# Protocol for isolating CD163+ Kupffer cells from human liver resections

Armando Andres Roca Suarez, Marie-laure Plissonnier, Maud Michelet, Anaëlle Dubois, Sarah Heintz, Maria Saez-Palma, Marion Delphin, Isabelle Bordes, Jennifer Molle, Audrey Diederichs, et al.

► To cite this version:

Armando Andres Roca Suarez, Marie-laure Plissonnier, Maud Michelet, Anaëlle Dubois, Sarah Heintz, et al.. Protocol for isolating CD163+ Kupffer cells from human liver resections. STAR Protocols, 2024, 5 (4), pp.103359. 10.1016/j.xpro.2024.103359. hal-05021470

HAL Id: hal-05021470

https://hal.science/hal-05021470v1

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Protocol

Protocol for isolating CD163+ Kupffer cells
from human liver resections

<figure><img src="image_2.png" /></figure>

The liver microenvironment contains a wide variety of monocyte and macrophage populations. Here, we present a protocol for the specific isolation of liver-resident macrophages, known as Kupffer cells (KCs), from human liver resections. We describe steps for dissociating human liver tissues, separating non-parenchymal cells into fractions by a 2-phase iodixanol gradient, and positive selection of KCs based on the expression of CD163. We then provide instructions for validating the procedure by immunofluorescence to detect CD163.

Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Armando Andres
Roca Suarez, Marie-
Laure Plissonnier,
Maud Michelet, ...,
Massimo Levrero,
Barbara Testoni,
Fabien Zoulim

armando-andres.
roca-suarez@inserm.fr
(A.A.R.S.)
barbara.testoni@inserm.fr
(B.T.)
fabien.zoulim@inserm.fr
(F.Z.)

Highlights

Protocol for the
isolation of human
Kupffer cells (KCs)

Validation of the
procedure by IF
detection of CD163

Isolated KCs are
suitable for functional
and perturbation
studies

Liver dissociation
technique is
compatible with the
isolation of additional
cell types
Protocol

Protocol for isolating CD163⁺ Kupffer cells
from human liver resections

Armando Andres Roca Suarez,<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,<fnref n="8" />,* Marie-Laure Plissonnier,<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> Maud Michelet,<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

Anaëlle Dubois,<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> Sarah Heintz,<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> Maria Saez-Palma,<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> Marion Delphin,<fnref n="4" /> Isabelle Bordes,<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

Jennifer Molle,<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> Audrey Diederichs,<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> Mélanie Rodà,<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> Emmanuel Combe,<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

Léa Mougené,<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> Guillaume Giraud,<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> Xavier Grand,<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> Michel Rivoire,<fnref n="3" />,<fnref n="5" /> Massimo Levrero,<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,<fnref n="6" />,<fnref n="7" />

Barbara Testoni,<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,* and Fabien Zoulim<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,<fnref n="6" />,<fnref n="9" />,*

SUMMARY

The liver microenvironment contains a wide variety of monocyte and macro-
phage populations. Here, we present a protocol for the specific isolation of
liver-resident macrophages, known as Kupffer cells (KCs), from human liver re-
sections. We describe steps for dissociating human liver tissues, separating
non-parenchymal cells into fractions by a 2-phase iodixanol gradient, and posi-
tive selection of KCs based on the expression of CD163. We then provide instruc-
tions for validating the procedure by immunofluorescence to detect CD163.
For complete details on the use and execution of this protocol, please refer to
Roca Suarez, Plissonnier, et al.<fnref n="1" />

BEFORE YOU BEGIN

The use of single-cell technologies has provided in recent years the opportunity to appreciate the wide variety of cell types present in the human liver microenvironment. <fnref n="2" />⁻<fnref n="4" /> This heterogeneity has been particularly explored in the hepatic myeloid compartment, which in addition to the embryonically-derived liver-resident Kupffer cells (KCs), presents a whole spectrum of monocytes and monocyte-derived macrophages. <fnref n="5" /> KCs can be differentiated from other hepatic cell populations by the expression of surface protein markers such as CD163, V-set and immunoglobulin domain containing 4 (VSIG4) and folate receptor beta (FOLR2). <fnref n="3" /> In addition to their central role in immune defense, KCs are characterized by the expression of genes involved in iron and cholesterol homeostasis. <fnref n="5" />

This STAR protocol presents a method for the specific isolation of human KCs based on the expres-
sion of CD163 (Figure 1). The protocol provides detailed information related to the validation of the
isolation procedure by immunofluorescence (IF) (Figure 2) and the conditions for KC culture. We
have previously employed this method for characterizing the inflammatory response induced via

<figure><img src="image_3.png" /></figure>

<footnote n="1">¹INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon (CRCL), Lyon, France</footnote>

<footnote n="2">²University of Lyon, Université Claude-Bernard (UCBL), Lyon, France</footnote>

<footnote n="3">³The Lyon Hepatology Institute EVEREST, Lyon, France</footnote>

<footnote n="4">⁴The Francis Crick Institute, London, UK</footnote>

<footnote n="5">⁵INSERM U1032, Centre Léon Bérard (CLB), 69008 Lyon, France</footnote>

