A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Immunology and Infection
 

Summary

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Cite this Article

Copy Citation | Download Citations

Kitani, H., Takenouchi, T., Sato, M., Yoshioka, M., Yamanaka, N. A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells. J. Vis. Exp. (51), e2757, doi:10.3791/2757 (2011).

Abstract

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.

After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.

In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

Protocol

1. Perfusion of the Liver

  1. Anesthetize an adult male Sprague-Dawley rat (at 10 to 15 weeks-old; body weight 150-200 g) by an intraperitoneal injection of a sodium pentobarbital solution (50 mg sodium pentobarbital / kg body weight).
  2. Place the animal in a stainless pan and spray 70% ethanol onto its abdomen and thorax.
  3. Make a small cut and peel off the skin. Open the abdomen by the cut with a pair of scissors.
  4. Confirm the location of portal vein, pass a surgical thread under the portal vein and loosely clump.
  5. Clump the distal part of the inferior vena cava and mesenteric vein to prevent blood reflux into the liver. Open the thoracic cavity by cutting the diaphragm with scissors, and expose the heart.
  6. Insert a plastic catheter (2 mm diameter) into the portal vein and tighten the thread firmly around the catheter.
  7. Connect the catheter to a perfusion system which is loaded with washing solution (Ca2+-Mg2+free HBSS containing 0.5 mM EGTA, 10 mM HEPES and 4.2 mM NaHCO3 ; pH 7.2) prewarmed at 37 °C.
  8. Begin perfusion in situ at a rate of 10 ml/min. At the same time, make a cut in right atrium wall. Continue perfusion for 10 min.
  9. Switch the perfusion solution to collagenase solution [HBSS containing 10mM HEPES and 4.2mM NaHCO3 supplemented with Type I collagenase (0.05%) and trypsin inhibitor (50 μg/ml); pH 7.5], and perfuse for 10 - 20 min at a rate of 10 ml/min until the liver slightly swells and shows the sign for disintegration of the liver structure under serous membrane.

2. Preparation of Parenchymal Hepatocyte-rich Cell Fraction

  1. Remove and carefully transfer the liver into a sterile beaker containing 25 ml of collagenase solution. Mince into small pieces by scissors.
  2. Add 75 ml of cold MEM into the resultant cell suspension. After gentle pipetting, filtrate through a cell strainer (100 μm)to remove connective tissues and undigested tissue fragments.
  3. Transfer the filtrate into 50 ml conical tubes and centrifuge at 50 x g for 1 min at 4°C (brake off).
  4. Carefully discard the supernatant. Resuspend the pellet with cold MEM.
  5. Repeat 2.4) three times.

3. Mixed Primary Culture of Liver Cells

  1. Suspend the cells obtained in 2.5) in the growth medium composed of DMEM containing 10% heat inactivated FCS, supplemented with 100 μM β-mercaptoethanol, 10 μg/ml insulin, 100 μg/ml streptomycin and 100 U/ml penicillin, and seed into five to ten tissue culture flasks (surface area: 75 cm2) at a density of 6.7 x 104 cells / cm2 (5 x 106 cells / flask / 12 -15 ml of medium).
  2. Incubate the culture flasksat 37°C in an atmosphere of 5% CO2- 95% air.
  3. Replace the growth medium every 2 to 3 days intervals.

