sulfite reagent</td>
			<td>Sigma-Aldrich</td>
			<td>S5133</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>ThermoFisher Scientific</td>
			<td>25200056</td>
			<td>Phenol red</td>
		</tr>
		<tr>
			<td>Vybrant MTT Cell Viability Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>V13154</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion Solution 1 (pH 7.4, filter sterilized)&#160;</td>
			<td></td>
			<td></td>
			<td>Stable at 4 &#176;C for 2 months</td>
		</tr>
		<tr>
			<td>EBSS</td>
			<td>Fisher Scientific</td>
			<td>14155063</td>
			<td>Complete to 200 mL</td>
		</tr>
		<tr>
			<td>EGTA (0.5 M)</td>
			<td>Bioworld</td>
			<td>40520008-1</td>
			<td>200 &#181;L</td>
		</tr>
		<tr>
			<td>HEPES (pH 7.3, 1 M)</td>
			<td>ThermoFisher Scientific</td>
			<td>AAJ16924AE</td>
			<td>2 mL&#160;</td>
		</tr>
		<tr>
			<td>Perfusion Solution 2 (pH 7.4, filter sterilized)&#160;</td>
			<td></td>
			<td></td>
			<td>Stable at 4 &#176;C for 2 months</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H20 (1.8 mM)</td>
			<td>Sigma</td>
			<td>C7902-500G</td>
			<td>360 &#181;L of 1 M stock&#160;</td>
		</tr>
		<tr>
			<td>EBSS (no calcium, no magnesium, no phenol red)</td>
			<td>Fisher Scientific</td>
			<td>14-155-063</td>
			<td>Complete to 200 mL</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>ThermoFisher Scientific</td>
			<td>AAJ16924AE</td>
			<td>2 mL&#160;</td>
		</tr>
		<tr>
			<td>MgSO4&#183;7H20 (0.8 mM)</td>
			<td>Sigma</td>
			<td>30391-25G</td>
			<td>160 &#181;L of 1 M stock</td>
		</tr>
		<tr>
			<td>Perfusion Solution 3&#160;</td>
			<td></td>
			<td></td>
			<td>Prepared fresh prior to use</td>
		</tr>
		<tr>
			<td>Solution 2</td>
			<td></td>
			<td></td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Liberase</td>
			<td>Roche</td>
			<td>5401127001</td>
			<td>0.094 Wunsch units/mL</td>
		</tr>
		<tr>
			<td>Mouse Anesthetic Cocktail&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acepromzine</td>
			<td></td>
			<td></td>
			<td>0.25 mg/mL final concentration</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td></td>
			<td></td>
			<td>7.5 mg/mL final concentration</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td></td>
			<td></td>
			<td>1.5 mg/mL final concentration</td>
		</tr>
		<tr>
			<td>Software</td>
			<td>URL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Benchling</td>
			<td>https://www.benchling.com/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>https://imagej.nih.gov/ij/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TIDE: Tracking of Indels by Decomposition</td>
			<td>https://tide.nki.nl/</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

electroporation-mediated delivery of cas9 ribonucleoproteins and mrna into freshly isolated primary mouse hepatocytes

该协议描述了一种快速有效的方法，用于分离原代小鼠肝细胞，然后电穿孔介导的CRISPR-Cas9作为核糖核蛋白（RNP）和mRNA递送。使用三步逆行灌注方法分离原代小鼠肝细胞，结果高产率高达50×每肝106 个细胞，细胞活力>85%。该方案提供了肝细胞电镀、染色和培养的详细说明。结果表明，电穿孔提供了89%的高转染效率，这是通过小鼠肝细胞中绿色荧光蛋白（GFP）阳性细胞的百分比和>35%的适度细胞活力来测量的。
为了证明这种方法的实用性，将靶向羟基苯基丙酮酸二加氧酶基因的CRISPR-Cas9电穿孔到初级小鼠肝细胞中作为原理验证基因编辑，以破坏与肝脏遗传代谢疾病（IMD）相关的治疗基因。RNP的靶向编辑率更高，为78%，而mRNA的编辑效率为47%。使用白蛋白测定 法在体外 评估肝细胞的功能，该测定表明将CRISPR-Cas9作为RNP和mRNA递送导致原代小鼠肝细胞中具有相当的细胞活力。该协议的一个有希望的应用是为影响肝脏的人类遗传疾病生成小鼠模型。

从肝脏中分离出可平板的原代肝细胞 肝脏灌注和肝细胞分离的整个过程如图 1所示。在该实验中，使用了野生型，8-10周龄的C57BL6 / 6J小鼠。该程序预计每只小鼠产生20-50个×106 个细胞，存活率在85%至95%之间。如果活力<70%，则应遵循全唇处理以去除死细胞。新鲜分离的肝细胞应接种在胶原 I 包被或含氮组织培养板上。在电镀的3-12小时内，预计肝细胞将?...

