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°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’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 μm) collagenase (100 mg/kg rat weight) should be added to Buffer 2 in a total volume not to exceed 60 mL at 37°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’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 μm mesh followed by filtration through a 30 μm 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 ≥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°C.</li>
 <li>Aspirate the buffer and resuspend all the pellets in 5 mL RPMI/BSA at 4°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°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°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°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°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°C, 5% CO2, RPMI/0.25% BSA with 40 ng/mL VEGF, 100 U/mL Penicillin, 100 μg/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 ≥97% and SEC purification is typically ≥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="Liver sinusoid diagram; hepatocytes, space of Disse, Kupffer cells, liver structure study." data-altupdatedat="1747911167000" data-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="Liver perfusion process diagram: O2 supply, pump, flasks, funnel for flushing and digestion flows." data-altupdatedat="1747911167000" data-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="Hepatocyte protein separation, SEC, diagram; 315-kDa and 190-kDa isoforms, electrophoresis results." data-altupdatedat="1747911167000" data-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="Organ distribution bar chart of ¹²⁵I-SA-bHep uptake; CPM/mg in liver, spleen, pancreas, others." data-altupdatedat="1747911167000" data-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="Endocytosis graph of ^125I-SA-b-Hep, CPM/mg protein, with and without cold Hep, SEC vs HC cells." data-altupdatedat="1747911167000" data-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="Electrophoresis gel result showing protein bands at 130 kDa under lanes 1 and 2, indicating separation." data-altupdatedat="1747911167000" data-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°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="Cell culture microscope images; A) Low magnification; B) High magnification; cell morphology study." data-altupdatedat="1747911167000" data-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.

<table><tbody><tr><td colspan="4">&lt;ul&gt; &lt;li&gt;Buffer 1: 142 mM NaCl 6.7 mM KCl, 10 mM HEPES, 1.5% BSA, pH 7.4&lt;/li&gt; &lt;li&gt;Buffer 2 (perfusion buffer): 67.0 mM NaCl, 6.7 mM KCl, 4.8 mM CaCl&lt;sub&gt;2&lt;/sub&gt;-H2O, 101.0 mM HEPES, BSA 1.5%, pH 7.4.&lt;/li&gt; &lt;li&gt;Buffer 3: 137 mM NaCl, 4.7 mM KCl, 0.7 mM MgSO&lt;sub&gt;4&lt;/sub&gt;, 1.2 mM CaCl&lt;sub&gt;2&lt;/sub&gt;-2H&lt;sub&gt;2&lt;/sub&gt;O, 10 mM HEPES, 1.5% BSA, pH 7.4&lt;/li&gt; &lt;li&gt;RPMI/BSA: 15g/L BSA in RPMI&lt;/li&gt; &lt;li&gt;TRIS-buffered Saline (TBS): 154 mM NaCl, 10 mM Trizma Base salt.&lt;/li&gt; &lt;li&gt;Collagenase: Collagenase A (#11088785103) from Roche, Collagenase Type I (#LS004691) from Worthington Biochemical or Collagenase I (#C0130) or IA (#C9891) from Sigma-Aldrich.&lt;/li&gt; &lt;/ul&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;All salts are from Sigma-Aldrich&lt;/p&gt;</td></tr><tr><td>20L water bath</td><td>ThermoFisher</td><td>2231</td><td>Water is about 45 &amp;deg;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></td></tr><tr><td>Catheter </td><td>BD Biosciences</td><td>381444</td><td></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></td></tr><tr><td>Bovine Serum Albumin</td><td>SeraCare</td><td>AP-4510-01</td><td></td></tr><tr><td>100 &amp;mu;m mesh</td><td>Spectrum labs</td><td>146488</td><td></td></tr><tr><td>30 &amp;mu;m mesh</td><td>Spectrum labs</td><td>146506</td><td></td></tr><tr><td>RPMI 1640</td><td>Invitrogen</td><td>21870</td><td></td></tr><tr><td>Polyethylene glycol</td><td>Spectrum labs</td><td>PO107</td><td></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 ...

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JoVE Journal

Biology

19.0K Views.  University of Nebraska, Lincoln. De studie van de lever sinusoïdale endotheelcellen (SEC) moeten worden uitgevoerd met primaire cellen afkomstig van het dier als geen cel-lijnen bestaan. Deze methode is gebaseerd op de lever spijsvertering en differentiële centrifugatie voor SEC zuivering voor latere kweken en experimenteren.

