An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

This study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the Animal Welfare Act under the supervision of our institutional Animal Care and Use Committee. Animal protocols were conducted according to institutional ethical guidelines of the Charité Berlin, Germany, and were approved by the Landesamt für Gesundheit und Soziales and comply with the ARRIVE guidelines.

1. Diet-induced Animal Model of Steatohepatitis

1. Transfer mice (C57Bl/6J, male) to non-SPF housing at the age of 4 weeks and expose them continuously to a broad range of environmental pathogens/antigens. Guarantee the exposure by daily handling of the laboratory animals as antigens are distributed by air passage in this way.
2. Maintain mice (C57Bl/6J, male, 12 weeks old) in open caging systems with conventional filters on a 12 h:12 h light/dark cycle at a temperature of 22 °C. Do not irradiate or autoclave experimental diet and bedding. Access animal housing facility without mask or hairnet. Open doors between animal rooms. Do not use an airshower.
3. Guarantee daily handling of the laboratory animals and switch rooms on a regular basis so that antigens are distributed by air passage. Complement dirty housing with daily non-specific microbial exposure to antigens from bedding of mammalian laboratory animals housed in rooms next to the laboratory mice.
4. Measure proportions of CD44^–CD62L^– effector memory CD4⁺ and CD8⁺ T cells in blood and spleen using flow cytometry (as described in refefence¹³).
   NOTE: In this regard we defined an increase of 20% of effector memory CD8⁺ T cells from CD8⁺ T cells as evidence of sufficient microbial exposure.
5. Start HFD (60 kJ% from fat, 19 kJ% from proteins and 21 kJ% from carbohydrates and ad libitum consumption of water with 6% sucrose content with five-week old C57Bl/6J male mice for 7-15 weeks. The HFD should contain 60% kJ from fat (as described above) in order to induce the development of insulin resistance throughout the course of the experiment.
   NOTE: Hematoxylin staining should be performed and histological characteristics as hepatocyte ballooning, Mallory Denk bodies, immune cell infiltration and macrovesicular steatosis must be found to demonstrate NASH-like liver pathology (as shown in reference¹³).

2. Preparation of Reagents and Solutions

1. Prepare 70% ethanol, phosphate buffered saline (PBS, without calcium and magnesium) supplemented with 0.5% BSA and ACK (Ammonium-Chloride-Potassium) lysis buffer.
2. Staining buffer
   1. Dissolve 10 mL of fetal calf serum (FCS) in 500 mL of PBS to obtain 2% FCS PBS. Place staining buffer on ice before use.
   2. Store solution in a plastic bottle at 4 °C.
3. Collagenase solution for adipose tissue digestion
   1. Dissolve 2.5 g of bovine serum albumin (BSA) in 500 mL of PBS to obtain a 0.5% BSA/PBS.
   2. Dissolve 74.5 g of CaCl₂ in 10 mL of 0.5% BSA/PBS to obtain a 10 mM CaCl₂ solution.
   3. Add 1 mg of collagenase type II (see Table of Materials) to each mL of 0.5% BSA with 10 mM CaCl₂ PBS.
   4. Prepare 3 mL of collagenase digest solution per g of adipose tissue sample. Prepare fresh collagenase solution for each isolation.
4. Collagenase solution for liver tissue digestion
   1. Dissolve 2.5 g of BSA in 500 mL of Hank´s Balanced Salt Solution (HBSS) to obtain 0.5% BSA HBSS.
   2. Add 10 mL of FCS in 500 mL of 0.5% BSA/HBSS to obtain 2% FCS 0.5% BSA/HBSS.
   3. Add 0.5 mg of collagenase type IV (see Table of Materials) to each mL of 2% FCS 0.5% BSA/HBSS.
   4. Add 0.02 mg of DNase per mL of 2% FCS 0.5% BSA/HBSS collagenase solution.
   5. Prepare 13 mL of collagenase digest solution per liver tissue sample.
   6. Prepare fresh collagenase solution for each isolation.

