Purification and Isolation of Hepatic Stellate Cells

Abstract

Quiescent human hepatic stellate cells (HSCs) serve as important reservoirs of fat-soluble vitamins in the body, namely vitamin A. In an activated form, HSCs are the drivers of fibrosis following chronic liver injury. In non-alcoholic steatohepatitis (NASH) specifically, activated HSCs are drivers of induction and progression of fibrogenesis. Isolation and purification of HSCs from donor liver samples provides an avenue to study HSCs and their fibrotic capabilities. Manual and chemical digestion of donor liver via dissection and Pronase, collagenase, and DNAse treatment creates a suspension of non-parenchymal liver cells. Quiescent HSCs can be further isolated from this suspension by density-gradient centrifugation in a 6%, 8%, 12%, and 15% arabinogalactan medium. After collection of HSCs from the low-density layers of the gradient, they can be grown on uncoated plastic. Rodent HSCs can also be isolated via density-gradient centrifugation. To isolate activated HSCs, liver tissue explants or established immortalized HSC lines can be utilized. Here, we described protocols for isolation of human and rodent HSCs.

1. Introduction

Human hepatic stellate cells (HSCs) constitute approximately 5–8% of all liver cells while storing 50–80% of the body’s vitamin A [1,2]. They primarily reside in the space of Disse between sinusoidal endothelium and parenchymal cells, and are also called vitamin A-storing cells, fat-storing cells, or lipocytes due to their retinyl palmitate reserves in cytoplasmic lipid droplets [2]. In addition to maintaining vitamin A homeostasis, HSCs have been shown to produce components of the extracellular matrix including collagen and adhesive glycoproteins [2]. Their capacity for cellular contraction makes HSCs key regulators of hepatic blood flow by controlling sinusoidal vascular tone [3]. HSCs influence the differentiation, proliferation, and morphogenesis of other hepatic cells during liver development and regeneration following injury [4].

HSCs play a key role in pathological states, including hepatic fibrosis, cirrhosis, and even cancer. Quiescent HSCs are activated to differentiate into myofibroblasts, losing their vitamin A-storing capabilities and proliferating to drive fibrosis following chronic liver injury [5,6]. Activation is mediated by extracellular inflammatory signals and oxidative stress [4]. As a result, hepatic stellate cells, particularly in the activated form, are considered key therapeutic targets to treat viral infections, alcoholic liver disease, and non-alcoholic steatohepatitis (NASH) that could progress to fibrosis. Purification and isolation of HSCs in vitro is critical for future clinical research on therapeutics.

This chapter discusses isolation and purification of human HSCs for further laboratory evaluation using density-gradient centrifugation. Purification of rodent HSCs is also briefly discussed, as rat and mouse models are often used to study NASH. Other, more novel methods of isolation including liver tissue explants and established cell lines are also briefly discussed [7].

2. Materials

2.1. Isolation of Quiescent Human HSCs

1. Liver tissue: 400–600 g wedges of donor liver.

2. Heat lamp with angled sterile tray or alternative comparable setup to warm liver tissue to 37 °C.

3. 1 L Leibovitz (L-15) medium bath to submerge liver tissue wedges during warming.

4. 1.5 L L-15 medium with 2 IU/mL heparin for perfusion into liver tissue wedges.

5. Perfusion pump (flow rate set to 60 mL/min) with Teflon catheter or tuberculin syringe suture.

6. 18-gauge Teflon catheter with 60 mL sterile syringe for manual perfusion.

7. 400 mL Ham’s modification of Dulbecco’s modified Eagle medium (Ham’s/DMEM) as base solution for 700 mg Pronase.

8. 400 mL Ham’s/DMEM as base solution for 80 mg collagenase.

9. 100 mL Ham’s/DMEM as base solution for 40 mg Pronase and 0.4 mg DNAse.

10. Surgical scissors to dissect liver capsule.

11. Water bath set at 250 rpm for tissue agitation.

12. Sterile cotton gauze for filtration.

13. 40 mL polypropylene tubes compatible with centrifuge.

