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.