npj | imaging Article

https://doi.org/10.1038/s44303-024-00013-7

Nondestructive, longitudinal, 3D oxygen

imaging of cells in a multi-well plate using pulse
electron paramagnetic resonance imaging
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1,4 1,4 2 2 1,3
Safa Hameed , Navin Viswakarma , Greta Babakhanova , Carl G. Simon Jr. , Boris Epel &
Mrignayani Kotecha 1

The use of oxygen by cells is an essential aspect of cell metabolism and a reliable indicator of viable
and functional cells. Here, we report partial pressure oxygen (pO2) mapping of live cells as a reliable
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indicator of viable and metabolically active cells. For pO2 imaging, we utilized trityl OX071-based
pulse electron paramagnetic resonance oxygen imaging (EPROI), in combination with a 25 mT
EPROI instrument, JIVA-25™, that provides 3D oxygen maps with high spatial, temporal, and pO2
resolution. To perform oxygen imaging in an environment-controlled apparatus, we developed a
novel multi-well-plate incubator-resonator (MWIR) system that could accommodate 3 strips from a
96-well strip-well plate and image the middle 12 wells noninvasively and simultaneously. The MWIR
system was able to keep a controlled environment (temperature at 37 °C, relative humidity between
70%–100%, and a controlled gas flow) during oxygen imaging and could keep cells alive for up to
24 h of measurement, providing a rare previously unseen longitudinal perspective of 3D cell
metabolic activities. The robustness of MWIR was tested using an adherent cell line (HEK-293 cells),
a nonadherent cell line (Jurkat cells), a cell-biomaterial construct (Jurkat cells seeded in a hydrogel),
and a negative control (dead HEK-293 cells). For the first time, we demonstrated that oxygen
concentration in a multi-well plate seeded with live cells reduces exponentially with the increase in
cell seeding density, even if the cells are exposed to incubator-like gas conditions. For the first time,
we demonstrate that 3D, longitudinal oxygen imaging can be used to assess cells seeded in a
hydrogel. These results demonstrate that MWIR-based EPROI is a versatile and robust method that
can be utilized to observe the cell metabolic activity nondestructively, longitudinally, and in 3D. This
approach may be useful for characterizing cell therapies, tissue-engineered medical products, and
other advanced therapeutics.

Cell and gene therapy, tissue engineering, and regenerative medicine hold T cell therapies16–18, musculoskeletal regeneration19–22, and almost all tissue
promise to make breakthroughs in many medical fields, such as cancer, regeneration and growth. Multiple approaches are being employed across
type I diabetes, sickle cell disease, musculoskeletal damage, trauma, and the fields to overcome hypoxia and to keep cells viable and functional for
wound healing1–4. These fields rely on autologous or allogeneic functional therapy purposes before, during, and after implantation. Another chal-
and viable cells for repair and regeneration5–7. Although promising, many lenge is the assessment of cell viability without destroying the product.
hurdles remain to utilize the full potential of these advanced therapies8. Currently, no method can provide a quantitative measure of viable cells
Hypoxia is a major bottleneck. It has been shown that adequate oxyge- without destroying them in the process. The issue is exacerbated if the cells
nation is the key to the success of beta cell replacement therapies9–15, CAR- are encapsulated in a scaffold. Ideally, the metabolic activities of cells

1
Oxygen Measurement Core, O2M Technologies, LLC, 2201 W Campbell Park Dr, Chicago, IL 60612, USA. 2Biosystems and Biomaterials Division, National
Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD 20899, USA. 3Department of Radiation and Cellular Oncology, The University of
Chicago, 5841 S Maryland Ave, Chicago, IL 60637, USA. 4These authors contributed equally: Safa Hameed, Navin Viswakarma.
e-mail: [email protected]

