大鼠肝细胞的分离和小学文化

Biology

Your institution must subscribe to JoVE's Biology section to access this content.

Fill out the form below to receive a free trial or learn more about access:

 

Summary

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

Cite this Article

Copy Citation | Download Citations | Reprints and Permissions

Shen, L., Hillebrand, A., Wang, D. Q. H., Liu, M. Isolation and Primary Culture of Rat Hepatic Cells. J. Vis. Exp. (64), e3917, doi:10.3791/3917 (2012).

Please note that all translations are automatically generated.

Click here for the english version. For other languages click here.

Abstract

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。

Protocol

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。

  1. 准备灌注缓冲汉克的平衡盐溶液中添加以下 (HBSS中,没有的Ca 2 +镁+,请参阅表1):2 + MgCl 2的 0.9毫米,EDTA的0.5毫米,与肝素钠至25日毫米。
  2. 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。
  3. 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。
  4. 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。
  5. 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。

2。大鼠肝脏隔离灌注

  1. 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。
  2. 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。
  3. 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。
  4. 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。
  5. 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。
  6. 流量增加至25毫升/分钟的速率。应该成为肝脏颜色苍白。
  7. 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。
  8. <李>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。
  9. 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。

3。肝细胞隔离

  1. 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。
  2. 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。
  3. 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
  4. 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。
  5. 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。
  6. 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。
  7. 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。
  8. 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。

4。肝细胞培养

  1. 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。
  2. 在一个典型的准备与活细胞> 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%。
  3. 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。
  4. 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。
  5. 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)。
  6. 每隔2天更换培养基中,如果需要的话。

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。

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)。

图1
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。

图2
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。

试剂名称 公司 目录编号
的HBSS(无钙2 +和Mg 2 +) Invitrogen公司 14174
HBSS中(与Ca 2 +和Mg 2 +) Invitrogen公司 14025
威廉姆斯的中等é Invitrogen公司 12551-032
collegenase二沃辛顿 LS004176
细胞过滤器(100微米) 屋宇署 352360
HepatoZYME-SFM Invitrogen公司 17705
Percoll密度西格玛 P4937
angiocath(18号) 屋宇署 381705

表1。

Subscription Required. Please recommend JoVE to your librarian.

Discussion

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%编制细胞与细胞之间的88〜96%的存活率。以下是其他的关键步骤:

  1. 任何协议描述细胞培养,最关键的环节是由细菌或真菌病原体的污染,以避免使用严格的无菌技术11。
  2. 桥粒,还作为黄斑粘着称为,是细胞结构,细胞与细胞之间的粘附专业。桥粒的完整性需要钙,它打破了由EDTA和钙自由媒体。酶的胶原酶能分解的桥粒,导致肝细胞的分离。因此,灌注使用的Ca 2 +免费中等和随后的Ca 2 +丰富的培养基中的Ca 2 +依赖胶原酶消化。
  3. 适当胶原酶治疗为肝细胞的准备是绝对关键。应保持在25毫升/分钟的流量Perfussion缓冲区II加collegenase二 。不成功的肝细胞培养的常见原因包括:1)贫困细胞分离,这可能是由于充分加热到37°C和/或血流速度缓慢灌注缓冲区; 2)细胞死亡,这可以变成d是由于过度胶原酶消化。
  4. 改变后的第一个4小时的文化传媒时要加倍小心,肝仍然比较脆弱,可以很容易损坏或中断直接接触;仅下降一侧以及吸管,从来没有直接对细胞的顶部。

Subscription Required. Please recommend JoVE to your librarian.

Disclosures

没有利益冲突的声明。

Acknowledgments

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)。

References

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

Comments

23 Comments

  1. Dear Dr.L. Shen,
    I have watched your viedo about " Isolation and Primary Culture of Rat Hepatic Cells"which was perfectly made. I and our young collegues in the department would like to have full protocol of this work. Is it possible to send it by email. ?
    Thank you for your kind collaboration.
    Regards
    Prof.Dr.Cengiz Baycu
    Head of dept. of Histology&Embryology
    University of Osmangazi Medical Faculty

    Reply
    Posted by: Cengiz B.
    September 13, 2012 - 3:25 AM
  2. Yes, please let me know your e-mail address and then I will send you the protocol. Thanks for your interest.

