Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Summary

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.

Abstract

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' 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.

Introduction

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 diaphragm^2,3. 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 leukocytes^5,6. 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 disease^13,14. 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.

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Protocol

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

1. 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.
2. Make 50 µg/mL collagen type I in 0.02 mol/L acetic acid. Dilution depends on the concentration of the lot.
3. Coat 10 cm culture dishes with 3 mL of collagen solution. Incubate at room temperature (RT) for 1 h.
   NOTE: When using a different dish or plate for culture, the volume of coating solution needs to be adjusted based on culture area, generally use 6-10 µg/cm².
4. Remove excess fluid from the coated surface, and wash with Phosphate-buffered saline (PBS) 3 times. Allow the dishes to air dry.
5. If the collagen solution is not sterile, sterilize the collagen-coated dishes by exposure to ultraviolet (UV) light for 10 min in a sterile tissue culture hood.

2. Equipment setup

1. Set up the heated and humidified recirculating perfusion apparatus as shown in Figure 1B.
2. Rinse the perfusion system using 10% bleach for 5 min followed by sterile water for another 10 min.
3. Drain the rinsing liquid as much as possible before perfusing the collagenase solution.
4. Infuse collagenase solution through the perfusion system (as shown in Figure 1B) to prewarm it at 37 °C. Set the pump rate at speed 1 as shown on the speed control dial (equal to 40 mL/min).
5. Keep this speed the same throughout the whole procedure. Reuse the collagenase solution run in a closed circuit during the day of the LSEC isolation.
   ​NOTE: The pump speed is fixed at 1 to avoid mechanical pressure that could perturb LSECs biological condition.

3. Surgical procedure

1. Weigh the mouse.
2. Inject 90 mg/kg body weight of ketamine and 10 mg/kg bodyweight of xylazine intraperitoneally (IP).
3. Once the mouse is nonreactive to painful stimuli, secure the mouse to the surgical surface, as shown in Figure 1B.
4. Spray the mouse abdomen with 70% ethanol. Using surgical scissors, make an incision of ~5 cm long starting from the lower part of the abdomen up to the xiphoid process.
5. Next, make two lateral cuts using small iris scissors on each side of the abdomen to fully expose the abdominal organs.
6. Place the sheath of the intravenous (IV) catheter under the animal's back to lift and level the abdomen.
7. Using a regular curved dressing forceps, gently pull the intestines and the stomach off to the left of the animal.
8. Place a 5-0 surgical suture under the inferior vena cava (IVC) just below the exposed left kidney. Tie a loose hitch in the suture.
9. Place another 5-0 surgical suture around the hepatic portal vein (PV) just above the splenic vein branching point off the hepatic PV. Tie a loose hitch in the suture.
10. Using the PV suture as tension, insert the 20 G IV catheter in the hepatic PV 1 cm below where it branches to the right and left hepatic PV.
11. 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.
12. With some of the Buffer A (Table 1) in an intravenous (IV) bottle positioned above the surgical area, use an IV line, and attach it to the catheter. Flush the liver with this solution while avoiding air entry into the system.
13. Tie off the suture around the inferior vena cava below the kidney. This will allow the liver to retro perfuse.
14. Cut the IVC below the suture to allow the animal to bleed out.
    NOTE: This step must be performed quickly to avoid congestion of the liver.
15. Secure the IV catheter with the PV suture.
16. Once perfusing, cut away the stomach, intestines, spleen, and other entrails attached to the liver.
17. 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.
18. Carefully remove the IV line and hook up the collagenase solution in the recirculating chamber.
    NOTE: Steps 3.16-3.18 need to be done within 5 min, so the liver will not be perfused too long with Buffer A. Be very careful not to allow any air bubbles into the liver.
19. Allow the liver to perfuse until the capsule becomes mottled and appears mushy (10-15 min or more, period varies depending on the different lots of collagenase).
20. While liver is in perfusion, make sure the animal is deceased before disposing it in a necropsy bag.
21. Once digested, remove the liver from the chamber and place it in a 10 cm Petri dish with about 20 mL of serum-free Dulbecco's Modified Eagle Medium (DMEM).
22. Gently pick apart the liver with a couple of pipette tips and discard the biliary tree.
23. Filter the liver suspension through a 70 µm cell strainer into a 50 mL conical tube.
24. Centrifuge the cell suspension at 50 x g for 2 min at RT. Collect the supernatant containing nonparenchymal hepatic cells.
    ​NOTE: Hepatocytes are precipitated in the pellet, which can be further purified using gradient centrifugation as previously described¹⁵.