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should be assessed via a non-destructive, 3D, and longitudinal method with a single exponential recovery fitting (Fig. 1c) and imaged using a 3D
before and after implantation. radial acquisition scheme and static gradients (Fig. 1d). Filtered back
The use of oxygen by cells and tissues is a fundamental aspect of basic projection (FBP) reconstruction is utilized for image reconstruction31–36.
redox biology. Oxygen consumption is the major component of cellular Trityl, which does not consume oxygen during oxygen reporting or enter
respiration and is a direct indication of viable and functional cells9. The cells, is an ideal molecule to report extracellular pO237. EPROI has high
amount of oxygen utilized by cells varies across species, cell types, and sensitivity (<1 torr) under hypoxic conditions and lower sensitivity
metabolic pathways9,23. Therefore, oxygen measurement in situ can provide (~3–5 torr) at 21% oxygen24. The reduced sensitivity at high oxygen
key information on cell health. Fluorescence-based oximetry can provide conditions is due to reduced signal-to-noise ratio (SNR) caused by
single-point oxygen measurements for cell-containing constructs. However, increased line broadening of the trityl EPR line, leading to increased error
oxygen imaging has the advantage of providing spatiotemporal information in the fit for the T1 calculation27. Fig. 1e provides the relationship between
regarding local oxygen concentrations24. Oxygen imaging methods, such as R1 and pO2 for materials used in the current study (PBS, culture medium,
blood-oxygen-level dependent magnetic resonance imaging (BOLD MRI) and VitroGel). For the past two decades, EPROI has been primarily used
and photoacoustic imaging, rely on the vascular structure or paramagnetic for cancer hypoxia research with few notable results, such as the
properties of hemoglobin making them unsuitable for in vitro oxygen demonstration of oxygen-guided radiation therapy in three different
measurements. tumor models and rabbit VX-2 tumor pO2 imaging using a human-sized
Pulse electron paramagnetic resonance (EPR) oxygen imaging EPROI instrument38–43. Recently, the applications of EPROI have been
(EPROI) is an emerging oxygen imaging method that provides 3D partial extended to the assessment of biomaterials, engineered grafts, and cell
pressure of oxygen (pO2) with high spatial, temporal, and pO2 encapsulation devices13,14,24,30,44–48.
resolution24–30. EPROI, where unpaired electrons are probed, is similar to In this work, our goal was to develop an incubator-resonator for a
MRI (can be called eMRI or electron MRI), but with a few key differences: multi-well plate and perform oxygen imaging of live cells while using a
(a) it uses a lower magnetic field (typically 9 to 42 mT) due to the 658 times custom-built incubator to provide physiological conditions (temperature,
higher magnetic moment of electrons compared to protons, and (b) it humidity, CO2) during imaging. Although EPROI is not restricted to any
requires exogenous contrast agents due to the extremely short lifetimes specific size or shape of an object, the multi-well plate was chosen because it is
and relaxation rates of EPR-sensitive endogenous probes in the body. For a standard consumable in biological labs and allows multiple replicates to be
the past two decades, the trityl radical OX071 (Fig. 1a, chemical formula: assessed simultaneously to increase statistical power. We used our recently
C52H39D24O18S12, molecular weight: 1384.97) has been used for EPROI introduced 25 mT instrument, JIVA-25™, for oxygen imaging. The instru-
because of its useful EPR properties (stable with narrow line width and ment has a 10 cm bore gap and is suitable for oxygen imaging in vitro and in
long relaxation time, in the range of 1–10 μs) and suitable biophysical rodents13,14,24,49,50. Most EPROI resonators for in vitro and in vivo oxygen
properties (high-water solubility, non-toxic nature)24,31. The spin-lattice imaging are cylindrical. The largest EPROI resonators to date are 32 mm in
relaxation rate (R1 = 1/T1) of trityl OX071 and its linear relationship with diameter for mice (JIVA-25TM) and 57 mm for a rabbit leg (for a 9 mT
pO2 are utilized for oxygen imaging27. The R1 is measured using the human-size EPROI instrument)43,44,51. Larger resonators are a challenge
inversion recovery electron spin echo (IRESE) (Fig. 1b) pulse sequence because the signal-to-noise ratio (SNR) drops as a square root of volume

Fig. 1 | Schematic of EPROI. a Ball-and-stick model of oxygen sensitive spin probe, static gradients that are used for image acquisition. e R1 vs pO2 calibration curves for
OX071. Carbon atoms are depicted in grey, hydrogen in white, deuterium in green, the materials used in the current project. The slopes and intercepts are: 109.664 torr/
oxygen in red, and sulfur in gold. b Inversion recovery electron spin echo pulse Ms−1, 0.181 Ms−1; 123.3941 torr/Ms−1, 0.1537 Ms−1; 113.484 torr/Ms−1, 0.130 Ms−1
sequence. c single exponential recovery fit of the signal as a function of delay, T, that for PBS, DMEM cell culture medium, and VitroGel, respectively.
is used to calculate R1 (= 1/T1). d radial k-space acquisition scheme shown in 2D with

