We would like to thank Dr. Paul Weigel at the Univ. of Oklahoma for the use of monoclonal antibody 30 for detection of the HARE/Stabilin-2 receptor and Janet Weigel for her technical assistance. This research is funded by University of Nebraska Research Funds.

1. Excision of the liver (adopted from P.O. Seglen12 and R. Blomhoff13 with modifications 14,15)
<ol>
 <li>Add 10 mL 30% isoflurane in polyethylene glycol to a petri dish in a closed 5.5 L chamber. It is optimal to wait 10-15 min after addition of isoflurane to create a stabilized atmosphere within the chamber.</li>
 <li>Place a rat in the sealed chamber and allow it to become anesthetized. Full effects of the anesthesia are apparent when the animal becomes limp, unresponsive, and exhibits deep breathing.</li>
 <li>Place 2 mL of isoflurane in polyethylene glycol in some cotton balls at the bottom of a 30 mL syringe tube.</li>
 <li>Place the rat on its back on a diaper-covered tray and place the syringe tube over the snout.</li>
 <li>Immediately confirm deep anesthesia by wetting the abdomen with 70% ethanol. Do not let the animal die by isoflurane overdose.</li>
 <li>Using bandage scissors and forceps, expose the entire abdominal cavity.</li>
 <li>Locate the vena porta and, using forceps, draw two strands of surgical silk or polyester thread (ligature) beneath the vena porta immediately above the mesenteric branch.</li>
 <li>Withdraw the forceps and tie a loose overhand knot.</li>
 <li>Using forceps underneath the vena porta and gently pulling back to straighten out the vein, cannulate the vein with an Insyte Autoguard catheter (18 GA, 1.3 x 300 mm, BD Biosciences) or similar catheter. The catheter should go several mm beyond the loop formed by the thread.</li>
 <li>Retract and remove the needle by releasing the spring-loaded trigger on the catheter. Blood should start seeping up into the catheter indicating proper ligature placement.</li>
 <li>Tighten down the overhand knot and secure it with another overhand knot.</li>
 <li>Sever either the inferior vena cava or descending aorta (aorta abdominalis) to allow blood to drain from the circulatory system.</li>
 <li>Begin flushing the liver with oxygenated TBS at 37&deg;C to remove the blood (blanching) at a flow rate of 20 mL/min. The liver should turn from a reddish purple to a loam color.</li>
 <li>While the liver is flushing, excise the liver by cutting away the GI tract and connective tissue. It is important not to lacerate the liver or puncture the Glisson&#x2019;s capsule.</li>
 <li>Place the liver on a plastic net over a funnel that allows buffers to be collected and recirculated. Buffers at this point should not be recirculated, but flow into the waste beaker. Flushing should not last more than 10 min. (Fig. 2A). Prepare the endocytosis solution required in step 2.1.</li>
</ol>
2. Internalization of 125I-SA-b-Hep (or other suitable labeled ligand)
<ol>
 <li>To 50 mL RPMI media in a small beaker, add to a final concentration 0.05% BSA and 0.01 mCi 125I-SA-b-Hep or other appropriately labeled ligand for endocytosis.</li>
 <li>Attach this beaker to the apparatus to allow for oxygenation.</li>
 <li>Turn off the pump, switch the inlet tubing, and then turn on the pump at 20 mL/min.</li>
 <li>As the labeled RPMI approaches the liver, turn off the pump and allow excess fluid in the funnel and tubing drain.</li>
 <li>Place the drain tubing into the media beaker to allow recirculation of labeled media and then restart the pump.</li>
 <li>Allow fluids to recirculate through the liver for no more than 1 hour.</li>
 <li>During this internalization step, be sure the temperature in the liver is stable by covering it and prepare the collagenase for digestion.</li>
</ol>
3. Digestion of the liver by collagenase digestion
<ol>
 <li>Freshly dissolved and filtered (0.45 &mu;m) collagenase (100 mg/kg rat weight) should be added to Buffer 2 in a total volume not to exceed 60 mL at 37&deg;C. The volume is dependent on the length/internal volume of the tubing and should be adjusted accordingly.</li>
 <li>Stop the pump and exchange the inlet tubing from the media beaker to the TBS.</li>
 <li>Flush the liver for 2 min at 50 mL/min which allows for loosening of the desmosomal cell junctions which are calcium dependent.</li>
 <li>While the liver is flushing, exchange the media beaker with the beaker containing collagenase on the apparatus.</li>