<footnote n="6">⁶Department of Hepatology, Croix Rousse Hospital, Hospices Civils de Lyon, Lyon, France</footnote>

<footnote n="7">⁷Department of Internal Medicine - DMISM and the IIT Center for Life Nanoscience (CLNS), Sapienza University, Rome, Italy</footnote>

<footnote n="8">⁸Technical contact</footnote>

<footnote n="9">⁹Lead contact</footnote>

<footnote>*Correspondence: armando-andres.roca-suarez@inserm.fr (A.A.R.S.), barbara.testoni@inserm.fr (B.T.), fabien.zoulim@inserm.fr (F.Z.)
https://doi.org/10.1016/j.xpro.2024.103359</footnote>
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Liver dissociation and isolation of CD163⁺ KCs by magnetic separation</figcaption></figure>

(A) Expression of CD163 protein in each cell population of the human liver microenvironment. Data obtained from Guilliams et al., Cell 2022.³

(B) KC isolation method based on a 2-phase iodixanol gradient and CD163⁺ selection.

(C) Placement of tubes in selected vessels in order to perform washing and enzymatic dissociation of the liver tissue.

(D) Mechanical dissociation and filtering of liver tissues.

(E) Dissociated liver tissues following centrifugation, where the NPC fraction can be observed in the supernatant above the hepatocyte pellet.

(F) 2-phase iodixanol gradient following centrifugation, showing a separation between HSCs (top layer) and the monocytes/macrophages + LSECs fraction (bottom layer).

(G) Experimental setup for the magnetic selection of CD163⁺ KCs, showing a pre-separation filter (yellow) on top of a LS column that is attached to a MidiMACS separator.

(H) Microscopic image of human KCs cultured for 24 h following isolation. HSCs, hepatic stellate cells; KCs, Kupffer cells; LSECs, liver sinusoidal endothelial cells; NPC, non-parenchymal cells; PHH, primary human hepatocytes.
<figure><img src="image_5.png" /><figcaption>**Figure 2. Validation of the KC isolation procedure by IF**</figcaption></figure>

(A) IF microscopy image of KCs, showing positive staining for nuclear DNA (DAPI, blue), CD68 (red) and CD163 (green).

(B) Percentage of CD163⁺/CD68⁺ KCs among total cells obtained following the isolation procedure (three independent experiments, two-three fields each). Bars represent mean ± SD. Images obtained from Roca Suarez, Plissonnier et al., Gut 2024.¹ IF, immunofluorescence; KCs, Kupffer cells.

Toll-like receptor 8 (TLR8) signaling (Figure 3),¹ but the isolated KCs can be used to perform a wide variety of functional and perturbation studies.

**Institutional permissions**

Liver cells employed for the description of this method were isolated from hepatic resections obtained in collaboration with the surgical department of the Centre Léon Bérard (French ministerial authorization AC2013-1871). Readers would need to acquire similar permissions from their corresponding institutions.

**Preparation of materials for liver dissociation**

Timing: 30 min (hands on)

1. Prepare the different media that are suitable for long term storage as described below.

<figure><img src="image_6.png" /><figcaption>**Figure 3. Induction of an inflammatory response via TLR8 signaling in KCs**</figcaption></figure>

(A) Treatment of KCs with the TLR8 agonist SLGN (150 nM, 24 h) induces the upregulation of monocyte genes and the downregulation of KC markers and TFs (n = 3, FDR<0.05, GEO: GSE240054).

(B) Concentration of cytokines present in culture supernatants from KCs stimulated with SLGN or DMSO (n = 4, Mann-Whitney test). Bars represent mean ± SEM. Data obtained from Roca Suarez, Plissonnier et al., Gut 2024.¹ AQP9, aquaporin 9; CCL, C-C motif chemokine ligand; CD5L, CD5 molecule like; DMSO, dimethyl sulfoxide; DNMT3A, DNA methyltransferase 3 alpha; EREG, epiregulin; FOLR2, folate receptor beta; ID3, inhibitor of DNA binding 3; IFN, interferon; IL, interleukin; KCs, Kupffer cells; S100A, S100 calcium binding protein A; SLC, solute carrier family; SLGN, selgantolimod; SPIC, Spi-C transcription factor; TFs, transcription factors; TFEC, transcription factor EC; TIMD4, T cell immunoglobulin and mucin domain containing 4; TNF-α, tumor necrosis factor alpha; TLR8, Toll-like receptor 8; VSIG4, V-set and immunoglobulin domain containing 4.
**Note:** These include KC culture medium, liver transport medium, liver dissociation buffer 1, liver dissociation buffer 2 and collagenase stop solution.

2. Provide the surgical department with liver transport medium beforehand.

3. Before receiving the liver resection, prepare a water bath at 54°C and a hot plate at 37°C.

4. Keep liver dissociation buffers 1 and 2 in the water bath at 54°C.

5. Keep a distilled water bottle, a PBS 1x bottle, a sterile tray and a sterile bowl at 37°C.

6. Prepare several 20-mL syringes and 50-mL falcon tubes, two cell scrapers and one scalpel.

7. Clean the pump and its tubes first with 70% EtOH and then with distilled water.

8. Prepare an autoclaved 0.5 L-glass bottle with a funnel and nylon filter.

**Note:** In addition, consider the required time to autoclave the glass bottle, metal bowl and tray, as well as providing the surgical department with transport medium.

KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td><strong>Antibodies</strong></td><td></td><td></td></tr><tr><td>Anti-CD163 (dilution: 5 µg/mL)</td><td>Abcam</td><td>ab87099</td></tr><tr><td>Anti-CD68 (dilution: 1 µg/mL)</td><td>Abcam</td><td>ab955</td></tr><tr><td>Rabbit IgG Ctrl (dilution: 5 µg/mL)</td><td>Abcam</td><td>ab171870</td></tr><tr><td>Mouse IgG Ctrl (dilution: 1 µg/mL)</td><td>Abcam</td><td>ab170190</td></tr><tr><td>Anti-rabbit Alexa 488 (dilution: 1/1,000)</td><td>Cell Signaling Technology</td><td>4412S</td></tr><tr><td>Anti-mouse Alexa 594 (dilution: 1/800)</td><td>Invitrogen</td><td>A-11032</td></tr><tr><td>Human ProcartaPlex (96 well) Mix&amp;Match 10-plex</td><td>Thermo Fisher Scientific</td><td>PPX-10-MXWCXKA</td></tr><tr><td><strong>Biological samples</strong></td><td></td><td></td></tr><tr><td>Liver tissue (non-tumor) from patients undergoing surgical resection</td><td>Centre Léon Bérard</td><td>Authorization: AC2013-1871</td></tr><tr><td><strong>Chemicals, peptides, and recombinant proteins</strong></td><td></td><td></td></tr><tr><td>MACS bovine serum albumin (BSA) stock solution</td><td>Miltenyi Biotec</td><td>130-091-376</td></tr><tr><td>autoMACS rinsing solution</td><td>Miltenyi Biotec</td><td>130-091-222</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Gibco</td><td>15140122</td></tr><tr><td>Gentamicin (10 mg/mL)</td><td>Gibco</td><td>15710049</td></tr><tr><td>Leibovitz's L-15 medium, no phenol red</td><td>Gibco</td><td>21083027</td></tr><tr><td>MEM non-essential amino acids (NEAA) solution (100x)</td><td>Gibco</td><td>11140-035</td></tr><tr><td>RPMI 1640 medium, GlutaMAX supplement</td><td>Gibco</td><td>61870010</td></tr><tr><td>ACK lysing buffer</td><td>Gibco</td><td>A1049201</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787-50ML</td></tr><tr><td>Trypan blue solution</td><td>Sigma</td><td>T8154-100ML</td></tr><tr><td>Collagenase from Clostridium histolyticum, type IV</td><td>Sigma</td><td>C5138-1G</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>D2650-100ML</td></tr><tr><td>HEPES, free acid</td><td>Euromedex</td><td>10-110-C</td></tr><tr><td>EGTA</td><td>Euromedex</td><td>1310-D</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Euromedex</td><td>1112-A</td></tr><tr><td>Calcium chloride (CaCl<sub>2</sub>)</td><td>Carl Roth</td><td>CN93.1</td></tr><tr><td>Di-sodium hydrogen phosphate dihydrate (Na<sub>2</sub>HPO<sub>4</sub> 2H<sub>2</sub>O)</td><td>Merck</td><td>106576</td></tr><tr><td>PBS 1x</td><td>Eurobio Scientific</td><td>CS1PBS01-01</td></tr><tr><td>Potassium chloride (KCl)</td><td>VWR</td><td>26764.298</td></tr><tr><td>HyClone FetalClone II serum</td><td>Cytiva</td><td>SH30066.03</td></tr><tr><td>OptiPrep density gradient medium</td><td>BioVision</td><td>M1248-250</td></tr><tr><td>DAPI</td><td>Cell Signaling Technology</td><td>4083</td></tr><tr><td>Paraformaldehyde (PFA) 16% solution</td><td>Electron Microscopy Sciences</td><td>15710</td></tr><tr><td>Dako fluorescent mounting medium</td><td>Dako</td><td>S3023</td></tr><tr><td>Selgantolimod (SLGN)</td><td>Gilead Sciences, Inc.</td><td>GS-9688</td></tr></tbody></table>