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(SNR ∝ 1/sqrt(V))24,43,44. To investigate the oxygen concentration of cells in a with no cells (CM), 5000 cells per well (5 K), and 50,000 cells per well (50 K).
multi-well plate in a controlled environment, we designed an environment- The cells were arranged in the middle 12 wells as shown in Fig. 3a. Figure 3b
controlled multi-well plate incubator resonator (MWIR) that was rectan- shows the representative pO2 maps from these middle 12 wells at the
gular for the best filling factor. beginning of the experiment. Interestingly, the wells with 50 K cells had
The ability of MWIR to perform oxygen imaging of live cells was lower oxygen tension than CM or 5 K cells, even though all wells were
tested using an adherent cell line (HEK-293), a nonadherent cell line individually supplied with 19.95% O2 gas flow. The lower oxygen tension for
(Jurkat cells), and Jurkat cells seeded in a hydrogel. For the experiments wells with 50 K cells represents a higher oxygen demand. Figure 3c provides
with cells, two identical sets of wells were prepared by seeding cells into strip the box plots of pO2 values vs cell density at t = 0. The difference in pO2
wells. One set was used for oxygen imaging in the MWIR, while the second values between CM and 5 K cells (p-value = 0.0002) and between 5 K cells
set was placed inside the standard biological-oxygen demand (BOD) and 50 K cells (p-value < 0.0001) is statistically significant, showing that pO2
incubator as a control. We performed oxygen imaging of cells with varying maps can be used to assess cell viability.
densities to assess the sensitivity of the method. We performed longitudinal We performed the oxygen imaging experiments for 2 h, 4 h, 8 h, and
oxygen imaging to assess the change in cell metabolism as a function of 24 h and compared cell viability for the MWIR versus BOD using the MTT
time. We also assessed dead cells as a negative control. We demonstrate assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for
oxygen imaging of live cells as a method to non-invasively assess their cellular dehydrogenase activity. The pO2 images and cell viability using
viability in 2D and 3D culture. MTT assay for MWIR versus BOD are similar for all time points and no
significant differences were observed (Fig. 3d). The pH values (Fig. 3e) and
Results cell morphology (Fig. 3f) of cells in the MWIR system were also consistent
Our first goal was to design hardware for environmental control during with the BOD incubator. These results demonstrate that EPROI imaging in
oxygen imaging of cells in 96-well strip-well plates by controlling tem- the MWIR does not affect cell viability or metabolic activity during a 24 h
perature, CO2, and humidity. Recently, we introduced a 25 mT experiment.
(720 MHz resonance frequency) EPROI instrument JIVA-25™, and we Next, control experiments using dead HEK-293 cells were performed
designed and tested a multi-well plate incubator resonator (MWIR) for to verify oxygen imaging results. The same three seeding densities and
the instrument. Figure 2 shows the schematics and components of seeding patterns were used as in Fig. 3a. The cells were killed either by
MWIR. JIVA-25™ has a 10 cm air gap between the magnet poles where exposing them to 4% paraformaldehyde (PFA) for 30 min or to 70 °C for
the MWIR was to be placed. The MWIR has three components: (a) a 15 min, as confirmed by morphological assessment. The pO2 images of dead
rectangular resonator for oxygen imaging (Fig. 2a) (b) a multi-well plate cells were taken immediately after killing the cells. As expected, the differ-
enclosure to keep the cells at desired high humidity and gas (95% ence in pO2 maps of dead cells with 0 (CM), 5000 (5 K), and 50,000 (50 K)
air + 5% CO2) conditions (Fig. 2b, c), and (c) heating control to main- cells per well is less pronounced but statistically significant compared to the
tain the temperature of 37 °C (not shown). live cells in both sets, as shown in Fig. 4a, c (live cells), b & d (dead with PFA)
Resonators are special volumetric units in magnetic resonance and c & g (dead with heat-shock). Note that the medium was also treated
instruments that confine radiofrequency energy and allow it to interact with with heat or PFA for dead cell experiments. The MTT assay (Fig. 4g–i)
the studied object52–54. Typical EPROI resonators are cylindrical. However, shows no significant difference between the MWIR and BOD. Figure 4j–l
to accommodate a multi-well plate, we designed and tested a rectangular shows trypan blue staining for assessing cell morphology and viability. Some
2-gap loop-gap resonator of 42 mm L × 38 mm W × 26 mm H (Fig. 2a). cells are still alive after PFA treatment (Fig. 4k), which could explain the
This is one of the largest volume resonators used in the field of EPROI. The lower pO2 in the wells for this treatment. It is possible that dead cells and the
cross-section of this resonator can accommodate three strip wells of a 96- treatment of the medium with PFA or heat-shock change the medium
well strip-well plate (Fig. 1D). Only the middle 12 wells (3 × 4) fit inside the viscosity and oxygen permeability in this medium, which is reflected by the
volume of the resonator are imaged. The rest of the wells, while being statistically significant difference in pO2 values for the dead cells. These
maintained at the constant temperature, humidity, and gas conditions, are results confirm that pO2 imaging is a sensitive tool that can differentiate
outside the imaging volume. between live and dead cells.
The second component (Fig. 2b, c) is the sealed multi-well plate Next, the sensitivity of oxygen imaging to cell density was assessed.
enclosure, with precisely controlled delivery of the necessary gas mixtures. HEK-293 cells were seeded at six different densities: 0 (CM), 10,000 (10 K),
For all experiments, the environment was maintained at a humidified state 25,000 (25 K), 50,000 (50 K), 75,000 (75 K), and 100,000 (100 K) cells per
with 95% air plus 5% CO2. The system can accommodate any other gas well. Figure 5a shows the arrangement of cells in the wells with two replicates
conditions, such as 5% O2. An integrated two-channel (hot-cold) control of each cell density. Figure 5b shows the representative pO2 maps of these
system was implemented to maintain the temperature at 37 °C. Ducts and wells and confirms that pO2 mapping can differentiate between these cell
air directors were built into the MWIR to enable air circulation from all sides densities. The box plot at t = 0 shown in Fig. 5c shows that the wells with
of the plate enclosure (Fig. 2b, c). higher cell densities had lower pO2, and the difference between different cell
The MWIR can image the 12 middle wells of 3 single strips from a 96- densities is statistically significant. Figure 5d shows how the average pO2
well stripwell plate (Fig. 2d), while keeping the temperature constant at evolved over the course of 4 h. As cells differentiate, proliferate, become
37 °C ± 1 °C (Fig. 2e) and relative humidity (RH) between 70% and 100% dormant, or die, their oxygen consumption rate changes, which can be
(Fig. 2f) by controlling the flow rate of the gas mixture between 3.75 sccm reflected in pO2 values. Most interesting are the time trends of 50 K, 75 K,
(standard cubic centimeter per min) to 30 sccm. A high gas flow rate and 100 K cells per well. We can see that within 4 h, the mean pO2 values of
maintained high humidity, but the system became susceptible to con- wells with 50 K cells matched the pO2 of wells with 100 K cells, where cell
densation in some of the wells, which affected cell viability beyond 8 h. dormancy or cell death was probably happening because of overcrowding.
Reducing the gas flow rate eliminated condensation without compromising The pO2 for 25 K cells dropped over time, indicating increased metabolic
cell viability. Therefore, a gas flow rate of 3.75 sccm was used for all activity in these wells. A comparison of the MTT assay for cells in the MWIR
experiments with cells. The MWIR was tested for its ability to provide a with cells in the BOD incubator shows no significant difference in cell
controlled environment for oxygen imaging using PBS in the middle viability except for 100 K cells per well (Fig. 5e). These experiments
12 stripwells (Fig. 2g). demonstrate the ability of EPROI to observe longitudinal cellular oxygen
For experiments with cells, two identical sets of wells were prepared by consumption and to differentiate between six different cell densities. One
seeding HEK-293 cells into strip wells. One set was used for oxygen imaging limitation of pO2 imaging technology could be when cells change their
in the MWIR, while the second set was placed inside the BOD incubator as a metabolism due to the environment; they may show similar pO2 as is the case
control. Three cell densities were used in quadruplicate: control medium for 50 K, 75 K, and 100 K cells in Fig. 5d. A similar cell morphology was

