Name: affordable oxygen microscopy-assisted biofabrication of multicellular spheroids
Uploaded: 2022-04-06T13:20:00
Duration: 13 min 21 s
Description: Watch this Scientific Journal Video about Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids at JoVE.com

This work has been supported by the Special research fund grant of Ghent University (BOF/STA/202009/003) and EU FP7 ITN Program &#34;Chebana&#34;, grant agreement no. 264772 (for AVK). Data is available upon request. G code for bioprinting is available for sharing.

The authors have nothing to disclose.

Significance. Tissue oxygenation measurement gives an insight on cell and tissue-specific metabolism, tissue growth and development, viability, tumorigenesis and cell migration, and interaction with microbes and other pathogens. The presented method gives a new tool for better understanding of the physiology of multicellular spheroid cultures: analysis of oxygenation with ratiometric nanoparticle O2 probes and provides means for assessing hypoxia, visualizing the reliance on particular energy production pathways and complements modeling studies and alternative metabolic imaging approaches43,44 on a widely available low-cost fluorescence microscope. Ratiometric measurements can be performed on a fluorescence widefield (pulsed LED excitation preferred), laser-scanning confocal, light sheet and two-photon microscopes, ideally equipped with T and O2 incubator chambers. Importantly, presented O2 microscopy measurement can be easily combined with the live multi-parameter analysis of various physiological parameters and cell tracers (in case of heterocellular spheroid cultures), viability (live/dead staining), lipid metabolism (lipid-sensing fluorescent probes), dynamics of pH (dyes, nanoparticles, and fluorescent proteins) or [Ca2+], performed in available fluorescence spectral channels or in parallel, giving an expanded view on real-time cell function in spheroids, bioprinted constructs, and potential tissue transplants.
Spheroid cultures find use in studies of cancer cells, tumor and stem cell niche microenvironment, tumoroids, drug efficiency and toxicity screening, and indeed as tissue building blocks for tissue biofabrication and self-assembly45. They can be generated from multiple cell types by a variety of different approaches46. The popularity of the spheroid model necessitates their standardization from the point of view of research integrity and improvement of data analysis, which was recently attempted by development of the first spheroid database8.
Our protocol is expected to help better understand live spheroid metabolism, standardize this experimental model, and subsequently improve their long-term stability, reproducibility, and viability within bioprinted and implantable materials.
Modifications. This protocol describes the use of micropatterned agarose low-adherent surface (micromold-based formation29) for high yield generation of O2 probe-loaded spheroids for oxygenation analysis. The alternative methods of spheroid production such as hanging drop, application of ultra-low attachment plates, lipid coating, or free-floating formation are also compatible with the suggested O2 probe loading protocol. The presented protocol was optimized for hDPSC spheroids and co-culture spheroids of hDPSC with HUVEC (1:1). Other cell lines are certainly applicable15,17,47,48; however, some protocol optimization may be required due to the different cell adhesion properties, culture conditions, metabolic substrate requirements, and cell compatibility with nanoparticle staining. The choice of a suitable ratiometric nanoparticle-based O2-sensitive probe has to be done depending on specific cell model and according to the probe photobleaching properties at the used microscopy set-up (intensity and type of the light source, spectral sensitivity of the camera) and availability of appropriate excitation/emission filters for corresponding reference and O2-sensitive spectral channels of the probe.
Some ratiometric O2 probes are commercially available2. Alternatively, they can be prepared in- house by co-precipitation of reference and O2-sensitive dyes with a polymer as described elsewhere24,25,49. For each individual model, the preliminary tests should be done in order to determine the appropriate probe and optimal microscopy conditions and to minimize the effect of probe photobleaching or photoinduced O2 consumption during ratiometric measurements. Previous studies of O2-sensing nanoparticles demonstrated their low cell toxicity, allowing application in a wide range of staining concentrations (1-20 &#181;g/mL). As some cationic nanoparticles can self-aggregate during storage, we recommend keeping their loading concentration as low as possible to minimize their potential effect on spheroid formation. Optionally, the nanoparticle suspension can be filtered (0.2 &#181;m) or cleared by centrifugation (10,000 x g, 5 min) prior to use to remove all aggregates from suspension.
Although the BioCAD and HMI software are specific for the presented bioprinter, this protocol is applicable to other bioprinters, as the preparation of the bioink, assembly, filling of a standard cartridge, and the design of a porous hydrogel scaffold are provided. Furthermore, the bioprinting parameters such as printing rate and temperature should be comparable for different extrusion-based bioprinters. Printing pressure and strut diameter are dependent on the needle type and diameter, bioink composition, temperature, and resulting viscosity but they can be a starting point to optimize printing parameters to bioprint GelMA-based bioinks with different bioprinters, though some bioprinters use spindles instead of air pressure to extrude the bioink50.
