Collagenase type 4

A two-step collagenase perfusion method was used to isolate and purify rat hepatocytes (19). Briefly, FLR dissected from DEN-treated rats 48 h after ALPPS were perfused with calcium-free Hanks balanced salt solution (HBSS) and then perfused with HBSS containing calcium and collagenase type IV (Gibco, 17104019). Livers were minced and filtered through cotton gauze to liberate the hepatocytes. The hepatocytes were suspended in 7 mL of Earle's minimum essential medium with 10% fetal bovine serum (FBS), 2% penicillin/streptomycin, 1% glutamine, and 1% nonessential amino acids. The hepatocytes were purified from nonparenchymal cells and nonviable hepatocytes by Percoll density gradient centrifugation at 1000 g for 10 min at 4 °C.
HSCs were isolated from the FLR of DEN-treated or PBS-treated rats 48 h after ALPPS by enzymatic digestion of the liver with collagenase type IV (Gibco, 17104019), Pronase (Roche, 10165921001), and DNase (Sigma, DN25) followed by centrifugation of the crude cell suspension through a density gradient medium (Nycodenz) 20 (link). 6-8 × 10⁶ cells from each rat FLR were cultured in Dulbecco's Modified Eagle Media (DMEM) with 10% FBS, 2% penicillin/streptomycin, and 1% glutamine. HSCs and hepatocytes were cultured at 37 °C in a humidified incubator with 5% CO₂.

Testis tissues were obtained from patients with OA in which case the spermatogenesis was thought to be normal via surgery. A total of 27 patients were included in this work (tab. S3).
Two-step enzymatic digestion was applied to isolate germ cells from testis. The testis tissues were cut into pieces and incubated with 10 mL Enzyme I (2 mg/mL type IV Collagenase (17104-01, Gibco) and 10 μg/mL DNase I (D4527, Sigma) in DMEM (SH30243.01B, HyClone)) in oscillating water bath at 34 °C, 100 rpm for 10–15 min. The seminiferous tubules were collected via removing the supernatant after sedimentation for 10 min at room temperature and incubated with 10 mL Enzyme II (mg/mL type IV Collagenase (17104-01, Gibco), 2 mg/mL Hyaluronidase (H3506, Sigma), 2 mg/mL Pancreatin (T8003, Sigma) and 10 μg/mL DNase I (D4527, Sigma) in DMEM (SH30243.01B, HyClone)) in oscillating water bath at 34 °C, 100 rpm for 10–15 min. The suspension was centrifuged at 300 g for 5 min and the supernatant was then removed. The precipitant was re-suspended using DF12-FBS (10% FBS in DF12) followed by being filtered using a 40 μM cell strainer and planted in a 10 cm dish which was coated with gelatin.

After measurement of airway resistance and collection of BALF, lungs were perfused with PBS, lung tissues were cut into pieces using a gentleMACS Dissociator (Miltenyi Biotec) and treated with collagenase type 4 (Thermo Fischer Scientific) plus DNAse I (Roche). The lymphocyte-enriched fraction was collected at the 35–70% interface of Percoll gradients (GE Healthcare). Cells were immediately stained or stimulated for 4 h with with 10⁻⁸ M PMA and 1 μg ml⁻¹ ionomycin, in the presence of 10 μg ml⁻¹ brefeldin A (all from Sigma-Aldrich).

After cervical dislocation, tumours and lymph nodes (draining and non-draining) were harvested in digestion medium. Tumours were digested in HBSS containing 1 mg/ml Collagenase Type IV (ThermoFisher) and 0.1 mg/ml DNase I (Sigma). Lymph nodes were digested in HBSS containing 1 mg/ml Collagenase Type D (Sigma) and 0.1 mg/ml DNase I (Sigma). Tumours were cut into small pieces and digested for 45 min at 37°C. The cell suspension was passed over a 70 µm cell strainer and washed in PBS + 1% FCS + 2 mM EDTA. For analysis of tumour cells expressing MHC-I, PD-L1 and CD200, 2 × 10⁶ cells were used for flow cytometry staining. For isolation of tumour-infiltrating immune cells (TIICs), immune cells were enriched using Ficoll-Paque (Cytiva) density gradient centrifugation. Lymph nodes were pierced and torn using forceps, incubated for 30 min at 37°C in the digestion buffer and passed over a 70 µm cell strainer.