Osteogenic media

Human mesenchymal stem cells (MSC, Lonza) were allowed to grow until they reached 70% confluency and then seeded onto a 6-well plate to initiate differentiation processes. The cells were cultured in MSC growth media (low glucose DMEM, 10% FBS, 5% Pen/Strep, Gibco), adipogenic media (Lonza), or osteogenic media (Lonza) for one week. adipogenic media was rotated between induction and maintenance every 3 days. Cells were then lifted off the substrate with 0.25% trypsin (Sigma Aldrich) and seeded onto glass-bottom dishes (Fig. 1D) and imaged using the multi-modal SLIM system. At the end of imaging, to confirm cell lineage, the cells were fixed with 4% paraformaldehyde (PFA) for 20 minutes and incubated in 60% isopropanol for 5 min followed by immersion in Oil Red O working solution (3:2; 300 mg/mL Oil Red O in isopropanol:DI water, Sigma Aldrich) for 10 min and then BCIP/NBT (Sigma Aldrich) for 10min (Fig 5C).

hMSCs (Lonza) were cultured in MSCGM (Lonza) and seeded (10,000 cells/cm²) on hydrogel surfaces. Cells were cultured for up to 21 days in osteogenic media (Lonza). After 3 days of culture in osteogenic media, cells were incubated in 2 μM calcein and 4 μM ethidium homodimer for 30 minutes for Live/Dead staining and imaged on a Zeiss fluorescence microscope. At 14 days hMSCs were lysed and assayed for alkaline phosphatase activity (ALP) by incubating with MUP substrate. hMSCs were scraped in PBS, transferred to cold 50 mM Tris-HCl and sonicated to lyse the cells. The total protein content for each lysate sample was determined using a BCA assay kit (Thermo Scientific). Samples were diluted to the same total protein content before assaying for ALP. Samples and ALP standards were loaded into a 96-well plate, then incubated with MUP substrate at 37 °C for 1 hour and read at 360 nm excitation/465 nm emission. Mineral deposition at 21 days post-induction was assayed by Alizarin Red staining and extraction. Cells were fixed in 10% formalin, rinsed in water and incubated in 2% Alizarin Red solution for 20 minutes. After 4 washes in water, the stained cells were scraped in 10% acetic acid and heated to 85 °C for 10 minutes. The supernatant was collected after centrifugation, neutralized with 10% ammonium hydroxide and read at 405 nm.