1Division of Gastroenterology, Hepatology & Nutrition, Department of Pediatrics, The Children's Hospital of Philadelphia, 2Department of Medicine, University of Pennsylvania
Wen, J. W., Olsen, A. L., Perepelyuk, M., Wells, R. G. Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion. J. Vis. Exp. (64), e3669, doi:10.3791/3669 (2012).
Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.
The entire procedure is carried out at room temperature unless otherwise specified.
1. Preparation of Enzyme Solutions
2. Preparation of Perfusion Equipment
3. Perfusion of the Liver
4. Preparation of the Biliary Tree
5. Isolation of the Portal Fibroblast Fraction
6. Representative Results
Primary portal fibroblasts isolated from adult rat liver are shown at 1, 3 and 7 days after isolation (Fig. 3). Notice that the cells are elongated, with morphology typical of fibroblasts. Cells can be stained with anti-elastin antibody (CL55041AP, Cedarlane Labs, Burlington, NC) to confirm purity. Portal fibroblasts undergo myofibroblastic differentiation in culture. This can be demonstrated by immunostaining for α-smooth muscle actin (α-SMA; A2547, Sigma, St. Louis, MO).
|0.3% collagenase solution||Type 2 collagenase||150 mg|
|HBSS with Ca2+/Mg2+||500 ml|
|Solution #1 (Pronase)||Bovine serum albumin||50 mg|
|Collagenase type 2||25 mg|
|Fetal bovine serum||1.5 ml|
|Solution #2||Bovine serum albumin||50 mg|
|(Hyaluronidase)||Collagenase type 2||25 mg|
|Fetal bovine serum||1.5 ml|
|Solution #3||DNase||3 mg|
|RPMI medium 1640||50 ml|
Table 1. Enzyme solutions.
Figure 1. Perfusion set up. A. Water circulator for warming column. B. Column containing perfusion media. C. Stopcock controlling pump outflow. D. Pump inflow. E. Variable-speed peristaltic pump. F. Vacuum flask with Bio-Clean detergent. G. Heat lamp. H. Water bath for warming perfusion media.
Figure 2. In situ perfusion. A. IV catheter inserted into the portal vein, secured with suture and connected to the perfusion tubing via stopcock. B. Glass pipette connected to vacuum flask and wall suction to remove perfusion fluid draining out of transected IVC. I. Intestines. L. Liver.
Figure 3. Phase contrast and immunofluorescence stain of rat portal fibroblasts in culture. Portal fibroblasts cultured for 1, 3, and 7 days after isolation were fixed and stained for elastin, α-SMA and DAPI.
Portal fibroblasts play an important role in biliary fibrosis. Here we describe a modification of the original protocol published by Kruglov and Dranoff 8 for isolating portal fibroblasts from rat liver, providing a straightforward way to study this cell population in vitro.
This approach uses protease digestion and size-based filtration. The primary advantage of the method is that a relatively pure population of cells can be obtained without passage in culture, enabling the study of gene expression or functional cell behavior several days after isolation. This technique also offers the potential for isolating cells from both healthy and diseased livers.
One of the most critical steps in this protocol is the enzymatic digestion of the hepatic parenchyma. To ensure successful digestion, it is important to confirm that blood is completely drained out of the liver during the initial perfusion by making sure that there is even blanching of the liver. To facilitate this, it is possible to use a cotton swab to gently massage the liver while perfusing. Over-digestion of the liver parenchyma results in a low yield of viable cells, while under-digestion will make it difficult to separate the liver parenchyma from the biliary tree. Since preparations of pronase have variability in enzymatic activity between different lots, optimization of the amount used may be needed when there is low yield of viable cells.
This protocol may be modified for isolating portal fibroblasts from mouse liver or young rat liver by using a smaller gauge IV catheter to cannulate the portal vein, decreasing the volume of perfusion solutions in proportion to animal weight, and decreasing the liver perfusion rate to about 10 ml/min.
Portal fibroblasts in culture undergo myofibroblastic differentiation in 10-14 days, as evidenced by α-SMA expression 9. Various markers have been used to distinguish portal fibroblasts from hepatic stellate cells, including fibulin-2, IL-6, elastin, nucleoside triphosphate diphosphohydrolase-2 (NTPDase2), cofilin-1 and neuronal proteins such as synaptophysin 2, 6, 10, 11. We have found that elastin is a good marker for portal fibroblasts, even after myofibroblastic differentiation, while NTPDase2 is lost after portal fibroblasts have undergone culture after several days 9. Therefore, we typically confirm isolation of portal fibroblasts by immunofluorescence staining for elastin.
The limitation of this technique is that, immediately after portal fibroblast isolation, there is a small fraction of contaminating cells including Kupffer cells and biliary cells. Portal fibroblasts will outgrow these contaminating cells within 2-3 days, however, yielding a relatively pure population (>98%) 9.
Since portal fibroblasts in culture undergo myofibroblastic differentiation on tissue culture plastic, we typically study these cells within 7 days of isolation. Cells are maintained in portal fibroblast culture media containing 10% fetal bovine serum, as described in the methods section. However, these cells will survive in growth media containing 2% fetal bovine serum for several days. We typically do not use low serum media until at least 24 hours after isolation. Once portal fibroblasts undergo myofibroblastic differentiation, they can be passaged several times and kept as frozen stocks of myofibroblasts.
No conflicts of interest declared.
This work was funded by NIH R01 DK05823 (to R.G.W.), F32 DK083213 (to J.W.W.), F30 DK081265 (to A.L.O.), and by a grant from the Fred and Suzanne Biesecker Pediatric Liver Center (to R.G.W.).
|Type 2 Collagenase||Worthington Biochemical (Lakewood, NJ)||LS004177|
|Pronase||Roche (Indianapolis, IN)||70290120|
|Hyaluronidase||Sigma (St. Louis, MO)||H3506|
|DMEM/F12||Invitrogen (Carlsbad, CA)||11320|
|HBSS without Ca2+/Mg2+||Mediatech (Manassas, VA)||21-021-CM|
|HBSS with Ca2+/Mg2+||Mediatech||21-020-CM|
|RPMI Medium 1640||Invitrogen||11875|
|Bovine Serum Albumin||Sigma||A2153|
|Fetal Bovine Serum||Gemini Bio (West Sacramento, CA)||900-108|
|Nitex Nylon Mesh 30 μm||Genesee Scientific (San Diego, CA)||57-105|