    Min

    lium@uc.edu

    Reply
    Posted by: Min L.
    November 8, 2012 - 10:44 AM
  3. my email:sxm1987²010@163.com,thank you very much

    Reply
    Posted by: shi x.
    November 9, 2012 - 2:53 AM
  4. Dear Dr.L. Shen,
    I have watched your viedo about " Isolation and Primary Culture of Rat Hepatic Cells"which was perfectly made. I and our young collegues in the department would like to have full protocol of this work. Is it possible to send it by email. ?
    Thank you for your kind collaboration.
    Regards
    Kita
    Department of ZheJiang University

    Reply
    Posted by: li y.
    November 7, 2012 - 11:18 PM
  5. Yes, please let me know your e-mail address and then I will send you the protocol. Thanks for your interest.

    Min Liu, Ph.D.
    lium@uc.edu

    Reply
    Posted by: Min L.
    November 8, 2012 - 10:45 AM
  6. I am Urmil Dhru from University of Maryland Baltimore requesting you to send the detail and complete protocol on my yahoo account Urmil_Dhru@yahoo.com or my umaryland account udhru@smail.uamryland.com

    Reply
    Posted by: Urmil D.
    June 27, 2014 - 12:24 PM
  7. Dear Dr.L. Shen,
    I have watched your viedo about " Isolation and Primary Culture of Rat Hepatic Cells"which was perfectly made. I and our young collegues in the department would like to have full protocol of this work. Is it possible to send it by email. ? My email: zhuhaossmu@hotmail.com.
    Thank you for your kind collaboration.
    Regards

    Hao Zhu
    Department of Anatomy
    Shanghai Jiao Tong University

    Reply
    Posted by: Hao Z.
    December 19, 2012 - 8:30 PM
  8. Dear Dr.L.Shen,
    I am very intereted in the video and protocol of "Isolation and Primary Culture of Rat Hepatic Cells" . Would you please send a full copy of video and protocol to me? My email: ldc1983king@yahoo.com.cn
    Thank you very much.
    Best wishes!

    Li Daochuan
    Department of Public health
    Sun yat-sen University

    Reply
    Posted by: li d.
    December 20, 2012 - 8:56 PM
  9. Dear Dr.L.Shen,
    I am very intereted in the video and protocol of "Isolation and Primary Culture of Rat Hepatic Cells" . Would you please send a full copy of video and protocol to me? My email: yipeng511@163.com
    Thank you very much.
    Best wishes!

    Yi Peng
    Department of Life Science and Technology
    China Pharmaceutical University

    Reply
    Posted by: Yi P.
    April 7, 2013 - 1:34 AM
  10. Dear Dr.L.Shen,
    I am very intereted in the video and protocol of "Isolation and Primary Culture of Rat Hepatic Cells" . Would you please send a full copy of video and protocol to me? My email: nagh0²14@naver.com
    Thank you very much.
    Best wishes!

    Gun Hyung Na
    Department of Surgery
    Catholic University of Korea

    Reply
    Posted by: Gun Hyung N.
    April 10, 2013 - 9:41 AM
  11. Dear Dr.L. Shen,
    I have watched your viedo about " Isolation and Primary Culture of Rat Hepatic Cells"which was perfec.
    I need a copy of it if it is possible please for my student learning about the liver perfusion.
    Dr Hamed zeinvand
    department of toxicology
    tehran university of medical science
    Iran

    Reply
    Posted by: hamed z.
    May 4, 2013 - 3:05 AM
  12. Dear Dr.L.Shen,
    I am very instereted in the video and protocol of "Isolation and Primary Culture of Rat Hepatic Cells" . Would you please send a full copy of video and protocol to me? My email: dickg@126.com
    Thank you very much.
    Best wishes!

