Isolation and Primary Culture of Rat Hepatic Cells

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Summary

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 × 108 cells per preparation with cell viability between 88 ~ 96%.

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

Abstract

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 μm pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Protocol

1. Preparation

All buffers are freshly prepared using sterile technique and filter sterilized using a Corning 0.22 μm filter.

  1. Prepare Perfusion buffer I by adding the following to Hank's Balanced Salt Solution (HBSS, without Ca2+ and Mg2+, see Table 1): Mg2+ (MgCl2) to 0.9 mM, EDTA to 0.5 mM, and HEPES to 25 mM.
  2. Prepare Perfusion buffer II by adding the following to HBSS (with Ca2+ and Mg2+, see Table 1): HEPES to 25 mM.
  3. Prepare Perfusion buffer II plus collagenaseII: Dissolve collagenase II (1000 U) with 300 ml Perfusion buffer II and keep the solution warm in water-bath before perfusion. This solution should be used within 30 min because the activity of collagenase II decreased with the time.
  4. Prepare William's complete Medium: Add the following to Williams' Medium E: L-glutamine to 2 mM, fetal bovine serum (FBS) to 5%; insulin to 100 nM, dexamethasone to 100 nM, penicillin to 100 IU/ml and streptomycin to 100 mg/ml.
  5. These buffers should be warmed for 30 minutes in the water bath at 42 °C, an optimal temperature corresponding to an outlet temperature at the cannula of 37 °C.

2. Rat Perfusion for Liver Isolation

  1. The perfusion system consists of the pump, autoclavable silastic tubing and a water bath (see Figure 1). Pre-set the flow rate of the peristaltic perfusion pump to 10 ml/min.
  2. Anesthetize an adult rat (300 g body weight) with intraperitoneal (ip) ketamine (87 mg/kg body weight) plus xylazine (13 mg/kg). Depth of anesthesia should be monitored by toe pinch. When the rat no longer responds to noxious stimulus, shave abdominal hair and prep the abdomen with betadine and ethanol. Enter through a midline incision.
  3. Expose the hepatic portal vein by carefully moving the viscera to the right outside of the abdominal cavity, and insert an 18-gauge angiocath into the hepatic portal vein (see Figure 1).
  4. Connect the perfusate tubing to the needle and initiate the infusion in situ at a low flow rate (10 ml/min) with pre-warmed (37 °C) Perfusion Buffer I.
  5. If performed properly, the liver should instantly begin to blanch. Once successful cannulation is confirmed, make a cut at inferior vena cava (IVC) to allow efflux (Figure 1). A further test for successful cannulation can be performed by applying light pressure with sterile swab on the IVC; all lobes of the liver should quickly begin to swell.
  6. Increase the rate of flow to 25 ml/min. The liver should become pale in color.
  7. Switch the perfusion solution to Perfusion buffer II plus collagenase II with no interruption of flow for an additional 6 minutes.
  8. Periodically (5-10 times during digestion) apply pressure with swab to the IVC for 5-second intervals. The liver will swell, leading to enhanced hepatic cell dissociation, in turn reducing total digestion time, and increasing final yield.
  9. After collagenase perfusion, liver should begin to look mushy. Dissect the liver free, place in a pre-chilled sterile beaker with 20 ml William's complete Medium, and then take it to tissue cell culture hood.

3. Hepatocyte Cell Isolation

  1. Within the cell culture hood, use a cell scraper to gently disperse the cells into William's complete Medium within a sterile Petri dish.
  2. Filter the cell dispersion through a 100 μm pore size cell strainer into a 50 ml Conical tube in order to remove connective tissues and undigested tissue fragments.
  3. Suspend cells in 40 ml William's complete Medium and centrifuge at 50 x g for 3 min at 4 °C.
  4. Aspirate the supernatant, and gently re-suspend cells in 40 ml cold William's complete Medium to wash cells. Repeat centrifugation.
  5. Aspirate the supernatant, and gently re-suspend cells with 25 ml William's complete Medium. Add 25 ml 90% Percoll solution in PBS into the tube and gently mix.
  6. Centrifuge at 200 x g for 10 min at 4 °C. Aspirate the dead cells from the top of the gradient because the viable cells remain at the bottom of the Percoll gradient.
  7. Suspend the cell pellet in 30 ml warm William's complete Medium, and then repeat centrifugation and re-suspend the cell pellet in 20 ml warm William's complete Medium.
  8. Count the cells within the cell suspension using a hemocytometer and determine cell viability by trypan blue staining.

