Human Hepatic Macrophages Isolation from A small Piece of Liver Tissue

Materials and Reagents

1. Collagenase II (sigma C6885): prepare a stock concentration of 50 mg/mL in RPMI media (i.e., dissolve 0.5 g in 50 mL of RPMI media), filter through a 0.22 μm filter and make aliquot of 500 μL. Store aliquots at -20 ℃.

2. DNase I (Roche 10,104,159,001): prepare a stock concentration of 10 mg/mL in RPMI media, filter through a 0.22 μm filter, and make aliquots of 500 μL. Store aliquots at -20 ℃.

3. Media: Sterile RPMI media (ThermoFisher cat # 21875034).

4. Sterile complete media (RPMI media +10% FCS + 0.5%pen/strep).

5. 90% Percoll: For a final volume of 50 mL, add 5 mL of 10×PBS to 45 mL of Percoll (GE Healthcare cat # 15-0891-01).

6. 25% Percoll: For a final volume of 40 mL, add 30 mL of 1×PBS to 10 mL of 90% Percoll.

7. 50% Percoll: For a final volume 30 mL, add 15 mL of 1×PBS to 15 mL of 90% Percoll.

8. Percoll gradient: In a 50 mL centrifuge tube, add 20 mL of 25% Percoll and underlay slowly 14.5 mL of 50% Percoll, at the bottom of the tube using a 10 mL plastic pipette.

Equipment

1.Sterile scalpel.

2. Refrigerated centrifuge.

3. Cell strainer(Ucallm).

4.Petri dishes (Ucallm).

5. 50 mL centrifuge tubes (Ucallm).

Procedure

A small piece of liver tissue is freshly collected by the surgeon from volunteers obtained by a small needle is inserted through the abdominal skin into the liver to collect a tissue sample. Before staring the procedure, thaw DNase and collagenase on ice, warm up the water bath to 37 ℃, and bring media and PBS to room temperature.

1.Transfer the liver tissue into a petri dish.

2. Cover the tissue with 1 mL (see Note 1) of RPMI media.

3. Cut into smaller pieces (using a scalpel).

4. Transfer the pieces and media into 50 mL tubethen add 1 mL of RPMI media (total media is now 2 mL, see Note 1).

5. Add collagenase: 25 μL/mL (＝50 μL for 2 mL total volume) (stock Concentration＝10 mg/mL. Final concentration≈0.25 mg/mL).

6. Add DNase 20 μL/mL (＝40 μL for 2 mL total volume) Stock concentration＝10 mg/mL. Final concentration≈0.2 mg/mL).

7. Transfer the tube into water bath≈37 ℃ for 30 min with fast shaking.

8. Take out of the tube from the water bath and add 10 mL of complete media (containing FBS) to stop the enzymatic digestion.

9. Filter by passing the cell suspension through the cell strainer(100 or 75 μm). Keep the cell suspension on ice.

10. Centrifuge for 3 min at 50×g at 4℃.

11. Keep the supernatant that contains hepatic macrophages that will be used for the Percoll gradient separation.

12. Slowly layer the supernatant on the Percoll gradient with a 10 mL pipette and spin down at 1500×g for 30 min without 

13. Carefully collect the middle interphase (white cell ring) and transfer it into a new 50 mL tube.

14. Wash the cells twice by adding PBS up to a final volume of 20 mL and centrifuging at 640×g for 10 min at 4 ℃.

15. Resuspend in RPMI complete media and plate the cells for 30 min. Wash the cells with PBS and add fresh media to remove the nonadherent cells.

16. Harvest the cell for further analysis.

Notes

1.These volumes are calculated based on liver border biopsy (usually taken during abdominal surgery). If the starting material is from a needle biopsy, volumes need to be scaled down.

References

1.Morgantini, C. (2020). Kupffer Cell Isolation from Human Biopsies. In: Aouadi, M., Azzimato, V. (eds) Kupffer Cells. Methods in Molecular Biology, vol 2164. Humana, New York, NY.https://doi.org/10.1007/978-1-0716-0704-6_2

2. Graham, C.T., Gordon, S. & Kubes, P. A historical perspective of Kupffer cells in the context of infection. Cell Tissue Res (2024). https://doi.org/10.1007/s00441-024-03924-4

Manufacturer’s Link

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

Roche: https://www.roche.com

Thermo Fisher Scientific: https://www.thermofisher.com

GE HealthCare: https://www.gehealthcare.com

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