 <li>Immediately after flushing, begin the digestion with collagenase in a circulatory loop at a flow rate of 20 mL/min for up to 15 min. Since collagenase batches vary by lot number, variations in the amount and time of digestion need to be optimized for every new lot of collagenase. Collagenase is also calcium dependent. Turn off the pump and switch the buffer and gas lines from Flask A to Flask B. Transfer the effluent line from the waste container to Flask B (Fig. 2B).</li>
 <li>Stop the pump, remove the catheter and transfer the liver to a dish containing 20-30 mL Buffer 1.</li>
 <li>Peel back the Glisson&#x2019;s capsule of the liver and shake the cells out in the liquid.</li>
 <li>As the liquid becomes opaque with cellular material, transfer the cells through a 100 &mu;m mesh followed by filtration through a 30 &mu;m mesh and then into a 50 mL conical on ice.</li>
 <li>Add more Buffer 1 to the liver and continue shaking cells from the liver matrix, transferring the liquid to the mesh filters and conical.</li>
 <li>Repeat steps 3.7 to 3.9 until no more cells can be dislodged with reasonable shaking of the liver.</li>
</ol>
4. Hepatocyte and SEC purification
<ol>
 <li>Centrifuge the 50 mL conicals containing cells at 150 x g for 3 min. to pellet the hepatocytes.</li>
 <li>Transfer the liquid fraction containing non-parenchymal cells to fresh 50 mL conicals on ice.</li>
 <li>Wash the hepatocytes be resuspending the pellet in Buffer 3 and repeating steps 4.1 and 4.2 three more times.</li>
 <li>At the end of the washes, the pellets are &ge;97% pure hepatocytes. To obtain the SECs, centrifuge all of the tubes containing pooled supernatant (containing HSCs, SECs, and KCs and some small hepatocytes) at 200 x g for 10 min. at 4&deg;C.</li>
 <li>Aspirate the buffer and resuspend all the pellets in 5 mL RPMI/BSA at 4&deg;C.</li>
 <li>Pool the cells together into 2 tubes and add RPMI/BSA up to a volume of 35 mL.</li>
 <li>Centrifuge the conicals at 100 x g for 3 min.</li>
 <li>Collect the top 25 mL of liquid and transfer to a clean 50 mL conical.</li>
 <li>Resuspend the pellet in the remaining 10 mL media and add back 25 mL of cold RPMI/BSA and centrifuge at 100 x g for 3 min.</li>
 <li>Repeat steps 3.8 and 3.9 once more, discard the cell pellet and lower 10 mL media.</li>
 <li>Centrifuge the supernatents to pellet SECs, KCs, and HSCs at 200 x g for 10 min. at 4&deg;C.</li>
 <li>Prepare three 50 mL tubes containing 20 mL 25% Percoll in PBS on ice. Underlay with 15 mL 50% Percoll in each tube.</li>
 <li>Resuspend the cell pellets in a total volume of 30 mL RPMI/BSA.</li>
 <li>Carefully overlay each gradient with 10 mL of cells and centrifuge at 900 x g for 20 min at 4&deg;C.</li>
 <li>SECs and KCs have very close buoyant densities and are at the 25/50% interface. HSCs are typically at the 25%/media interface due to their higher buoyancy as lipid storing cells. Any remaining hepatocytes and blood cells have the lower buoyancies and are pelleted. Aspirate from the top down near the 25/50% interface and discard this material.</li>
 <li>Collect the cells in the 25/50% interface and transfer to a fresh conical with cold RPMI. The final volume should be ~40 mL to dilute out the Percoll.</li>
 <li>Centrifuge the conical at 350 x g for 10 min at 4&deg;C to pellet the cells.</li>
 <li>Resuspend the cells in ~10 mL pre-warmed RPMI containing 100 U/mL Penicillin/100 g/mL Streptomycin and place cells in an acid-washed glass Petri dish or crystallizing dish.</li>
 <li>Incubate the cells for 15 min. at 37&deg;C in a tissue culture incubator.</li>
 <li>Rock or swirl the plate a little and then collect the SECs in the supernatant. The KCs adhere to the glass much more rapidly than SECs. Alternatively, SECs may be separated from KCs by immunopurification using anti-CD31 or anti-Stabilin2/HARE antibodies conjugated to magnetic beads.</li>
 <li>Viability and cell numbers may be assessed by trypan blue exclusion using a hemacytometer or with using an automated cell counter.</li>
 <li>Culture the SECs on fibronectin-coated plastic dishes at 37&deg;C, 5% CO2, RPMI/0.25% BSA with 40 ng/mL VEGF, 100 U/mL Penicillin, 100 &mu;g/mL Streptomycin or with hepatocyte conditioned media.</li>
 <li>Hepatocytes, KCs, and SECs may be assess for internalization of labeled material by the use of a gamma counter and normalizing to total cellular protein or by microscopy (if fluorescently labeled) using these culturing methods.</li>
</ol>
5. Representative results:
