Cellbind

293T cells (Thermo Fisher Scientific #R78007, female) were cultured for 48 h in CellBIND (Sigma Aldrich, St. Louis, MO, USA) culture dishes using Pro293 medium (Lonza, Basel, Switzerland) supplemented with 1% GlutaMAX Supplement (Life Technologies, Zug, Switzerland) and 1% (v/v) Penicillin-Streptomycin (10,000 U/mL, Life Technologies, Zug, Switzerland) in a CO2 incubator (New Brunswick Galaxy 170 S, Eppendorf, Schönenbuch, Switzerland) at 37°C, 5% CO2.

Inhibition of endosomal acidification was measured using LysoTracker Red DND-99 (ThermoFisher). Vero cells (ATCC) were seeded in 96-well clear CellBind plates (Sigma-Aldrich) 24 h prior to the experiment at a density of 40,000 cells/well in a 100 μL volume of complete DMEM supplemented with 10% inactive FBS and 1% of penicillin/streptomycin. On the day of the experiment, the medium was replaced with 100 μL of serum free DMEM. A volume of 0.4 μL of each compound from the Spectrum Collection library (MicroSource), consisting of 2,560 individual compounds formatted as 10 mM solutions in DMSO, was incubate with the cells at 37°C for 2 h. Following this, 0.1 μM final concentration of LysoTracker Red DND-99 (ThermoFisher) was added to each well and incubated for 30 min at 37°C. The cell medium was then replaced with 100 μL of FluoroBrite DMEM (ThermoFisher). Fluorescence at excitation/emission 574/594 nm was measured using an Envision plate reader (Perkin Elmer). Data were plotted using Prism 9 (GraphPad).

Following 72 hours storage, the released MAPC were counted and plated at 2 x 10⁴ cells/cm² in a CellBIND (Sigma-Aldrich) 24-well plate and incubated for 24 hours in a humidified incubator (37°C, 5% CO₂) before capturing images. Attached cell number was assessed using the alamar blue assay. After 1 hour of incubation at 37°C, fluorescence was taken at an excitation of 545 nm and emission of 590 nm using Varioskan^™ LUX Multimode Microplate Reader (ThermoFisher Scientific). Plates were then stained with methylene blue to assess cell number as previously described [31 (link)]. After washing with sterile PBS, absorption was measured at 650 nm using Infinite F50 plate reader (Tecan Life Sciences, UK). Cell number was assessed using a standard curve produced from non-stored cells. Attachment efficiency was calculated as the number of attached cells divided by the number of seeded cells. Functional cell recovery was calculated as the number of viable cells multiplied by the attachment efficiency.