Gibco glutamax supplement

Reagents were obtained from the following manufacturers: Advansta Corporation (San Jose, USA): WesternBright Sirius^®; Carl Roth GmbH & Co. KG (Karlsruhe, Germany): ROTI^®Stock 10× PBS, ROTI^®Stock 10× PBS-T, sodium chloride (≥99.5%), sodium dodecyl sulfate (SDS; ≥99.5%), and sulfuric acid; Promega GmbH (Mannheim, Germany): peroxidase-conjugated anti-human IgG Ab; GenScript (Leiden, Netherlands): SARS-CoV-2 Spike protein RBD (Omicron Variant, His Tag); Seramun Diagnostika GmbH (Heidesee, Germany): TMB substrate solution; SERVA Electrophoresis GmbH (Heidelberg, Germany): acrylamide/bis(acrylamide) (30% T, 2.67% C), BlueBlock PF 10×, Coomassie Brilliant Blue G-250, TEMED, and trypsin (sequencing grade, MS approved); Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany): ExtrAvidin-Peroxidase, imidazole (≥99.5%), 2-Mercaptoethanol (BioUltra), and polyethylenimin (PEI); Sino Biological (Eschborn, Germany): ACE-2 (His-tagged, biotinylated); Surmodics IVD, Inc. (Eden Prairie, USA): StabilZyme™ SELECT Assay diluent (Protein-free); Thermo Fisher Scientific (Waltham, MA, USA): goat anti-human IgA secondary Ab-HRP, Gibco DMEM, Gibco GlutaMAX Supplement, Gibco 100× MEM Non-Essential Amino Acids Solution, penicillin/streptomycin (10,000 U/mL), Gibco Fetal Bovine Serum (FBS), and SuperBlock^® (PBS).

The cell culture medium, Dulbecco’s modified Eagle’s medium (DMEM), was purchased from Biowhittaker/Lonza, penicillin-streptomycin from Cambrex/Lonza, Glutamine [Gibco^® GlutaMAX™ Supplement (35050061)], and heat inactivated fetal bovine serum (FBS, 10100147) was from Thermo Fisher Scientific. The primers were designed and purchased from Sangon Biotech, China. Mouse IL-1β enzyme-linked immunosorbent assay (ELISA) Kit (D720335-0096) and recombinant murine TNFα (C600052-0005) were purchased from Sangon Biotech, China. The Cell Counting Kit (CCK-8) (C0038) and Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit (C1063) were purchased from Beyotime Corporation, China. H₃R agonist methimepip (Met) (sc-204080) was from Santa Cruz Corporation, and ciproxifan (CPX) (C6492), H₃R blocker, was from SIGMA Inc. MCC950 (CP-456773, 210826-40-7, AbMole, China) was purchased from AbMole Inc.

Immortalized human skin epidermal keratinocytes HaCaT were kindly provided by Professor Norbert E. Fusenig of Deutsches Krebsforschungszentrum (Heidelberg, Germany) [18 (link)]. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% FBS, 1% Gibco^® GlutaMAX™ Supplement (Thermo Fisher Scientific, Tokyo, Japan), in a 5% CO₂-atmosphere at 37 °C.

To obtain stem cells from human exfoliated deciduous teeth (SHED), human deciduous incisors were obtained from a 7-year-old child at the Nippon Dental University Hospital at Tokyo under approved guidelines set by the Committee of Ethics, the Nippon Dental University School of Life Dentistry at Tokyo (authorization number: NDU-T2012-35, August 13, 2015) [19 (link)]. Similar to our previous reports, the incisors were washed three times in ice cold PBS (−) and then cut into halves from the tooth cervix with a diamond disc. Dental pulp tissues were carefully moved out from the pulp cavity. After washing three times with growth medium MEM-α (Thermo Fisher Scientific, Tokyo, Japan) containing 20% FBS, 100 units/mL penicillin, 10 mg/mL streptomycin and 1% Gibco^® GlutaMAX™ Supplement (Thermo Fisher Scientific), the dental pulp tissues were minced into 1- to 3-mm² fragments, plated on 10-cm dishes with the growth medium, and cultured at 37 °C in a humidified tissue culture incubator with 5% CO₂ and 95% O₂. After 7–10 days of cultivation, the plastic-adherent confluent cells were treated with 0.05% trypsin containing 1 mM EDTA for 5 min to harvest pure mesenchymal cells. SHED were passaged and continuously subcultured and maintained in the complete growth medium. SHED from third to 14 passages were used in the experiments [19 (link)].

This study was approved by the Medical Research Ethics Committee of the University of Malaya Medical Centre (MREC ID No: 2021518-10145). Wharton’s jelly-derived mesenchymal stem cells (WJMSCs) isolated from the human umbilical cord were used to conduct biocompatibility analysis on the fabricated bioscaffold. Initially, the collected umbilical cord was immersed in 70% ethanol for 30 s followed by rinsing twice with PBS. Then, the inner lining of the umbilical cord tissue, known as Wharton’s jelly, was cut into approximately 5 mm in diameter and rinsed with PBS to remove the blood clots. Next, the tissue was transferred into a T25 flask containing complete media comprising 90% DMEM/F-12 (ATCC), 10% Gibco Foetal Bovine Serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA, Europe approved, South American Origin), 1% of Gibco antibiotic–antimycotic solution (100×, Thermo Fisher Scientific), and 1% of Gibco Glutamax supplement (Thermo Fisher Scientific) and incubated at 37 °C in a humid atmosphere of 5% CO₂. The isolated cells were trypsinised and grown until passage 3.

To obtain human dental pulp cells, lower third molars were obtained from adults (17–26 years old) at the Nippon Dental University Hospital at Tokyo under approved guidelines set by the Committee of Ethics, the Nippon Dental University School of Life Dentistry at Tokyo (authorization number: NDU-T2012-35, 13 August 2015). Dental pulp tissues (Figure 1b) were minced into 1- to 3-mm² fragments, plated on 10-cm dishes with the complete growth medium (MEM-α (Thermo Fisher Scientific, Tokyo, Japan) containing 20% FBS, 100 units/mL penicillin, 10 mg/mL streptomycin and 1% Gibco^® GlutaMAX™ Supplement (Thermo Fisher Scientific)), and cultured at 37 °C in a humidified tissue culture incubator with 5% CO₂ and 95% O₂. After 7–10 days, the plastic-adherent confluent cells were treated with 0.05% trypsin containing 1 mM EDTA for 5 min to harvest pure mesenchymal cells. The dental pulp cells (DPCs) were passaged and continuously subcultured and maintained in the complete growth medium. DPCs from third to seventh passages were used in the experiments.