Hepatocyte purification is &ge;97% and SEC purification is typically &ge;95% using this method. Excision of the abdominal cavity, cannulation of the portal vein, and blanching of the liver all should occur within a minute for best results. HARE/Stabilin-2 is a specific receptor on liver SECs and is not expressed in other liver cell types. In this sample, cell lysates of both hepatocytes and SECs were separated by 5% SDS-PAGE and probed with a monoclonal antibody against both isoforms of Stabilin-2 (Fig. 3).
Unfractionated heparin injected into the blood stream is cleared by the liver16. Clearance is primarily performed by liver SECs in contrast to hepatocytes and KCs17. The Stabilin-2/HARE receptor is the main clearance receptor that binds and internalizes heparin in SECs18. In our example here, we labeled heparin with a biotin tag on the carboxylate group of GlcNAc/NS. The biotin was conjugated with iodinated streptavidin for detection of internalized heparin proteoglycan. Injection of the labeled heparin followed by exsanguination 30 minutes post-injection allows us to monitor which organs internalize this form of heparin. In this short time interval, the liver is the primary clearance organ (Fig. 4).
To more clearly understand which cell type is responsible for clearance, we added 125I-SA-b-Hep to RPMI media supplemented with 0.05% BSA and allowed it to circulate through the liver for 20 min. Following collagenase digestion and cellular purification, the vast majority of labeled heparin is internalized by SECs in contrast to hepatocytes (Fig. 5). 
<img src="/files/ftp_upload/3138/3138fig1.jpg" alt="Figure 1"/> Figure 1. Basic liver architecture. SECs form the walls of the sinusoids and are normally fenestrated (small clusters of circles). Stellate cells (HSCs) are found in the Space of Disse in contrast to Kupffer cells (KCs) which are normally present in the microvasculature of the sinusoids. Hepatocytes occupy the other side of the Space of Disse.
<img src="/files/ftp_upload/3138/3138fig2.jpg" alt="Figure 2"/> Figure 2. Schematic of the perfusion apparatus. A) Set-up of apparatus during the flushing/washing of the sinusoids of the liver. TBS is in a 1 L flask. B) A 125 mL flask containing ~60 mL Buffer 2 with collagenase is used for digestion of the liver in a closed circuit. The effluent line is also changed from the waste container to flask B through a notch cut in the rubber stopper. The oxygen line is not submerged into the buffer containing collagenase (flask B) to avoid foaming. The stoppers on each flask are notched to allow efficient transfer of the effluent line and to prevent increased pressure from the oxygen gas.
<img src="/files/ftp_upload/3138/3138fig3.jpg" alt="Figure 3"/> Figure 3. Hepatocyte preparations are not contaminated with SECs. Equal amounts of cell lysates from purified batches of hepatocytes (lane 1) and SECs (lane 2) were separated by 5% SDS-PAGE and blotted to nitrocellulose. Monoclonal antibody #30 which is specific against both isoforms (315 kDa and 190 kDa) of HARE/Stabilin-2 was used to probe the lysates.
<img src="/files/ftp_upload/3138/3138fig4.jpg" alt="Figure 4"/> Figure 4. Distribution of labeled heparin in organs. Rats were injected via the lateral tail vein with 125I-SA-b-hep wait for 30 minutes and then exsanguinated. Blood was collected so as to not to give any organs artificially high readings. Counts per minute (CPM) of each organ were normalized against the weight of the organ in grams. 
<img src="/files/ftp_upload/3138/3138fig5.jpg" alt="Figure 5"/> Figure 5. Amount of labeled heparin in hepatocytes and SECs. RPMI media containing 125I-SA-b-Hep was allowed to circulate through an intact liver for 20 min followed by collagenase digestion and cellular purification. Equal amounts of hepatocyte and SEC cell protein lysates were quantified with the Bradford Assay and counted by a gamma counter.
<img src="/files/ftp_upload/3138/3138fig6.jpg" alt="Figure 6"/> Figure 6. Kupffer cells are separated from SECs by adhesion on glass. Cells were placed in an acid-washed glass crystallizing dish and allowed to adhere for 15 min at 37&deg;C, 5% CO2. The supernatent containing non-adhered cells was gently swirled and placed in a centrifuge tube. Lysates from both adhered cells on glass (lane 1) and from non-adhered cells (lane 2) were separated by 8% SDS-PAGE, blotted, and probed with an antibody against CD163 which is specific for Kupffer cells.
<img src="/files/ftp_upload/3138/3138fig7.jpg" alt="Figure 7"/> Figure 7. SECs plated on fibronectin coated plastic 6-well dishes were incubated overnight in RPMI supplemented with 0.1% BSA. Phase contrast images were taken at (A) 100x and (B) 200x magnification.

