Mrc 5 fibroblasts

Human fetal lung fibroblasts (MRC5) were purchased from ATCC and umbilical vein cells (HUVECs) were either freshly isolated from donors or purchased from Clonetics, Lonza respectively. The MRC5 were grown in minimum essential medium (MEM, Invitrogen) supplemented with glutamine, 10% fetal bovine serum (FBS) and standard Penicillin and Streptomycin (PS). The HUVEC were cultured in EBM-2 endothelial basal medium supplemented with the EGM-2 Single Quots (Clonetics, Lonza). NIH/3T3 mouse embryonic fibroblast cells were purchased from ATCC and they were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS) and standard Penicillin and Streptomycin (PS). MycoAlert mycoplasma detection kit (Clonetics, Lonza) was used to verify that cells were mycoplasma-free. The HCMV clinical strain VR1814 (a kind gift from Dr Giuseppe Gerna, University of Pavia, Italy), the HSV-1 (a kind offer from Prof. Maria Masucci’s lab, Karolinska Institute, Sweden), and the MCMV Smith strain (kindly provided by Dr. Frank Stassen, University of Maastricht, Netherlands) were used for the study. The VR1814 virus was propagated in HUVECs and titrated [60 (link)] in MRC5 fibroblasts (ATCC), and frozen at -80°C.

CD31^–CD45^–EPCAM⁺HTII-280⁺ alveolar epithelial cells were resuspended in CK+DCI medium, 3D medium (DMEM/F12 supplemented with 10% FBS, penicillin/streptomycin 1,000 U/mL, 1 mM HEPES, 1 mM l-glutamate, and insulin/transferrin/selenium from Sigma), or SAGM as shown at a density of 5 × 10³ cells/50 μL and then mixed with 3D Matrigel (Corning) containing MRC5 fibroblasts (ATCC CCL-171) at a density of 50 × 10³ cells/50 μL. A total of 100 μL of the suspension was seeded on a 0.4 mm–pore cell culture insert in a 24-well supported format (Corning). After polymerization of Matrigel, 500 μL of CK+DCI medium was added to the bottom chamber. Medium was supplemented with 10 μM Y-27632 for the first 48 hours. Medium was changed every other day. Cultures were maintained at 37°C in a humidified incubator (5% CO₂).

MRC5 fibroblasts (ATCC) and MeWo cells (Sloan Kettering Institute) were grown in minimum essential medium (MEM) (Life Technologies) supplemented with 7% fetal bovine serum, l-glutamine, nonessential amino acids, and penicillin-streptomycin. When the cells were ∼90% confluent, they were inoculated with trypsin-dispersed infected cells at a ratio of one infected cell to eight uninfected cells (18 (link)). The titer of the inoculum was 1 million infectious foci/ml. The virus strain was the completely sequenced low-passage-number VZV-32 strain (50 (link)).