Critical steps and troubleshooting. One of the critical steps is the choice of O2 probe, which is based on the following considerations: first, the available fluorescence microscope set-up (i.e., compatible excitation light source, filters for excitation and emission, camera sensitivity and spectral transmittance and numerical aperture of the objective), which can limit the number of available probes in respect to their photostability, brightness and potential phototoxicity. It is also important to note that some types of immersion oil (in case of oil-immersion objectives) can interfere with red and infrared fluorescence signals. We see the photostability tests as highly important and that they must be performed during initial set-up and preliminary tests. Potential spectral crosstalk between the fluorescent channels and different dyes must be considered. Indeed, the potential dark and photoinduced toxicity of the O2 probe and other selected dyes, in relation to the specific cell model, have to be evaluated during protocol optimization51. The specific issue for nanoparticles can be their self-aggregation, which can be mitigated by optimizing their working concentration, composition of the staining medium (e.g., serum content), sonication of nanoparticles before mixing with medium or by using of O2-probe pre-stained and washed cells for spheroid formation instead of probe loading during the spheroid formation and compactization procedure.
We did not observe problems associated with the light penetration depth with described spheroid models, but this has to be considered when choosing ratiometric intensity signals, size of spheroid and biofabricated constructs (tissue grafts) and optimizing cell staining with respective dyes. MMIR1 probe having closely matching red and near-infrared emission wavelengths is expected to provide the lowest background and best tissue light penetration, compared to other red/blue or green-emitting fluorescent biosensor probes.
Production and handling of ratio images (and potentially spectral unmixing or deconvolution) can be done in a vendor provided software or in the opensource options such as ImageJ, Fiji52,53, napari (https://napari.org), MATLAB, and others. A critical step will be in subtracting background and measuring signal intensity changes in a linear range.
Calibration of O2 probe: Rotenone and antimycin A are inhibitors of complexes I and III of the mitochondrial electron transport chain, respectively, blocking cell respiration54,55,15 and inducing dissipation of O2-gradients in spheroids and their equilibration with the environmental O2. Thus, changing the environmental O2 concentration (i.e., 20%, 15%, 10%, 5%, 2.5%, 1%, 0% O2) will allow for ratiometric intensity or phosphorescence lifetime calibration of the O2-sensitive probe in cells. Typically, O2-controlled incubators are not able to achieve absolute 0% O2 and in certain situations, a quick test of response of probe to deoxygenation is required. In such cases, 25-100 &#181;g/mL of glucose oxidase or Na2SO3/K2HPO4 mixture (50 mg/mL for each compound for 10x solution in distilled water) have to be added to the sample. Importantly, if the cell line has a significant degree of non-mitochondrial O2 consumption, consider this when performing inhibition of respiration and calibration experiments.
Limitations and future research. The main limitation of the proposed method and the ratiometric detection in general, is the calibration and conversion of observed ratio levels into the actual O2 levels, which due to intrinsic differences in hardware and user image acquisition settings would make the ratio calibration instrument- and cell-specific. Importantly, for a widefield fluorescence microscope and described setup, the ratio images are reflecting combined projections rather than ideal optical sections and O2 calibration would not make sense. On the other hand, we show that semi-quantitative ratio measurements provide statistically significant and easy-to-measure comparative phenotyping of spheroids produced from various sources and having differences in oxygenation.
The future research in making more accessible and affordable microscopy approaches designed for live 3D objects such as SPIM, light sheet, theta, two-photon and luminescence lifetime imaging combined with the machine learning56,57,58,59,19, will help bringing multiparametric O2 imaging to even more quantitative applications.

Molecular oxygen (O2) is one of the key metabolites regulating cell and tissue viability, function, and death. Under physiological conditions, local tissue oxygenation is dynamically regulated by tissue vascularization, blood flow, and cell metabolism, in some cases leading to the formation of O2 gradients, hypoxic microenvironment, and/or oxidative stress1,2. Cells sense the O2 gradients through the direct involvement of the molecular O2&#160;in signaling function (via hypoxia-inducible factor (HIF)-mediated signaling, histone lysine demethylases KDM, and by other means), changes in cellular redox potential (reactive O2 species sensing through Nrf2/Keap1 signaling or iron-regulatory proteins), and can subsequently adjust their metabolism, proliferation, potency, and differentiation3. Thus, heterogeneity of cell and tissue oxygenation, its gradients, and associated phenomena are important players in tissue development and homeostasis. Different tissues and cell types often require different &#8216;normal&#8217; physiological O2 levels, which define&#160;the special tissue architecture with cells positioned according to the tissue O2 microgradients4. Some cell types are sensitive to O2 decrease (e.g., neurons, hepatocytes, pancreatic islet cells, or muscle cells)5,6, while others can withstand extreme hypoxia and form steep O2 gradients (e.g., in the intestinal and colon epithelia)7. With the progress in tissue bioengineering and biofabrication, the necessity of O2 quantification in microaggregates and spheroid 3D tissue constructs becomes important. One of the concerns is the standardization of the multicellular spheroid model physiological parameters, which depend on the method of spheroid generation and culture conditions8. In addition, uncontrolled application of O2 releasing biomaterials or O2 perfusion in microtissues lacking functional vascularization can be toxic to cells, lead to reprogramming of their metabolism, and decreased survival in the post-transplantation period9,10. The credibility of the O2 measurement approach and optimization of the oxygenation environment to reach the efficient culture of physiologically relevant microaggregates was recently proved by mathematical modeling of pluripotent stem cells-derived liver spheroids used as an example11. Another field where the knowledge of tissue O2 levels is recognized is cancer therapy12,13. Heterogeneous tumor hypoxia can impair successful anticancer therapy. Measuring and controlling the exact oxygenation levels during patient treatment or tumor hypoxia modeling would allow improvement of individual and personalized therapy strategies14. Thus, quantitative monitoring of dynamic oxygenation in biofabricated constructs and 3D tumor models is a prominent tool for physiological analysis of their respiration activity, metabolism, and optimization of tissue culture, manufacturing conditions, or basic studies of the hypoxia-mediated therapeutic response.
