Name: isolation and characterization of mouse primary liver sinusoidal endothelial cells
Uploaded: 2021-12-16T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells at JoVE.com

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the NIH (1RO1DK122948 to SHI) and the NIH Silvio O. Conte Digestive Diseases Research Core Centers P30 grant mechanism (DK084567). Support was also provided to KF by the Japan Society for the Promotion of Science (JSPS) Overseas Research Fellowships. We would also like to acknowledge Dr. Gregory J. Gores and Steven Bronk for their original design and optimization of the collagenase perfusion apparatus.

Animal protocols were conducted as approved by the Institutional Animal Care and Use Committee (IACUC) of Mayo Clinic. Eight-week-old C57BL/6J male mice were purchased from Jackson Laboratory. Mice were housed in a temperature-controlled 12:12-h light-dark cycle facility with free access to diet.
1. Preparation of collagen-coated culture dish or plate
<ol>
	<li>To make 50 mL of 0.02 mol/L acetic acid, add 0.6 mL of glacial acetic acid to 49.4 mL of H2O.</li>
	<li>...

<ol>
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In the current manuscript, we describe a protocol for LSEC isolation from mouse liver consisting of two-step collagenase perfusion and subsequent magnetic-activated cell sorting (MACS). This protocol consists of the following three steps: (1) Perfusion through the PV with a calcium-free buffer followed by a collagenase-containing buffer to achieve liver cell dispersion; (2) Exclusion of hepatocytes with low-speed centrifugation; and (3) MACS-based positive selection of LSECs from nonparenchymal cells (NPCs) using anti-CD...

Liver sinusoidal endothelial cells (LSECs) line the hepatic sinusoid walls and are the most abundant nonparenchymal cells in the liver1. LSECs are distinguished from other capillary endothelial cells elsewhere in the body by the presence of fenestrae and the lack of a classical basement membrane or a diaphragm2,3. Hence, the LSECs possess distinctive phenotypic and structural characteristics that enhance their permeability and endocytic capacity to eliminate a variety of circulating macromolecules, including lipids and lipoproteins. LSECs play a pivotal role in the crosstalk between parenchymal and nonparenchymal cells, such as stellate cells and immune cells. LSECs are key in maintaining liver homeostasis by keeping the stellate cells and Kupffer cells in a quiescent status4. LSECs modulate the composition of hepatic immune cells populations by mediating adhesion and trans-endothelial migration of circulating leukocytes5,6. During acute and chronic liver injury7, including ischemia-reperfusion injury (IRI)8, nonalcoholic steatohepatitis (NASH)9, and hepatocellular carcinoma (HCC), LSECs undergo phenotypic changes known as capillarization characterized by defenestration and formation of basement membrane10. These phenotypic changes in LSECs are associated with LSECs dysfunction and the acquisition of prothrombotic, proinflammatory, and profibrogenic properties.
Several methods for the isolation of LSECs from mouse liver have been developed11. Some techniques depend on separating nonparenchymal and parenchymal cells followed by density gradient centrifugation to purify the LSECs from nonparenchymal fractions. The limitation of this method is the presence of contaminating macrophages in the final steps of LSECs isolation, which could affect the purity of the isolated LSECs12. This protocol is based on collagenase perfusion of the mouse liver and CD146+ magnetic beads positive selection of nonparenchymal cells to purify LSECs. LSECs isolated using this method show high purity and preserved morphology and viability. These LSECs are optimum for functional studies, including permeability and adhesion assay, as well as downstream studies for pathways of interest. Moreover, with the growing interest in generating big datasets in both clinical research and discovery science, these high-quality LSECs isolated from both healthy and diseased livers with nonalcoholic steatohepatitis (NASH) or other conditions can be pooled or used individually, allowing multi-omics data generation and comparison between health and disease13,14. In addition, the isolated LSECs can be employed to develop two-dimensional as well as three-dimensional in vitro models like organoids to decipher the activated signaling pathway in LSECs and their intercellular communication with other liver cells under different noxious stimuli and in response to various therapeutic interventions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2.0-inch 20 G Intra Venous (IV) catheter</td>