4. Separation of nonparenchymal hepatic cells and LSECs purification

NOTE: Purify the CD146⁺ LSECs using the immunomagnetic beads, following the manufacture's instructions.

1. Centrifuge the supernatant at 300 x g for 5 min at 4 °C.
2. Collect the cell pellet and resuspend in 1 mL of isolation buffer (Table 1), determine the cell number using an automatic cell counter following the manufacturer's instructions.
3. Centrifuge the cell suspension at 300 x g for 10 min at 4 °C, aspirate the supernatant completely.
4. Resuspend the pellet with 90 µL of isolation buffer per 10⁷ total cells.
5. Add 10 µL of CD146 magnetic beads per 10⁷ total cells. Mix well and incubate for 15 min at 4 °C.
6. To wash the cells, add 1-2 mL of isolation buffer per 10⁷ cells, centrifuge at 300 x g for 10 min, and then aspirate the supernatant completely.
7. Resuspend up to 10⁹ cells in 500 µL of isolation buffer (Table 1).
   NOTE: For higher cell numbers, scale up buffer volume accordingly.
8. Prepare the separation column, rinse it with 3 mL of isolation buffer.
9. Apply the cell suspension onto the column stacked with 70 µm pre-separation filters.
10. Wash the column with the 3 mL of isolation buffer 3 times.
    NOTE: When washing the column, the isolation buffer should be added as soon as the column reservoir is empty.
11. Remove the column from the separator and place it on a 15 mL centrifuge tube. Pipette 5 mL of isolation buffer onto the column.
12. To recover the magnetic beads labeled cells, firmly push the plunger into the column to flush out the cells.
13. Centrifuge at 300 x g for 5 min at 4 °C. LSECs are ready for microscopic examination and downstream analysis.

5. LSECs immunophenotyping and purity assessment by flow cytometry

1. Determine the cell numbers of isolated LSECs using an automatic cell counter following the manufacturer's instructions.
2. Centrifuge the cells at 300 x g for 5 min, aspirate the supernatant completely.
3. Take 1 x 10⁶ cells/tube and resuspend with 90 µL of staining buffer (Table 1).
4. Add 10 µL/tube of mouse FcR block and 1 µL of viability dye, incubate at 4 °C for 10 min.
5. Stain the cells with a combination of CD45, CD146, and stabilin-2 antibodies diluted at 1:50 with staining buffer. Incubate at 4 °C for 20 min.
6. Wash the cells with 5 mL of staining buffer and centrifuge at 300 x g for 10 min, then aspirate the supernatant completely.
7. Resuspend the cells with 300 µL of staining buffer and run through the flow cytometer.

6. LSECs culture and examination

1. Seed 1 x 10⁶ cells/well in a 6-well plate and culture it with endothelial cells growth medium consisting of 5% fetal bovine serum (FBS), 1% endothelial cells growth supplement, and 1% primocin solution.
2. Examine the isolated LSECs by light microscopy. Acquire bright-field images with a 10x magnification.

7. LSECs morphology and fenestrae examination by scanning electron microscopy

1. Pre-coat the cell culture inserts (3 µm pore size) with collagen solution.
2. Culture isolated LSECs (120,000 cells) on the insert with endothelial cells growth medium in a 24-well plate at 37 °C warm and humidified atmosphere. Allow cells to settle down and adhere for 2 h.
3. For cell fixation, add an equivalent volume of prewarmed at 37 °C Trump's fixative to the cell culture media.
4. After incubation for 10 min, replace 50% diluted Trump's fixative with undiluted Trump's fixative.
5. Fix the cells in Trump fixative for 2 h, then incubate for 1 h in 1% osmium tetroxides.
6. Proceed with samples dehydration, drying in a critical point drying device, mounting, sputter coating, and examination using a scanning electron microscope.

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Results

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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Discussion

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...

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Materials

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