Name: isolation of rat portal fibroblasts by in situ liver perfusion
Uploaded: 2012-06-29T12:00:00
Duration: 7 min 39 s
Description: Watch this Scientific Journal Video about Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion at JoVE.com

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.).

The entire procedure is carried out at room temperature unless otherwise specified.
1. Preparation of Enzyme Solutions
<ol>
 <li>Prepare enzyme solutions according to Table 1 and sterile filter using a 0.22 &mu;m filter. Keep solution 1 and solution 2 at room temperature and solution 3 at 4 &deg;C until time of use. All solutions and equipment coming into contact with isolated cells must be sterile.</li>
</ol>
2. Preparation of Perfusion Equipment
<ol>
 <li>Prime the perfusion system with HBSS minus Ca2+/Mg2+, which has been warmed to 37 &deg;C. Note: To ensure that the perfusate is 37 &deg;C when it arrives at the liver, HBSS is warmed in a water bath set to 37 &deg;C. After it passes through the perfusion pump, it goes through a jacketed glass condenser that is connected to a water circulator set to 40 &deg;C (Fig. 1).</li>
 <li>Sterilize surgical tools in a beaker with 70% ethanol.</li>
</ol>
3. Perfusion of the Liver
<ol>
 <li>Anesthetize an adult male Sprague-Dawley rat by intraperitoneal injection of Nembutal (50-100 mg/kg body weight as necessary to obtain adequate anesthesia).</li>
 <li>Place the animal in a supine position on a plastic tray and fix the limbs to the tray with tape.</li>
 <li>Clean the abdomen with 70% EtOH.</li>
 <li>Make a small incision in the skin on the abdomen in the midline. Dissect the skin away from the fascia by making a large U-shaped cut on the abdomen. Cut the abdominal muscle with scissors following the same shape to expose the peritoneal cavity.</li>
 <li>Expose the portal vein by using 2 cotton swabs to gently move the abdominal viscera to the right side.</li>
 <li>Pass two sterile 2.0 silk sutures underneath the portal vein and tie loosely.</li>
 <li>Cannulate the portal vein with a 16-18 gauge IV catheter and secure the catheter in place by tightening the loosely tied sutures from the previous step (Fig. 2).</li>
 <li>Inject diluted Heparin through the IV catheter (1 ml of Heparin 5000 USP units/ml diluted with 4 ml of HBSS minus Ca2+/Mg2+). The liver should blanch.</li>
 <li>Connect the IV catheter to the perfusion system. Perfuse with HBSS minus Ca2+/Mg2+ at a rate of 20 ml/min for 10 minutes. Transect the IVC inferior to the liver to allow drainage of blood and perfusate. Aspirate pooled blood from the abdominal cavity.</li>
 <li>Change the perfusion fluid to a 0.3% collagenase solution (150 mg of type 2 collagenase in 500 ml HBSS plus Ca2+/Mg2+) and perfuse for 25 minutes. Keep the liver moist by covering it with the previously dissected abdominal flap. Put a Petri dish cover over the abdominal flap and position a heat lamp over the area to maintain the intra-abdominal temperature at approximately 37 &deg;C.</li>
</ol>
4. Preparation of the Biliary Tree
<ol>
 <li>Remove the liver by lifting it out from the abdominal cavity with forceps and place it in a sterile tissue culture dish filled with cold Leibovitz's L-15 media.</li>
 <li>Working in the tissue culture hood, peel off the liver capsule and gently tease the liver parenchyma apart with forceps. Move the liver through several dishes filled with Liebovitz's L-15 media while continuing to tease away the parenchyma, until finally left with the isolated biliary tree. The liver parenchyma is tan colored, whereas the remaining biliary tree is white.</li>
 <li>Place the isolated biliary tree into a 50 ml falcon tube filled with 10 ml of penicillin/streptomycin (10,000 IU/ml penicillin and 10,000 &mu;g/ml streptomycin); leave on ice for 15 minutes.</li>
</ol>
5. Isolation of the Portal Fibroblast Fraction
<ol>
 <li>Place the biliary tree into a sterile 50 ml falcon tube and mince with sterile scissors.</li>
 <li>Add a small amount of enzyme solution #1 into the tube (about 5 ml) and continue to mince the biliary tree until it reaches the consistency of a slurry.</li>
 <li>Pour the slurry into a sterile glass bottle. Wash out falcon tube with the remaining solution #1, then pour it into the glass bottle. Incubate at 37 &deg;C with shaking at 100 rpm for 30 minutes.</li>
 <li>Filter the slurry through a beaker covered with a single layer of nylon mesh (30-micron pore size).</li>
 <li>Transfer any tissue remaining on top of the mesh to a sterile glass bottle by washing the mesh with enzyme solution #2. Start by pouring half (25 ml) of the solution into a sterile 15 cm Petri dish. Carefully remove the mesh from the beaker by holding the edges of the mesh without contaminating the center area, then inverting the mesh and dipping it into the solution in the Petri dish to remove any remaining tissue on the mesh. Discard the mesh. Transfer the solution in the Petri dish into a sterile glass bottle. Rinse the Petri dish with the remaining 25 ml solution #2 and transfer it to the same glass bottle. Incubate at 37 &deg;C with shaking at 100 rpm for 30 minutes.</li>
 <li>In the meantime, take the filtrate in the beaker and pour into a 50 ml falcon tube. Centrifuge at 1600 rpm (460 x g) for 5 minutes at room temperature.</li>
 <li>Aspirate media and resuspend the pellet in 25 ml of solution #3.</li>
 <li>Take the solution from step 5.5 and filter it into a second sterile beaker covered with 30-micron mesh.</li>
 <li>Discard the mesh. Centrifuge the filtrate at 1600 rpm for 5 minutes at room temperature. Aspirate and resuspend the pellet with 25 ml of solution #3 as was done in step 5.7.</li>
 <li>Combine the 2 fractions from steps 5.7 and 5.9 and centrifuge again at 1600 rpm for 5 minutes at room temperature.</li>
 <li>Aspirate media and resuspend the pellet in 10 ml of portal fibroblast culture media (DMEM/F12 supplemented with 10% FBS, 1% penicillin/streptomycin, 0.3% gentamycin, and 0.2% fungizone).</li>
 <li>Count cells for viability and plate in tissue culture dishes. Depending on the desired application, 0.5 to 5 x 106 cells per 10-cm tissue culture plate are appropriate. From an adult Sprague-Dawley rat, we typically obtain 2 to 10 million viable cells.</li>
 <li>Replace the culture media the next day to remove non-adherent debris. Thereafter, replace culture media every 2 days.</li>
</ol>
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 &alpha;-smooth muscle actin (&alpha;-SMA; A2547, Sigma, St. Louis, MO).
<table border="1">
 <tbody>
 <tr>
 <td rowspan="2">0.3% collagenase solution</td>
 <td>Type 2 collagenase</td>
 <td>150 mg</td>
 </tr>
 <tr>
 <td>HBSS with Ca2+/Mg2+</td>
 <td>500 ml</td>
 </tr>
 <tr>
 <td rowspan="7">Solution #1 (Pronase)</td>
 <td>Bovine serum albumin</td>
 <td>50 mg</td>
 </tr>
 <tr>
 <td>Collagenase type 2</td>
 <td>25 mg</td>
 </tr>
 <tr>
 <td>Pronase</td>
 <td>18 mg</td>
 </tr>
 <tr>
 <td>DNase</td>
 <td>3 mg</td>
 </tr>
 <tr>
 <td>DMEM/F12</td>
 <td>47.5 ml</td>
 </tr>
 <tr>
 <td>Pen/Strep</td>
 <td>1 ml</td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>1.5 ml</td>
 </tr>
 <tr>
 <td>Solution #2</td>
 <td>Bovine serum albumin</td>
 <td>50 mg</td>
 </tr>
 <tr>
 <td rowspan="6">(Hyaluronidase)</td>
 <td>Collagenase type 2</td>
 <td>25 mg</td>
 </tr>
 <tr>
 <td>Hyaluronidase</td>
 <td>22 mg</td>
 </tr>
 <tr>
 <td>DNase</td>
 <td>3 mg</td>
 </tr>
 <tr>
 <td>DMEM/F12</td>
 <td>47.5 ml</td>
 </tr>
 <tr>
 <td>Pen/Strep</td>
 <td>1 ml</td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>1.5 ml</td>
 </tr>
 <tr>
 <td rowspan="2">Solution #3</td>
 <td>DNase</td>
 <td>3 mg</td>
 </tr>
 <tr>
 <td>RPMI medium 1640</td>
 <td>50 ml</td>
 </tr>
 </tbody>
</table>
Table 1. Enzyme solutions.
<img alt="Figure 1" src="/files/ftp_upload/3669/3669fig1.jpg" /> 
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.
<img alt="Figure 2" src="/files/ftp_upload/3669/3669fig2.jpg" /> 
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.
<img alt="Figure 3" src="/files/ftp_upload/3669/3669fig3.jpg" /> 
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, &alpha;-SMA and DAPI.

