作者要感谢技术援助乔希贝斯福德先生和晓敏李博士。这项工作是支持的，一部分由NIH资助（DK70992，DK92779到ML）。

1。准备所有缓冲区新鲜配制使用无菌操作技术，使用康宁0.22微米过滤器和过滤消毒。 <ol><li>准备灌注缓冲汉克的平衡盐溶液中添加以下我 （HBSS中，没有的Ca 2 +和镁+，请参阅表1）：镁2 + MgCl 2的 0.9毫米，EDTA的0.5毫米，与肝素钠至25日毫米。 </li><li>准备加入以下的HBSS（ 灌注缓冲II与Ca 2 +和Mg 2 +， 请参阅表1）：肝素钠至25毫米。 </li><li>准备灌注缓冲加collagenaseII：溶于300毫升灌注缓冲区第二胶原酶II（000 U），并保持在水浴之前灌注的解决方案的温暖。此解决方案应在30分钟内，因为Ⅱ型胶原酶的活性随时间下降。 </li><li>准备威廉的完全培养基中添加以下威廉姆斯中等电子邮件：L-谷氨酰胺2毫米，胎牛血清（FBS），5％，至100纳米的胰岛素，地塞米松至100纳米，青霉素100 IU / ml和链霉素100毫克/毫升。 </li><li>这些缓冲区应加热30分钟水浴42℃，最佳温度，出口温度在37套管°C。 </li></ol> 2。大鼠肝脏隔离灌注<ol><li>灌注系统由泵，高压灭菌的硅胶管和水浴（ 见图1）。预先设定的蠕动灌注泵流量10毫升/分钟。 </li><li>麻醉成年大鼠（300 g体重）腹腔内（IP），氯胺酮（87毫克/公斤体重）加甲苯噻嗪（13毫克/千克）。应监测麻醉深度的脚趾掐。当老鼠不再响应伤害性刺激，剃腹毛，并准备用优碘和乙醇的腹部。通过正中切口进入。 </li><li>暴露小心移动到外面的腹腔内脏，并插入到肝门静脉（ 见图1）18号angiocath的肝门静脉。 </li><li>连接灌流针管，并开始在低流量（10毫升/分钟）与预温（37℃） 灌注缓冲我在原地输液。 </li><li>如果执行得当，肝脏应立即开始发白。一旦确认插管成功，在切下腔静脉（IVC），让流出（ 图1）。插管成功的一个进一步的测试，可以用无菌棉签上腔静脉压光;肝叶应迅速开始膨胀。 </li><li>流量增加至25毫升/分钟的速率。应该成为肝脏颜色苍白。 </li><li>没有额外的6分钟的流量中断切换II PLUSⅡ型胶原酶灌注缓冲灌注液。 </li> &lt;李&gt;定期在消化过程中的（5-10倍）适用于与拭子压力，下腔静脉5秒的时间间隔。肝脏会膨胀，导致增强肝细胞分离，反过来降低总的消化时间，增加最终产量。 </li><li>胶原酶灌注后，肝应该开始寻找糊状。解剖肝在预冷的无菌烧杯，20毫升威廉完整的中等的地方，然后把它带到组织细胞培养罩。 </li></ol> 3。肝细胞隔离<ol><li>在细胞培养罩，使用细胞刮刀轻轻地分散到威廉的完全培养基无菌培养皿内的细胞。 </li><li>通过孔径100微米到50毫升锥形管细胞滤网过滤细胞分散，以消除结缔组织和未消化的组织碎片。 </li><li>暂停细胞在4°C为3分50 XG40毫升威廉完整的中等和离心机</li><li>吸出上清液，轻轻重新挂起40毫升冷威廉的完全培养基中的细胞洗涤细胞。重复离心。 </li><li>轻轻吸上清，重新悬浮细胞25毫升威廉完整的中等。添加到管在PBS 25毫升90％的Percoll溶液，轻轻混匀。 </li><li>在4°C离心10分钟在200 XG吸因为活细胞在Percoll梯度底部从梯度上方的死细胞。 </li><li>暂停30毫升温水威廉的完全培养基中的细胞沉淀，然后重复离心和重新挂起20毫升温水威廉的完全培养基中的细胞沉淀。 </li><li>细胞内暂停使用血球计数细胞，并确定由台盼蓝染色细胞活力。 </li></ol> 4。肝细胞培养<ol><li>温暖威廉完整的中等首选浓度稀释的细胞，如2.5×10 5细胞/毫升。板所需的体积的细胞，细胞培养板，如。 5×10 5细胞/毫升/井，6口井/板;或2.5×10 5细胞/ 1.5毫升/孔，12孔/板。联合国涂层板是肝细胞培养的罚款。 </li><li>在一个典型的准备与活细胞&gt; 85％，细胞密度应达到约60-70％的汇合点，它允许细胞与细胞接触，同时保持足够的空间，肝细胞生长充分发挥其细胞大小和产量的最终合流90-95％。 </li><li>以形成单层甚至肝，换句话说，在井的中部地区，以尽量减少对细胞的倾向聚合（最可能是由于细胞培养箱内的空气流动），使板内保持前在孵化器中放置30分钟，细胞培养罩。 </li><li>培养的细胞在37°C在95％空气和5％CO2培养箱气氛。 4小时培养后，细胞能保持在相同的含血清培养基或更换培养基，血清-F稀土介质，如HepatoZYME-SFM（ 见表1）。无血清培养基，有助于维持细胞形态与激素没有不利影响时，用无血清培养基。 </li><li>让细胞恢复和增长至少隔夜实验之前。我们建议使用24小时内的细胞试验，因为这可能有助于保护关键酶的功能（例如，细胞色素P450）。 </li><li>每隔2天更换培养基中，如果需要的话。 </li></ol> 5。代表结果所描述的条件定期产生1.0×10 8％准备从一个肝细胞的细胞丰收。台盼蓝排斥测量肝细胞的生存能力，持续88〜96％范围内。 在图2中所描述的肝细胞聚集和后4小时直播的形式集群。在典型的单层增长最孤立的细胞扁平化和传播。 24小时，定义边缘的细胞，细胞表面是相当顺利，可见脂滴。细胞有一至三年的核仁，是圆形，位于细胞的中心，原子核显示细胞间的大小一致（ 图2）。 <img alt="图1" src="/files/ftp_upload/3917/3917fig1.jpg" /> 图1。肝脏灌注图。一个18号angiocath的是肝门静脉插入，然后灌流管连接针。一旦确认插管成功，使下腔静脉（IVC）在截止到允许流出。 <img alt="图2" src="/files/ftp_upload/3917/3917fig2.jpg" /> 图2。培养肝细胞，随着时间的推移（4小时至24小时），放大倍数×200的形态。培养24小时后，分布在典型的单层生长的细胞，细胞之间的连接处是线性的。 <table bordeR =“1”&gt; <tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>的HBSS（无钙2 +和Mg 2 +） </td><td> Invitrogen公司</td><td> 14174 </td></tr><tr><td> HBSS中（与Ca 2 +和Mg 2 +） </td><td> Invitrogen公司</td><td> 14025 </td></tr><tr><td>威廉姆斯的中等é </td><td> Invitrogen公司</td><td> 12551-032 </td></tr><tr><td> collegenase二</td><td>沃辛顿</td><td> LS004176 </td></tr><tr><td>细胞过滤器（100微米） </td><td>屋宇署</td><td> 352360 </td></tr><tr><td> HepatoZYME-SFM </td><td> Invitrogen公司</td><td> 17705 </td></tr><tr><td> Percoll密度</td><td>西格玛</td><td> P4937 </td></tr><tr><td> angiocath（18号） </td><td>屋宇署</td><td> 381705 </td></tr></tbody></table> 表1。

