Dispase type 2

The tumor organoids were isolated as previously described [10 (link)]. Briefly, cancer tissues were incubated with collagenase type II (Sigma-Aldrich, Louis, MO, USA), dispase type II (Roche Applied Science, Mannheim, Germany), and Y-27632 (BioVision, Mountain View, CA, USA) for 1 h at 37°C. Isolated cells were washed with PBS and centrifuged at 300 ×g for 3 min. The cells were then embedded in Matrigel (growth factor reduced, phenol red free; Corning, NY, USA) and seeded in 4-well plates, followed by the addition of the culture medium. The composition of the CRC organoid culture medium was 1 × B27 (Gibco, Grand Island, NY, USA), 1.25 mM N-acetyl cysteine (United States Pharmacopeia, Rockville, MD, USA), 50 ng/mL human epidermal growth factor (BioVision), 50 ng/mL human Noggin (Peprotech, Rocky Hill, NJ, USA), 10 nM gastrin (Sigma-Aldrich), 500 nM A83-01 (BioVision), and 100 mg/mL primocin (InvivoGen, San Diego, CA, USA). To prevent anoikis, 10 μM Y-27632 was added to the culture medium during the first 2-3 days. When organoids were >200 µm, they were passaged by pipetting using the Gentle Cell Dissociation Reagent (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer's instructions.

Human dermal fibroblasts were isolated from the foreskin of a 5 y. o. male as described before⁶⁸ . The tissue was washed 3 times in PBS (Gibco, Carlsbad, CA, USA) and digested on the dermal side in 2.8 ml/60 cm² dispase type II (Roche, Basel, Switzerland) and incubated for 20 h at 4 °C. The epidermis and dermis was separated using a forceps, the dermal tissue was placed in a tissue culture plate and 1.5 ml FGM (Gibco, Carlsbad, CA, USA) was added. The enzymatic reaction was stopped by Trypsin (all Sigma-Aldrich, St. Louis, MO, USA) with 10 ml FGM consisting of DMEM medium with 10% FCS, 1% glutamine and 1% Pen/Strep (all Gibco, Carlsbad, CA, USA). The cells’ suspension was filtered using a cell strainer and washed with 5 ml PBS. Cells were centrifuged at 130 g for 5 min at 25 °C, the supernatant discarded and suspended in 10 ml PBS. The medium was changed every other day and cultured for 4 days at 37 °C. The purity was regularly controlled optically and reached approx. 90%. The cells were suspended in a collagen matrix NMSC model on the 18 mm diameter microscope cover glass (VWR, Darmstadt, Germany). The fibroblasts were cultured for 26 h at 37 °C before the TPE-FLIM measurements.

Mouse DRG neurons were isolated as described previously [20 (link), 21 (link)] with small modifications. Cervical, thoracic and lumbar spinal regions were exposed, the roof of the vertebral canal was removed, and DRG were collected. Ganglia were digested consecutively with papain and collagenase type 2/neutral protease solutions to obtain a single DRG cell suspension. Papain and Collagenase type II (CLS2) were from Worthington; Dispase type II was from Roche. DRG cell cultures were plated on round glass coverslips (EMS) coated with Poly-D-Lysine (Sigma) and Laminin (Sigma). The serum-free DRG medium consisted of Neurobasal A (Gibco), B-27 Supplement (Gibco), 2mM L-Glutamine-Pen-Strep (Gemini) and 2mM GlutaMAX Supplement (Gibco).