Watch this Scientific Journal Video about Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes at JoVE.com

Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes

该协议描述了从肝脏中分离原代小鼠肝细胞并将CRISPR-Cas9电冲为核糖核蛋白和mRNA的技术，以破坏与肝脏遗传性代谢疾病相关的治疗靶基因。所描述的方法导致电穿孔后的高生存能力和高水平的基因修饰。

电穿孔介导的Cas9核糖核蛋白和mRNA递送到新鲜分离的初级小鼠肝细胞中

This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 ...

我们技术的主要优点是，它是一种简单、快速且可重复的方法，用于编辑小鼠原代肝细胞中的基因，作为生成体外和体内疾病模型的一种方式。使用我们的技术，有可能敲低分离的肝细胞中的靶基因，然后将其移植回患者体内，用基因编辑的肝细胞替换患病细胞 缺乏经验的研究人员可能会在整个灌注过程中难以插管并保持导管稳定。建议用户在开始之前仔细阅读并练习该过程和故障排除方法。展示肝脏灌注和肝细胞分离的将是临床设施经理Tina Parker和研究生研究助理Ilayda Ates。电穿孔程序将由我实验室的研究生研究助理Callie Stuart演示。在麻醉的手术平面下。通过使用剪刀在腹部皮肤上做一个U形横向切口开始肝脏灌注，并通过侧面扩大孔。继续打开肋骨的皮肤。用剪刀切开腹膜，将腹腔暴露到胸腔。小心不要切伤任何器官。使用镊子的背面或剪刀的钝面将肠子向右侧移动。识别门静脉和下腔静脉。启动泵以通过导管冲洗预热的灌注溶液。停止泵并取出导管。然后将连接到导管的针尖以与静脉成10至20度角插入下腔静脉，从导管上取下针头。然后以平行于静脉的运动轻轻地将导管推入静脉。将导管连接到导管，而无需将导管进一步移动到静脉中。以每分钟两毫升的流速启动泵。寻找肝脏立即变白，作为灌注成功的标志。用剪刀剪开门静脉。逐渐将流速增加到每分钟五毫升。一旦灌注溶液流过三个仔细监测肝脏的弹性。要确定肝脏是否软化，请用棉签或镊子轻轻按压肝脏，并检查肝脏上是否形成压痕。注意不要过度灌注，以免细胞活力丧失。一旦肝脏在30至50毫升灌注溶液后软化，三次流过，停止泵，取出导管和胆囊。小心不要溢出其内容物。然后仔细解剖肝脏。将肝脏放入含有冰冷DME M和10%FBS培养基的100毫米培养皿中。轻轻旋转培养皿以去除可能的血栓。将肝脏转移到含有冰冷DME M和10%FBS培养基的新培养皿中。要从肝囊中释放细胞，请将培养皿放在冰上，然后使用两对无菌镊子，轻轻撕开所有叶上的肝囊。使用细胞提升器在培养基中轻轻旋转肝脏以释放肝细胞。继续这个动作，直到肝脏变得非常小，悬浮液变成棕色和不透明。从平板上取出肝组织残留物，并使用设置为低速的25毫升血清移液器，小心地将细胞悬液转移到在冰上预冷并装有100微米细胞过滤器的50毫升锥形管中。将新鲜冰冷的DMEM和10%FBS培养基加入培养皿中，以洗掉并从表面收集剩余的细胞。然后，转移到50毫升锥形管中。重复此步骤，直到收集所有细胞。要从非实质细胞中洗涤和纯化肝细胞，请将收集在50毫升锥形管中的细胞以50 RCF在4摄氏度下离心5分钟，低减速或没有任何中断。丢弃含有碎片和非实质细胞的超纳顿。然后通过轻轻旋转管将沉淀重新悬浮在剩余的超级纳顿中。颗粒重悬后加入30毫升新鲜冰冷DMEM和10%FBS培养基。通过打开电穿孔设备上的电源按钮开始制备 CRISPR CAS 9 底物培养基、细胞和电穿孔仪。然后通过按X按钮和向下箭头按钮设置电穿孔程序，直到程序T 0 28出现在屏幕上。通过将1.5毫升PM添加到六个孔胶原蛋白一包被板中，为电穿孔肝细胞制备目标孔。将板在 37 摄氏度的培养箱中孵育，直到准备使用。在PCR分裂试管中，按照文本手稿中所述制备CAS 9 RMP和mRNA复合物。将mRNA SG RNA混合物储存在冰上直至电穿孔。另外，制备含有一微克E G F P MRNA的管，以使用荧光显微镜验证电穿孔成功。在 100 RCF 下在 4 摄氏度下离心电穿孔反应两分钟。从细胞沉淀中取出上钠，并沿管壁侧面每次反应加入100微升核离子溶液。用手轻轻摇动试管，将肝细胞重新悬浮在电穿孔溶液中。