A number of methods enable for multiplexed (or multi-parameter) analysis of cell oxygenation in spheroid and microaggregate tissue constructs. Pioneered with neurospheres and tumor spheroids15,16,17, the phosphorescence lifetime imaging microscopy (PLIM)-based approach enables for direct quantification of cell oxygenation in live microtissues and establishing its correlation with cell viability, proliferation, or distribution of the functional cell types. Despite the power of the approach, PLIM microscopy upgrade is still not widely available even among the popular microscopy vendors18,19. Fortunately, a good number of cell-penetrating O2-sensitive nanoparticle probes can also be used for the fluorescence intensity-based ratiometric measurement mode15,17,20,21,22,23,24. Typically, the probe would display two emission wavelengths, i.e., O2-sensitive and insensitive (reference), which can be measured on a fluorescence microscope, high-content screening imager, or microplate reader. Such O2-sensing nanoparticles can be produced by precipitation technique with the biocompatible polymers, O2-sensing, and reference dyes spanning visible and near-infrared spectra, or they can be purchased commercially2,25. Since the analysis of thick 3D microtissues would benefit from the use of red-shifted fluorescent dyes, the ratiometric O2-sensing nanoparticle probe multi-modal infrared number 1 (MMIR1), consisting of O2-sensing PtTPTBPF26 and reference aza-BODIPY27 dyes impregnated in a positively charged polymethacrylate-based copolymer was constructed28. With this design, an O2-sensitive signal (near-infrared, excitation (exc.) = 635 nm, emission (em.) = 760 nm; Isens) is affected by the [O2] concentration via phosphorescence quenching process, while the red reference dye intensity (exc. = 580 nm, em. = 650 nm; Iref) remains unaffected (Figure 1A). Thus,&#160;Iref / Isens ratio (R) in stained cells can enable O2 calibration22.
Here, we describe a semi-quantitative approach for monitoring live cell oxygenation in spheroids and bioprinted constructs, helping to estimate O2 gradients in endpoint and kinetic measurements. Such O2 imaging can be performed with other types of ratiometric O2 probes (Figure 1B) and depending on the number of available light sources and fluorescence filters, can be used with other dyes to gain information on live/dead cells, mitochondrial function, and tracing other cell types. Advanced measurement modes such as fluorescence lifetime imaging microscopy (FLIM) can also be used19. The biggest challenge with the use of cell staining dyes and O2-sensitive nanoparticles is the optimization of the staining protocol. In the case of the MMIR1 probe and related nanoparticles, cells are either pre-stained or co-incubated during the process of spheroid formation. In the described protocol, O2 probe-stained spheroids were generated on low-adherent agarose surface29,30,31,32 (Figure 1C), which enables for subsequent spheroid-based bioprinting and multi-parameter analysis of O2 and cell death. To illustrate the applicability of the approach, the oxygenation levels in homo- or hetero-cellular (formed with the addition of human umbilical vein endothelial cells, HUVEC) human dental pulp stem cell (hDPSC) spheroids were compared before and after bioprinting, using conventional fluorescence microscope.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>Also available from Sigma</td>
		</tr>
		<tr>
			<td>12 well cell-culture plates, sterile</td>
			<td>Greiner bio-one</td>
			<td>665-180</td>
			<td>Similar products are also available from Sarstedt, Corning and other companies.</td>
		</tr>
		<tr>
			<td>12 Well Chamber slide, removable</td>
			<td>Ibidi</td>
			<td>81201</td>
			<td>Also available from Grace Bio-Labs, ThermoFisher Scientific and others</td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Nerbe plus</td>
			<td>02-502-3001</td>
			<td>Similar products are also available from Sarstedt, Corning, VWR and other companies</td>
		</tr>
		<tr>
			<td>3cc Cartridge, UV secure, luer lock</td>
			<td>RegenHU</td>
			<td>C-033CC-UV</td>
			<td>Also available from CelIink and others</td>
		</tr>
		<tr>
			<td>3D Discovery Bioprinter</td>
			<td>RegenHU</td>
			<td>N/A</td>
			<td>Bioprinter equiped with an extrusion based printhead, heating mantle, UV-LED curing lamp, 3D Discovery HMI software (CAD/CAM software with direct machine control) and BioCAD software (version 1.1-12)</td>
		</tr>
		<tr>
			<td>6 well cell-culture plates, sterile</td>
			<td>Greiner bio-one</td>
			<td>657160</td>
			<td>Similar products are also available from Sarstedt, Corning, VWR and other companies</td>