    Reply
    Posted by: yufan g.
    July 2, 2013 - 4:41 PM
  13. Reply
    Posted by: su x.
    August 2, 2013 - 10:51 PM
  14. Dear Dr.L. Shen,
    I have watched your viedo about " Isolation and Primary Culture of Rat Hepatic Cells"which was perfect.
    Would you please send a full copy of protocol to my email (raghava@suven.com).

    Mr Raghava Choudary Palacharla,
    Drug Metabolism and pharmacokinetics.
    Hyderabad, INDIA - 500055

    Reply
    Posted by: Raghava Choudary P.
    August 5, 2013 - 7:25 AM
  15. Hello Dr L Shen,

    Thank you for your work in producing this protocol. I would very much like to have a full copy of this protocol. Best wishes. sylvie.coulibaly@wmich.edu.

    Reply
    Posted by: Sylvie C.
    August 27, 2013 - 6:30 PM
  16. Dear Hello Dr L Shen,

    I would be glad and appreciate if you kindly email me this protocol video and pdf.

    Best Wishes
    Waqar Saeed

    Waqar.saeed33@gmail.com

    Reply
    Posted by: blue v.
    September 3, 2013 - 10:59 PM
  17. Dear Dr.L.Shen,
    I am very intereted in the video and protocol of "Isolation and Primary Culture of Rat Hepatic Cells" . Would you please send a full copy of video and protocol to me? My email: atulwavhal@gmail.com
    Thank you very much.
    Best wishes!

    Reply
    Posted by: Atul W.
    November 21, 2013 - 10:56 PM
  18. Dear Dr.L. Shen,
    I have watched your viedo about " Isolation and Primary Culture of Rat Hepatic Cells"which was perfectly made. I and our young collegues in the department would like to have full protocol of this work. Is it possible to send it by email. ? My email: mbenyamina.2001@yahoo.fr
    Thank you for your kind collaboration.
    Regards.
    Departement of Biotechnology
    University of Oran. Algeria

    Reply
    Posted by: benyamina m.
    March 3, 2014 - 3:22 PM
  19. Dear Dr.L. Shen,
    I have watched your viedo about " Isolation and Primary Culture of Rat Hepatic Cells"which was perfectly made. I and our young collegues in the department would like to have full protocol of this work. Is it possible to send it by email. ? My email: mbenyamina.2001@yahoo.fr
    Thank you for your kind collaboration.
    Regards.
    Departement of Biotechnology
    University of Oran. Algeria

    Reply
    Posted by: benyamina m.
    March 3, 2014 - 5:29 PM
  20. Dear Dr. Shen,
    Very useful protocol for me, since Im trying to isolate hepatocytes from sheep liver. I have been facing problem since cells are not attaching to collagen coated plates. Kindly guide me on these aspects and also send the complete protocol (both text and video) to my e mail ID: bdpunith@gmail.com.

    Thanks and Regards,
    Punith B D
    NIANP
    Bangalore, India

    Reply
    Posted by: Punith B.
    July 21, 2014 - 8:38 AM
  21. Dear Dr.L.Shen,
    I am very intereted in the video and protocol of "Isolation and Primary Culture of Rat Hepatic Cells" . Would you please send a full copy of video and protocol to me? My email: delongn@mcmaster.ca
    Thank you for sharing!
    Best wishes!

    McMaster University
    Canada

    Reply
    Posted by: Nicole D.
    September 4, 2014 - 2:19 PM
  22. Dear Dr.L.Shen,
    I am very interested in your video and I want to prepare hepatocyte cells accordingly. I want to ask which brand/catalog of insulin do you use for preparing the medium?

    Reply
    Posted by: Wenjie W.
    October 10, 2014 - 2:15 AM
  23. why the steps showed in the video is different from what written in the protocol? in the video no Percoll used, but in protocol you used Percoll

    Reply
    Posted by: Xia D.
    May 22, 2018 - 6:58 AM

Post a Question / Comment / Request

You must be signed in to post a comment. Please or create an account.

Usage Statistics