Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

All experimental procedures were conducted with the approval of the ethics and animal care committees of Homburg University Medical Center.

1. Preparation of Materials and Buffers

1. Freshly prepare all buffers required for liver digestion using sterile components and a laminar hood to avoid bacterial contamination.
2. Prepare the collection buffer (CB) by mixing 49.5 mL of RPMI medium and 0.5 mL of fetal bovine serum (FBS; low endotoxin, heat inactivated) to achieve a 1% (v/v) solution. Store the solution on ice until further usage.
   NOTE: Approximately 25 mL of CB is necessary to digest one whole liver.
3. Prepare the digestion buffer (DB) by using the following ingredients: RPMI medium, 1% (v/v) FBS (low endotoxin, heat inactivated), collagenase P (0.2 mg/mL), DNase-I (0.1 mg/mL), and dispase (0.8 mg/mL).
   NOTE: Approximately 25 mL of DB is necessary to digest one whole liver. Pre-warm the DB in the 37 °C water bath before use.
4. Reconstitute the enzymes upon arrival in Hanks' balanced salt solution (HBSS; collagense P and dispase) or in DNase-I buffer (50% (v/v) glycerol, 1 mM MgCl₂, and 20 mM Tris-HCl; pH 7.5), aliquot it, and store it at -20 °C. Store the DNase-I buffer at 4 °C and use it within two months.

2. Preparation of Liver Single-cell Suspension

1. Euthanize untreated wild-type mice by cervical dislocation in accordance with the local ethics and animal care committees.
2. Place the mice on a dissection board and wet the fur with 70% ethanol. Using scissors, open the abdomen with a midline incision of the skin, followed by a Y-incision towards the limbs. Open the peritoneum up to the sternum using the scissors. In order to uncover the liver, displace the intestine gently to the right side using a cotton swab.
3. With the help of scissors and forceps, remove the liver lobes, leaving the gall bladder behind, and avoid contamination with connective tissue. Weigh the liver and place it on ice in a Petri dish containing HBSS.
4. Place the liver lobes on a dry Petri dish and cut the liver tissue into homogeneous cubes approximately 2 mm a side by using a scalpel. Transfer the pieces into a 15-mL conical centrifuge tube.
5. Add 2.5 mL of DB to the 15-mL conical centrifuge tube containing the liver pieces and place it in a 37 °C water bath to start the digestion process (a healthy liver takes 60-70 min and a cirrhotic liver takes 80-90 min).
   NOTE: If one entire liver is being digested, the liver should be separated into two 15-mL conical centrifuge tubes to ensure good cell viability.
6. Prepare a new 15-mL conical centrifuge tube to collect released liver cells. Place a polyamide 100-µm filter mesh on the top of the tube and wet the mesh with 800 µL of CB. Place the conical centrifuge tube on ice.
7. Mix the samples in the 37 °C water bath after 5 and 10 min in order to support the digestion process by shaking the 15-mL conical centrifuge tube containing the liver pieces.
8. 15 min after starting the digestion, gently mix the liver pieces using a 1,000-µL pipette with a cut tip that enables the liver pieces to pass through easily. Place the tubes back into the water bath and allow the pieces to settle for 2 min.
9. Remove the supernatant containing the disseminated cells (typically 2x 700 µL) and add it to the tube prepared in step 2.6. Replace the removed supernatant with DB (2x 700 µL) and place it back into the 37 °C water bath.
10. Repeat the procedure described in step 2.8 (typically at 30, 40, 50, 55, and 60 min) until approximately 60-70 min have passed since the start of the digestion. From 40 min onwards, the remaining liver pieces should be small enough to pass through an uncut 1,000-µL pipette tip.
    NOTE: Healthy liver is digested fully within 60-70 min, while fibrotic liver typically needs 80-90 min. By this time point, the liver tissue should not be visible in the conical 15-mL centrifuge tube containing the liver pieces, and all released cells should be transferred into the tube prepared in step 2.6.
11. At the end of the digestion process, collect the cells and centrifuge them for 8 min at 180 x g and 4 °C (using reduced acceleration/4 and deceleration/2).
12. Resuspend the cell pellet in 1 mL of ammonium-chloride-potassium (ACK) lysis buffer and incubate it for 1 min at room temperature in order to lyse the red blood cells. Stop the reaction by adding 5 mL of CB, and then centrifuge the cells for 8 min at 180 x g and 4 °C (using reduced acceleration/4 and deceleration/2). Resuspend the cells in 4 mL of CB and store them on ice. Count the cells, as described in step 3.
    NOTE: The cell pellet is loose, and therefore the supernatant is pipetted away instead of being decanted.

