Oxygen levels within live spheroids cultured with/without PFC-MPs
To better understand MP effects on oxygen levels in our spheroid model, we used RuDPP and fluorescent microscopy building upon our earlier validation experiments without cells. For the spheroid system, we mixed cell suspension with different amounts of RuDPP to find an optimum concentration required for the formation of spheroids with RuDPP(Figure S1A). Although the working concentration of dye in our system is low, we were able to utilize this concentration to directly image intracellular oxygen levels. To study cellular oxygen levels throughout spheroids we made 3D images of z-stacks and looked at the fluorescence intensity throughout the whole spheroid as shown inFigure 3A . We observed cells at higher oxygen tensions (shown in black) in spheroids cultured with PFC-MPs, demonstrating that MPs improve oxygen levels in the spheroids. Quantitative cellular measurements corresponding O2% were performed via a calibration curve based on RuDPP in a controlled oxygenated environment(Figure S1C). To perform this, we focused on individual cells and the intensity of those cells along a line drawn in a central confocal plane of spheroids. As shown in Figure 3B, the addition of MPs increased oxygen levels from 7 to 10%, emphasizing the validity of our approach. In related work, partial pressure of oxygen (PO2) levels in human colorectal spheroids with a diameter of ∼600 μm were measured using electron spin resonance (ESR) microscopy, and PO2 values were reported in the range of 50-60 mmHg which is equivalent to 6.6-7.9% oxygen at inner regions.[29] In vitro experiments are usually performed in incubators that maintain a PO2 of ~142 mmHg (18.7% oxygen), whereas cells in our body do not experience a PO2 greater than 100 mmHg (13% oxygen). Despite lower oxygen tensions in vivo , cells can tolerate these levels and easily survive because of short transport distances from supply via blood vessels.[30] When it comes to avascular 3D cell aggregates, especially larger ones, we expect lower PO2 due to greater distances and mass transport limitations. The Hypoxyprobe™ (pimonidazole hydrochloride) method is often used to study hypoxia responses in spheroids;[31] however, the method is an indirect endpoint test with no quantitative analysis in terms of oxygen tensions. Using RuDPP is advantageous for live and kinetic imaging since the fluorescence lifetime of RuDPP is largely insensitive to pH, ion concentrations, and cellular contents, making it suitable for cell culture applications.[32] Previous work has shown that MCF-7 cells labeled with RuDPP at a concentration of 20 µg.mL−1 maintained 90% viability after 24h,[33] confirming its biocompatibility.