4. Hepatocyte Culture

  1. Dilute cells with warm William's complete Medium to preferred concentration, e.g. 2.5 x 105 cells/ml. Plate cells at a desired volume on cell culture plates, e.g. 5 x 105 cells / 2 ml / well, 6 wells/plate; or 2.5 x 105 cells / 1.5 ml / well, 12 wells / plate. Un-coated plate is fine for hepatic cell cultures.
  2. In a typical prep with > 85% viable cells, the cell density should reach approximately 60-70% confluence, which allows for cell-cell contact while maintaining sufficient space for the hepatocytes to grow to their full cell size and yield a final confluence of 90-95%.
  3. In order to form an even monolayer of hepatocytes, in other words, to minimize the tendency for cells to aggregate in the central area of the well (most likely due to the air-flow inside of cell incubator), allow the plates to remain inside the cell culture hood for 30 minutes before placing them in a incubator.
  4. Culture the cells at 37 °C in a humidified atmosphere of 95% air and 5 % CO2. After 4-h culture, the cells could either remain in the same serum-containing medium or replace the medium with serum-free medium, e.g. HepatoZYME-SFM (see Table 1). The serum-free medium helps to maintain cell morphology with no adverse effects from hormones when a serum-free medium is used.
  5. Allow cells to recover and grow at least overnight prior to experimentation. We recommend to use the cells for test within 24 hr because this may help preserve the function of critical enzymes (e.g., P450s).
  6. Replace the growth medium at 2 day intervals, if needed.

5. Representative Results

The conditions described regularly generate cell harvests of 1.0 x 108 cells per preparation from one rat liver. The viability of the hepatocytes measured by trypan blue exclusion was consistently within the range of 88 ~96%.

As depicted in Figure 2, the hepatocytes aggregate and form cluster after 4 hr seeding. Most isolated cells flatten and spread in typical monolayer growth. By 24 hr, the edges of cells are defined, surface of cells is fairly smooth, and lipid droplets are visible. The cells have one to three nucleoli, which are round, located at the center of the cells, and the nuclei display consistent size among cells (Figure 2).

Figure 1
Figure 1. A diagram of liver perfusion. An 18-gauge angiocath is inserted into the portal vein of liver, then a perfusate tubing is connected to the needle. Once successful cannulation is confirmed, make a cut at inferior vena cava (IVC) to allow efflux.

Figure 2
Figure 2. Morphology of cultured hepatocytes over time (4 hr to 24 hr), magnification x 200. After cultured for 24 hr, the cells spread in typical monolayer growth, and the junctions between the cells are linear.

Name of the reagent Company Catalog number
HBSS (without Ca2+, and Mg2+) Invitrogen 14174
HBSS (with Ca2+, and Mg2+) Invitrogen 14025
Williams's Medium E Invitrogen 12551-032
Collegenase II Worthington LS004176
Cell strainer (100 μm) BD 352360
HepatoZYME-SFM Invitrogen 17705
Percoll Sigma P4937
Angiocath (18-gauge) BD 381705

Table 1.

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Discussion

Primary culture of hepatocytes is an in vitro model widely used to study various aspects of liver physiology and pathology. For example, primary culture is used to assess the expression and function of drug-metabolizing enzymes including cytochromes P450, drug metabolism, drug-drug interactions, and the mechanisms of cytotoxicity and genotoxicity 3-7. The protocol describing isolation and culture of rat hepatic cells is adapted from the previous reports of Aiken et al. 8, and others 2,9,10 with modifications. The conditions described consistently generate viable hepatocytes up to 1.0 x 108 cells per preparation with cell viability between 88 ~ 96%. The following are other critical steps:

  1. As with any protocol describing cell culture, the most critical aspect is to avoid contamination from bacterial or fungal pathogens by using strict aseptic techniques 11.
  2. Prior to perfusion, proper care should be taken to avoid perforating the back wall of the portal vein.
  3. Desmosome, also known as macula adherens, is a cell structure specialized for cell-to-cell adhesion. The integrity of the desmosome requires calcium, and it is broken down by EDTA and calcium-free media. The enzymes collagenase can dissociate the desmosome, leading to the isolation hepatic cells. Therefore, perfusion uses Ca2+ free medium and subsequently Ca2+ rich medium containing Ca2+ dependent collagenase, for digestion.
  4. Appropriate collagenase treatment is absolutely crucial for hepatocyte preparation. The flow rate of Perfussion buffer II plus collegenase II should be kept at 25 ml/min. Common causes of unsuccessful hepatocyte culture include: 1) poor cell dissociation, which could be due to perfusion buffers inadequately warmed to 37° C and/or slow perfusion speed; and 2) cell death, which could be due to excessive collagenase digestion.
  5. Be cautious when changing the media after the first 4-h culture, as the hepatocytes are still relatively fragile and can be easily damaged or disrupted by direct contact; pipette only down the side of the well, and never directly on top of the cells.

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Disclosures

No conflicts of interest declared.

Acknowledgments

The authors would like to thank Mr. Josh Basford and Dr. Xiao-min Li for technical assistance. This work was supported in part by NIH grants (DK70992 and DK92779 to M.L.).

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

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