14. Fresh Eagle’s minimal essential medium (MEM) to serve as base for 0.2 mg/mL DNAse solution.

15. Arabinogalactan density gradient: 1.5 mL each of 6%, 8%, 12%, and 15% arabinogalactan prepared by dissolving arabinogalactan in distilled water and layered across 40 mL polypropylene tubes compatible with centrifuge.

16. Centrifuge machine: must be capable of 50,490 × g for 25 min.

17. Sterile pipettes.

18. Medium 199 containing 20% serum (10% calf and 10% horse) layered on uncoated 35-mm plastic petri dishes.

2.2. Isolation of Rodent HSCs

1. Rat liver tissue.

2. Gey’s balanced salt solution (GBSS) bath for perfusion into liver tissue.

3. Perfusion pump (flow rate set to 10 mL/min) with Teflon catheter or tuberculin syringe suture.

4. 0.05% Pronase in GBSS.

5. 0.05% collagenase in GBSS.

6. Surgical scissors to dissect liver capsule.

7. 100 mL GBSS as base for 0.02% Pronase and 0.05% collagenase solution.

8. Sterile cotton gauze for filtration.

9. Centrifuge machine: must be capable of 1400 × g for 17 min, compatible with elutriation procedure setup (see Note 1).

10. 40 mL polypropylene tubes compatible with centrifuge.

11. Metrizamide density gradient: 10 mL of 18% Metrizamide, and 10.5 mL of 13% Metrizamide prepared by diluting 30% Metrizamide stock solution with GBSS and layered across 40 mL polypropylene tubes compatible with centrifuge.

12. Sterile pipettes.

13. Elutriator rotor with separation chamber for centrifugal elutriation procedure: must be capable of 18 mL/min flow rate and compatible with centrifuge machine.

14. Siliconized centrifuge tubes.

3. Methods

3.1. Isolation of Quiescent Human HSCs

This method (Fig. 1) is described by Friedman et al. [8] for isolation of quiescent human HSCs from liver tissue (see Note 2).

1. Collect 400–600 g tissue wedges from human liver (see Note 3).

2. Warm tissue to 37 °C using a heat lamp directed toward an angled tray and incubating in L-15 medium for 15–20 min. While incubating in solution, simultaneously perfuse tissue wedge with warm L-15 medium with 2 IU/mL heparin both via pump (60 mL/min flow rate) and manually (200 mL/min). A perfusion pump should be ligated to a portal vein opening in the tissue wedge via Teflon catheter or tuberculin syringe suture. Manual perfusion should also be via portal vein opening, performed with 18-gauge Teflon catheters attached to 60 mL sterile syringes (see Note 4).

3. Perfuse wedges in Pronase (700 mg dissolved in 400 mL Ham’s/DMEM) for 15–20 min. Subsequently perfuse wedges in collagenase (80 mg in 400 mL Ham’s/DMEM) for 30 min. Use the same perfusion pump setup (60 mL/min flow rate) as the previous step but discard any remaining L-15 medium prior to the Pronase/collagenase perfusion. Liver should be adequately softened at the conclusion of this step.

4. Separate softened liver tissue away from capsule. Recommended method is to dissect the capsule with surgical scissors and manually agitate or scrape away the tissue. Perform tissue agitation in a 37 °C water bath (250 rpm for 30 min) (see Note 5).

5. Make a solution of agitated or scraped tissue by suspending cells in 40 mg Pronase and 0.4 mg DNAse in 100 mL Ham’s/DMEM. Subsequently filter solution through cotton gauze.

6. Place tissue solution in 40 mL polypropylene tubes. Wash four times via centrifugation (500 g for 7 min at 37 °C) in fresh MEM with 0.2 mg/mL DNAse (see Note 6).

7. Prepare poly-clear tubes for density-gradient centrifugation. Layer tubes with 1.5 mL each 6%, 8%, 12%, and 15% arabinogalactan solution.

8. Add tissue suspension to arabinogalactan tubes and centrifuge at 50,490 × g for 25 min at 37 °C to layer all non-parenchymal cells.