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Fig. 2 | Schematics of the MWIR apparatus. a A rectangular resonator without air removal from MWIR showing that the temperature was maintained at 37 °C.
directors and covers. b a cross-section of multi-well plate enclosure and the reso- f Relative humidity plot shows that a gas flow rate of 30 sccm sets the humidity at
nator, air flow is shown using yellow direction arrows. c a resonator with air director 100% while 3.75 sccm sets the humidity at 70%. g Representative pO2 maps (slice #4
(left) and cover (right) installed; a two-channel air intake is visible. d Three 8-well at ~ 3.2 mm from the bottom of the well) of the middle 12 wells (columns C to F)
stripwells from a 96-well plate with columns C to F containing 150 μL of culture filled with PBS during reoxygenation with 95% air + 5% CO2 after N2 bubbling.
medium with 1 mM OX071. The 12 wells outlined in green were used for pO2 SCCM (standard cubic centimeter per min).
imaging. e Thermal image of the multi-well plate enclosure immediately after

observed for cells at all seeding concentrations after 4 h in the MWIR or and 100,000 (100 K) cells per well, triplicate for each cell density) were
BOD (Fig. 5f). made. One was placed in the MWIR and the other in the BOD incubator
The experiments in Fig. 5 show that cell viability was similar for the and, after 4 h, pO2 images were acquired for both sets. Figure 6a shows
MWIR and BOD, even though low pO2 was observed for high-cell the arrangement of cells in the middle 12 wells in the plate, and Fig. 6b, c
density wells (50 K, 75 K, 100 K). This raised a question of whether the shows the representative pO2 maps of the cells in MWIR and in the BOD
high cell density wells in the BOD incubator were also experiencing low incubator for 4 h, respectively. Interestingly, similar to MWIR, the cells
pO2. This was tested by the following experiment. Two identical sets of in the BOD incubator also experienced low pO2 for high cell densities.
plates with four cell densities (0 K (CM), 10,000 (10 K), 50,000 (50 K), Figure 6d shows the pO2 values for MWIR and BOD. In all cases, there

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Fig. 3 | Oxygen imaging of HEK-293 cells in MWIR. a The pattern of cell seeding viability measured by MTT assay after 24 h of imaging shows no significant dif-
for control (CM), 5 K, and 50 K cells per well (n = 4). Each well had 150 μL of ference between cells in the MWIR and BOD incubator at 5 K and 50 K densities
medium. b Representative pO2 maps of wells (slice #4, ~3.2 mm from the bottom of (p > 0.05). e pH of the medium from wells that were in MWIR and in the incubator
the well) in the transverse plane at t = 0 h (first imaging experiment). c Box plot of after 24 h. f Cell morphology after 24 h imaging from MWIR and BOD.
pO2 for each cell density; differences are statistically significant (p ≤ 0.001). d Cell