(Continued on next page)
<table><thead><tr><th colspan="3">Continued</th></tr><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Critical commercial assays</td></tr><tr><td>CD163 MicroBead kit (containing CD163-biotin and anti-biotin microBeads)</td><td>Miltenyi Biotec</td><td>130-124-420</td></tr><tr><td colspan="3">Deposited data</td></tr><tr><td>RNA-seq from human KCs treated with the TLR8 agonist SLGN</td><td>GEO-NCBI</td><td>GSE240054</td></tr><tr><td colspan="3">Software and algorithms</td></tr><tr><td>EVOS FL Auto 2 imaging system software</td><td>Thermo Fisher Scientific</td><td>v2.0.2094.0</td></tr><tr><td>ProcartaPlex analysis software</td><td>Thermo Fisher Scientific</td><td>v3.1.0</td></tr><tr><td>ImageJ</td><td>National institutes of Health</td><td>v1.54f</td></tr><tr><td colspan="3">Other</td></tr><tr><td>LS columns</td><td>Miltenyi Biotec</td><td>130-042-401</td></tr><tr><td>Pre-separation filters (30 µm)</td><td>Miltenyi Biotec</td><td>130-041-407</td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td></tr><tr><td>MidiMACS separator</td><td>Miltenyi Biotec</td><td>130-042-302</td></tr><tr><td>Transfer pipettes</td><td>VWR</td><td>612-1747</td></tr><tr><td>Coverslips</td><td>VWR</td><td>631-0149</td></tr><tr><td>24-well culture plates</td><td>Biologix</td><td>07-6024</td></tr><tr><td>50-mL polypropylene tubes</td><td>Falcon</td><td>352070</td></tr><tr><td>Luer-Slip 20-mL syringes</td><td>Terumo</td><td>SS+20ES1</td></tr><tr><td>SuperFrost microscope slides</td><td>Thermo Scientific Menzel</td><td>17234884</td></tr><tr><td>Eclipse Ts2R-FL inverted research microscope</td><td>Nikon</td><td>Ts2R-FL</td></tr><tr><td>KOVA Glassic slide 10 with grid chamber</td><td>Kova International</td><td>87144F</td></tr><tr><td>Precision model GP 02 water bath</td><td>Thermo Fisher Scientific</td><td>TSGP02</td></tr><tr><td>C-MAG HP 10 hot plate</td><td>IKA</td><td>0003582000</td></tr><tr><td>Peristaltic dispensing pump IPC</td><td>Ismatec</td><td>C.P.78001-10</td></tr><tr><td>Nylon filter membrane 37 x 37 cm</td><td>Buisine</td><td>03.60/35</td></tr><tr><td>Glass media round bottle (0.5 L)</td><td>Cole-Parmer</td><td>FV-34503-40</td></tr><tr><td>Polypropylene utility funnel</td><td>Cole-Parmer</td><td>FV-06122-10</td></tr><tr><td>Stainless steel mixing bowl</td><td>Cole-Parmer</td><td>FV-07301-00</td></tr><tr><td>Stainless steel laboratory tray</td><td>Fisher Scientific</td><td>10537931</td></tr><tr><td>Non-filtered pipette tips (200 µL)</td><td>Fisher Scientific</td><td>21-403-457</td></tr><tr><td>Refrigerated centrifuge 5910 Ri</td><td>Eppendorf</td><td>5943000131</td></tr><tr><td>Tubing/MicroPerpex, 2 m, i.d., 3.0 mm, o.d., 5.0 mm, silicone</td><td>Cytiva</td><td>80106594</td></tr><tr><td>Stainless steel disposable scalpel</td><td>Paramount Surgimed Ltd.</td><td>PDSS23</td></tr></tbody></table>

## MATERIALS AND EQUIPMENT

<table><caption>KC culture medium</caption><thead><tr><th>Reagent</th><th>Final concentration</th><th>Amount</th></tr></thead><tbody><tr><td>RPMI 1640 medium</td><td>N/A</td><td>445 mL</td></tr><tr><td>Penicillin-Streptomycin</td><td>1% (v/v)</td><td>5 mL</td></tr><tr><td>HyClone FetalClone II serum</td><td>10% (v/v)</td><td>50 mL</td></tr><tr><td><strong>Total</strong></td><td>-</td><td><strong>500 mL</strong></td></tr></tbody><tfoot><tr><td colspan="3">Store at 4°C, for up to 6 months.</td></tr></tfoot></table>

<table><caption>Liver transport medium</caption><thead><tr><th>Reagent</th><th>Final concentration</th><th>Amount</th></tr></thead><tbody><tr><td>PBS 1x</td><td>N/A</td><td>490 mL</td></tr><tr><td>Penicillin-Streptomycin</td><td>1% (v/v)</td><td>5 mL</td></tr><tr><td>Gentamicin</td><td>1% (v/v)</td><td>5 mL</td></tr><tr><td><strong>Total</strong></td><td>-</td><td><strong>500 mL</strong></td></tr></tbody><tfoot><tr><td colspan="3">Store at 4°C, for up to 6 months.</td></tr></tfoot></table>
## Liver dissociation buffer 1

<table><thead><tr><td>Reagent</td><td>Final concentration</td><td>Amount</td></tr></thead><tbody><tr><td>NaCl</td><td>137 mM</td><td>24 g</td></tr><tr><td>KCl</td><td>2.7 mM</td><td>0.6 g</td></tr><tr><td>Na<sub>2</sub>HPO<sub>4</sub> 2H<sub>2</sub>O</td><td>0.7 mM</td><td>0.35 g</td></tr><tr><td>HEPES</td><td>25 mM</td><td>18 g</td></tr><tr><td>EGTA</td><td>0.5 mM</td><td>0.57 g</td></tr><tr><td>Distilled water</td><td>N/A</td><td>3 L</td></tr><tr><td>Total</td><td>-</td><td>3 L</td></tr></tbody></table>

Adjust pH to 7.4, pass through a 0.2-µm filter and store at 4°C, for up to 3 months.