4. Selective Isolation of Macrophages

  1. After 12 days of culture, macrophages proliferate on the cell sheet. These cells are suspended by reciprocal shaking of the culture flasks at 80 - 120 strokes per minute for 30 min at 37 °C.
  2. Transfer the culture medium into 100 mm non-tissue culture grade plastic dishes. Pool the medium from two T75 flasks into one plastic dish. Refill culture flasks with the growth medium and return them to the incubator.
  3. Incubate plastic dishes for 30 min at 37 °C in an atmosphere of 5% CO2- 95% air. Macrophages readily attach to non-tissue culture grade plastic dishes under this incubation process, whereas other contaminating cells do not.
  4. Aspirate the medium and gently rinse the plastic dish with PBS. Repeat this step three times.
  5. Add 1 ml of TrypLE Express solution into the dish, and incubate for 10min at 37 °C in an atmosphere of 5% CO2- 95% air.
  6. Add 9 ml of the growth medium. Gently scrape off the attached macrophages by a cell scraper.
  7. Transfer the cell suspension into a 15 ml conical tube and centrifuge at 180 x g for 5 min at 25°C (brake on).
  8. Discard the supernatant and add 1 ml of the growth medium. Dissociate into single cells by vigorous pipetting. Enumerate the cell number by a hemocytometer-counting.
  9. Isolation steps can be repeated with the same culture flasks at two to three day intervals for more than two weeks.
  10. Isolated macrophages can be cultured in the growth medium described in 3.1.

5. Representative Results

An example of a mixed primary culture of adult rat liver cells is shown in Figure 2. Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells. Then, around day 6 to 12, phase contrast-bright, round macrophage-like cells vigourouly proliferate on the fibroblastic cell sheet. By shaking of the culture flasks, macrophages are readily suspended into the culture medium, and subsequent transfer and incubation in plastic dishes result in selective adhesion of macrophages(Figure 3). The macrophage-like cells can be harvested as early as day 8, and the numbers reached maximal levels on days 12 to 14. More than 106 cells can be harvested from a T75 culture flask repeatedly at 2 to 3 day intervals for more than two weeks, enabling a total cell yield per flask of 107 per T75 culture flask (Figure 3). The purities of the isolated macrophages were 95 to 99%, as evaluated by flowcytometry or immunocytochemistry with rat macrophage-specific antibodies,such as ED-1 (Figure 4), ED-3, OX-41 (Figure 4) and Iba19.The isolated cells show typical characteristics of macrophages, such as active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.

Figure 1
Figure 1. A scheme for selective isolation and purification of macrophage-like cells from mixed primary culture of rat liver cells.

Figure 2
Figure 2. Primary culture of rat liver cells and proliferation of macrophage-like cells. Arrow indicates a multinuclear giant cell. Scale bar = 100 μm. Reprinted from the Journal of Immunological Methods, Vol.360, 47-55 (2010) 9 with permission from Elsevier.

Figure 3
Figure 3. Selective isolation of macrophage-like cells by the shaking and attachment method. Cells were suspended into the culture medium by shaking the flasks, subsequently transferred into non-tissue culture grade plastic dishes, and incubated at 37 °C. As early as 10 min after plating, macrophage-like cells attached to the dish surface, while other contaminating fibroblastic cells remained suspended. After rinsing with PBS, a highly purified macrophage population was obtained. These cells gained typical macrophage morphology after 40 min of culture, and mitotic cells were frequently observed (arrows). Subsequent changes in cell numbers recovered from flasks at different culture periods are shown. Values are an average±SD from three to five flasks. Experiments were repeated three times and data are indicated by different symbols. Scale bar =100 μm. Reprinted from the Journal of Immunological Methods, Vol.360, 47-55 (2010)9 with permission from Elsevier.

Figure 4
Figure 4. Immunocytochemical staining of macrophage-like cells with monoclonal antibodies against rat macrophages.

Figure 5
Figure 5. Phagocytosis of FITC-labeled microbeads by macrophage-like cells.

Discussion

Here, we report a simple and efficient method to obtain macrophages from the mixed primary cultures of adult rat liver cells. Our method relies first on the novel proliferative activity of macrophages in the mixed culture of rat liver cells, and then subsequent isolation and purification of these cells on the basis of their biological characteristics as macrophages9. It may be possible the macrophages proliferated in the mixed primary culture of adult liver cells might have originated from Kupffer or related cells that contaminated into the parenchymal hepatocytes fraction. The macrophages might have responded to specific environmental changes caused by transformation of parenchymal hepatocyte10 and other fibroblastic cells in the mixed primary cultures.