3. Determination of the Cell Count Using Flow Cytometry

NOTE: For determining the cell counts, an automated cell counter or, ideally, the flow cytometry-based cell quantification described below is suggested instead of the classical Neubauer chamber-based method. The liver single-cell suspension described in step 2 contains parenchymal and non-parenchymal cells (NPC) with greatly differing sizes and granularities. The proper exclusion of cellular debris together with the gating-on forward scatter, side scatter (FSC-SSC) characteristic of NPCs when using flow cytometry ensures the success of the described protocol¹².

1. Prepare an aliquot of the liver cell suspension (20 µL) and add 174 µL of phosphate-buffered saline (PBS) and 6 µL of propidium iodine (PI; end concentration 0.375 µg/mL). Add counting beads that allow for the quantification of the cells.
2. Gate on SSC-A-FSC-A to avoid debris and further exclude doublets using FSC-H and FCS-A.
   NOTE: It is important to follow the gating strategy depicted in Figure 2.
3. Gate out PI-positive dead cells, measure 30 µL from your samples, and record the events. Follow the manufacturer's guideline to calculate the cell count.
   NOTE: Follow steps 4 for flow cytometry measurement, steps 5 and 6 for magnetic based progenitor cell isolation, and steps 7 for flow cytometry sort of progenitor subpopulations.

4. Staining of the Liver Single-cell Suspension for the Fow Cytometry Analysis of Progenitor Subsets

Table 1.

Table 2.

1. Place an aliquot of the cell suspension from step 2.11 into a reaction tube (1.5 mL) containing 2.5 x 10⁵ cells, determined as described in step 3.
2. Centrifuge the cells for 3 min at 300 x g and 4 °C using a microcentrifuge.
   NOTE: At this point, a vacuum pump can be utilized to carefully remove the supernatant.
3. Resuspend the cell pellet in Fc-block mix and incubate it for 5 min on ice. Prepare Fc-block mix with a total volume of 50 µL per stain. Add 10 µL of FcR-blocking reagent, 1 µL of purified anti-CD64 (1:100; 0.5 µg/stain), and 40 µL of staining buffer (ST buffer: HBSS, 1% (v/v) FBS, and 0.01% sodium azide) per stain.
   NOTE: Sodium azide is highly toxic. Use appropriate personal protective equipment, such as nitrile gloves, a laboratory coat, and a face mask.
4. Add 50 µL of staining mix to the cells (the total volume of cells is now 100 µL) and incubate the cells with the antibody mix, protected from light and for 20 min on ice.
5. Prepare the antibody mix with a total volume of 50 µL per stain. For 50 µL of ST buffer, add the following conjugated antibodies for basic staining: CD45 (0.5 µL; 1:200; 0.1 µg/stain), CD31 (0.5 µL; 1:200; 0.25 µg/stain), ASGPR1 (1 µL; 1:100; 0.2 µg/stain), podoplanin (1 µL of a 1:14 dilution; 1:1,400 end dilution; 0.014 µg/stain), and CD133 (3 µL; 0.09 µg/stain).
   NOTE: Table 1 summarizes the antibody clones and dilutions utilized for basic stains and for additional surface markers of the various subsets. Prepare extra cell suspensions for the antibody mix containing the corresponding isotype controls and/or fluorescence minus one controls (FMOs), as depicted in Table 2.
6. Add 400 µL of ST buffer to each sample and centrifuge the cells for 4 min at 300 x g and 4 °C.Discard the supernatant and resuspend the cells in 100 µL of secondary antibody mix containing 0.125 µL of donkey anti-goat IgG (1:800; 0.25 µg/stain) and 0.25 µL of fluorescent-conjugated streptavidin (1:400; 0.25 µg/stain) in 100 µL of ST buffer.
7. Incubate the cells while protected from light and with the antibody mix for 20 min on ice. Add 400 µL of ST buffer to each sample and centrifuge the cells for 4 min at 300 x g and 4 °C. Discard the supernatant and resuspend the cells in 300 µL of ST buffer containing 0.25 µg/mL PI.
   NOTE: The cell viability of progenitor cells decreases with the time; therefore, it is suggested to measure fresh samples as soon as possible.

