Isolation of Human Hepatic Stellate Cells

Materials and Reagents

All materials must be filtered with a 0.20 μm sterile filter.

1.Enzyme H solution: Added Three milliliters of Dulbecco’s modifed Eagle’s medium (DMEM) was added to the lyophilized Enzyme H powder and mixed them evenly by shaking gently. Aliquots were cryopreserved at −20 ℃. Repeated freezing and thawing were avoided. (Miltenyi Biotec, 130-093-237, Germany)

2.Enzyme R solution: DMEM (2.7 ml) was added to the lyophilized Enzyme R powder. After mixing, the solution aliquots were cryopreserved at −20 ℃. Repeated freezing and thawing were avoided. (Miltenyi Biotec, 130-093-237, Germany)

3.Enzyme A solution: One milliliterl of DMEM was added to the lyophilized Enzyme a powder; vigorous shaking was avoided while mixing. The aliquots were cryopreserved at −20 ℃. (Miltenyi Biotec, 130-093-237, Germany)

4.DNase I (Roche, Switzerland)

5.Nycodenz Separation Solution (density 1.131 g/ml, Beijing Solarbio Science & Technology Co., Ltd.)

6.RPMI 1640 medium (Gibco, USA)

7.DMEM/F12 medium (Gibco, USA)

8.Gey’s balanced salt solution (GBSS, Sigma, USA)

9.Phosphate-bufered saline (PBS, Gibco, USA)

10.Penicillin-streptomycin (Gibco, USA)

11.Fetal bovine serum (FBS, Gibco, USA)

12.Trypan blue (Beyotime Biotechnology, China)

Equipment

1.Standard laboratory equipment

2.Precooled centrifuges (5810R, Eppendorf, Germany)

3.Sterile surgical instruments and preparation tools

4.Sterile 15ml plastic tubes (Ucallm, China)

5.Sterile 50ml plastic tubes (Ucallm, China)

6.Pasteur pipettes (Ucallm, China)

7.Standard cell culture room equipped with a sterile hood

8.Petri dishes(Ucallm, China)

9.70μm cell strainer (Ucallm, China)

10.Cell counting chambers (Biologix, 07–2104, China)

11.Inverted fluorescence microscope (NIKON, Ti-S, Japan)

12.Gentle MACS tissue processor (Miltenyi Biotec, 130-093-235, Germany)

13.Gentle MACS C Tube (Miltenyi Biotec, 130-093-237, Germany)

Procedure

1. Preparation of a single cell suspension from human hepatic tissue

1.1 Surgically removed fresh normal liver tissues were processed and collected in an ice box at 4 ℃ under aseptic conditions.

1.2 The tissues were quickly transported to the cell culture chamber. The specimen was        

placed in a Petri dish and washed 5 times with a sterile PBS solution to remove residual blood from the tissue surface.

1.3 The connective tissues were trimmed away as much as possible, and the tissues were cut into blocks approximately 1 cm in diameter. The bile ducts, and blood vessels were trimmed away as much as possible using ophthalmic scissors, and the tissue were blocks were further chopped into small approximately 1-mm pieces and placed into a glass Petri dish.

1.4 The premixed working solution (100μl of penicillin-streptomycin, 250μl of Enzyme H, 150μl of Enzyme R, and 20 μl of Enzyme A) was added to 5 ml of DMEM and mixed well to make the mixed-enzyme solution (required for 2–4 g of tissues).

1.5 To prepare the single cell suspension of human liver tissue, the mixed-enzyme solution was prewarmed in a 37 ℃ water bath for 10 min before adding it to the tissues. The enzymatic treatment was carried out in the GentleMACS^™ tissue processor at 37 ℃ for 15 minutes with constant shaking using the h_tumor_01.01 program.

2.Isolation of human hepatic cells

2.1 The enzymatic digestion was stopped by diluting the enzyme solution with 10ml of DMEM containing penicillin-streptomycin and FBS. The solution was filtered through a SmartStrainer filter (70 μm), and the filter was rinsed with DMEM containing penicillin-streptomycin and FBS after cell filtration.

2.2 The filtered cell suspension was centrifuged at 50 g for 7minutes (4 ℃) to isolate the hepatocytes, which were contained in the pellets.

2.3 The supernatant was centrifuged again at 450 g for 8 minutes (4 ℃); then, the supernatant was discarded, and the pellet containing the HSCs was collected.

2.4 The pellet was suspended in 7 ml of DMEM and centrifuged at 450 g for 7 minutes (4 ℃). The supernatant was removed as much as possible. One hundred fifty microliters of DNase solution and 10 ml of 18% Nycodenz were added sequentially to resuspend the pellet.

3. Nycodenz density gradient centrifugation

3.1 Five milliliters of the cell suspension in the 18% Nycodenz solution was added to each of two 15 ml centrifuge tubes. Then, 3 ml of 12% Nycodenz and 3 ml of 8.2% Nycodenz were added gently. After further mixing with 2ml of GBSS, the solutions were subjected to horizontal centrifugation at 1450g for 22min (4 ℃) without braking.

3.2 After the centrifugation was completed, the HSCs were aggregated at the interface of the 8.2% Nycodenz solution and the GBSS solution and the 18% and 12% Nycodenz solutions.

3.3 The cells were carefully collected into another centrifuge tube and then evenly mixed in 4ml of 1×PBS. The solution was centrifuged at 150 g for 8min at 4 ℃. The resulting supernatant was discarded, and the cell pellet was resuspended in RPMI 1640 medium containing FBS, followed by trypan blue staining and cell counting.

References

1.Zhai, X., Wang, W., Dou, D. et al. A novel technique to prepare a single cell suspension of isolated quiescent human hepatic stellate cells. Sci Rep 9, 12757 (2019). https://doi.org/10.1038/s41598-019-49287-7

2.Shang, L., Hosseini, M., Liu, X.et al. Human hepatic stellate cell isolation and characterization. J Gastroenterol 53, 6–17 (2018). https://doi.org/10.1007/s00535-017-1404-4

Manufacturer’s Link

Miltenyi Biotec: https://www.miltenyibiotec.com

Roche: https://www.roche.com

Solarbio: https://www.solarbio.com

Thermo Fisher Scientific: https://www.thermofisher.com ›home ›brands ›gibco

Sigma-Aldrich: https://www.sigmaaldrich.com

Beyotime: https://www.beyotime.com

Eppendorf: https://www.eppendorf.com

biologixlab.com: https://www.biologixlab.com

Nikon USA: https://www.nikonusa.com

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