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...

Watch this Scientific Journal Video about Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells at JoVE.com

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. ...

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