Dispase

Leydig TM3 cells were obtained from ATCC (UK). Merck supplied the dibutyryl cyclic AMP (Bt2cAMP), Neutral Red, GPX4 inhibitor RSL3, Ebselen, horse serum, FBS, BSA, Hepes, Penicillin/Streptomycin, Luteinizing Hormone (LH), human Chorionic Gonadotropin (hCG), Percoll, Collagenase D, Dispase, DNase I, X-TremeGENE transfection reagent, Insulin-Transferrin-Sodium Selenite Supplement (TIS), Complete Mini (a Roche mixture of protease inhibitors) and Dulbecco’s modified Eagle’s/Ham’s nutrient F12 (DME/F12) medium. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol were obtained from Avanti Polar Lipids. ThermoFisher Scientific provided M-199 medium, Opti-MEM medium, Soybean Trypsin Inhibitor (STI), DPBS, HBSS, EGF, C11-BODIPY(581/591), Mitotracker Deep Red, and primary rabbit antibodies against StarD1 and StarD4. A testosterone assay kit and primary rabbit antibodies against GPx4 and β-actin were obtained from Abcam. A horseradish peroxidase-conjugated IgG secondary antibody was from Cell Signaling Technology. Gel electrophoresis consumables were from BioRad. 7-OOH was prepared by dye-sensitized photoperoxidation of Ch, as described [34 (link)]. Stock solutions of 7-OOH in 2-propanol were standardized for hydroperoxide content by iodometric analysis [22 (link)] and stored at −20 °C.

All human PDAC organoid lines were generated from established patient-derived xenograft (PDX) PDAC tumor models or from fresh primary PDAC tumor tissue as previously described with some modifications [1 (link), 29 (link)]. To date, 8 PDAC organoid lines have been established (S1 Table). Six PDAC organoid lines were generated from PDX PDAC tumors (Panc129, Panc193, Panc269, Panc271, Panc272, and Panc320) whereas two lines were generated directly from primary pancreatic tumor tissue (Panc308 and Panc368). Briefly, PDAC tumor tissue was washed vigorously and minced with surgical scissors. The fragments were then digested with digestion media [Advanced DMEM/F12 (Gibco) containing 1% fetal bovine serum (FBS), 10mM HEPES (Gibco), 2mM GlutaMax (Gibco) and 1% Penicillin/Streptomycin (Gibco) with 1.5mg/ml collagenase (C2139 Sigma), 125ug/ml dispase, 0.1mg/ml DNAse1 (EMD Millipore) and 10μM Y27632] at 37°C for 30 min. The digested cells were washed with wash media (DMEM containing 10% FBS, 10mM HEPES, 2mM GlutaMax, 1% Penicillin/Streptomycin and 10μM Y27632) to inactivate the digestive enzymes, and then filtered through a 100μm strainer to removed large undigested fragments prior to culture. Isolated pancreatic cells were embedded in Basement Membrane Extract (BME, Biotechne 9mg/ml) and medium was refreshed every 2–3 days.

Samples were obtained from surgical procedures. Tumor tissues were cut to very small pieces, which were incubated overnight in a medium containing dispase, collagenase (Merck), and supplemented with penicillin G, kanamycin sulphate, streptomycin sulphate, and gentamycin at standard concentrations (100μg/ml) (Merck). Then the detached cells were washed and seeded in culture flasks.
Melanoma primary cultures were propagated in the following culture media: Ham’s-F10, Ham’s F10 supplemented with 100μM of tyrosine, RPMI 1640, and DMEM. The media corresponding to each of these conditions were used for the experiments. All these culture media were supplemented with 10% heat-inactivated fetal calf serum (FCS), and with L-glutamine, penicillin, and streptomycin at standard concentrations (Thermo Fisher Scientific) in a humidified air with 5% CO2 at 37°C. Cell culture medium was renewed every 2–3 days. Once the cells were at or near confluence they were subcultured. Melanoma primary cultures were regularly checked for mycoplasma contamination using MycoAlert^® Mycoplasma Detection Kit (Lonza).