<ol>
	<li>Berry, M. N., Friend, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yield+preparation+of+isolated+rat+liver+parenchymal+cells:+a+biochemical+and+fine+structural+study.">High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study.</a> J. Cell Biol. 43, 506-520 (1969).</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 Cell Biol. 13, 29-83 (1976).</li><li>Saito, K., Kobayashi, K., Mizuno, Y., Fukuchi, Y., Furihata, T., Chiba, K., Schmidt, M., Schmitz, H. J., Baumgart, A., Guedon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisome+proliferator-activated+receptor+alpha+(PPARalpha)+agonists+induce+constitutive+androstane+receptor+(CAR)+and+cytochrome+P450+2B+in+rat+primary+hepatocytes.">Peroxisome proliferator-activated receptor alpha (PPARalpha) agonists induce constitutive androstane receptor (CAR) and cytochrome P450 2B in rat primary hepatocytes.</a> Drug Metab. Pharmacokinet. 25, 108-111 (2010).</li><li>Gomez-Lechon, M. J., Donato, M. T., Castell, J. V., Jover, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hepatocytes+in+primary+culture:+the+choice+to+investigate+drug+metabolism+in+man.">Human hepatocytes in primary culture: the choice to investigate drug metabolism in man.</a> Curr. Drug Metab. 5, 443-462 (2004).</li><li>Goncalves, L. A., Vigario, A. M., Penha-Goncalves, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+isolation+of+murine+hepatocytes+for+in+vitro+malaria+liver+stage+studies.">Improved isolation of murine hepatocytes for in vitro malaria liver stage studies.</a> Malar. J. 6, 169 (2007).</li><li>Bu, S. Y., Mashek, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+long-chain+acyl-CoA+synthetase+5+mediates+fatty+acid+channeling+between+anabolic+and+catabolic+pathways.">Hepatic long-chain acyl-CoA synthetase 5 mediates fatty acid channeling between anabolic and catabolic pathways.</a> J. Lipid Res. 51, 3270-3280 (2010).</li><li>Aiken, J., Cima, L., Schloo, B., Mooney, D., Johnson, L., Langer, R., Vacanti, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+in+rat+liver+perfusion+for+optimal+harvest+of+hepatocytes.">Studies in rat liver perfusion for optimal harvest of hepatocytes.</a> J. Pediatr. Surg. 25, 140-144 (1990).</li><li>Jauregui, H. O., McMillan, P. N., Hevey, K., Naik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+analysis+of+lectin+binding+to+adult+rat+hepatocyte+cell+surfaces.">A quantitative analysis of lectin binding to adult rat hepatocyte cell surfaces.</a> In Vitro Cell. 24, 401-412 (1988).</li><li>Klaunig, J. E., Goldblatt, P. J., Hinton, D. E., Lipsky, M. M., Trump, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+liver+cell+culture.+II.+Primary+culture.">Mouse liver cell culture. II. Primary culture.</a> In Vitro. 17, 926-934 (1981).</li><li>Hay, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operator-induced+contamination+in+cell+culture+systems.">Operator-induced contamination in cell culture systems.</a> Dev. Biol. Stand. 75, 193-204 (1991).</li></ol>

