Mrc 5

Heparan sulfate (HS) and enoxaparin sodium (EX) were kindly supplied by Techdow Pharma S.r.l Assago Milanofiori (MI) Italy.; MRC5 (Lung Normal Fibroblast Cells ATCC^® CCL171™) and HCT-8 (Human Colon Adenocarcinoma Epithelial Cells HRT-18 CCL-244™) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured as follows.
MRC5 cells were cultured in DMEM high glucose medium supplemented with 2 mM l-glutamine, 100 U/mL penicillin–streptomycin mixture, and 10% fetal bovine serum (FBS), at 37 °C, in a 5% CO₂ humidified incubator. HCT-8 cells were cultured in RPMI-1640 medium supplemented with 2 mM l-glutamine, 100 U/mL penicillin–streptomycin mixture, and 10% of FBS, at 37 °C, in a 5% CO₂ humidified incubator. Adherent sub-confluent cell monolayers were prepared in growth medium (2% FBS) in 96-well plates for cytotoxicity assays and viral inhibition tests.
HCoV-229E and HCoV-OC43 strains were obtained from ATCC, passaged, and amplified on MRC5 and HCT8 cells, respectively.

MRC-5 (ATCC CCL-171, American Type Culture Collection, Manassas, VA, USA), HeLa (ATCC CCL-2), and Ca Ski (ATCC CRL-1550) cells were cultured in Eagle’s Minimum Essential Medium (EMEM; Sigma-Aldrich) or RPMI-160 (Sigma-Aldrich), respectively, supplemented with 10% inactivated fetal bovine serum (FBS), 2 mM L-glutamine, and 100 μg streptomycin-100 U penicillin at 37 °C in a 5% CO₂ atmosphere until confluent. HeLa cells contain HPV-18 sequences, whereas Ca Ski cells contain an integrated HPV16 genome as well as sequences related to HPV18. The CMV strain AD-169 (ATCC VR-538) was propagated in MRC-5 cell line in EMEM supplemented with 2% inactivated FBS, L-glutamine, and antibiotics.

BHK-21 (ATCC CCL-10), C6/36 (ATCC CRL-1660), Vero (ATCC CCL-81), HuH-7 (from Duke Cell Repository), HEK293T (ATCC CRL-3216), and MRC-5 (ATCC CCL-171) cells were purchased from the American Type Culture Collection (ATCC) unless otherwise stated. BHK-21 and C6/36 were cultured in RPMI 1640 (Gibco); Vero, HuH-7, and HEK293T were cultured in DMEM media (Gibco); MRC-5 was cultured in EMEM (ATCC), all of which were supplemented with 9% fetal calf serum (HyClone). All cell lines tested negative for mycoplasma. Wild-type ZIKV strains, namely PF13/251013-18⁶² (KX369547, at fourth passage in Vero, gift from Didier Musso at Institut Louis Malardé), H/PF/2013⁶³ (KJ776791, from European Virus Archive) and Paraiba (KX280026, gift from Pedro F. C. Vasconcelos at Instituto Evandro Chagas) ZIKV strains were passaged on C6/36. YF17D virus was obtained from passage of Stamaril (Sanofi Pasteur) in Vero as previously described¹⁶ . After infection, cells are maintained in maintenance media which contain 2% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Viral titers were determined by plaque assay on BHK-21 and/or Vero. All experiments performed with ZIKV were based on plaque titers obtained on Vero while YF17D was titered on BHK-21.

The human esophageal fibroblasts cell lines (FEE4-T and BEF-T) were cultured in Dulbecco’s modified Eagle’s medium and Medium 199 (4:1 ratio) supplemented with 10% fortified bovine calf serum and gentamicin (50 μg/mL). Human colon fibroblast CCD-18Co (ATCC CRL-1459) and human lung fibroblast MRC-5 (ATCC CCL-171) were cultured in Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum. For individual experiments, fibroblasts were equally seeded, maintained in full growth media, and kept in log phase growth until ready to use. We used two non-neoplastic, telomerase-immortalized, esophageal squamous cell lines (EoE1-T and EoE2-T) that were created by our laboratory using esophageal mucosal biopsy specimens from patients who had EoE, as previously described.[2 (link)] Cells were maintained in monolayer culture at 37°C in humidified air with 5% CO2 in growth medium co-cultured with a fibroblast feeder layer as previously described.[30 (link)] For individual experiments, epithelial cells were equally seeded into standard culture dish without fibroblast feeder cells and maintained in growth medium.

To verify the long-term transplantation safety of MSCs in healthy and ACLF mice, we performed a 24-week tumorigenicity study as previously described elsewhere [30 (link)]. Healthy 7-week-old C57BL/6J male mice (with or without ACLF induction) received 1 × 10⁷ MRC-5 (negative control; ATCC, Manassas, VA; #CCL-171), 1 × 10⁷ ES-D3 (positive control; #CRL-11632), or 1 × 10⁷ hADMSCs (MSCs group, with or without pIRES-Nrf2-DKK1 plasmid pre-transfection) (12 mice per group for healthy and 18 mice for ACLF group). After 24 weeks, or when the mice exhibited severe symptoms of dyspnea and minimal activity, they were sacrificed to assess the extent of tumor formation.