<ol>
	<li>Parola, M., Marra, F., Pinzani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibroblast+-+like+cells+and+liver+fibrogenesis:+Emerging+concepts+in+a+rapidly+moving+scenario.">Myofibroblast - like cells and liver fibrogenesis: Emerging concepts in a rapidly moving scenario.</a> Mol. Aspects Med. 29, 58-66 (2008).</li><li>Dranoff, J. A., Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portal+fibroblasts:+Underappreciated+mediators+of+biliary+fibrosis.">Portal fibroblasts: Underappreciated mediators of biliary fibrosis.</a> Hepatology. 51, 1438-1444 (2010).</li><li>Tuchweber, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+phenotypic+modulation+of+portal+fibroblasts+in+the+early+stages+of+cholestatic+fibrosis+in+the+rat.">Proliferation and phenotypic modulation of portal fibroblasts in the early stages of cholestatic fibrosis in the rat.</a> Lab Invest. 74, 265-278 (1996).</li><li>Beaussier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prominent+contribution+of+portal+mesenchymal+cells+to+liver+fibrosis+in+ischemic+and+obstructive+cholestatic+injuries.">Prominent contribution of portal mesenchymal cells to liver fibrosis in ischemic and obstructive cholestatic injuries.</a> Lab Invest. 87, 292-303 (2007).</li><li>Kinnman, N., Housset, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peribiliary+myofibroblasts+in+biliary+type+liver+fibrosis.">Peribiliary myofibroblasts in biliary type liver fibrosis.</a> Front Biosci. 7, d496-d503 (2002).</li><li>Kinnman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myofibroblastic+conversion+of+peribiliary+fibrogenic+cells+distinct+from+hepatic+stellate+cells+is+stimulated+by+platelet-derived+growth+factor+during+liver+fibrogenesis.">The myofibroblastic conversion of peribiliary fibrogenic cells distinct from hepatic stellate cells is stimulated by platelet-derived growth factor during liver fibrogenesis.</a> Lab Invest. 83, 163-173 (2003).</li><li>Uchio, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+retinol-binding+protein-1+expression+and+modulation+during+in+vivo+and+in+vitro+myofibroblastic+differentiation+of+rat+hepatic+stellate+cells+and+portal+fibroblasts.">Cellular retinol-binding protein-1 expression and modulation during in vivo and in vitro myofibroblastic differentiation of rat hepatic stellate cells and portal fibroblasts.</a> Lab Invest. 82, 619-628 (2002).</li><li>Kruglov, E. A., Jain, D., Dranoff, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+primary+rat+liver+fibroblasts.">Isolation of primary rat liver fibroblasts.</a> J. Investig. Med. 50, 179-184 (2002).</li><li>Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor-beta+and+substrate+stiffness+regulate+portal+fibroblast+activation+in+culture.">Transforming growth factor-beta and substrate stiffness regulate portal fibroblast activation in culture.</a> Hepatology. 46, 1246-1256 (2007).</li><li>Bosselut, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+proteomic+features+of+two+fibrogenic+liver+cell+populations:+hepatic+stellate+cells+and+portal+myofibroblasts.">Distinct proteomic features of two fibrogenic liver cell populations: hepatic stellate cells and portal myofibroblasts.</a> Proteomics. 10, 1017-1028 (2010).</li><li>Dranoff, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecto-nucleoside+triphosphate+diphosphohydrolase+NTPDase2/CD39L1+is+expressed+in+a+novel+functional+compartment+within+the+liver.">The ecto-nucleoside triphosphate diphosphohydrolase NTPDase2/CD39L1 is expressed in a novel functional compartment within the liver.</a> Hepatology. 36, 1135-1144 (2002).</li></ol>

