C9891

Animals were deeply anesthetized with a mixture of hypnorm (Vm21757/4000; Vetapharma), midazolam (Hameln Pharmaceuticals), and isotonic saline (25%, 25%, 50% v/v. 0.3 ml/100 g) and killed by cervical dislocation. The TG were removed, cut into smaller sections with microscissors, placed in 5 mg/ml collagenase suspension (C9891; Sigma-Aldrich) in Ham’s F12 growth medium (21765-029; Gibco Life Technologies, Invitrogen) supplemented with 1% penicillin/streptomycin (15140; Gibco Life Technologies, Invitrogen) and incubated (37°C) for 15 min. Following incubation, the collagenase solution was centrifuged (280 g) for 5 min. The pellet was re-suspended in 1 ml 0.125% trypsin (15090-046; Gibco Life Technologies, Invitrogen) and placed in the incubator for 5–10 min. prior centrifugation (5 min./280 g). The sectioned and enzymatically digested ganglia were then mechanically dissociated into a homogenous solution by repeated pipetting. The cell solution was added to uncoated culture flasks before being placed in an incubator (37°C – 95% air/ 5% CO₂). The growth medium was changed after 3 and 24 hrs, and then continuously every 2–3 days prior to the experimental procedure.

One week after the labeling procedure, the rats were again anesthetized with isoflurane, euthanized by decapitation, and DRGs from Th11-L2 were collected [9 (link), 20 (link)]. Neurons were isolated by mechanical and enzymatic dissociation, as described previously [20 (link)]. The ganglia were incubated with collagenase IA (2 mg/ml C9891, Sigma Aldrich, Munich, Germany in DMEM, PAA Laboratories GmbH, Linz, Austria) for 1 h in 5% CO₂ at 37 °C. Enzymatic dissociation was terminated by replacing collagenase-containing DMEM with fresh DMEM + cultural medium (DMEM + , i.e., DMEM plus 10% FCS, 1% penicillin/streptomycin, and 0.1% insulin). Tissue digestion was stopped by FCS. Ganglia were triturated using sterile Pasteur pipettes (Sigmacote®; Sigma-Aldrich, Munich, Germany) to dissociate individual cells. After centrifugation at 100 rcf, cells were resuspended in 10 ml DMEM + and centrifuged once more. The pellet was resuspended in 1.8 ml DMEM⁺, and cells were plated on glass coverslips coated with poly-L-lysine. The coverslips were cultured in DMEM⁺ for one day before electrophysiological experiments.

Tumor tissues were obtained by radical nephrectomy or partial nephrectomy. They were cut into 1–2-mm-thick sections before digestion with collagenase type IV (1 mg/mL, C9891, Sigma-Aldrich) and Y-27632 (HY-10071, MCE, 10 μM) for 1 h. The suspension was filtered through a 70-μm mesh. The pellet was resuspended in 200 μL of Basement Membrane Extract (BME, 3533-001-02, R&D) and seeded into prewarmed 12-well plates. After the BME solidified, human renal cancer organoid medium was added for culture.