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All heMSC-microcarrier constructs derived from either spinner or shake flask cultures and cell-only pellets derived from tissue culture plastic (at 70% confluency) were generated by seeding cells attached to microcarriers or cells only in clear round-bottom ultra-low attachment 96-well plates (Corning) at 1 construct or 1 pellet per well in MSC growth medium at day 0 of differentiation. After 1 day, they were then differentiated in chondrogenic differentiation medium containing DMEM-high glucose (Gibco), 1 mM sodium pyruvate (Gibco), 100 nM dexamethasone (Sigma), 0.1 mM l-ascorbic acid-2-phosphate (Sigma), 1% vol/vol ITS + 1 (Sigma), l-proline (Sigma), 1% vol/vol penicillin/streptomycin (Gibco), and 100 ng/ml recombinant human BMP2 (CHO-derived; R&D Systems), with a medium change every 2 to 3 days for a total of either 21 days for the screening study or 28 days for all other experiments.
The differentiating culture was tested at day 21 for the screening study and weekly for 28 days for all other experiments.

Tails from bovines were obtained from a local abattoir. As we used leftovers of the slaughterhouse, no approval of an ethical committee was required according to Chinese regulations. IVD isolation and cultivation were carried out as previously described on day 0 [19 (link)]. In brief, after dissecting the surrounding soft tissue, individual IVDs with endplates were resected with a band saw. The endplates were rinsed with phosphate-buffered saline (PBS) using an APEXPULSE Disposable Pulse Lavage system (Apex, Guangzhou, China). Then, the IVDs were washed with PBS containing 10% penicillin/streptomycin (Gibco, Waltham, MA) for 15 minutes on a shaking table. The IVDs were then incubated with Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, Munich, Germany) supplemented with 2% fetal calf serum (FCS), 1% penicillin/streptomycin, 1% ITS+1 (Sigma-Aldrich), 50 μg/ml L-ascorbic acid (Sigma-Aldrich), and 0.1% Primocin (InvivoGen, San Diego, CA, USA) in a humidified (85%) atmosphere with 5% CO₂ at 37°C. All IVDs were cultured under physiological loading (0.02–0.2 MPa; 0.2 Hz; 1 hour/day) within a custom-designed bioreactor from day 1 (Figure 1(a)). The culture medium was replaced once a day after physiological loading. IVDs from the same tail were randomly assigned to different groups. IVDs subjected to only physiological loading culture were assigned to control (CON) groups.

Semi-confluent cells (~ 80%) were first washed in PBS, followed by the addition of RV43 (MOI20) or H3N2 (MOI5 or 4x10⁵pfu/ml) in a final volume of 500 μl bronchial epithelial cell basal media (BEBM; LONZA Clonetics Walkersville USA) with 0.1% insulin-transferrin-sodium selenite liquid media supplement (ITS+ 1 Sigma-Aldrich Co, Castle Hill, NSW, Australia). During infection, pNECs were incubated either at room temperature with shaking (RV43) or 37 °C without shaking (H3N2). After one hour, virus-containing media was removed, cells were washed twice with PBS and 1 ml of fresh BEBM with 0.1% ITS was added to each well. All experiments were performed in duplicate, or where possible, triplicate. After 48 h of incubation at 33 °C (RV43) or 35 °C (H3N2), supernatant was collected, briefly centrifuged to remove any cellular debris and stored at -80 °C for subsequent cytokine/chemokine and viral analyses.

For chondrogenesis in pellet cultures, isolated hMSCs (5 × 10⁵) were resuspended and spun in a 15-mL polypropylene tubes, centrifuged at 600g for 6 min and resuspended in chondrogenic medium consisting of high glucose DMEM containing 1 mM sodium pyruvate, 100 nM dexamethasone, 1% ITS + 1 (Sigma) [insulin (5 mg/mL), transferrin (5 ng/mL), and sodium selenite (5 ng/mL), 1% PS and bovine serum albumin (BSA; 1 mg/mL)], 40 μg/ml proline, and ascorbic acid (50 μg/mL) (all from Sigma-Aldrich, Poole, U.K., http://www.sigmaaldrich.com), and 10 ng/mL recombinant human transforming growth factor (TGF)-beta3 (Peprotech, Rocky Hill, NJ, USA). Cells were centrifuged at 600g for 5 min to form pellets. The pellets were cultured in a total of 1 mL chondrogenic medium per tube and incubated at 37 °C in 5% humidified CO₂. The medium was changed every 2 to 3 days and pellets were centrifuged with every medium change.
Pellets were harvested at 7 and 21 days of culture and were washed with PBS to remove medium then transferred to RNase-free 1.5-mL micro centrifuge tubes for RNA isolation. Others were harvested at 21 days for histological and immunohistochemical analyses.

Adipogenic, chondrogenic, and osteogenic differentiation of SHED and ASC were carried out as previously described at the third passage [19 (link)]. The cultures were initiated at a density of 1000 cells/cm² in six-well plates and grown until confluence and then subjected to differentiation into adipogenic, chondrogenic, and osteogenic lineages. Briefly, the adipogenic lineage was initiated by inducing the cells with 10% FBS, 200 μM indomethacin, 0.5 mM 3-isobutyl-1-methylxanthine (IMBX), 10 μg/mL insulin, and 1 μM dexamethasone (all from Sigma-Aldrich). Lipid droplets were visualized using oil red staining (Sigma-Aldrich). For chondrogenic differentiation, cells were cultured in medium supplemented with ITS+1 (Sigma-Aldrich), 50 μM L-ascorbic acid 2-phosphate, 55 μM sodium pyruvate (Invitrogen), 25 μM L-proline (Sigma-Aldrich), and 10 ng/mL transformation growth factor-β (TGF-β; Sigma-Aldrich). Assessment of proteoglycan accumulation was visualized by Alcian Blue staining (Sigma-Aldrich). Osteogenic differentiation was stimulated in a 3-week culture period in medium supplemented with 10% FBS, 10–7 M dexamethasone, 10 mM glycerol phosphate (Fluka, Buchs, Switzerland), and 100 μM L-ascorbic acid 2-phosphate. The assessment of calcium accumulation was visualized using von Kossa staining (Sigma-Aldrich).