没有利益冲突的声明。

小学文化的肝细胞在体外模型广泛应用于肝的生理和病理研究的各个方面。例如，小学文化，是用来评估药物代谢酶的表达和功能，包括细胞色素P450药物代谢，药物相互作用，细胞毒性和遗传毒性3-7机制。适应从以前的报告艾肯等人描述大鼠肝细胞的分离和文化协议。8，和其他修改2,9,10。所描述的条件不断生成可行肝可达1.0×10 8％编制细胞与细胞之...

isolation and primary culture of rat hepatic cells

原发性肝细胞培养是一个有价值的工具，已被广​​泛用于肝功能疾病，病理生理，药理及其他相关学科的基础研究。基于完整的肝细胞分离两步胶原酶灌注的方法，首先介绍了Berry和朋友在1969年1，自那时以来，经过了多次修改。最常用的技术，描述了1976年2 Seglenin。从本质上讲，是由非循环胶原酶灌注，通过门静脉麻醉的成年大鼠分离肝细胞。离体细胞，然后通过100微米的孔大小网眼尼龙过滤网过滤，培养上板。经过4小时的文化，取代含血清或无血清培养基，如额外的时间来培养，HepatoZYME-SFM培养基。这些程序需要手术和无菌培养，可以更好的视频显示比文字的步骤。 ĤERE，我们通过视频和书面协议，允许在可行的肝细胞生成大量的一贯记录这些程序的详细步骤。

Watch this Scientific Journal Video about Isolation and Primary Culture of Rat Hepatic Cells at JoVE.com

Isolation and Primary Culture of Rat Hepatic Cells

原发性肝细胞提供了一个宝贵的工具来评估生化，分子，在生理有关的实验系统和代谢功能。我们描述了可靠的协议，对大鼠原位肝灌注，一贯产生，最多to1.0×10的可行肝<sup&gt; 8</sup每准备&gt;细胞与细胞之间的88〜96％的存活率。

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-and-primary-culture-of-rat-hepatic-cells

Research

JoVE Journal

Biology

55.4K 次观看.  University of Cincinnati College of Medicine. 原发性肝细胞提供了一个宝贵的工具来评估生化，分子，在生理有关的实验系统和代谢功能。我们描述了可靠的协议，对大鼠原位肝灌注，一贯产生，最多to1.0×10的可行肝 8细胞与细胞之间的88〜96％的存活率。

视频: Isolation and Primary Culture of Rat Hepatic Cells

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.

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 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.

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 ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

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 week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic 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 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.

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 ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

来自新生儿和成年鼠皮肤的原代小鼠角化细胞的分离和培养

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

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.

大鼠肾上腺皮质细胞的原代培养及非甾体功能的测定

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 Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

微型猪原主主动脉内皮细胞的隔离与培养

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

成年小鼠和大鼠心肌细胞的隔离、转染和长期培养

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

哺乳动物原色素上皮细胞的分离、培养和基因工程用于非病毒基因治疗

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

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

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

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 ...