Cnt prime epithelial culture medium

The urothelial cancer cell lines (UCCs) VM-Cub1, RT112, SW1710, and UM-UC-3 were provided by Dr. M. A. Knowles (Leeds, UK), Dr. J. Fogh (New York, NY, USA) and Dr. B. Grossmann (Houston, TX, USA) or by the DSMZ (Braunschweig, Germany). They were cultured in DMEM GlutaMAX-I (Gibco, Life Technologies, Darmstadt, Germany) supplemented with 10% fetal calf serum (Biochrom, Berlin, Germany) at 37 °C in a humidified atmosphere of 5% CO₂. As a benign urothelial control, we used the HBLAK cell line ([25 (link)], spontaneously immortalized from primary human bladder epithelial cells; obtained from CELLnTEC, Bern, Switzerland), which were cultured in CnT-Prime Epithelial Culture Medium (CELLnTEC, Bern, Switzerland). All cell lines were authenticated by DNA fingerprint analysis. Normal urothelial cells (UP) were cultured as described [25 (link)] with informed consent of the donors and approval by the Ethics Committee of the Medical Faculty of the Heinrich-Heine-University, study number 1788.
Cytokines TGFβ (Human, recombinant TGF-β1, Miltenyi Biotec, Bergisch Gladbach, Germany, reconstituted in water) and TNFα (Human, recombinant TNFα, Sigma-Aldrich, St. Louis, MO, USA, dissolved in phosphate buffered saline (PBS)) were used at 5 ng/mL and 20 ng/mL, respectively.

Human gingival epithelial cells (hGECs) were purchased from CELLnTEC advanced cell systems AG (Stauffacherstrasse, Bern, Switzerland). hGECs were cultured in CnT-prime epithelial culture medium (CELLnTEC advanced cell systems AG, Stauffacherstrasse) at 37 °C in 5% CO₂. To induce differentiation of hGECs, the cells were seeded onto cell culture inserts in a 12-well plate (ThinCert^TM, Greiner Japan, Tokyo, Japan) using CnT-Prime 3D barrier medium (3D-medium, CELLnTEC advanced cell systems AG, Stauffacherstrasse). Briefly, the cells were seeded at 5.0 × 10⁵ cells in the upper chamber with 0.5 mL of 3D-medium. Additional 5 mL of 3D-medium was added to the lower chamber. For the down-regulation of the Col4a6 gene, 5 nM of siRNA targeting the Col4a6 gene (StelthTM SiCol4a6; Life Technologies) was transfected into hGECs using Lipofectamine RNAiMAX (Life Technologies), according to the manufacturer’s instructions. StelthTM RNAi Negative Control High GC Duplex (Life Technologies) was used as the negative control. Transfected hGECs were seeded at 2.0 × 10⁶ cells in the upper chamber with 0.5 mL of 3D-medium, and 5 mL of 3D-medium was added to the lower chamber. After 24 h, 3D-media of both chambers were aspirated, and 4 mL of 3D-medium was added only to the lower chamber, and the cells were cultured for additional 7 days.

The Ca9-22 human oral epithelial cell line was purchased from the RIKEN BRC Cell Bank (Tsukuba, Japan). Ca9-22 cells were maintained in Dulbecco’s modified Eagle’s medium (GibcoBRL, Grand Island, NY, USA) containing 10% fetal bovine serum (Hyclone Laboratories, Logan, UT, USA), penicillin (100 U/ml), and streptomycin (100 μg/ml) in a humidified atmosphere containing 5% CO₂ at 37°C, which was replaced every 3 days. Human primary gingival epithelial cells (HGECs; CELLnTEC, Bern, Switzerland) were maintained in CnT-Prime epithelial culture medium (CELLnTEC), which was replaced every 5 days, in a humidified atmosphere containing 5% CO₂ at 37°C. For experiments, cells were seeded into 12-well plates at 1×10⁵ cells/ml, incubated for 48 h, primed with 50 ng/ml recombinant human (rh) interferon (IFN) γ (PeproTech Rocky Hill, NJ, USA) for 12 h, and then treated with P. gingivalis 1690 (penta-acylated lipid A) LPS (0–1 μg/ml; Astarte Biologics, Bothell, WA, USA). It is conceivable that gingival epithelial cells in inflamed gingival tissues are already primed (with IFN-γ produced by inflammatory lymphoid cells) for secretion of various inflammatory cytokines in response to bacterial components such as LPS [24 (link), 25 (link)]. Therefore, we pretreated cells with rhIFN-γ [26 (link)]. We confirmed that IFN-γ treatment does not affect ANGPTL2 induction (data not shown).