## Liver dissociation buffer 2

<table><thead><tr><td>Reagent</td><td>Final concentration</td><td>Amount</td></tr></thead><tbody><tr><td>NaCl</td><td>137 mM</td><td>16 g</td></tr><tr><td>KCl</td><td>2.7 mM</td><td>0.4 g</td></tr><tr><td>Na<sub>2</sub>HPO<sub>4</sub> 2H<sub>2</sub>O</td><td>0.7 mM</td><td>0.23 g</td></tr><tr><td>HEPES</td><td>25 mM</td><td>12 g</td></tr><tr><td>CaCl<sub>2</sub></td><td>5 mM</td><td>1.1 g</td></tr><tr><td>Distilled water</td><td>N/A</td><td>2 L</td></tr><tr><td>Total</td><td>-</td><td>2 L</td></tr></tbody></table>

Adjust pH to 7.4, pass through a 0.2-µm filter and store at 4°C, for up to 3 months.

## Collagenase stop solution

<table><thead><tr><td>Reagent</td><td>Final concentration</td><td>Amount</td></tr></thead><tbody><tr><td>Leibovitz's L-15 medium</td><td>N/A</td><td>485 mL</td></tr><tr><td>HyClone FetalClone II serum</td><td>2% (v/v)</td><td>10 mL</td></tr><tr><td>MEM NEAA (100x)</td><td>1% (v/v)</td><td>5 mL</td></tr><tr><td>Total</td><td>-</td><td>500 mL</td></tr></tbody></table>

Store at 4°C, for up to 6 months.

* **Collagenase solution:** Add 110 mg of collagenase to 300 mL of Liver dissociation buffer 2.

Prepare fresh before tissue processing.

**Note:** Lyophilized collagenase aliquots can be weighted and stored at -20°C.

* **16% OptiPrep solution:** Mix 7.9 mL of OptiPrep stock (60%) with 22.1 mL of PBS 1x. Mix by pipetting. This volume is enough for the preparation of 3 gradients.

Prepare fresh before tissue processing and keep at 20°C-22°C.

* **9% OptiPrep solution:** Mix 9 mL of OptiPrep stock (60%) with 51 mL of PBS 1x. Mix by pipetting. This volume is enough for the preparation of 3 gradients.

Prepare fresh before tissue processing and keep at 20°C-22°C.

* **AutoMACS/BSA buffer:** Mix 2 mL of MACS BSA stock solution with 38 mL of autoMACS rinsing solution (final BSA concentration 0.5%).

Prepare fresh and keep cold (2-8°C).
* **4% PFA solution:** Mix 2 mL of PFA stock solution (16%) with 6 mL of PBS 1x.

Prepare fresh and keep at 20°C–22°C.

△ **CRITICAL:** PFA solution is toxic and irritant. We recommend utilizing a chemical hood when handling PFA. PFA must be disposed according to institutional guidelines.

* **PBS/BSA buffer:** Mix 5 mL of MACS BSA stock solution with 45 mL of PBS 1x. Final BSA concentration is 1%.

Prepare fresh and keep cold (2–8°C).

* **Permeabilization buffer:** Mix 25 mL of PBS/BSA buffer with 25 µL of Triton X-100. Mix by vortexing.

Prepare fresh and keep at 20°C–22°C.

## STEP-BY-STEP METHOD DETAILS

### Enzymatic and mechanical dissociation of the liver

© Timing: 45 min

Although this protocol focuses on the isolation of KCs (Figures 1A and 1B), primary human hepatocytes (PHH), hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs) can be isolated in parallel and cultured as previously described.⁶,⁷ Thus, the first step of the method is common to all these cell types and consists on the dissociation of liver tissues in order to separate hepatocytes from the non-parenchymal cell (NPC) fraction.

**Note:** As an example, this protocol allows to obtain 2–15 × 10⁸ hepatocytes, a number which is dependent on several factors. These are described in the limitations section below.

△ **CRITICAL:** Process the liver tissue as soon as possible (<1 h after resection). A longer interval between the surgical procedure and dissociation can lead to a decrease in cell viability.

1. Tissue wash with PBS in order to eliminate blood:

a. Place the liver resection on a metal tray and identify exposed vessels that could allow to perfuse a large portion of the liver tissue.

b. Using a 20-mL syringe filled with PBS (pre-warmed at 37°C), perfuse the liver and select 2–3 vessels that once perfused make the tissue increase in volume.

**Note:** Perform this step slowly in order to avoid leakage. If too much resistance is encountered, examine alternative vessels to perfuse instead.

c. Eliminate as much blood as possible by performing several PBS washes. Stop once the flushed PBS is clear.

d. Discard flushed blood/PBS from the metal tray.