Freshly obtained LMMP preparations (see Dissection of Longitudinal Muscle Myenteric Plexus section) were incubated for 10 min at 37°C with collagenase type II from Clostridium histolyticum (10 mg/ml), dispase (60 μg/ml) and DNase I (10 μg/mL, all purchased from Merck). Tissue debris were filtered through cell strainers and cells were stained for flow cytometry analysis. Alternatively, cells were purified by density gradient centrifugation through Ficoll-Hypaque (20 minutes at 600xg at room temperature; Merck). Mononuclear cells (10⁷ cells/mL) were:

The clinical organization of the PDL was selected from the root PDL of patients with periodontitis in our hospital (approximately 1 mm³). After the PDL tissue of the patient was extracted, 2 mg/mL collagenase and 4 mg/mL dispase (Merck, Darmstadt, Germany) in a ratio of 1:1 were added and digested at 37 ℃ for 40 min. Single-cell suspension culture was carried out in α-MEM growth medium containing 10% fetal bovine serum (Thermo Fisher Scientific, CA, USA) and 1% penicillin/streptomycin (Thermo Fisher Scientific). The conditions of the incubator were 37 ℃ and moist air with 5% CO₂ added. PDLSCs were subcultured using 0.25% trypsin. The PDLSCs used in this study were all 2nd to 5th generation.
Osteogenic differentiation induction: The OriCell® Human Related Stem Cell Osteogenic Differentiation Induction Kit was used to induce osteogenic differentiation of PDLSCs. The experimental operation was carried out in full accordance with the manufacturer's instructions.

Excised pulp tissue was digested in a solution containing 3 mg/ml collagenase type I (Merck KGaA, Darmstadt, Germany) and 4 mg/ml dispase (Merck KGaA, Darmstadt, Germany) for 1 h at 37°C. After digestion, 5 volumes of α-MEM medium containing 10% FCS were added. This solution was centrifuged at 120 × g for 10 min, at room temperature. The precipitated material was resuspended in α-MEM medium and filtered through filters with pores of 70 μM. This procedure resulted in a single-cell suspension for in vitro culture. The cells were seeded into culture flasks with α-MEM, supplemented with 10% FCS, 100 μM l-ascorbic acid, 2 mM l-glutamine and penicillin (100 U/mL)/streptomycin (100 mg/ml) and incubated at 37°C with 5% CO₂. In the initial stage, after adhering to culture flasks, cells grew slowly. Cell colonies were identified after 10–14 days, with their fibroblastoid appearance, which showed to be dependent on the cell density obtained in initial plating. After reach 70% of confluence, cells were considered ready to proceed with experiments. Accordingly, isolated DPSCs were randomly assigned to the following experimental groups: Group 1–LPS stimulus and laser irradiation; Group 2–LPS stimulus and no laser irradiation; Group 3–Control group, no LPS or laser treatment.

Obtained oral mucosa tissue was briefly washed in a medium with added streptomycin, penicillin and Amphotericin B (LG-DMEM, 12320032; P&S, 15140122; Amphotericin B, 15290018, everything Gibco) cut into small pieces and then immersed in the mixture of collagenase and dispase (Merck, 10269638001, 5 mg/ml) overnight. The following day, lamina propria was mechanically separated from the epithelium, cut into pieces of approx. 0.5 mm and plated in six-well plates with growth medium comprising LG-DMEM, 10% FBS, 1% Glutamax and antibiotics. The plates were kept at 37 °C and 5% CO₂ in a humidified culture incubator. The growth medium was partially changed every third day. 15 days following the plating, cells growing from the pieces of tissue have been noticed and were allowed to cover 60–70% of the plate surface. When cells reached the desired confluence, they were detached using 0.25% trypsin and seeded in new plates. For these experiments, cells between the 2nd and the 7th passage were used. For all the experiments described in this article, cells originating from the same donor were used.