		</tr>
		<tr>
			<td>Antimycin A from Streptomyces sp.</td>
			<td>Sigma-Aldrich</td>
			<td>A8674-25MG</td>
			<td>Used as inhibitor of complex III of the mitochondrial electron transport chain, blocking cell respiration</td>
		</tr>
		<tr>
			<td>B-Braun Tip Cap, luer lock</td>
			<td>RegenHU</td>
			<td>TC-BB-B</td>
			<td>Also available from Regemat, Cellink and others</td>
		</tr>
		<tr>
			<td>Cell view cell culture dish, PS, 35/10mm, glass bottom, 1 compartment, sterile</td>
			<td>Greiner bio-one</td>
			<td>627861</td>
			<td>For microscopy of bioprinted spheroids</td>
		</tr>
		<tr>
			<td>Collagen from human placenta, type IV</td>
			<td>Sigma</td>
			<td>C5533</td>
			<td>For the preparation of 0.07mg/mL Collagen and 0.03mg/mL Poly-D-lysine coated microscopy dishes</td>
		</tr>
		<tr>
			<td>D(+)-Glucose</td>
			<td>Merck</td>
			<td>8342</td>
			<td>Prepare 1M stock solution, 1/100 for preparation of imaging medium (final concentration 10mM)</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (DMEM), phenol red-, glucose-, pyruvate- and glutamine-free</td>
			<td>Sigma-Aldrich</td>
			<td>D5030-10X1L</td>
			<td>For preparation of imaging medium</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium 2</td>
			<td>PromoCell</td>
			<td>C-22011</td>
			<td>Also available from Lonza and others</td>
		</tr>
		<tr>
			<td>Endothelial Growth SupplementMix</td>
			<td>PromoCell</td>
			<td>C-39216</td>
			<td>Also available from Lonza and others</td>
		</tr>
		<tr>
			<td>FCCP, Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone</td>
			<td>Sigma-Aldrich</td>
			<td>C2920-10MG</td>
			<td>Used as mitochondrial uncoupler</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270-098</td>
			<td>Also available from Sigma</td>
		</tr>
		<tr>
			<td>GelMA</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>GelMA was synthesized by the PBM group (Prof Dr Peter Dubruel and Sandra Van Vlierberghe, University of Gent) [https://www.sciencedirect.com/science/article/pii/S0142961213011782] but are also available from Xpect Inx</td>
		</tr>
		<tr>
			<td>Glucose-oxidase from Aspergillus nige (10000 u)</td>
			<td>Sigma-Aldrich</td>
			<td>G7141</td>
			<td>Used to test the response of probe to deoxygenation</td>
		</tr>
		<tr>
			<td>GlutaMAX 100x Supplement</td>
			<td>Gibco</td>
			<td>35050-038</td>
			<td>Dilution 1/100 for preparation of imaging medium (final concentration 2mM)</td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td>Dilution 1/100 for preparation of imaging medium (final concentration 10mM)</td>
		</tr>
		<tr>
			<td>Hoechst 34580</td>
			<td>Invitrogen (Life Technologies)</td>
			<td>H21486</td>
			<td>Also available from Sigma, BDBiosciences and others</td>
		</tr>
		<tr>
			<td>Human dental pulp stem cells (hDPSC)</td>
			<td>Lonza</td>
			<td>PT-5025</td>
			<td>Also available from ATCC or other vendors</td>
		</tr>
		<tr>
			<td>Human umbilical vein endothelial cells (HUVEC)</td>
			<td>Lonza</td>
			<td>CC-2517</td>
			<td>Also available from ATCC or other vendors</td>
		</tr>
		<tr>
			<td>KH2PO4 (potassium dihydrogen phosphate)</td>
			<td>Merck</td>
			<td>4873</td>
			<td>For preparation of sodium sulfite solution (5 mg / mL Na2SO3 and 5 mg / mL KH2PO4). Prepare fresh from 10x concentrated stock solution daily.</td>
		</tr>
		<tr>
			<td>Li-TPO</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Li-TPO-L was synthesized by the PBM group (Prof Dr Peter Dubruel and Sandra Van Vlierberghe, University of Gent)&#160; [https://link.springer.com/referenceworkentry/10.1007%2F97 
			8-3-319-45444-3_15) , https://asmedigitalcollection.asme.org/nanoengineeringmedical/article/6/2/021001/376814/Hybrid-Tissue-Engineering-Scaffolds-by-Combination] but comparable photo-initiators are available from Sigma</td>
		</tr>
		<tr>
			<td>MEM Alpha Medium + Glutamax Minimum essential medium</td>
			<td>Gibco</td>
			<td>32561-029</td>
			<td>Also available from Sigma and others</td>
		</tr>
		<tr>
			<td>Micro-patterned 3D-printed PDMS stamps, wells with a diameter 400 &#181;m, thickness, total well numbers 1585</td>
			<td>Self-fabricated</td>
			<td></td>
			<td>These stamps were self-fabricated by the Centre for Microsystems Technology (Professor Dr Jan Vanfleteren, University of Gent) but can also be obtained commercially from Merck (Z764094, Microtissues 3D Petri Dish micro-mold mixed spheroid kit)</td>
		</tr>
		<tr>
			<td>Na2SO3 (sodium sulfite)</td>
			<td>Sigma-Aldrich</td>
			<td>239321-500G</td>
			<td>For preparation of sodium sulfite solution (5 mg / mL Na2SO3 and 5 mg / mL KH2PO4). Prepare fresh from 10x concentrated stock solution daily.</td>