No conflicts of interest declared.

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 &alpha;-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 (&gt;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.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Type 2 Collagenase</td>
 <td>Worthington Biochemical (Lakewood, NJ)</td>
 <td>LS004177</td>
 </tr>
 <tr>
 <td>Pronase</td>
 <td>Roche (Indianapolis, IN)</td>
 <td>70290120</td>
 </tr>
 <tr>
 <td>Hyaluronidase</td>
 <td>Sigma (St. Louis, MO)</td>
 <td>H3506</td>
 </tr>
 <tr>
 <td>Deoxyribonuclease</td>
 <td>Sigma</td>
 <td>D4527</td>
 </tr>
 <tr>
 <td>DMEM/F12</td>
 <td>Invitrogen (Carlsbad, CA)</td>
 <td>11320</td>
 </tr>
 <tr>
 <td>HBSS without Ca2+/Mg2+</td>
 <td>Mediatech (Manassas, VA)</td>
 <td>21-021-CM</td>
 </tr>
 <tr>
 <td>HBSS with Ca2+/Mg2+</td>
 <td>Mediatech </td>
 <td>21-020-CM</td>
 </tr>
 <tr>
 <td>Leibovitz's L-15</td>
 <td>Invitrogen</td>
 <td>11415</td>
 </tr>
 <tr>
 <td>RPMI Medium 1640</td>
 <td>Invitrogen</td>
 <td>11875</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>Sigma</td>
 <td>A2153</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Gemini Bio (West Sacramento, CA)</td>
 <td>900-108</td>
 </tr>
 <tr>
 <td>Nitex Nylon Mesh 30 &mu;m</td>
 <td>Genesee Scientific (San Diego, CA)</td>
 <td>57-105</td>
 </tr>
 </tbody>
</table>

isolation of rat portal fibroblasts by in situ liver perfusion

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.

Watch this Scientific Journal Video about Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion at JoVE.com

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

isolation-of-rat-portal-fibroblasts-by-in-situ-liver-perfusion

Research

JoVE Journal

Biology

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Isolation and Primary Culture of Rat Hepatic Cells

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 &mu;m 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.

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 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

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.

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...