2. Tissue wash with liver dissociation buffer 1:

a. Place the metal tray containing the liver resection on a pre-warmed plate previously heated to 37°C.

b. Set the pump to 30 mL/min and place the tubes in a flask containing liver dissociation buffer 1 (500 mL). Seal the container with parafilm in order to limit potential contaminations.
**Note:** It is useful to keep a second 500-mL bottle of liver dissociation buffer 1 in the water bath at 54°C, in case more than 500 mL are required.

c. Place a pipette tip (without filter) at the end of each tube and start the pump in order to eliminate air bubbles from the tubes.

d. Place the tubes in previously selected vessels and perform washing of the tissue. Although the water bath is set to 54°C, the buffer will decrease in temperature as it passes through the tubes and be close to 37°C once in the tissue (Figure 1C).

**Note:** Optimal temperature of the water bath should be determined and adapted by considering the length of the pump tubes. The goal is to obtain a buffer temperature of 37°C as it enters the tissue, which will favor the collagenase action in the following step.

3. Enzymatic dissociation of the liver:

a. Decrease the pump speed to 20 mL/min and place the tubes inside a flask containing collagenase solution in order to perfuse the liver tissue.

b. Verify the liver consistency every few minutes. Collagenase digestion takes ≈ 10 min.

c. Stop perfusion with the collagenase stop solution once the liver tissue has a soft and elastic consistency, as compared to its condition before perfusion.

**Note:** It is important to carefully monitor the time of collagenase digestion. Longer times can result in cell damage and short ones in inefficient tissue dissociation.

4. Mechanical dissociation of the liver:

a. Place the liver in a metallic bowl and cut it open with a scalpel.

b. Pour collagenase stop solution (≈100 mL) and mechanically dissociate the tissue using cell scrapers.

c. Pass the dissociated tissues through a funnel with a nylon mesh and collect the filtered cell suspension in a glass bottle (Figure 1D).

5. Separation of hepatocytes from the NPC fraction:

a. Distribute the filtered cell suspension into 50-mL tubes, fill as many as required and centrifuge at 130 g for 1 min at 20°C (acceleration 6, deceleration 2).

b. Recover the supernatant containing the NPC fraction.

c. Further process or discard the hepatocyte pellet (Figure 1E).⁶

## Gradient separation of the NPC fraction

### Timing: 2 h

Following liver dissociation and isolation of hepatocytes, supernatants containing NPCs is recovered in order to perform gradient separation of cell fractions. As described below, we employ a 2-phase iodixanol gradient that allows separation of cells into three main fractions, the top one containing HSCs, the middle one containing monocytes/macrophages + LSECs, and the pellet containing remaining hepatocytes and other NPCs (Figure 1F).⁸

6. Prepare the 2-phase iodixanol gradient in a 50-mL tube.

a. Add 10 mL of OptiPrep 16% solution.

b. Add 20 mL of OptiPrep 9% solution on top of the 16% layer. Perform this step very slowly in order that the two layers do not mix.

c. Leave at 20°C–22°C while preparing the NPC fraction.

7. Split the supernatant recovered after liver dissociation (NPC fraction) into several 50-mL tubes.

8. Centrifuge at 800 g for 10 min at 20°C (acceleration 9, deceleration 9).

9. Discard the supernatant.
10. Resuspend the pellet of each tube in 5 mL of KC culture medium by pipetting.

11. Pool 4 tubes in order to have a final volume of 20 mL.

12. Very slowly, add the 20 mL of cell suspension on top of the 2-phase iodixanol gradient.

13. Centrifuge at 800 g for 30 min at 20°C without brakes (acceleration 9, deceleration 0).

△ CRITICAL: It is crucial to remove the brakes from the centrifuge before this step (deceleration 0), otherwise separation of the NPC fraction will not be successful. Acceleration/deceleration settings should be adapted according to the centrifuge model employed.

14. Using a 25-mL pipette, eliminate or further process the first layer of cells that contains HSCs.⁷

15. Recover the second layer of cells that is near the 10 mL-mark, using a transfer pipette. Try to recover as little OptiPrep as possible and transfer to a 50-mL tube.

**Note:** Pool samples into a single 50-mL tube if several gradients were prepared.

16. Fill the remaining of the 50-mL tube with KC culture medium and mix by inverting.

17. Centrifuge at 800 g for 10 min at 20°C (acceleration 9, deceleration 9).

18. Discard the supernatant.

19. Resuspend cells in 1 mL of PBS.

20. Add 5 mL of ACK lysing buffer and incubate for 5 min at 20°C-22°C. This step is performed in order to lyse red blood cells.

21. Add 9 mL of PBS and centrifuge at 800 g for 10 min at 20°C (acceleration 9, deceleration 9).

22. Discard supernatant.

23. Resuspend cells in 50 mL of KC culture medium and recover 10 µL of cell suspension in order to assess cell number and viability.

24. Centrifuge at 800 g for 10 min at 20°C (acceleration 9, deceleration 9).

25. Mix 10 µL of cell suspension with 10 µL of trypan blue and determine cell number and percentage of viable cells using a counting chamber.