			<td>Terumo, SOmerset, NJ, USA</td>
			<td>SR-OX2051CA</td>
			<td></td>
		</tr>
		<tr>
			<td>2&#8211;3-inch perfuion tray with a hole in the center</td>
			<td></td>
			<td></td>
			<td>customized; made in house</td>
		</tr>
		<tr>
			<td>405/520 viability dye</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-110-205</td>
			<td></td>
		</tr>
		<tr>
			<td>4-inch regular curved dressing forceps</td>
			<td>Fisher Brand</td>
			<td>FS16-100-110</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 Perma-Hand silk suture</td>
			<td>Ethicon, Raritan, NJ, USA</td>
			<td>A182H</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-stabilin-2 (Mouse) mAb-Alexa Fluor&#195; 488</td>
			<td>MBL International, Woburn, MA, USA</td>
			<td>D317-A48</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA stock</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD146 (LSEC)-PE, anti-mouse</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-118-407</td>
			<td></td>
		</tr>
		<tr>
			<td>CD146 (LSEC) MicroBeads, mouse</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-092-007</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD45-Viogreen, anti-mouse</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-110-803</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen type I</td>
			<td>Corning, Corning, NY, USA</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>Gibco, Waltham, MA, USA</td>
			<td>17101-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial cells growth medium</td>
			<td>ScienCell Research Laboratories, Carlsbad, CA, USA</td>
			<td>211-500</td>
			<td></td>
		</tr>
		<tr>
			<td>FcR blocking reagent, mouse</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo software, version 10.6</td>
			<td>Becton, Dickinson and Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hardened Fine scissors</td>
			<td>F.S.T, Foster city, CA, USA</td>
			<td>14091-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated (37 &#176;C) and humidified recirculating perfusion apparatus equipped with Oxygen injection at a rate of 10psi.</td>
			<td></td>
			<td></td>
			<td>customized; made in house</td>
		</tr>
		<tr>
			<td>Hitachi S 4700 scanning electron microscope</td>
			<td>Hitachi Inc, Pleasanton, CA, USA</td>
			<td>SEM096</td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS pre-separation filters (70 &#956;m)</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-095-823</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS rinsing buffer</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-091-222</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Smart Strainer (70 &#956;m)</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-098-462</td>
			<td></td>
		</tr>
		<tr>
			<td>MACSQunt flow cytometer</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell Cell Culture Insert</td>
			<td>Millipore Sigma, Burlington, MA, USA</td>
			<td>PITP01250</td>
			<td></td>
		</tr>
		<tr>
			<td>Nexcelom cell counter</td>
			<td>Nexcelom bioscience, Lawrence, MA, USA</td>
			<td>Cellometer Auto T4 Plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare, Chicago, IL, USA</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>F.S.T, Foster city, CA, USA</td>
			<td>14001-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Very small curved dressing forceps</td>
			<td>F.S.T, Foster city, CA, USA</td>
			<td>11063-07</td>
			<td></td>
		</tr>
	</tbody>
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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.