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No conflicts of interest declared.

Anesthesia of the rat by the hanging drop method in which isoflurane vapor induces unconsciousness is the preferred method for our studies. A vaporizer may also be used instead of a syringe with cotton to maintain the animal outside the chamber, but the surgical procedures are so quick, it is not required. Polyethylene glycol is added to the isoflurane to decrease the vapor pressure and evaporation rate. Too much isoflurane vapor will induce death too quickly. If the animal dies before the liver is cannulated and blanched with TBS, pooling blood may clot in the liver and prevent efficient washing of the sinusoids and digestion with collagenase. The addition of heparin may be useful as an anticoagulant but is not used in these studies since we use labeled heparin as our probe.
Gey's buffered saline solution (GBSS) is often substituted by other laboratories for the purification of SECs. While it is useful for SEC purification, hepatocytes do not tolerate GBSS as well as Buffers 1-3 and viabilities are not nearly as high. When measuring endocytosis, it is good practice to measure background levels in the organ and in purified hepatocytes. 
This method is one of two for efficient purification of SECs following the liver perfusion with collagenase. The other method separates the non-parenchymal cells by elutriation centrifugation 19 instead of Percoll gradient. The advantages for using elutriation over Percoll gradients are slightly higher cell viabilities and numbers. The downside is that elutriation centrifugation requires a specialized rotor, dedicated centrifuge, and peristaltic pump together which costs tens of thousands of dollars. This method only requires the use of a refrigerated table top centrifuge and peristaltic pump which is standard in many laboratories.
Separation of SECs and KCs by selective adhesion is a standard method20-22. While it is true that SECs will adhere to the glass and plastic, the key point is to use acid-washed clean glass with no more than a 15 minute incubation at 37&deg;C at 5% CO2. KCs will always adhere faster than SECs and it is advisable to gently wash the KCs with media to extract any remaining SECs that have become loosely attached. Pronase and other proteases are also omitted from the collagenase digestion steps in this protocol to increase viabilities of the cells. Proteases digest extracellular receptors and may inhibit cellular adhesion in the final purification steps.
The method presented here has a wide variety of applications. Although we show the end result in which heparin is initially taken up for clearance, perfusion of the liver for obtaining primary hepatocytes, KC, SECs, and HSCs may be useful for a number of studies involving metabolic pathways, immunoregulation, scavenger activities, and other physiological studies. The most difficult part of this procedure is good cannulation, digestion of the liver, and handling of the liver without having the catheter slip from the portal vein.