This study was approved by the institutional review board of Kyoto Prefectural University of Medicine. All experimental procedures were conducted in accordance with the tenets of the Declaration of Helsinki. Written informed consent was obtained from all patients after they were given a detailed explanation of the purpose of the research and the experimental protocols.
Human conjunctival epithelium was harvested from conjunctival tissue obtained during ocular surface reconstruction for SJS/TEN in the chronic stage or conjunctival chalasis surgery. Samples were immersed overnight at 4 ºC in 1.0 U/ml⁻¹ purified dispase (Roche Diagnostic Ltd., Basel, Switzerland)^{15 (link)} for comprehensive miRNA analysis, and quantitative miRNA PCR. For transfection with the miRNA inhibitor and RT-qPCR, we cultured primary human conjunctival epithelial cells (PHCjECs) using a modified, previously-described method^{16 (link)}. Briefly, detached epithelial cells were grown in CnT-prime epithelial culture medium (CELLNTEC, Bern, Switzerland) and 1% antibiotic–antimycotic solution. Cell colonies usually became obvious within 3 to 4 days. After reaching 80% confluence in 7 to 10 days, the cultured PHCjECs were used in the subsequent procedures.

For most experiments, six different UCCs overexpressing TagGFP2 or UTX-TagGFP2 (VM-CUB1, RT112, SW1710, T24, 639-V and KU-19-19) were used. Parental UCCs were obtained from the DSMZ (Braunschweig, Germany) and Dr. H.B. Grossmann (Houston, TX, USA). For comparison, we investigated the spontaneously immortalized normal human urothelial cell line HBLAK (provided by CELLnTEC, Bern, Switzerland) [21 (link)]. Cells were cultured and treated in DMEM GlutaMAX-I (Gibco, Darmstadt, Germany) supplemented with 10% fetal calf serum (Biochrom, Berlin, Germany), except for HBLAK cultured in CnT-Prime Epithelial Culture Medium (CELLnTEC, Bern, Switzerland; HBLAK) and KU-19-19 in RPMI-1640 (Gibco), at 37 °C and 5% CO₂. STR (short tandem repeat) profiling via DNA fingerprint analysis was performed for all cell lines. KDM6A and KMT2C/D genotypes were obtained from the CCLE database and ascertained by targeted PCR and Sanger sequencing. For individual cell lines, such as HBLAK, whole-exome sequencing data from other own projects [21 (link)] was used to elucidate the mutation status.

VK2/E6E7 kindly gifted from Dr. Chen (Tongji Medical College) is a vaginal epithelial cell. The cells were maintained and passaged in CnT-Prime epithelial culture medium (CELLnTEC, Switzerland) at 37°C with 5% CO₂. The adhesion test was conducted as described previously (Gao et al., 2019 (link)), with slight modifications. 1.0 × 10⁵ cells/well of C. albicans were inoculated into VK2/E6E7 monolayer cultures in a 24-well plate with or without KBN at 37°Cwith 5% CO₂ for 1.5 h. All the mixtures of cell monolayer and fungal cells were fixed with 4% paraformaldehyde for 15 min, stained with 30 μg/mL CFW (Sigma-Aldrich, United States) for 15 min, and then photographed under a fluorescent microscope.

Human gingival epithelial progenitors (HGEPs) and primary keratinocytes derived from healthy gingival epithelium were purchased from CELLnTEC Advanced Cell Systems (Basel, Switzerland) and cultured in CnT-Prime epithelial culture medium (CELLnTEC Advanced Cell Systems) at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. HGEPs were spread onto 100 mm tissue culture plates at a density of 4.0 × 10⁴ cells/mL. Arecoline (arecoline hydrobromide) was purchased from Sigma-Aldrich (St. Louis, MO). Following overnight incubation, the HGEPs were treated with arecoline at a concentration of 50 μg/mL. The concentration of arecoline used in this study was as described in previous experiments [24 (link)]. Briefly, arecoline at the concentration of 50 μg/mL had no cytotoxic effect on the cells stimulated, even for a prolonged period, the method of alternating between 3 days with 50 μg/mL of arecoline and 3 days without arecoline for 1 month was selected. The untreated samples were used as controls (Fig. 2).
Fig. 2

Flow chart of cell culture. Human gingival epithelium progenitors (HGEPs), cells were treated with arecoline at a concentration of 50 μg/mL. The culture media was replaced every 3 days, alternating media with and without arecoline for 30 days. Untreated samples were used as controls. DDW, double-distilled water; ARE, arecoline