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

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Biochemistry

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

Metabolic diseases such as diabetes, pre-diabetes, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) are becoming increasingly common. Ex vivo liver perfusions allow for a comprehensive analysis of liver metabolism using nuclear magnetic resonance (NMR), in nutritional conditions that can be rigorously controlled. As in silico simulations remain a primarily theoretical means of assessing hormone actions and the effects of pharmaceutical intervention, the perfused liver remains one of the most valuable test beds for understanding hepatic metabolism. As these studies guide basic insights into hepatic physiology, results must be accurate and reproducible. The greatest factor in the reproducibility of ex vivo hepatic perfusion is the quality of surgery. Therefore, we have introduced an organized and streamlined method to perform ex vivo mouse liver perfusions in the context of in situ NMR experiments. We also describe a unique application and discuss common issues encountered in these studies. The overall purpose is to provide an uncomplicated guide to a technique we have refined over several years that we deem the golden standard for obtaining reproducible results in hepatic resections and perfusions in the context of in situ NMR experiments. The distance to the center of the field for the magnet as well as the inaccessibility of the tissue to intervention during the NMR experiment makes our methods novel.

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

The protocol describes a straightforward method of resectioning an intact mouse liver for metabolic studies through portal vein perfusion.

Metabolic diseases such as diabetes, pre-diabetes, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) are becoming ...

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. ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

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 ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Noninvasive Intraocular In Vivo Imaging of Engrafted Liver Spheroids in Mice

In Vivo Imaging of Liver Spheroids Engrafted in the Anterior Chamber of the Mouse Eye

Biomedical studies of the liver in mammals are hindered by the lack of methods for in vivo noninvasive longitudinal imaging at cellular resolution. Until now, optical imaging of the liver in situ is possible by intravital imaging, which offers high-resolution imaging at the cellular level but cannot be performed multiple times and, therefore, longitudinally in the same animal. Noninvasive imaging methods, such as bioluminescence, allow repeated imaging sessions on the same animal but do not achieve cell resolution. To address this methodology gap, we have developed a platform for noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. In the workflow described in this study, primary mouse liver spheroids are generated in vitro and transplanted into the anterior chamber of the eye of recipient mice, where they engraft on the iris. The cornea acts as a natural body window through which we can image the engrafted spheroids by conventional confocal microscopy. The spheroids survive for months in the eye, during which the cells can be studied in contexts of health and disease, as well as being monitored in response to different stimuli over repeated imaging sessions using appropriate fluorescent probes. In this protocol, we provide a breakdown of the necessary steps to implement this imaging system and explain how to best harness its potential.

Author Spotlight: Harnessing Mouse Eye Chambers for Noninvasive Liver Spheroid Studies

Here, we describe a platform that allows noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. The workflow spans from generating spheroids from primary liver cells to transplantation into the mouse eye and in vivo imaging at cellular resolution by confocal microscopy.

Biomedical studies of the liver in mammals are hindered by the lack of methods for in vivo noninvasive longitudinal imaging at cellular resolution. Until ...

Rat Liver Perfusion and Primary Hepatocytes Isolation: An Old Procedure Crucial for Cutting-Edge 3D Organoids Culture

Primary hepatocytes are a commonly used tool for in vitro liver-related studies. However, the maintenance of these cells has always been a challenge due to the rapid loss of morphology, viability, and functionality in culture. A recent approach to long-term culture is the generation of three-dimensional (3D) organoids, an in vitro tool that can recapitulate tissues in a dish based on the marvelous ability of the liver to regenerate itself. Published protocols have been designed to obtain long-term functional 3D organoids from primary adult hepatocytes (Hep-Orgs). The 3D organoid cutting-edge tool requires the ability to isolate cells from adult tissue, and this initial step is crucial for a high-quality final result. The two-step collagenase perfusion, introduced in the 1970s, is still a valid procedure to obtain single hepatocytes. The present article aims to describe all the crucial steps of the surgical procedure, thereby optimizing the primary hepatocytes isolation procedure in the rat model. Moreover, particular attention is paid to the PREPARE guidelines to increase the likelihood of successful procedures and ensure high-quality results. A detailed protocol allows researchers to speed up and optimize the downstream work to establish 3D organoids from primary adult rat hepatocytes. Compared to 2D hepatocytes, Hep-Orgs were still viable and in active proliferation at Day 15, demonstrating a long-term potential.

This protocol presents an optimized two-step collagenase liver perfusion technique in a rat model and shows the use of isolated hepatocytes for in vitro long-term culture of 3D organoids.

Primary hepatocytes are a commonly used tool for in vitro liver-related studies. However, the maintenance of these cells has always been a challenge due ...

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

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