5. Magnetic Microbead-based Enrichment of Progenitor Cells

1. Transfer an aliquot of the liver single-cell suspension containing 1.5-2 x 10⁶ cells, based on the cell count determined as described in step 3, into a new 15-mL conical centrifuge tube.
2. Centrifuge the cells for 8 min at 180 x g and 4 °C (using reduced acceleration/4 and deceleration/2).
   NOTE: Typically, one healthy C57Bl/6 mouse liver (or 1 g of liver tissue) will need to be separated into three 15-mL conical centrifuge tube.
3. Resuspend the cells in 400 µL of HBSS 0.5% (v/v) bovine serum albumin (BSA) and add 40 µL of anti-CD31 microbeads and 30 µL of anti-CD45 microbeads. Incubate the cells for 15 min at 4 °C.
   NOTE: Do not exceed the incubation time, as the purity of the separation will be significantly reduced.
4. Add 5 mL of HBSS 0.5% (v/v) BSA to the cells and centrifuge them for 8 min at 180 x g and 4 °C (using reduced acceleration/4 and deceleration/2).
5. Resuspend the cell pellet in 100 µL of dead cell removal beads and incubate them at room temperature for 15 min. In the meantime, prepare an LS separation column and calibrate it with 3 mL of HBSS 0.5% (v/v) BSA.
   NOTE: Do not exceed the incubation time, as the purity of the separation will be significantly reduced.
6. Add 900 µL of HBSS 0.5% (v/v) BSA to the cells, filter them using 100-µm polyamide filter mesh, and load them on the LS separation column.
   NOTE: Load one entire liver onto one LS separation column.
7. Wash the column three times with 3 mL of HBSS 0.5% (v/v) BSA and collect the flow-through.
8. Centrifuge the flow-through for 8 min at 180 x g and 4°C (using reduced acceleration/4 and deceleration/2).
9. Resuspend the cell pellet either in rinsing buffer (RB) (PBS and 2 mM ethylenediaminetetraacetic acid (EDTA)) containing 0.5% (v/v) BSA or ST buffer, depending upon the further experimental procedure.

6. Magnetic Microbead-based Automated Cell Purification of Progenitor Cell Subsets Combined from Multiple Livers

NOTE: Since progenitor cell subsets represent rare cell populations, combining cells from multiple livers is often needed to achieve sufficient cell numbers for further experiments. As an example, CD133⁺ and gp38⁺ cell separation is described below.