are no significant differences between MWIR and BOD (except consumption rate (OCR). For Jurkat cells in VitroGel, the metabolic
10 K, *p ≤ 0.05). activity of cells is reduced by 17.3% for 50 K cells and by 4% for 5 K cells
The above experiments were performed using HEK-293 adherent compared to Jurkat cells in medium, providing a quantitative assessment
cells that were attached to the bottom of the well. Next, we performed of the impact of cell seeding on viable cells.
oxygen imaging of a non-adherent cell line, Jurkat cells, using MWIR. Cell Finally, we demonstrate nondestructive 3D imaging of cells seeded
densities of 0 K (CM) 5 K, and 50 K cells per well were used in the in a scaffold using EPROI. Figure 9a shows the schematic of the three
arrangement shown in Fig. 7a. Representative pO2 maps (Fig. 7b) and box cell densities (0 K (CM), 5 K and 50 K cells per well). Figure 9b shows
plots (Fig. 7c) at t = 0 show higher pO2 values for these cells compared to three slices of HEK-293 cells at 3.21 mm (Bottom), 3.57 mm (Middle),
HEK-293 (Fig. 3b, c), indicating different metabolic activity for Jurkat and 3.92 mm (Top) from the bottom. The three slices represent a
cells. The pO2 heterogeneity may be due to the Jurkat cells growing in complete 3D volume of the 150 μL of medium in each well. A gradient is
aggregates. The MTT viability assay (Fig. 7d), pH test (Fig. 7e), and cell visible from the bottom to the top of each well, especially for the wells
morphology (Fig. 7f) after 4 h of oxygen imaging show that there is no with 50 K HEK-293 cells, which are adhered to the bottom of the well, as
significant difference between the Jurkat cells in MWIR versus the BOD shown in Fig. 9c for three selected wells denoted as A, B, and C in Fig. 9b.
incubator, and EPROI using the MWIR is a noninvasive and robust Figure 9d shows the change in the pO2 values for control, 5 K and 50 K
method. cells, going from the bottom of the well to the top. Figure 9e shows the
A significant issue for assessing tissue-engineered medical products change in pO2 at t = 0 and t = 4 h for HEK-293, Jurkat, and Jurkat in
is that once the cells are seeded into a hydrogel scaffold, there is no way to VitroGel for the 50 K cells. There is a statistically significant difference
track their metabolic activity without destroying the construct. To between pO2 at the bottom and top of the well (p ≤ 0.05) for all con-
address this issue, we performed oxygen imaging of Jurkat cells seeded in ditions except Jurkat at 4 h.
a VitroGel, a commercially available glucose-based hydrogel. The cell
seeding scheme with three densities, 0 K (CM), 5 K, and 50 K cells per Discussion
well is shown in Fig. 8a. Figure 8b shows the representative pO2 maps. In this study, oxygen imaging of live cells was performed using trityl-
The box plot in Fig. 8c shows that the pO2 values were significantly OX071-based EPROI in an environment-controlled system, MWIR,
different for the wells with 5 K and 50 K cells. Interestingly, the pO2 that could accommodate 12 wells of a standard 96-well strip-well plate.
values are higher for Jurkat cells in VitroGel (Fig. 8c) compared to Jurkat The MWIR provided constant temperature, gas intake, and humidity
cells in culture medium (Fig. 3c), suggesting that Jurkat cells have in each well to maintain normal cell activity during the oxygen imaging
reduced metabolic activity upon cell-seeding or that cells are lost during of wells. While it is possible to perform point oxygen measurements
the VitroGel encapsulation process. using fluorescence-based probes, they are not suitable for 3D oxygen
Figure 8d shows the normalized pO2 values for HEK-293, Jurkat, and imaging. In addition, they are less sensitive at higher oxygen
Jurkat in VitroGel as a function of cell seeding density. The normalized concentrations24. Once the MWIR system was established, oxygen
pO2 values were calculated by dividing the mean pO2 of wells with cells by imaging of HEK-293 cells was demonstrated using three cell densities:
the mean pO2 of wells with control medium. The normalized pO2 of control (0 K), 5 K, and 50 K cells per well. We show that for metabo-
Jurkat cells in VitroGel is higher than Jurkat cells in medium or HEK-293 lically active cells, the pO2 values of the wells are significantly different
cells. HEK-293 cells have the lowest pO2 or the highest oxygen between different cell densities. After 24 h, the viability, pH and

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Fig. 4 | Comparison of pO2 of live and dead HEK-293 cells. The cells were seeded in 5 K: p = 0.0008; 5 K vs 50 K: p < 0.0001. f (Heat-shock) CM vs 5 K: p = 0.2598; 5 K vs
the same pattern shown in Fig. 3a. Representative pO2 maps and box plots of pO2 50 K: p < 0.0001. Note that the medium was also treated with PFA or heat for the
values as a function of cell density (a, d) live cells (same as Fig. 3a), (b, e) dead cells dead cell experiments. g–i MTT assay of live and dead cells after imaging shows no
(killed with 4% PFA), and (c, f) dead cells (killed with heat-shock). A significant significant difference between MWIR and BOD (p > 0.05). j–l Trypan blue exclusion
difference in the pO2 of 5 K and 50 K cells per well is observed for all three cases. assay for the confirmation of cell death before imaging. Some cells were still alive
d (live cells) CM vs 5 K: p = 0.0002; 5 K vs 50 K: p < 0.0001. e (PFA dead cells) CM vs when killed using 4% PFA (live cells are indicated by red arrows).

morphology of the cells that were in MWIR were similar to the cells that the cell density at higher cell densities55,56. EPROI with the MWIR does
were in the BOD incubator, indicating that oxygen imaging did not not have this issue and could differentiate between different cell den-
interfere with the metabolic activity of the cells. A well-known lim- sities. To test the sensitivity of the method, oxygen imaging of HEK-
itation of MTT assay is that it doesn’t follow a linear relationship with 293 cells was performed with six different cell densities (Fig. 5) and