**Note:** This step can be modified according to the cell counting method employed in each laboratory.

26. Discard supernatant after centrifugation.

## Isolation of CD163⁺ KCs by magnetic separation

### Timing: 2 h

Once the cell fraction containing monocytes/macrophages + LSECs has been recovered, we proceed to the selection of liver-resident KCs. This part of the protocol is based on the positive-selection of cells expressing CD163, a marker that differentiates this population from other macrophages present within the liver microenvironment.³

27. Resuspend the cell pellet (still present in the 50-mL falcon from step 23) in 80 µL of cold auto-MACS/BSA buffer per 10⁷ total cells.

28. Add 20 µL of CD163-Biotin per 10⁷ total cells.

29. Mix well by pipetting and incubate for 10 min at 2–8°C.

30. Add 2 mL of cold autoMACS/BSA buffer per 10⁷ cells and centrifuge at 300 g for 10 min at 20°C (acceleration 9, deceleration 9).

31. Discard the supernatant.

32. Resuspend the cell pellet in 80 µL of cold autoMACS/BSA buffer per 10⁷ total cells.

33. Add 20 µL of anti-biotin MicroBeads per 10⁷ total cells.

34. Mix well and incubate for 10 min at 2–8°C.
35. Place LS columns in the MACS MultiStand with the MidiMACS separator (Figure 1G).

36. Place a 30 µm pre-separation filter on top of the LS column.

37. Place a 50-mL tube under the LS column.

38. Rinse the pre-separation filter and LS column by directly adding 3 mL of autoMACS/BSA buffer into the middle of the filter.

**Note:** It is important to wet the pre-separation filter completely in order to facilitate passage of cells in suspension.

39. Apply the cell suspension onto the pre-separation filter and wait until the column reservoir is empty.

**Note:** In our experience, it is better to limit the number of cells to no more than $10^8$ per column in order to prevent it from clogging. Instead, the cell suspension can be divided into multiple columns.

40. Wash the column with 3 mL of cold autoMACS/BSA buffer and wait until the column reservoir is empty. Repeat this step two more times.

41. Remove the column from the MidiMACS separator and place it on a 50-mL tube.

42. Pipette 5 mL of autoMACS/BSA buffer onto the column and flush out the cells by firmly pushing the plunger into the column.

**Optional:** Observe the cells at the microscope in order to assess the presence of hepatocytes and the need to pass the cell suspension through a second magnetic column.

43. Add 15 mL of KC culture medium and mix by inverting the tube.

**Note:** The volume to resuspend the pellet can be adjusted as required in order to obtain a cell concentration that allows counting.

44. Recover 10 µL of cell suspension in order to assess cell number and viability, as described in step 25.

45. Centrifuge the cell suspension at 300 g for 10 min at 20°C (acceleration 9, deceleration 9).

46. Discard the supernatant and resuspend the pellet with KC culture medium at a concentration of $6 \times 10^5$ cells/mL.

**Optional:** Prepare several wells of a 24-well plate with glass cover slips in order to validate the obtained KC population by IF.

47. Plate 500 µL of cell suspension ($3 \times 10^5$ cells) in each well of a 24-well plate and incubate for 20 min at 37°C with 5% CO$_2$.

48. Aspirate medium containing debris and non-adherent cells and replace it with 500 µL of KC culture medium.

49. Incubate cells for 12 h at 37°C with 5% CO$_2$.

**Note:** Following cell plating and culture, KCs should have the elongated shape and large processes that characterize their morphology (Figure 1H).

## Validation of the KC isolation procedure by IF

### Timing: 15 h

We determine purity of the cell population by assessing the expression of CD163 by IF. In parallel, we perform staining for CD68 as a general myeloid marker (Figure 2A). We favor this approach
considering 1) the potential of CD163 for ectodomain shedding and 2) the current lack of commercial flow cytometry antibodies detecting the intracellular domain of CD163.⁹ Therefore, it is necessary to include a permeabilization step during the procedure, as the anti-CD163 IF antibody recognizes the intracellular portion of the protein.

50. Remove cell culture medium and wash the platted KCs with PBS.

51. Add 500 µL of 4% PFA solution to each well and incubate for 10 min at 20°C-22°C.

52. Wash cells with ice-cold PBS. Repeat this step two more times.

53. Incubate cells with 500 µL of permeabilization buffer for 1 h at 20°C-22°C.

54. Wash cells with ice-cold PBS. Repeat this step two more times.

55. Incubate cells with primary antibodies (anti-CD163 - 5 µg/mL, anti-CD68 - 1 µg/mL) or isotype controls (Rabbit IgG Ctrl - 5 µg/mL, Mouse IgG Ctrl - 1 µg/mL) diluted in PBS/BSA buffer for 12 h at 4°C in a humidified chamber.

56. Wash cells with ice-cold PBS. Repeat this step two more times.

57. Incubate cells with secondary antibodies (anti-rabbit Alexa488 - 1/1000, anti-mouse Alexa594 - 1/800) diluted in PBS/BSA buffer for 1 h at 20°C-22°C in the dark.