Experimental schematics and equipment set up: 
In this protocol, mouse liver was digested using a closed perfusion circuit, then nonparenchymal cells and hepatocytes were separated by low-speed centrifugation at 50 x g for 2 min. Primary LSECs were isolated using CD146 magnetic beads selection from the nonparenchymal fraction. The experimental schematics are shown in Figure 1A. The cannula was placed through the PV while the inferior vena cava was tied up to en...

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Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

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. ...

Here, we demonstrate our protocol of primary mouse liver sinusoidal cell isolation or LSECs isolation. The protocol consists of liver collagenase perfusion and low-speed ultracentrifugation for non-parenchymal cell purification and CD146 magnetic bead selection. Our LSEC isolation method is highly efficient and applicable to healthy and diseased mouse liver.The isolated LSECs display high purity and preserve the structure and function. LSECs isolated using this protocol are optimum for functional and molecular studies and multi-omics data generation. This protocol will be helpful for studying intercellular communication in the liver.Begin by coating 10-centimeter culture dishes with three milliliters of collagen solution. Then, incubate the dishes at room temperature for one hour. After an hour, remove the excess fluid from the coated surface.Wash the dishes with PBS three times and allow them to air dry. Set up the heated and humidified recirculating perfusion apparatus. Rinse the perfusion system using 10%bleach for five minutes.Then, rinse the system again with sterile water for another 10 minutes. Drain the rinsing liquid as much as possible before perfusing with collagenase solution. Infuse the collagenase solution through the perfusion system to prewarm it to 37 degrees Celsius.Set the pump rate to speed one on the speed control dial. Keep the speed the same throughout the whole procedure. Weigh the mouse for the procedure.Then secure it to the surgical surface. Spray the mouse abdomen with 70%ethanol. Using surgical scissors, make an incision of approximately five centimeters starting from the lower part of the abdomen up to the xiphoid process.After that, make two lateral cuts using small iris scissors on each side of the abdomen to expose the abdominal organs fully. Place the sheath of the IV catheter under the animal's back to lift and level the abdomen. Using a regular curved dressing forceps, gently pull the intestines and the stomach off to the left of the animal.Then, place a 5-0 surgical suture under the inferior vena cava, or IVC, just below the exposed left kidney. Tie a loose hitch in the suture. Next, place another 5-0 surgical suture around the hepatic portal vein.Place the suture just above the splenic vein branching point of the hepatic portal vein. Again, tie a loose hitch in the suture. Using the portal vein suture as tension, insert the 20-gauge IV catheter in the hepatic portal vein one centimeter below where it branches to the right and left hepatic portal vein.Then, slide the catheter up the vein, but keep it below the branching area. Allow the blood to travel down the catheter until it begins to drip out. Secure the IV catheter with the portal vein suture.Use an IV line to attach an IV bottle with buffer A to the catheter. Then, flush the liver with this solution while avoiding air entry into the system. Tie off the suture around the IVC below the kidney.After that, cut the IVC below the suture to allow the animal to bleed out. Once perfusing, cut away the stomach, intestines, spleen and other entrails attached to the liver. Then, cut away the diaphragm and the major vessels from the thoracic cavity.Remove the liver from the animal and place it on the perfusion tray. Carefully remove the IV line and hook up the collagenase solution in the recirculating chamber. Allow the liver to perfuse until the capsule becomes modeled and appears mushy.This should typically take 10 to 15 minutes. Once digested, remove the liver from the chamber and place it on a 10-centimeter Petri dish with about 20 milliliters of serum-free DMEM. Gently pick apart the liver with a couple of pipette tips and discard the biliary tree.After that, filter the liver suspension through a 70-micrometer cell strainer into a 50-milliliter conical tube. Centrifuge the cell suspension at 50 times G for two minutes at room temperature. Then, collect the supernatant containing non-parenchymal hepatic cells.Centrifuge the supernatant at 300 times G for five minutes at four degrees Celsius. After that, collect the cell pellet and resuspend it in one milliliter of isolation buffer. Determine the cell number using an automatic cell counter following the manufacturer's instructions.Next, centrifuge the cell suspension. Aspirate the supernatant entirely and resuspend the pellet with 90 microliters of isolation buffer per 10 million cells. Add 10 microliters of CD146 MicroBeads per 10 million total cells.Mix it well and incubate for 15 minutes at four degrees Celsius. To wash the cells, add one to two milliliters of isolation buffer per 10 million cells. Centrifuge the solution at 300 times G for 10 minutes.After that, aspirate the supernatant entirely and resuspend up to one billion cells in 500 microliters of isolation buffer. Next, prepare the separation column by rinsing it with three milliliters of isolation buffer. Apply the cell suspension onto the column stacked with 70-micrometer pre-separation filters.Wash the column with three milliliters of isolation buffer three times. After that, remove the column from the separator and place it on a 15-milliliter centrifuge tube. Pipette five milliliters of isolation buffer onto the column.To recover the magnetic bead-labeled cells, firmly push the plunger into the column to flush out the cells. Finally, centrifuge the cells at 300 times G for five minutes at four degrees Celsius. Isolated LSECs purity and surface markers were assessed with flow cytometry.Data analysis and gating strategy for gated LSECs and singlets are based on forward scatter and side scatter data. Isolated LSECs were stained with a viability dye and a combination of CD45, CD146 and Stabilin-2 antibodies. Viable LSECs were gated on CD45 negative population and analyzed for CD146 and Stabilin-2 expression.From this data, it was confirmed that the isolated LSECs had greater than 94%viability and greater than 90%purity. The LSEC-specific phenotype of the isolated cells was further confirmed using light microscopy and scanning electron microscopy. Isolated LSECs were cultured in the complete growth medium for six hours and examined by light microscopy.The white arrows in the SEM image indicate LSEC fenestrae, which is a characteristic morphological feature of LSECs. The critical steps to ensure complete perfusion of a liver tissue are proper catheter tape positioning, avoiding the introduction of air in the catheter, and the optimum timing of collagenase digestion. High-quality LSEC acquired using our protocol will facilitate identification of the molecular mechanism of LSEC function and will improve our understanding of their role in intercellular communication in the liver, in health and in disease.

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