Fibroblast medium

Primary cultured human airway fibroblasts (HLF) were prepared from second to fourth generation peripheral lung tissue in resected specimens. All procedures were approved by the Human Research Ethics Board (University of Manitoba) and all donors gave informed consent. As described in detail (Ghavami et al. 2010; Sharma et al. 2015), after microdissection to separate the lamina reticularis and submucosal compartment from encircling airway smooth muscle bundle, HLF were isolated by enzymatic dissociation. For all experiments, passage 3–7 of HLF were used and cells were cultured in fibroblast medium (Sciencell Research Laboratories, Carlsbad, CA). The medium was changed every 48 h. Transfection of CRISPLD2 siRNA and nontargeting siRNA (negative control) (siRNA universal non‐targeting control 1, Sigma‐Aldrich Corporation, St. Louis, MO) was performed using DharmaFECT 1 reagent according to the manufacturer's recommended protocol (Thermo Scientific, Lafayette, CO). The final concentration of siRNA was 25 nmol/L; siRNA sequences for CRISPLD2 knockdown were as follows: 5′‐GAACCAACAUCUAUGCAGA(dT)(dT)‐3′ and 5′‐UCUGCAUAGAUCUUGGUUC(dT)(dT)‐3. Primary HLF cells from five normal and five COPD patients (GOLD classification) are used for siRNA transfection. The suppression of CRISPLD2mRNA in HLF cultures was confirmed prior to each set of experiments.

Human dermal fibroblasts (hDF; ScienCell Research Laboratories, Carlsbad, CA, USA) were used in this study for the subsequent in vitro studies. The hDF were cultured in Fibroblast Medium (ScienCell Research Laboratories) with the respective recommended antibiotics. The cells were trypsinized using trypsin/EDTA, collected using a hemocytometer, and placed into GelMa solution at a density of 2 × 10⁶ cells/mL. Then, the GelMa hydrogels were fabricated using the methods described above and cultured in Fibroblast Medium.

Primary adult human pulmonary fibroblasts (ScienCell, CA, USA, 3310) were cultured in Fibroblast Medium (ScienCell, CA, USA, 2301) containing 2% FBS, 1% Fibroblast Growth Supplement (FGS) and 1%penicillin/streptomycin solution (P/S) in a humidified atmosphere of room air supplemented with 95% O₂/5% CO₂ at 37 °C.

J1.1 and ACH-2, derivatives of Jurkat and A3.01 T cell lines in which the HIV-1 LAV strain was latently infected, respectively [23 (link),24 (link)], were obtained from the NIH AIDS Reagent Program and were grown in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS). The parental Jurkat and A3.01 cell lines were obtained from American Type Culture Collection (ATCC) and were similarly grown in RPMI1640 medium. EA.hy926, a human umbilical vein endothelial cell (HUVEC)-derived endothelial cell line [25 (link)], and HS-5, a bone-marrow-derived stromal cell line [26 (link)], were from ATCC and were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS. HS-27A, another bone-marrow-derived stromal cell line (ATCC), was grown in RPMI1640 with 10% FBS. Primary FRC (ScienCell Research Laboratories) were grown in fibroblast medium (ScienCell Research Laboratories) supplemented with 1% fibroblast growth supplement and 2% FBS.

CI-huThyrEC human thyroid epithelial cells were purchased from InSCREENeX (cat. no. INS-CI-1017; Braunschweig, Germany). Primary human thyroid fibroblasts were purchased from ScienCell Research Laboratories (cat. no. 3730; Carlsbad, CA, USA). All the cells were grown according to suppliers’ instructions using the recommended media and other reagents (huThyrEC medium, cat. no. INS-ME-1017; InSCREENeX), epithelial cell coating solution (cat. no. INS-SU-1020; InSCREENeX), and fibroblast medium (cat. no. 2301; ScienCell Research Laboratories). Human recombinant TNF was purchased from PeproTech, Inc. (Cranberry, NJ, USA) and used at 25 ng/mL for the experiments. Human recombinant TGF-β1 was obtained from PeproTech and used at 10 ng/mL. In one set-up, thyroid epithelial cells were also cultured on depleted medium which consisted of huThyrEC basal medium with 1% fetal bovine serum, amphotericin B, penicillin, and streptomycin. The cells were seeded onto six-well plates and cultured until they reached a sufficient confluence. Medium containing TNF or TGF-β1 was added, and after the duration of the experiment the cells were lysed with the Tri reagent system (Sigma-Aldrich Corp., St. Louis, MO, USA). The experiments were repeated three times (N = 3) with two technical replicates per experiment, using the average of the two as the final value for the experiment.

The human prostate fibroblast cells and fibroblast medium were purchased from ScienCell (Cat # 4430 and SC-2301; Carlsbad, CA, USA). The human normal prostate smooth muscle cells and PriGrow X medium were purchased from abm (cat #T4079 and TM4079, Richmond, BC, Canada). HEK-293T, PZ-HPV-7, CA-HPV-10, LNCaP, PC-3, and DU145 cells were obtained from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) and cultured, as described previously [52 (link)]. The human prostate stromal myofibroblast WPMY-1 cells were purchased from the American Type Culture Collection (ATCC; CRL-2854; Manassas, VA, USA). The cells were cultured as instructed by the manufacturers. Fetal bovine serum (FBS) was purchased from HyClone Laboratories, Inc. (Logan, UT, USA); RPMI 1640 medium came from Life Technologies (Rockville, MD, USA) and Matrigel came from BD Biosciences (Bedford, MA, USA). TNFα and TGFβ were purchased from PeproTech (Cranbury, NJ, USA).

CAFs and paired normal fibroblasts (NFs) were isolated from tumor tissue and adjacent non-tumor tissue (more than 5 cm away from the tumor margin) from ESCC patients who underwent surgical resection at the Department of Thoracic Surgery, Zhongshan Hospital, Fudan University. Informed consent was obtained from all patients, and the study was approved by the Ethics Committee of Zhongshan Hospital, Fudan University. After washing with phosphate buffered saline (PBS), the tissues were cut and minced into small pieces (1–2 mm³) and seeded in a culture flask with Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS). Fibroblasts grew outwards from the explants and reached 80% confluence after 2 weeks. Homogeneous CAFs and NFs were cultured in fibroblast medium (Sciencell, Carlsbad, CA, USA) for later experiments.