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Fig. 5 | The sensitivity of pO2 imaging. a Six different cell densities of HEK-293 cells (32.15 ± 1.18 torr), (n = ~ 200–300 voxels per well, n = 2 per cell density). There is a
(CM (0 K), 10 K, 25 K, 50 K, 75 K, and 100 K cells per well) were seeded in the given statistically significant difference (p < 0.0001) in pO2 between CM vs 10 K, 10 K vs
pattern (n = 2). b A representative pO2 map at t = 0 h showing visual assessment of 25 K, 25 K vs 50 K, 50 K vs 75 K, and 75 K vs 100 K. d The change in pO2 as a function
cell metabolic activity. The image is taken ~3.2 mm above the bottom of the well. of time. e MTT assay after 4 h revealed no significant differences between MWIR and
c Box plot of pO2 as a function of cell density. The mean and standard error obtained BOD (p > 0.05), except for 100 K cells (p ≤ 0.05). f Morphological assessment of cells
from the pO2 map for CM (144.06 ± 4.82 torr), 10 K (116.61 ± 1.45 torr), 25 K in MWIR and BOD after 4 h of measurements.
(49.58 ± 0.74 torr), 50 K (27.97 ± 0.70 torr), 75 K (30.15 ± 0.94 torr), and 100 K

EPROI could differentiate between them. Longitudinal measurement These results confirm that pO2 imaging is a sensitive tool that can differ-
of pO2 was also demonstrated. Experiments with non-adherent Jurkat entiate between live and dead cells.
cells showed higher pO2 values compared to HEK-293 adherent cells. The oxygen imaging showed that cells seeded at high density
The higher pO2 values may indicate a lower oxygen consumption experienced low pO2 in both the MWIR and BOD incubator.
profile for these cells. This finding has serious significance as, across the world, many labs
EPROI is a 3D imaging method (Fig. 9) and was used to measure the may be subjecting their cells to low oxygen conditions without
difference in pO2 at different depths of cell culture wells and in 3D hydrogel realizing it.
scaffolds seeded with cells. The pO2 maps were heterogeneous in all cases, Finally, our experiments of Jurkat cells seeded in VitroGel (Fig. 8)
which could arise from the nonuniformity of oxygen consumption due to demonstrated the ultimate promise of this technology, which is to assess the
cell clustering within the wells. oxygen consumption and viability of cells seeded in a biomaterial without
We performed oxygen imaging with dead cells for comparison with destroying the construct. Currently, there are no other methods to non-
live cells. We used two methods for cell killing, 4% PFA and 70 °C heat- destructively assess oxygen consumption for cells in 3D for cells in tissue
shock. In both cases, the pO2 in wells with cells was higher than wells with engineered constructs.
live cells since dead cells do not consume oxygen. Interestingly, some cells Oxygen consumption is one of the many ways to assess cell via-
were still alive after PFA treatment, and correspondingly these wells had bility. Oxygen consumption can be determined by assessing the dif-
lower pO2 compared to the medium, showing the sensitivity of the method. ference in oxygen supplied versus the amount expelled. Our method is

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Fig. 6 | Assessment of pO2 for HEK- 293 cells cultured 4 h in the BOD incubator. (35.98 ± 0.99 torr) or BOD -CM (106.42 ± 1.00 torr), 10 K (110.69 ± 1.85 torr), 50 K
a The layout of the wells for 0 K (CM), 10 K, 50 K, and 100 K cells per well (n = 3). (37.10 ± 1.57 torr), 100 K (25.95 ± 1.05 torr), (n = 300–750 voxels per well, n = 3 per
Representative pO2 maps at 4 h of cells in (b) MWIR and (c) BOD incubator taken cell density). There is a significant difference (p < 0.0001) in pO2 between different
~ 3.2 mm from the bottom of the wells. d pO2 quantification between the MWIR and cell densities. The MWIR vs BOD p-values among wells are as follows: CM,
BOD incubator. The mean and standard error of pO2 are: MWIR- CM p = 0.1938; 10 K, p = 0.0179; 50 K, p = 0.5495; 100 K, p = 0.01863; MWIR: CM vs
(148.27 ± 2.97 torr), 10 K (91.77 ± 1.16 torr), 50 K (34.9 ± 0.95 torr), 100 K 10 K, p = 0.0015; 10 K vs 50 K, p = 0.00014; and 50 K vs 100 K p = 0.9415.

Fig. 7 | Oxygen imaging of Jurkat cells using MWIR. a The cell seeding pattern of and 5 K vs 50 K, p < 0.0001. d The MTT assay after 4 h of imaging showed no
Jurkat cells CM, 5 K, and 50 K cells per well (n = 4). b A representative pO2 map significant difference in cell viability between the MWIR and BOD incubator
taken at t = 0 h (~ 3.2 mm from the bottom of the well). c The box plot of pO2 values (p > 0.05). e, f pH and cell morphology confirm that the MWIR emulates incubator-
from t = 0 h. Differences in pO2 are statistically significant: CM vs 5 K, p = 0.0002 like conditions to enable in situ oxygen mapping of live cells.