1. Isolation of CD133⁺ progenitors
   1. After step 5.9, count the cells, as described in step 3, and resuspend up to 10⁶ cells (typically pooling 4-6 healthy liver samples) in 100 µL of RB.
   2. Add anti-CD64 1:100 and anti-CD16/32 1:100 to the cells and incubate them on ice for 5 min. Add 1:100 anti-CD133 biotinylated antibody to the cells and incubate them on ice for a further 10 min.
   3. Add 5 mL of RB and centrifuge for 8 min at 180 x g and 4 °C (using reduced acceleration/4 and deceleration/2).
   4. Resuspend the cells in 400 µL of RB and add 10 µL of anti-biotin microbeads. Incubate the sample for 15 min at 4 °C. Do not exceed the incubation time, as this reduces the purity of the samples.
   5. Add 5 mL of RB and centrifuge for 8 min at 180 x g and 4 °C (using reduced acceleration/4 and deceleration/2). Resuspend the pellet in 1 mL of RB.
2. Isolation of gp38⁺ progenitors
   1. After step 5.9, count the cells, as described in step 3, and resuspend up to 10⁶ cells (typically pooling 4-6 healthy liver samples) in 100 µL of RB.
   2. Add anti-CD64 1:100 and anti-CD16/32 1:100 to the cells and incubate them on ice for 5 min. Next, add 1:100 anti-gp38 biotinylated antibody to the cells and incubate them on ice for a further 10 min.
   3. Add 5 mL of RB and centrifuge for 8 min at 180 x g and 4 °C (using reduced acceleration/4 and deceleration/2).
   4. Resuspend the cells in 400 µL of RB and add 5 µL of anti-biotin microbeads. Incubate the sample for 15 min at 4 °C. Do not exceed the incubation time, as this reduces the purity of the samples.
   5. Add 5 mL of RB and centrifuge for 8 min at 180 x g and 4 °C (using reduced acceleration/4 and deceleration/2).
   6. Resuspend the cell pellet in 1 mL of RB.
3. Common steps after step 6.1 or 6.2
   1. Place the 15-mL conical centrifuge tube containing the magnetically labeled cell suspensions on a chill 5 rack with two additional empty tubes for collecting the positive and negative fractions. Place the chill rack on the separation platform of the separator.
   2. Use the positive selection program (Possel_d2) with an extensive washing step between each sample (rinse option).
      NOTE: Using column-based manual separation of cells instead of the automatic separator will reduce the purity of the sample.
   3. Collect the positive fraction and centrifuge for 8 min at 180 x g and 4 °C (using reduced acceleration/4 and deceleration/2).
   4. Resuspend the cell pellet either in RB or ST buffer, depending upon the further experimental procedure.
   5. For a purity check, take an aliquot of the cells and stain them as described in step 4.

7. Flow Cytometry Cell Sorting

NOTE: A high purity sort of any progenitor cell subset could be achieved with the protocol described below. The overall yield of cells is much lower than that described in step 6 and is best for gene expression analysis.

Table 3.

1. Acquire cells from the magnetic-based enrichment (described in step 5); count them, as described in step 3; and stain them for progenitor markers (CD133, gp38), CD31, ASGPR1 and CD45, as explained in step 4.
2. Prepare sorting medium (SM): phenol-red free Dulbecco's Modified Eagle's Medium (DMEM) containing 0.5% (v/v) BSA and 0.01% (v/v) sodium azide. Resuspend the stained cells in SM, transfer them into a polypropylene round-bottom tube, and place them on ice.
   NOTE: Sodium azide is highly toxic. Use appropriate personal protective equipment, such as nitrile gloves, a laboratory coat, and a face mask. Do not use sodium azide if the sorted cells are used for functional assays.
3. Use the appropriate software for the type of sorter used for the cell isolation. Open the software and follow the manufacturer's instructions to set up the sorting parameters and machine specifications.
   NOTE: The machine specifications are summarized in Table 3. While machine specifications could be different at various institutions, it is important to note that the reduction of sorting pressure and a larger nozzle size is necessary (45 psi and 85-µm nozzle) to increase the viability of the progenitor cell population and the quality of the RNA prepared from the sorted cells. It is important to use enriched liver progenitor cells for setting the compensation. Other liver or leukocyte populations have different auto-fluorescence and result in a suboptimal resolution of the stromal population and in a false gating strategy.
4. Calibrate the machine and place a 5-mL polypropylene round-bottom tube containing 350 µL of SM in the sort collection device. Start sample acquisition and sort. Collect 1,000 events of the subpopulation and stop the sample flow.
5. Take the tube out of the sorting device and measure the sorted cells on the flow cytometer. Identify the percentage of the sorted cell population present among the living cells. This percentage gives the purity of the sorted cells.
6. If sort purity exceeds 95%, place a 5-mL polypropylene round-bottom tube containing 350 µL of RLT lysis buffer in the sort collection device.
7. Start the sort and collect 7,000-10,000 events per stromal subpopulation.
8. Transfer the RLT lysis buffer containing the sorted cells in a DNase- and RNase-free reaction tube and store the samples at -80 °C until further analysis.