Nutristem complete media

MSCs were seeded into 48-well plates with a seeding density of 15,000 cells/per well. The day after, cells were infected by H1N1 IAV (A/FM/1/47-MA) at an MOI of 1 diluted in OptiMEM(100 μl/well). After 1 h, the infection media were removed, and cells were washed twice with PBS. MSCs were cultured in Nutristem complete media (Biological Industries) at 37°C for 24 h, 48 h, and 72 h. For LDH assay and testing virus titer, conditioned media of each group were collected at each time point, and then cells were washed with PBS and fixed using 4% paraformaldehyde (PFA). Lactate dehydrogenase (LDH) measurement was performed using the CytoTox 96^® Non-Radioactive Cytotoxicity Assay (Promega). For virus titer, IAV RNA was isolated from conditioned media using the QIAamp Viral RNA Mini RNA isolation kit (QIAGEN), and the qPCR was performed using QuantiTech Virus Kit (QIAGEN) and analyzed in CFX384 Thermocycler (Bio-Rad). Cell images were obtained using Incucyte S3, objective 20x (Sartorius). For nuclei counting of attached cells, DAPI (4′,6-diamidino-2-phenylindole, Thermo Fisher) staining was used to identify nuclei. The fluorescent images were captured and analyzed (Cellomics ArrayScan VTI HCS Reader, Thermo Fisher Scientific, CA, United States). For publication purposes, the brightness of all images was enhanced at the same percentage (+20%) to optimize visualization.

Bone marrow aspirates were obtained from a commercial vendor (Lonza) or at The Ottawa Hospital (with informed written consent and ethical approval granted by the Ottawa Health Science Network Research Ethics Board, REB ID: 20120929-01H). Human bone marrow-derived MSCs (n = 3 different donors; 2 females and 1 male) were cultured, characterized, and cryopreserved, as described previously (McIntyre et al., 2018 (link); Tan et al., 2019 (link)). MSCs have been characterized to meet all the criteria (plastic adherence, differentiation potential, and cell surface antigen expression) proposed by the International Society for Cellular Therapy (ISCT) (Dominici et al., 2006 (link)) as previously described (McIntyre et al., 2018 (link); Tan et al., 2019 (link)). MSCs were thawed and cultured in Nutristem complete media (Biological Industries) for 24–72 h prior to being lifted and plated for in vitro assays or in vivo studies. MSCs were cultured and maintained at 37°C in a humidified incubator containing 5% CO₂. All experiments used cells between passages 3 to 5.