		</tr>
		<tr>
			<td>O2 probes: MMIR1, SI-0.2+, SII-0.2+</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Can be prepared &#8216;in-house&#8217; using commercially available dyes, polymers and precipitation method [https://pubs.acs.org/doi/abs/10.1021/nn200807g https://onlinelibrary.wiley.com/doi/abs/10.1002/adfm.201201387 ]. Some nanoparticles are available commercially as discussed in [https://link.springer.com/article/10.1007/s00018-018-2840-x ]</td>
		</tr>
		<tr>
			<td>Pen Strep :Penicillin (10,000 U/mL) / streptomycin (10,000 &#956;g/mL) 100x solution</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Also available from Sigma . Apply in dilution 1:100</td>
		</tr>
		<tr>
			<td>Petri dishes, sterile</td>
			<td>Greiner bio-one</td>
			<td>633181</td>
			<td>Similar products are also available from Sarstedt, Corning, VWR and other companies</td>
		</tr>
		<tr>
			<td>Piston for 3cc cartridge</td>
			<td>RegenHU</td>
			<td>P-03CC-UV</td>
			<td>Also available from Cellink, Regemat and others</td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P6407-5mg</td>
			<td>For the preparation of 0.07mg/mL Collagen and 0.03mg/mL Poly-D-lysine coated microscopy dishes</td>
		</tr>
		<tr>
			<td>PVDF syringe filter 0.22 &#181;m</td>
			<td>Novolab</td>
			<td>A35149</td>
			<td>Similar products are also available from Sarstedt, Corning, VWR and other companies</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875-1G</td>
			<td>Used as inhibitor of complex I of the mitochondrial electron transport chain, blocking cell respiration</td>
		</tr>
		<tr>
			<td>Sodium pyruvate (100 mM)</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td>Dilution 1/100 for preparation of imaging medium (final concentration 1mM)</td>
		</tr>
		<tr>
			<td>SYTOX Green</td>
			<td>Invitrogen (Life Technologies)</td>
			<td>S7559</td>
			<td>Also available from Sigma, Promega and others</td>
		</tr>
		<tr>
			<td>Taper tip 22 gauge (conical PE needle</td>
			<td>Amada Myachi Europe</td>
			<td>22K62222</td>
			<td>Similar products are also available from RegenHU, Cellink, Regemat and others</td>
		</tr>
		<tr>
			<td>Tissue culture flask (75 cm2)</td>
			<td>VWR</td>
			<td>734-2313</td>
			<td>Similar products are also available from Sarstedt, Corning, VWR and other companies</td>
		</tr>
		<tr>
			<td>Ultrapure Agarose</td>
			<td>Invitrogen (Life Technologies)</td>
			<td>16500-500</td>
			<td>Other types of Agarose such as Agarose low melting point (A-9414, Sigma), Agarose for routine use (A-9539, Sigma)</td>
		</tr>
		<tr>
			<td>Widefield fluorescence inverted microscope</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td>Inverted fluorescence microscope IX81, with motorised Z-axis control, CoolLED pE4000 (16 channels, 365-770 nm), ORCA-Flash4.0LT (Hamamatsu) cMOS camera, glass warming plate Okolab, CellSens Dimension v.3 software and air objectives 4x/0.13 UPlanFLN and 40x/0.6 LUCPlanFLN. (Optional, for high-resolution imaging) 60x/1.0 LUMPLFLN water</td>
		</tr>
	</tbody>
</table>

affordable oxygen microscopy-assisted biofabrication of multicellular spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

The high throughput production of O2 probe pre-stained spheroids using micropatterned agarose method is schematically illustrated in Figure 1C showing examples of agarose microwells without and with the pre-stained spheroids. The efficiency of spheroid formation on agarose coating and their shape/sphericity can be cell-specific. For instance, human colon HCT116 cells never formed ideal spherical structures using agarose microwell method, in contrast to lipid-coated surface17, whereas hDPSCs alone and co-cultured with HUVEC always produced spheroids of highly reproducible shape and size proportional to cell composition, initial cell number, and duration of spheroid formation/growth. All the tested cell types efficiently accumulated O2 probe nanoparticles during spheroid formation (Figure 1B) and preserved this staining over at least 5 days, allowing their use for bioprinting and subsequent monitoring of the bioprinted tissue oxygenation (Figure 1D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63403/63403fig01.jpg" /> 