9. Recover HSCs from less than 6% to 6% or 6% to 8% interfaces of the resulting density gradient using sterile pipettes.

10. Wash once via centrifugation (500 × g for 7 min at 37 °C) in fresh MEM with 0.2 mg/mL DNAse.

11. Plate HSCs on uncoated 35 mm plastic petri dishes in Medium 199 containing 20% serum (10% calf and 10% horse). Change medium every 48 h during the growth phase. Typical spread takes 48–72 h, although proliferation in primary culture can occur after 7 days. HSCs can survive on plain plastic for up to 28 days [9].

12. Visualize HSCs via immunofluorescence (see Fig. 2), with visible lipid droplets in the perinuclear zone. Quiescent HSCs express α-smooth muscle actin (α-SMA) and Desmin cytoskeletal proteins, although human HSCs have a greater concentration of these epitopes than rodent HSCs [7]. Activated HSCs present in fibrotic clusters have also been shown to express glial fibrillary acidic protein (GFAP) [10,11]. Immunofluorescence can also be used to visualize HSCs not grown on primary culture (see Notes 7–10).

Fig. 1.

Flowchart of the basic process of isolation of human HSCs via density-gradient centrifugation

Fig. 2.

Immunofluorescence staining demonstrates differential expression of GFAP, Desmin, and α-SMA proteins in mouse and human primary HSCs. (Image source: Shang L, Hosseini M, Liu X, Kisseleva T, Brenner DA (2018) Human hepatic stellate cell isolation and characterization. J Gastroenterol 53(1):6–17)

3.2. Isolation of Rodent HSCs

The rodent model has been used extensively to study NASH, and isolation of rat HSCs via density-gradient centrifugation is a similar process to isolating human HSCs, with some key differences [12].

1. Warm liver to 37 °C by perfusing portal vein in situ with Gey’s balanced salt solution (GBSS) via pump (10 mL/min flow rate) for 6 min. During perfusion, excise tissue from lobular area of rat liver.

2. Perfuse tissue in Pronase (0.05% in GBSS) via pump (10 mL/min flow rate) for 30 min. Subsequently perfuse in collagenase (0.05% in GBSS) via pump (10 mL/min flow rate for 30 min). Liver should be adequately softened at the conclusion of this step.

3. Separate softened liver tissue away from capsule. Recommended method is to dissect the capsule with surgical scissors and mince the tissue.

4. Incubate the liver tissue pieces in Pronase and collagenase (0.02% and 0.05% respectively, in 100 mL GBSS) for 30 min at 37 °C. Subsequently filter solution through cotton gauze.

5. Centrifuge filtrate at 450 × g for 7 min. Resuspend cell pellet in 10 mL GBSS.

6. Prepare polypropylene tubes for density-gradient centrifugation. Layer tubes with 10 mL 18% Metrizamide and 10.5 mL 13% Metrizamide.

7. Add tissue suspension to Metrizamide tubes and centrifuge at 1400 × g for 17 min at 37 °C to layer all non-parenchymal cells.

8. Recover HSCs from the top layer of the resulting density gradient using sterile pipettes.

9. Wash once via centrifugation (450 × g for 5 min at 37 °C) in GBSS and resuspend pellet in 10 mL GBSS (see Note 1).

10. Perform first round of centrifugal elutriation at 864 × g at 4 °C, using GBSS as the elutriation fluid. Wash cells into separation chamber at flow rate of 16 mL/min for 30 s. Collect 100 mL of overflow liquid in siliconized centrifuge tubes.

11. Perform second round of centrifugal elutriation at 864 × g at 4 °C, using GBSS as the elutriation fluid. Wash cells into separation chamber at flow rate of 18 mL/min for 30 s. Collect another 100 mL of overflow liquid in siliconized centrifuge tubes.

12. Centrifuge fractions at 1000 × g for 10 min. Resuspend cell pellets in 10 mL GBSS or as needed for future studies.

13. Plate HSCs on uncoated 35 mm plastic petri dishes in Medium 199 containing 20% serum (10% calf and 10% horse). Change medium every 18–24 h after plating [9].

14. Rodent HSCs can also be visualized via immunofluorescence (Fig. 2), although human HSCs have a greater affinity for α-SMA, Desmin, and GFAP epitopes [7].