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Fig. 8 | Oxygen imaging of Jurkat cells seeded in VitroGel. a The cell seeding (p < 0.0001), but no significant difference between CM and 5 K cells (p = 0.6581).
pattern for 0 K (CM), 5 K, and 50 K cells per well (n = 4). b pO2 maps of the wells at d Plot of pO2 versus cell density (0 K, 5 K, and 50 K cells per well, n = 4) for HEK-293,
time t = 0 (~3.2 mm from the bottom of the wells). c Box plot of pO2 vs cell density at Jurkat, and Jurkat in VitroGel. Error bars are standard errors.
t = 0. There is a statistically significant difference between 5 K and 50 K cells

Fig. 9 | 3D oxygen imaging of HEK-293 cells, Jurkat cells and Jurkat cells in with the highest number of pixels in the “y” direction. d The pO2 at the bottom,
VitroGel. a The cell seeding pattern of 0 K (CM), 5 K, and 50 K cells per well (n = 4). middle, and top of the wells at t = 0 for CM, 5 K and 50 K HEK-293 cells. e The pO2
b Three slices from the bottom to top showing the change in pO2 over depth for for 50 K cells at the bottom, middle and top of the wells for HEK-293, Jurkat and
HEK-293 cells. c pO2 maps of three wells with 50 K cells denoted as A, B, and C in Jurkat seeded in VitroGel at t = 0 and 4 h. There is a significant difference in pO2
b for better visualization of vertical gradient. For each well, the central slice in the yz between the bottom of the well and the top of the well for all cases except for Jurkat
plane was cut and reported. The central slice was identified by choosing the slice cells at 4 h.

based on a different principle. EPROI with MWIR measures the oxygen oxygen concentrations. This provides a snapshot of cell metabolic
equilibrium in the medium where oxygen from the atmosphere is dif- activity in real time without destroying the sample. This equilibrium
fusing into the system from the top of the well while cells in the well provides a useful, noninvasive, 3D measure of cell viability and func-
consume oxygen, which causes a shift in the equilibrium to lower tionality that matters for cell therapy devices.