Figure 1: Principle of O2 probe function and its application for spheroid staining and bioprinting. (A)&#160;A simplified Yablonski diagram, explaining the principle of O2-sensing by the phosphorescent nanoparticle (NP) probes. The transfer of energy to molecular O2 during the forbidden triplet excited state (T) leads to a decrease of phosphorescence intensity inversely proportional to phosphorescence lifetime tau, &#964; (changes from &#964;1 at 0% O2 to &#964;2 at 21% O2), which is the time between excitation to singlet state (S1) and its return to the ground state (G0)42. Quantitative O2 measurement can be done by ratiometric measurement or by measuring &#964; (phosphorescence lifetime measurements).(B) An example of staining of human dental pulp stem cell (hDPSC) spheroids stained with different types of nanoparticle O2 probes, shown in transmission light, reference, and O2-sensitive dye fluorescence channels. Scale bar is 100 &#181;m. (C) Schematics of high-throughput O2 probe pre-stained spheroid generation using micropatterned agarose method. From left to right: PDMS silicon stamp placed in a well of the culture plate, an example of a microwell pattern in agarose (4x magnification), and an example of MMIR1 pre-stained spheroids produced from HCT116 human colon cancer cells on day 2 after seeding on micropatterned agarose (4x magnification). Scale bar is 100 &#181;m. (D) From left to right: a bioprinted waffle-construct and microscopy analysis of MMIR1 pre-stained spheroid bioprinted construct on days 1 and 5 after bioprinting. The fluorescence signal corresponds to O2-sensitive phosphorescent dye in MMIR1 nanoparticles (exc. = 635 nm/em. = 760 nm). Scale bar is 400 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63403/63403fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
On the described microscopy set-up (conventional widefield fluorescence microscope equipped with the light-emitting diode (LED) as a light source) red/near-infrared MMIR1 probe was found as the best in terms of photostability of its reference and sensitive dyes with the less than 5% decrease in brightness of their initial intensity ratio (R = Iref/Isens) after 12 cycles of continuous illumination. This allowed using MMIR1 probe for dynamic real-time study of rapid respiratory response in hDPSC spheroids upon treatment with mitochondrial uncoupler (FCCP) and inhibitor of complex I of electron-transport chain (rotenone) (Figure 2A,B). We found that in the stem cell-derived spheroids, FCCP displayed only mild uncoupling effect with a slight decrease of cell respiration within ~80 s after adding this drug, in agreement with previously reported metabolic features of DPSC40. On the other hand, rotenone strongly inhibited respiration leading to spheroids reoxygenation (reflected as the increase of Rp - spheroid periphery ratio and Rc - spheroid core ratio) and dissipation of the periphery-to-core O2 gradients within ~80 s after stimulation (Figure 2A). Two-point semi-calibration of MMIR1 probe ratio (R) at high (atmospheric) and low (in presence of sodium sulfite and glucose oxidase in imaging media) O2 levels confirmed the decrease of R, related to the decrease of oxygenation in spheroids with preliminary inhibited respiration by antimycin A/rotenone cocktail treatment (Figure 2C), illustrating how the measurement of MMIR1 probe R can be applied for semi-quantitative monitoring of long-term steady-state and quick oxygenation responses in 3D.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63403/63403fig02.jpg" /> 
Figure 2: Kinetic analysis of O2 probe response to different stimuli. (A) Representative result of hDPSC spheroid oxygenation imaging at rest and in response to FCCP and rotenone treatments. Scale bar is 100 &#181;m.&#160;(B)&#160;Kinetic response of MMIR1 intensity ratio to FCCP and rotenone treatment in comparison to the initial photobleaching intensity ratio kinetics (%).(C)&#160;Changes in the intensity ratio images of MMIR1 probe in hDPSC spheroids at oxygenated (antimycin A + rotenone addition) and deoxygenated (glucose oxidase addition) states.The color bar represents O2 probe intensity ratio (R = Iref / Isens) distribution across the image. <a href="https://www.jove.com/files/ftp_upload/63403/63403fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To illustrate MMIR1 probe application for live metabolic imaging, the comparative analysis of O2 gradients in homocellular hDPSC versus heterocellular hDPSC/HUVEC (1:1) spheroids was performed. Taking advantage of the availability of free blue and green fluorescence spectral channels co-staining with Hoechst 34580 (HXT) and SYTOX Green (SYTOX) was performed to demonstrate the application of O2 probe for multiparametric studies (Figure 3). Using the automated protocol of pixel-by-pixel intensity ratio calculations provided by the imaging software false-color ratio images of R distribution in spheroids were produced, visualizing real-time detected periphery-to-core O2 gradients in all types of spheroids: with the oxygenated periphery and hypoxic niches in the center (Figure 2 and Figure 3A). However, Rp and Rc values, as well as the steepness of O2 gradient in the middle cross-sections varied for different spheroid types: see the line profiles of the middle cross-sections in hDPSC versus hDPSC/HUVEC spheroids and hDPSC versus bioprinted hDPSC spheroids (Figure 3B,C). As there was no widely accepted way of describing O2 gradients, we introduced several parameters allowing easy comparison and detailed description of general spheroid oxygenation: Rp and Rc, and such characteristics of O2 gradients as a difference between the oxygenated and hypoxic zones as (Rp-Rc), and average changes of oxygenation per &#181;m as (Rp-Rc) / r, where r is a distance between periphery and hypoxic core, correlating in the middle cross-section with spheroid radius (Figure 3D). Statistical comparison of these parameters together with the data from necrotic dead cells visualized by SYTOX in spheroids, helped us to draw preliminary conclusions on the origin of the spheroid cell-type-specific O2 distribution gradients.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63403/63403fig03.jpg" /> 
Figure 3: Comparative analysis of oxygenation gradients in spheroids. (A) Representative examples of live microscopy of oxygenation (ratiometric fluorescence microscopy of MMIR1) and live/dead cell staining (Hoechst 34580, HXT, blue and SYTOX Green, SYTOX) in homocellular hDPSC spheroids before and after printing, and heterocellular hDPSC/HUVEC (1:1) spheroids. hDPSCs in all types of spheroids were from the same cell culture. Spheroids were pre-stained with 5 &#181;g/mL of MMIR1 probe for 2 days during their formation, and then used either for imaging or bioprinting (only hDPSC spheroids), with subsequent imaging analysis on day 1 after bioprinting. False-color images of the intensity ratio (Iref/Isens) images of MMIR1-stained spheroids correspond to their periphery-to-core O2 gradients. Scale bar is 100 &#181;m. The color bar represents O2 probe intensity ratio (R = Iref/Isens) distribution across the image. Numbers 1 and 2 of diameter cross-sections correspond to intensity profiles shown in B and C. (B,C) Comparison of intensity ratio profiles between homogeneous hDPSC versus heterogeneous hDPSC/HUVEC spheroids (B) and homogeneous hDPSC spheroids before and after bioprinting (C). Profiles were measured for cross-sections of spheroids shown on (A).&#160;(D) Schematic representation of parameters used for the description of the periphery-to-core O2-gradients in spheroids, where r is a radius of spheroid and Rp and Rc are intensity ratios of the periphery and core region of spheroid correspondingly. The color bar schematically represents distribution of O2 gradient from high (red) to low (blue) levels in ideally spherical spheroid. (E,F) Comparative analysis of periphery-to-core O2-gradients (Rp-Rc) / r values in hDPSC/HUVEC and hDPSC spheroids (E) and in hDPSC spheroids before and on day 1 after bioprinting (F). Statistical analysis was done for one experimental repeat (n = 18-23). Boxes correspond to standard deviations. Asterisks indicate the statistical difference between groups (at p = 0.05); ** = p &#60; 0.0005. <a href="https://www.jove.com/files/ftp_upload/63403/63403fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In comparison to homocellular hDPSC spheroids, hDPSC/HUVEC spheroids had significantly steeper gradients: higher values of (Rp-Rc) / r parameter and range (Rp-Rc; Figure 3F). At the same time, they displayed higher oxygenation of the periphery (Rp) and core (Rc) (Figure 4A, Table 1). Interestingly, they were also statistically larger (Figure 4A, Table 1), suggesting that such differences in oxygenation were caused by their different cellular bioenergetic profiles. These data are in agreement with the well-known metabolic features of HUVEC cells having generally lower respiration activity of cells with the strong reliance on glycolysis and pentose-phosphate pathways as the main bioenergetic sources of ATP and NAD(P)H41. At the same time, the pronounced periphery-to-core O2 gradient formed in heterogeneous spheroids is likely generated by hDPSC in their composition, characterized by hyperpolarized mitochondria and active electron-transport chain40 and, according to the results on hDPSC spheroids oxygenation, having strong respiration activity (Figure 3, Figure 4A, and Table 1). Thus, generally higher viability of hDPSC/HUVEC spheroids confirmed by the lower intensity of their dead cells staining with SYTOX (Figure 3A) is potentially linked to their higher oxygenation levels.