58. Wash cells with ice-cold PBS. Repeat this step two more times.

59. Incubate cells with 2 µg/mL of DAPI diluted in PBS for 1 min at 20°C-22°C in the dark.

60. Wash cells with PBS, followed by a second wash with water.

61. Add a drop of mounting medium to the cover slip and place it face down on a glass slide.

62. Seal coverslips with nail polish to prevent drying and movement under the microscope.

63. Proceed with imaging analysis or store at 4°C in the dark.

## EXPECTED OUTCOMES

This protocol allows to isolate 10⁻⁵⁰ × 10⁶ KCs from one surgical liver resection. The large majority of recovered cells are CD163⁺/CD68⁺ (Figure 2B). Cells isolated with this method can be used for a wide variety of functional studies. As an example, we have previously employed this protocol for characterizing the response of KCs to the selective TLR8 agonist SLGN.¹ In this context, we have shown that SLGN treatment (150 nM, 24 h) is able to trigger KC plasticity and promote the activation of antiviral programs over those associated with the KC identity. This can be observed by the upregulation of genes that characterize monocyte populations (e.g., *EREG*, *S100A12*), the downregulation of KC markers (e.g., *FOLR2*, *VSIG4*) and KC-specific transcription factors (e.g., *SPIC*, *ID3*) (Figure 3A). Moreover, these transcriptional changes are accompanied by the production of a variety of cytokines that include tumor necrosis factor alpha (TNF-α), interleukin (IL)-6 and interferon (IFN)-γ (Figure 3B).

## LIMITATIONS

The number and viability of isolated KCs is dependent on several factors, including size of the liver tissue, underlying patient clinical history, time between surgical resection and start of the dissociation procedure, and efficiency of the collagenase perfusion step. To illustrate this situation, our laboratory processes liver resections that weight between 70 to 250 g. However, it has been our experience that smaller samples are often less challenging to perfuse and dissociate, which results in higher cell yields at the end of the procedure, as previously reported.¹⁰

As described in the sections above, this protocol based on the positive selection of CD163⁺ cells is designed for the specific isolation of KCs. If readers are interested in capturing the monocyte/macrophage diversity present in the human liver, there are protocols based on negative selection that can be employed with this aim.¹¹

## TROUBLESHOOTING

### Problem 1

Liver tissue is difficult to perfuse with the collagenase solution and does not acquire a soft consistency (step 3).
### Potential solution

* Transfer the liver tissue to a metal bowl and open it with a scalpel in order to pour collagenase solution directly over it. Limit this step to only a few minutes and proceed with mechanical dissociation.

### Problem 2

Red blood cells are present after liver dissociation (step 5).

### Potential solution

* Perform additional washing steps of the liver resection until all the blood present has been flushed out (step 1).

### Problem 3

Magnetic column is clogged after applying the cell suspension (step 39).

### Potential solution

* Pipette up and down the cell suspension inside the column in order to facilitate cell passage. In case this method does not unclog the column, remove the cell suspension and pass it through a new pre-rinsed column.

### Problem 4

Hepatocytes are present after magnetic selection and KC elution (step 42).

### Potential solution

* Hepatocytes can be differentiated visually from NPCs due to their large size. If their presence is still observed after magnetic selection, pass the eluted cell suspension through a second magnetic column. This can improve cell purity, but can also decrease the number of recovered KCs.

### Problem 5

Low number of KCs recovered (step 44).

### Potential solution

* Verify that the incubation times, resuspension volume, amount of antibody used and cell count were correct.

## RESOURCE AVAILABILITY

### Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Fabien Zoulim (fabien.zoulim@inserm.fr).

### Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Armando Andres Roca Suarez (armando-andres.roca-suarez@inserm.fr).

### Materials availability

This study did not generate new unique reagents.

### Data and code availability

The accession number for the RNA-seq data reported in this paper is GEO: GSE240054.
## ACKNOWLEDGMENTS

This work was supported by the European Union's Horizon 2020 research and innovation program under grant agreement no. 847939 (IP-cure-B project), the "Agence Nationale pour la Recherche sur le SIDA et les hepatites virales et les maladies infectieuses emergentes" (ANRS-MIE) under grant agreement ECTZ206376, and the French National Research Agency (ANR) within the framework of the RHU cirb-RNA (ANR-17-RHUS-0003) and IHU EVEREST (ANR-23-IAHU-0008) as part of the program "Investissements d'Avenir." The graphical abstract and figures were created using Biorender.com.

## AUTHOR CONTRIBUTIONS

Conceptualization, F.Z., B.T., and M.L.; formal analysis, A.A.R.S., M.-L.P., M.M., A. Dubois, S.H., M.S.-P., M.D., I.B., J.M., A. Diederichs, M.R., E.C., L.M., G.G., and X.G.; funding acquisition, F.Z., B.T., and M.L.; investigation, A.A.R.S., M.-L.P., M.M., A. Dubois, S.H., M.S.-P., M.D., I.B., J.M., A. Diederichs, M.R., E.C., L.M., G.G., and X.G.; methodology, A.A.R.S. and M.-L.P.; resources, M.R.; supervision, F.Z., B.T., and M.L.; visualization, A.A.R.S., A. Dubois, and S.H.; writing – original draft, A.A.R.S.; writing – review and editing, all authors.

## DECLARATION OF INTERESTS

This work was performed as a scientific collaboration with Gilead Sciences, who provided the TLR8 agonist SLGN, in the frame of the EU-funded IP-cure-B project.

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