一旦肝细胞均匀分散在电穿孔溶液中，将100微升肝细胞悬浮液转移到含有CAS九个复合物的试管中。将条带孔的内容物转移到电穿孔容器中。将核兽医容器放入电穿孔装置上的槽中。通过按X按钮电穿孔肝细胞。电穿孔后，等待设备上弹出一个屏幕，上面写着：好的。再次按下 X 按钮并卸下容器。将电穿孔容器在冰上孵育15分钟。向电穿孔容器中加入500微升预武装PM中。将300微升电穿孔反应转移到预热板上的每个目的地孔中。将板在 37 摄氏度下孵育过夜。将膜基质加入冰冷的肝细胞维持培养基中，并通过上下移液10次混合。接种细胞24小时后，从指定用于基因编辑分析的孔中取出培养基。将覆盖混合物缓慢移液到细胞顶部，然后将板放回 37 摄氏度。在电镀后24小时，将条件培养基从指定用于MTT和白蛋白测定的孔中转移并储存在微离心管中，直到准备进行白蛋白测定。通过提取基因组DNA并按照文本手稿中所述设置PCR来继续分析靶标CAS九活性。在接种后3至12小时内，肝细胞粘附在平板上，细胞形态在24小时内呈现出典型的多边形或六边形外观在接种后24小时内使用高碘酸移试剂通过糖原染色来验证分离细胞的纯度。染色肝细胞的细胞质呈洋红色。肝细胞在电穿孔后24小时成像，平均89.8%的肝细胞为GFP阳性。在电穿孔后三天，CAS 9诱导HPD位点进行分析，结果显示用CRISPR Cas电穿孔的肝细胞中9个mRNA的靶插入缺失为47.4%，CAS 9个RNP的插入缺失为78.4%。MTT测定显示用CRISPR Cas九个mRNA和R n p处理的肝细胞的活力为35.4%和45.9%.与MTT结果一致，用CAS九RNA处理的肝细胞的标准化白蛋白水平为31.8%，CAS 9 r p的肝细胞的标准化白蛋白水平为34.5%。要记住的最重要的事情是，适当的插管会影响所有后续步骤和手术的成功。完成此过程后，可以移植肝细胞和小鼠以研究其植入。此外，还可以提取接种肝细胞的基因组DNA，分析CAS nine编辑的效率和特异性。

Research

JoVE Journal

Bioengineering

Self-reporting Scaffolds for 3-Dimensional Cell Culture

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

科研

生物工程

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

多孔硅微粒进行交付的siRNA治疗的

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

交货蛋白质，多肽或细胞不可渗透的小分子进入活细胞被孵化与核内体溶解试剂dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

微流体流钱伯斯再造使用血止血建模和血小板输注<em&gt;体外</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

纳米丝绸制造和药物输送应用

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

利用 CRISPR-Cas9 系统高效生成和编辑人胰细胞无饲养干细胞

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

CRISPR/Cas9 复合原注射液生产基因工程金黄叙利亚仓鼠

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

Handling and Assessment of Human Primary Prostate Organoid Culture

人原发性前列腺组织培养的处理与评价

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process ...

Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye

用于向小鼠眼睛输送生物制剂的封装细胞技术

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, ...