To illustrate the applicability of live microscopy imaging of spheroid oxygenation in biofabrication, MMIR1 O2-probe pre-stained spheroids were used for bioprinting in GelMA bioink with the following comparison of O2 gradients in hDPSC spheroids before and on day 1 after bioprinting (Figure 3A,C,F, Figure 4B, and Table 2). Bioprinted hDPSC spheroids had significantly oxygenated periphery (higher Rp) in comparison to spheroids, measured before bioprinting, while their core oxygenation had similar values (Figure 4B). Changes in their periphery oxygenation affect the range (an increase of (Rp-Rc) and steepness (increased (Rp-Rc)/r) of their periphery-to-core O2 gradients, which were statistically different from hDPSC spheroids measured before bioprinting (Figure 3F and Figure 4B). The dead cells staining was generally brighter in bioprinted spheroids, suggesting that decreased spheroid viability is involved in the alteration of spheroid oxygenation (Figure 3A).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63403/63403fig04.jpg" /> 
Figure 4: Comparative analysis of MMIR1-stained spheroids diameter and intensity ratio. (A) Comparison between hetero- hDPSC/HUVEC and homocellular hDPSC spheroids. (B) Comparison of homocellular hDPSC spheroids before and after bioprinting. Rp and Rc - intensity ratio at the periphery and the core of the spheroids correspondingly; (Rp-Rc) - the difference in intensity ratio, corresponding to the range of O2 gradient in spheroids. Statistical analysis was done for one experimental replicate (n = 18-23). Boxes correspond to standard deviations. Asterisks indicate the statistical difference between groups (at p = 0.05), where * = p &#60; 0.005 and ** = p &#60; 0.0005. <a href="https://www.jove.com/files/ftp_upload/63403/63403fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Type of spheroid (N)</td>
			<td>Diameter [&#181;m]</td>
			<td>Rp [a.u.]</td>
			<td>Rc [a.u.]</td>
			<td>Rp-Rc [a.u.]</td>
			<td>(Rp-Rc)/r [a.u./&#181;m]</td>
		</tr>
		<tr>
			<td>hDPSC / HUVEC (18)</td>
			<td>113.2 &#177; 10.6</td>
			<td>0.779 &#177; 0.034</td>
			<td>0.398 &#177; 0.055</td>
			<td>0.982 &#177; 0.051</td>
			<td>0.00677 &#177; 0.00091</td>
		</tr>
		<tr>
			<td>hDPSC (18)</td>
			<td>88.1 &#177; 9.9</td>
			<td>0.558 &#177; 0.025</td>
			<td>0.331 &#177; 0.034</td>
			<td>0.227 &#177; 0.032</td>
			<td>0.00520 &#177; 0.00090</td>
		</tr>
		<tr>
			<td>p-value</td>
			<td>1.5 x 10-8 *</td>
			<td>1.5 x 10-21 *</td>
			<td>1.3 x 10-4 *</td>
			<td>1.4 x 10-12 *</td>
			<td>9.2 x 10-6 *</td>
		</tr>
	</tbody>
</table>
Table 1. Spheroid diameter, intensity ratio at the core (Rc) and periphery (Rp) of the spheroids, the difference in intensity ratio (Rp-Rc) and (Rp-Rc) / r value in heterogeneous hDPSC/HUVEC and homogeneous hDPSC spheroids. p-value of a t-test on N spheroids demonstrates statistical difference and is indicated with an asterisk as well.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Type of spheroid (N)</td>
			<td>Diameter [&#181;m]</td>
			<td>Rp &#160;[a.u.]</td>
			<td>Rc [a.u.]</td>
			<td>Rp-Rc [a.u.]</td>
			<td>(Rp-Rc)/r [a.u./&#181;m]</td>
		</tr>
		<tr>
			<td>hDPSC (18)</td>
			<td>88.1 &#177; 9.9</td>
			<td>0.558 &#177; 0.025</td>
			<td>0.331 &#177; 0.034</td>
			<td>0.227 &#177; 0.032</td>
			<td>0.00520 &#177; 0.00090</td>
		</tr>
		<tr>
			<td>Bioprinted hDPSC (23)</td>
			<td>80.9 &#177; 12.5</td>
			<td>0.584 &#177; 0.023</td>
			<td>0.323 &#177; 0.038</td>
			<td>0.261 &#177; 0.039</td>
			<td>0.00658 &#177; 0.00144</td>
		</tr>
		<tr>
			<td>p-value</td>
			<td>0.054</td>
			<td>0.0024 *</td>
			<td>0.46</td>
			<td>9.9 x 10-4 *</td>
			<td>6.3 x 10-6 *</td>
		</tr>
	</tbody>
</table>
Table 2. Spheroid diameter, intensity ratio at the core (Rc) and periphery (Rp) of the spheroids, the difference in intensity ratio (Rp-Rc) and (Rp-Rc) / r value in homogeneous hDPSC spheroids before and after bioprinting. p-value of statistical analysis (N spheroids). The statistical difference is indicated with an asterisk.

Watch this Scientific Journal Video about Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids at JoVE.com

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

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