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Conclusions distilled water. This resultant suspension was vortexed gently, and pH
In the current project, EPROI was demonstrated for cells in 96-well was adjusted to 7.4 using 1.0 M NaOH. At pH 7.0, the trityl dissolved
plates to be compatible with current cell culture practices. However, completely. Finally, the volume was made to 10 ml with deionized-
EPROI can be extended to other geometries by adjusting the instru- double distilled water to reach to the 72.2 mM concentration and
ment’s bore size, resonator size and design. The use of EPROI to assess sterilized through a 0.22 μm syringe filter. Deoxygenation of the
cell viability and oxygen tension may lead to better devices for sterilized stock solution was done by bubbling 100% nitrogen gas via a
addressing unmet medical needs. This nondestructive oxygen imaging needle through the rubber septum for 0.5 h to 3 h, after which the vial
technique could be used to optimize therapeutic doses for cell therapies, was sealed and stored at −20 °C. The stock was diluted to the desired
optimize tissue engineered medical products, assess drug toxicity, and concentration in sterile phosphate buffered saline (PBS, pH 7.4,
provide a new and innovative view of the live cells in these systems. Gibco, ThermoFisher Scientific, Waltham, MA, USA).
EPROI is suitable for any tissue or organ of arbitrary sizes and may
become a standard tool for quality control in cell therapy and tissue Preparation of cell culture plate for pO2 imaging
engineering. Aliquots (1.5 uL) of sterile 100 mM OX071 stock were added to each well
containing 150 uL of medium, which gives a final concentration of 1 mM
Materials and methods OX071. Strips wells were then loaded onto the multi-well plate enclosure
We used two identical sets of 8-well strips from a 96-well strip well plate for and sealed before placing them inside MWIR for imaging.
all cell experiments. One set of three 8-well strips was used for oxygen
imaging experiments using the MWIR system in JIAV-25™, while the sec- pO2 calibration
ond set of three 8-well strips was placed in the BOD incubator as a control. The pO2 calibration of cell medium and VitroGel was performed in
At the end of the experiments, an MTT assay was performed on the middle 1 mM OX071 by placing the sample in a 10 mm tube in JIVA-25TM
12 wells in both sets to determine the cell viability in both sets. Student’s vertical 10 mm resonator at 37 °C. The sample was bubbled with
T test was used to calculate the statistical significance. nitrogen and air mixture at different gas concentrations to achieve the
final desired equilibrium oxygen concentration between 0% and 21%.
Cells and culture conditions The inversion recovery sequence (Fig. 1b) and spectroscopic (no ima-
Immortalized human embryonic kidney cell line HEK-293 (CRL-1573) ging) T1 provided average pO2 values and were used to obtain pO2
and T-cell leukemia cell line Jurkat (TIB-152) were purchased from versus R1 calibration (26, 29). The pO2 calibration details (Fig. 1e) are as
ATCC, Manassas, VA, USA. HEK-293 were cultured in high glucose follows: O2 relaxation rate in PBS at 0 mmHg was 0.181 Ms−1, with a
DMEM medium (Gibco, ThermoFisher Scientific, Waltham, MA, USA) slope of 109.664 torr/Ms−1, in DMEM cell medium at 0 mmHg was
while Jurkat cells were cultured in the RPMI-1640 (Gibco, Thermo- 0.1537 Ms−1, with a slope of 123.3941 torr/Ms−1. The O2 relaxation rate
Fisher Scientific, Waltham, MA, USA). Both medium were supple- in VitroGel made with DMEM cell medium at 0 mmHg was 0.130 Ms-1,
mented with fetal bovine serum (FBS, 10% by volume) and a mixture of with a slope of 113.484 torr/Ms−1.
penicillin 100 U/ml and streptomycin 100 μm/ml. Cells were seeded in
8-well strips in three densities: 0 cells (referred control medium, CM), pO2 measurement and image processing
5000 cells/well (5K), and 50,000 cells/well (50K) in a volume of 150 µL of Oxygen imaging experiments were performed using JIVA-25™ at the
medium per well in a set seeding pattern (Fig. 3a) as well as other resonance frequency of 720 MHz. The conditions in the MWIR were a
densities and patterns as demonstrated in the text. Wells without cells humidified gas mixture of 95% air and 5% CO2 at 37 °C throughout the
but with medium only (CM) served as an internal control. The dead cells pO2 measurements. To avoid contamination, the incoming gases were
were used as a negative control. No shaking culture was applied for filtered using inline filters, and autoclaved deionized water was used in
experiments using Jurkat cells. Duplicate sets of 8-well strips with the the humidifier. Any hardware that came into contact with the strip wells,
same seeding densities and seeding pattern were kept in the BOD such as the multi-well plate cover was cleaned with 70% (by volume)
incubator as a control for the MWIR. The MWIR for oxygen con- ethanol. pO2 images were acquired using the pulse inversion recovery
centration measurements and the BOD had a humidified atmosphere electron spin echo (IRESE) sequence27,30. Prior to acquiring pO2 maps,
containing 5% (by volume) CO2 at 37 °C. No medium exchange was an amplitude map image was acquired using an electron spin echo (ESE)
conducted during the measurements. sequence that shows the signal amplitude for each well. The imaging
parameters were: 90°/180° pulse lengths 60 ns, 16-phase cycles scheme
Jurkat cells in VitroGel with free induction decay (FID) suppression, spin-echo delay 400 ns,
Jurkat cell suspensions were cultured in 75-cm2 flasks in RPMI con- equal solid angle spaced 654 projections, 67 baselines, 1.5 G/cm gradient,
taining FBS with penicillin-streptomycin to a density of 1–2 × 106 cells/ 10-time delays from 410 ns to 15 μs (410 ns, 612 ns, 912 ns, 1.361 μs,
mL. For experiments, cells were split and counted on a hemocytometer. 2.03 μs, 3.029 μs, 4.518 μs, 6.74 μs, 10.055 μs, 15 μs), 55 μs repetition
Cells suspended in culture medium were mixed with VitroGel (glucose- time, overall, 10 min image duration. R1 (1/T1) images were recon-
based polysaccharide gel, TheWell Bioscience, Cat # VHM01) hydrogel structed using filtered back projection in an isotropic 64 × 64 x 64 cube,
matrix to achieve a final concentration of 5000 and 50,000 cells per field of view 5 cm × 5 cm x 5 cm with 0.78 mm cubic voxels. Approx.
0.05 mL suspended in 20% by volume VitroGel diluted with cell culture 6–7 slices from the bottom to the top of the well and about ~ 300–800
medium. Cells in VitroGel were transferred to wells of a 96-well plate at voxels per slice were collected from each well. The R1 maps were cal-
150 µL/well. After 15 min of incubation at room temperature inside the culated voxel by voxel by fitting data to the single exponential fit and
biosafety cabinet, 50 µl of additional complete RPMI medium was converted to pO2 maps using the calibration values obtained from the
added. Cells were incubated overnight in the BOD incubator, and pO2 calibration experiment.
imaging was performed.
Data collection
Preparation of trityl stock solution The wells of 96-well plates are cylindrical with a 12.15 mm height and
Trityl (Tris(8-carboxyl-2,2,6,6-tetra(2-(1-hydroxy-2,2-d2-ethyl)) 6.25 mm diameter (Fig. 9B, C). In a well, 150 µL of aqueous medium has a
benzo[1,2-d:4,5-d’]bis(1,3)dithiol-4-yl)methyl (OX071)) radical 5.98 mm height. A 50 mm cube of pO2 data containing 262,144 voxels was
(Fig. 1a) was used as an EPR oximetry probe for the measurement of collected that encompassed the 12 wells of the 96-well strip wells. To create
oxygen concentration. One gram of trityl powder (MW: 1384.96, pO2 images for figures, a mask was created using a spatial image that fits
University of West Virginia) was dissolved in 8 ml deionized-double within the bounds of the well. This mask was then transferred to pO2 maps

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Kotecha, M. Oxygen Imaging of a Rabbit Tumor Using a Human-Sized Competing interests
Pulse Electron Paramagnetic Resonance Imager. Mol. Imaging Biol., MK and BE disclose their ownership in O2M Technologies, LLC. All other
15, https://doi.org/10.1007/s11307-023-01852-3 (2023). authors declare no competing interest.
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Eng. Part C. Methods 28, 264–271 (2022). Correspondence and requests for materials should be addressed to
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in Tissue Engineering 129–147 (John Wiley & Sons, Inc, 2017).
47. Kotecha, M. et al. Noninvasive Absolute Electron Paramagnetic Publisher’s note Springer Nature remains neutral with regard to jurisdictional
Resonance Oxygen Imaging for the Assessment of Tissue claims in published maps and institutional affiliations.

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