<ul>
<li>Buffer 1: 142 mM NaCl 6.7 mM KCl, 10 mM HEPES, 1.5% BSA, pH 7.4</li>
<li>Buffer 2 (perfusion buffer): 67.0 mM NaCl, 6.7 mM KCl, 4.8 mM CaCl2-H2O, 101.0 mM HEPES, BSA 1.5%, pH 7.4.</li>
<li>Buffer 3: 137 mM NaCl, 4.7 mM KCl, 0.7 mM MgSO4, 1.2 mM CaCl2-2H2O, 10 mM HEPES, 1.5% BSA, pH 7.4</li>
<li>RPMI/BSA: 15g/L BSA in RPMI</li>
<li>TRIS-buffered Saline (TBS): 154 mM NaCl, 10 mM Trizma Base salt.</li>
<li>Collagenase: Collagenase A (#11088785103) from Roche, Collagenase Type I (#LS004691) from Worthington Biochemical or Collagenase I (#C0130) or IA (#C9891) from Sigma-Aldrich.</li>
</ul>
All salts are from Sigma-Aldrich
<table border="1">			
<tbody>
<tr>
	<td>Name</td>
	<td>Company</td> 		
	<td>Model/catalog #	</td>
 <td>Comment</td>
</tr>
<tr>
	<td>20L water bath</td>
	<td>ThermoFisher</td>
	<td>2231</td>
 <td>Water is about 45&#176;C to compensate for cooling within the tubing.</td>
</tr>
<tr>
	<td>Peristaltic Pump</td>
	<td>Cole-Parmer</td>
	<td>7553-70</td>
 <td>Masterflex series</td>
</tr>
<tr>
	<td>Refrigerated Centrifuge</td>
	<td>Sorvall</td>
	<td>Legend XTR</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Catheter		</td>
	<td>BD Biosciences</td>
	<td>381444</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Dessicator chamber</td>
	<td>Fisher</td>
	<td>08595E</td>
 <td>Use internal plate</td>
</tr>
<tr>
	<td>Percoll</td>
	<td>Sigma</td>
	<td>P4937</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Bovine Serum Albumin</td>
	<td>SeraCare</td>
	<td>AP-4510-01</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>100 &mu;m mesh</td>
	<td>Spectrum labs</td>
	<td>146488</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>30 &mu;m mesh</td>
	<td>Spectrum labs</td>
	<td>146506</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>RPMI 1640</td>
	<td>Invitrogen</td>
	<td>21870</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Polyethylene glycol</td>
	<td>Spectrum labs</td>
	<td>PO107</td>
 <td>&nbsp;</td>
</tr>
</tbody>
</table>

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.

Watch this Scientific Journal Video about In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion at JoVE.com

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.

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 ...

in-vivo-liver-endocytosis-followed-purification-liver-cells-liver

Research

JoVE Journal

Biology

18.9K Views.  University of Nebraska, Lincoln. 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.

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

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

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.

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.

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

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

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 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. ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...