3. Generation of Single cell Suspensions

1. Adipose tissue digestion
   1. Euthanize mice via isoflurane anesthesia followed by cervical dislocation. Spray the chest with 70% ethanol. Carefully make a 5-6 cm central incision through the integument and abdominal wall along the entire length of the rib cage to expose the pleural cavity and heart.
      NOTE: Do not damage underlying organs and keep scissors tips up.
   2. Inject at least 10 mL of 0.9% saline solution in the apex of the left ventricle using a 26 G needle.
      NOTE: Successful perfusion is noted by blanching of the liver.
   3. Open the peritoneal cavity with scissors and cut out the perigonadal fat pads on each side with fine curved scissors. Dissect gonadal tissues with fine curved scissors and weigh adipose tissue.
   4. Perform the mechanical dissociation using scissors and cut adipose tissue into fine pieces in a Petri dish at 4 °C. Transfer adipose tissue to 50 mL conical centrifuge tubes and rinse Petri dish with 1 mL of 0.5% BSA/PBS.
   5. Add 3 mL of adipose tissue digest solution (as prepared in step 2.3) per gram of adipose tissue. Incubate adipose tissue solution at 37 °C for 20 min under gentle shaking (200 rpm).
   6. Add 5 mL of 0.5% BSA/PBS per gram of adipose tissue and place on ice. Triturate the solution numerous times with a 10 mL serological pipette and pass it through a strainer (100 µm) with the aid of a plunger.
   7. Centrifuge at 500 x g for 10 min at 4 °C.
   8. Remove the floating adipocyte fraction by pipetting. Resuspend the cell pellet (stromal vascular fraction) in 1 mL of ACK lysis buffer (see Table of Materials). Add 10 mL of 2% FCS PBS.
   9. Centrifuge at 500 x g for 10 min at 4 °C.
   10. Decant supernatant and resuspend cell pellet in 250 µL of 2% FCS PBS.
   11. Count the number of viable cells on a hemocytometer using Trypan blue exclusion.
2. Liver tissue digestion
   1. Store liver in a conical centrifuge tube filled with PBS and transport on ice.
   2. Perform the mechanical dissociation using syringe stamps in a Petri dish at 4 °C. Put dissected liver tissue in a 50 mL conical centrifuge tube containing 10 mL of warm liver digest solution. Rinse the Petri dish with 3 mL of liver digest solution.
   3. Incubate liver tissue solution at 37 °C for 20 min under gentle shaking (200 rpm).
   4. Add 20 mL of HBSS. Triturate the solution numerous times with a 10 mL serological pipette and pass it through a strainer (100 µm) with the aid of a plunger.
   5. Centrifuge at 500 x g for 10 min at 4 °C. Decant the supernatant and resuspend the cell pellet in 20 mL of HBSS.
   6. Centrifuge at 30 x g for 1 min at room temperature to remove the hepatocyte matrix. Discard the cell pellet.
   7. Centrifuge the supernatant at 500 x g for 10 min at 4 °C. Resuspend the pellet in 10 mL of 33% low viscosity density gradient medium solution (see Table of Materials) in HBSS followed by centrifugation (800 x g; 30 min; room temperature; no brake).
   8. Resuspend the pellet in 1 mL of ACK lysis buffer and incubate for 4 min at room temperature. Then add 10 mL of HBSS.
      NOTE: To discard the supernatant aspirate the supernatant with interphase (hepatocytes) very carefully with a transfer pipette (as much as possible without taking the pellet with immune cells and erythrocytes).
   9. Pass cells through a 30 μm strainer in a 15 mL conical centrifuge tube and centrifuge at 500 x g for 10 min at 4 °C. Decant supernatant and resuspend cell pellet in 250 µL of 2% FCS PBS.
   10. Count the number of viable cells on a hemocytometer using Trypan blue exclusion.

4. Surface Staining

1. Prepare antibody mix for T-cell-subsets (Panel 1) and innate immune cells (Panel 2) as described in Table 1 and Table 2. Volumes are optimized for one sample (100 µL) regarding to antibody concentrations.
   NOTE: Lymphocytes and innate immune cells present differences in autofluorescence and should be analyzed separately.
2. Use up to 3 x 10⁶ cells in 100 µL in a polystyrene FACS tube for surface staining. To block Fc receptors add 10 µL of anti-CD16/CD32 antibody (diluted 1:100) and incubate for 10 min on ice. Use an unstained negative control sample to adjust side scatter (SSC) and forward scatter (FSC) to determine the location of the negative cell population.
3. Vortex and add the appropriate volume of antibody mixture for Panel 1 and Panel 2. Add 1 µL of viability dye to each sample to allow for live and dead cell discrimination. Incubate for 20 min at 4 °C and protected from light.
4. Wash two times with 2 mL of 2% FCS PBS and centrifuge at 300 x g for 5 min at 4 °C.
5. Resuspend the cell pellet in 300 µL of 2% FCS PBS and store at 4 °C until FACS analysis.
   NOTE: Before starting the measurement pass cells through 30 µm cell strainer into polystyrene FACS tube and vortex. Flow cytometry was performed with labeled cells stored in 2% FCS PBS at 4 °C for 1-3 h. Cells should be analyzed as soon as possible for optimized results.

5. Flow Cytometry Compensation, Acquisition, and Gating

1. Run unstained negative control sample to set the FSC and SSC and adjust voltages of the flow cytometer to detect leucocyte populations and to distinguish between debris and viable cells. Exclude debris and dead cells.
   NOTE: Viability of the cell suspensions from the liver might be lower than the viability from the cell suspensions from perigonadal adipose tissue due to an additional density separation step.
2. Run single stained control samples for multi-color compensation.
   NOTE: Antibody capture beads could also be used to adjust for spectral overlap if the number of cells of a cell population of interest is too low to compensate using cells. To detect possible autofluorescence signals of the cells fluorescence minus one (FMO) controls for every antibody are recommended but were not applied in this protocol.
3. Start the measurement, collect appropriate number of events (at least 50,000 events) and record experimental data.
4. Export FCS data files for analysis and set the gating strategy. Gate on CD45⁺ leucocytes to identify subsequent cell populations.