In conclusion, our method provides isolation of macrophage-like cells in sufficient number and purity from the primary culture of rat liver cellswithout using sophisticated equipment and technical skills. In addition, the isolation procedure can be repeated with the same culture flask for more than two weeks. This method might be applicable to other mammalian species.

Disclosures

All animals were kept and operated in the animal facility in the National Institute of Animal Health, according to the guidelines and regulations set forth by the Institution's Committee for animal experiments.

Acknowledgments

This work was funded by a research grant and a Grant-in-Aid from the Food Nanotechnology Project of the Ministry of Agriculture, Forestry, and Fisheries of Japan.

Materials

Name Company Catalog Number Comments
Sodium pentobarbital solution Kyoritsu Seiyaku Somnopentyl Injection
Hank’s balanced salt solution (HBSS) Invitrogen 14185-052
Ethylene glycol tetraacetic acid(EGTA) Sigma-Aldrich E-4378
Collagenase Wako Pure Chemical Industries, Ltd. 032-10534 Cell dissociation grade
(Lot check needed)
Trypsin inhibitor Sigma-Aldrich T-9128
Eagle’s minimum essential medium (MEM) Sigma-Aldrich M-4655
Dulbecco’s modified Eagle’s medium(DMEM) Sigma-Aldrich D-6459 High glucose-type
Fetal calf serum (FCS) Hyclone SH30070.03 Heat inactivated at 56°C for 30 min.
(Lot check needed)
TrypLE Express Invitrogen 12605
Dulbecco’s phosphate buffered saline (PBS) Invitrogen 14190 Ca2+-Mg2+ free
β-mercapt–thanol Sigma-Aldrich M-7522 Stock: 100 mM in distilled water
Insulin Sigma-Aldrich I-5500 Stock: 10 mg/ml in 0.1N HCl
Penicillin/ streptomycin Invitrogen 15070
Cell strainer BD Biosciences 352360 Mesh size: 100 μm
Tissue culture flask Sumitomo Bakelite Co., Ltd. MS-21250 Surface area: 75 cm2
Non-tissue culture plastic dishes BD Biosciences 351005
Cell scraper Corning 3010
Reciprocal shaker TAITEC NR-1

DOWNLOAD MATERIALS LIST

References

  1. Crispe, I. N. The liver as a lymphoid organ. Annu. Rev. Immunol. 27, 147-163 (2009).
  2. Roberts, R. A. Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis. Toxicol. Sci. 96, 2-15 (2007).
  3. Gao, B., Jeong, W. I., Tian, Z. Liver: An organ with predominant innate immunity. Hepatology. 47, 729-736 (2008).
  4. Janousek, J., Strmen, E., Gervais, F. Purification of murine Kupffer cells by centrifugal elutriation. J. Immunol. Methods. 164, 109-117 (1993).
  5. Olynyk, J. K., Clarke, S. L. Isolation and primary culture of rat Kupffer cells. J. Gastroenterol. Hepatol. 13, 842-845 (1998).
  6. Heuff, G., Meyer, S., Beelen, R. H. Isolation of rat and human Kupffer cells by a modified enzymatic assay. J. Immunol. Methods. 174, 61-65 (1994).
  7. Seglen, P. O. Preparation of isolated rat liver cells. Methods Cell Biol. 13, 29-83 (1976).
  8. Yoshioka, M. Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells. Vet. Immunol. Immunopathol. 58, 155-163 (1997).
  9. Kitani, H., Takenouchi, T., Sato, M., Yoshioka, M., Yamanaka, N. A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells. J. Immunol. Methods. 360, 47-55 (2010).
  10. Gorrell, Liver fibrosis: the hepatocyte revisited. Hepatology. 46, 1659-1661 (2007).

Comments

15 Comments

  1. Thank you for submitting this video. Very impressive.
    Have you ever tried this method for the mouse liver???

    Reply
    Posted by: Anonymous
    August 15, 2011 - 8:22 PM
  2. Yes. Macrophages proliferate on the mixed primary cultures of adult mouse liver cells. In addition, we applied this method to the bovine liver (Vet. Immunol. Immunopathol.140:341-345, ²011).

    Reply
    Posted by: Hiroshi K.
    August 17, 2011 - 4:23 AM
  3. Congratulations! Nice work!

    Reply
    Posted by: Anonymous
    September 8, 2011 - 9:09 AM
  4. Thank you for share your amazing work. Have you ever tried to isolate another type of cell with this technique ? Hepatocytes or stellate cells ?. If no, would you think this can be done ?.
    Many thanks in advance,

    Reply
    Posted by: Anonymous
    September 9, 2011 - 5:11 AM
  5. We have not systematically isolated another type of cells with our mixed cultures. However, a small number of epithelial-like cells often contaminate into liver macrophages. These cells proliferate and form large round colonies after two weeks of culture. These cells might be related to hepatoblasts, but should further be characterized.

    Reply
    Posted by: Hiroshi K.
    September 12, 2011 - 3:42 AM
  6. Great work! I am interested in trying this myself, And I am wondering--
    How essential is it that the collagenase be perfused through the vasculature.
    Is it possible to excise a section of a (mouse) liver, and treat with collagenase ex-vivo?
    Thanks very much in advance.

    Reply
    Posted by: Anonymous
    February 23, 2012 - 9:59 AM
  7. We have not done ex-vivo digestion of liver sections (or pieces) by collagenase. Although this method might give certain numbers of dissociated liver cells, the yield and viability will be less than that by perfusion method. Considering the small size of mouse liver, we recommend perfusion method.

    Reply
    Posted by: Anonymous
    February 27, 2012 - 6:52 PM
  8. Very nice methods, I just have a question for the shaker, should we must use reciprocal shaker, or we can use the normal orbital shaker? Thanks a lot

    Reply
    Posted by: yize l.
    May 16, 2013 - 3:35 PM
  9. Thank you for your interest on our work. You may use the orbital shaker, but be careful not to spill the medium over the neck of culture flasks.

    Reply
    Posted by: Hiroshi K.
    May 17, 2013 - 1:51 AM
  10. Great! can you provide detailed info regarding the fluorescent beads utilized in figure 5? thank for you kindness

    Reply
    Posted by: mariagrazia g.
    August 30, 2013 - 11:28 AM
  11. We use "Fluoresbrite Plain Microspheres (2.5% Solids-Latex),1.0μm YG" ( Cat. No. 17154) from Polysciences, Inc.
    Labeling procedure is described in J. Immunol. Methods 360, 47-55 (2010).

    Reply
    Posted by: Hiroshi K.
    September 1, 2013 - 8:42 PM
  12. i have prepared primary culture of hepatocyte. initially lots of hepatocyte we obtain . but noe this process is steady. so what should be done to increase the growth of hepatocye?please help me

    Reply
    Posted by: Gopi S.
    April 15, 2015 - 6:52 AM
  13. Viability of the hepatocytes is largely dependent on the condition of collagenase treatment. To increase the viability, you might reduce the duration of collagenase digestion (accepting lower yield). After digestion, cells should be suspended in cold medium and centrifuged at 4°C. Also, try younger animals.

    Reply
    Posted by: Hiroshi K.
    April 21, 2015 - 10:39 PM
  14. Thanks for providing such information . you describe the procedure to isolate macrophages. Can i apply this same procedure to isolate hepatocyte? Do needful

    Reply
    Posted by: Gopi S.
    April 22, 2015 - 1:30 AM
  15. We use the mixed primary culture of adult liver cells (mainly consisted of hepatocytes) to grow and isolate macrophages. Our protocol 1 and 2 (please see above) can be applied to isolate hepatocytes. You might consult with more detailed protocol published in JoVE, such as Lee, S. M. L., Schelcher, C., Demmel, M., Hauner, M., Thasler, W. E. Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure. J. Vis. Exp. (79), e50615, doi:10.3791/50615 (2013).

    Reply
    Posted by: Hiroshi K.
    April 22, 2015 - 2:31 AM

Post a Question / Comment / Request

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

Usage Statistics