This work was supported by the Special Research Fund (BOF) grants of Ghent University (BOF/STA/202009/003; BOF/IOP/2022/058), Research Foundation Flanders (FWO, I001922N) and the European Union, fliMAGIN3D-DN Horizon Europe-MSCA-DN No. 101073507.

1. Generation of multicellular spheroids
<ol>
	<li>Cell culture 
	NOTE: Cell cultures can be collected from the American Type Culture Collection (ATCC), Lonza, Sigma-Aldrich, or other vendors. ATCC provides all required handling information, including preferred growth&#160;media, subculturing procedures, biosafety level, growth rate, and STR profiles. Here, 500 cells/spheroid of human colon cancer cell line HCT116 is used in McCoy&#39;s 5A media (VWR, 392-0420) supplemented with 10% FBS and 1 mM S...

Multicellular Spheroid Formation for FLIM-Based Metabolic and Hypoxia Analysis

Multicellular spheroids are becoming a method of choice in the studies of tumor and stem cell niche microenvironments, drug discovery, and development of the &#39;tissue building blocks&#39; for biofabrication. Spheroids&#39; heterogeneous internal architecture, gradients of nutrients and oxygenation can mimic those of in vivo tissues and tumors in a relatively simplified and accessible setting. With the need for more methodological transparency26,28 and...

Multicellular spheroids represent a group of 3D tissue models obtained by the self-aggregation of cells and exhibiting a spherical shape. They are widely used to mimic cell-cell and cell-matrix interaction in vitro and to reproduce&#160;a 3D context within a multitude of cancer and stem cell-derived constructs. Several techniques are employed to reduce cell attachment and promote the aggregation. These include the hanging-drop method relying on the surface tension1; cell attachment repelling methods such as ultra-low attachment plates, micro-molds, and microwells2,3; acoustic wave-based approach4; flow-induced aggregation methods (spinner flasks, bioreactor, and microfluidic devices)5; magnetic particles-assisted formation6 and use of the aggregation-promoting synthetic and ECM-based matrices and scaffolds7,8,9.
In cancer research, development, and validation of new drug therapies, spheroids are an attractive model due to their ability to recapitulate the spatial diffusion-limited gradients of nutrients, waste products, and O2, often leading to the formation of a necrotic core, typical to the solid tumors10,11. These more reliable and sophisticated in vitro models challenge the need for extensive use of animal models (Food and Drug Administration [FDA] Modernization Act 2.012), according to the 3Rs principle of animal research (replacement, reduction, and refinement). In addition to cancer, spheroids find their application in stem cell research. For instance, pluripotent stem cells have the capacity to form embryoid bodies (EB), which can be used for the differentiation of induced pluripotent stem cells (iPSCs) towards specialized cell types that are challenging to obtain directly from patients, such as neural precursor cells13 or ovarian granulosa cells13,14. Furthermore, the formation of an EB is often the first step in the development of more complex organoid models, e.g., neural15, retinal16, cardiac17, liver18, stomach19, and intestinal organoids20. Factors including size, reproducibility, throughput, and downstream applications should be considered when choosing an appropriate spheroid formation method for the experiments.
The increased complexity of 3D culture can lead to higher variability compared to 2D culture. Factors such as nutrient composition21, media evaporation22, viscosity23, pH control24, spheroid formation method, and even the time in the culture25,26 can result in obtaining spheroids of varying morphology, sizes, viability, and different chemoresistance27,28. Recent research demonstrated that spheroid oxygen gradients are not always static and are affected by the formation method, spheroid size, and extracellular viscosity, affecting the spheroid heterogeneity29. To improve reproducibility and data accessibility on spheroids, the MISpheroID knowledge base has been developed26, identifying cell line, culture medium, formation method, and spheroids size as the minimal information for a reproducible result. Therefore, a detailed comparison was made of multiple high-throughput (SphericalPlate 5D, lab-made micromolds, and Microtissue molds) and low attachment methods (i.e., Biofloat and Lipidure-coated 96-well plates, both scaffold-free and scaffold-based) (Figure 1 and Table 1), including the well size (given an estimation of the maximum spheroid size), consumables used, preparation time and the possibility of monitoring spheroids without transporting them to microscopy dishes. The latter enables long-term studies, whereas spheroids produced with high-throughput methods often result in endpoint experiments. All methods except for the grids of the 5DspheriPlate do not bring unwanted autofluorescence, hereby enabling their direct use in microscopy.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66845/66845fig01.jpg" /> 
Figure 1: Spheroid formation methods explained. High-throughput methods such as the SphericalPlate 5D, which has integrated patented microwells in the plate, while the lab-produced micromolds and the MicroTissue molds use stamps to make multiple microwells in agarose (blue). Low-attachment plates such as Lipidure (Amsbio) and Biofloat (Sarstedt) use a non-adherent coating inhibiting cell-surface adhesion and promoting cell self-aggregation. <a href="https://www.jove.com/files/ftp_upload/66845/66845fig01large.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></td>
			<td>5D SpheriPlate</td>
			<td>Self-produced micromolds</td>
			<td>Microtissue</td>
			<td>Low attachment methods</td>
		</tr>
		<tr>
			<td>Number of spheroids/well&#160;</td>
			<td>750</td>
			<td>1589</td>
			<td>81</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Diameter well</td>
			<td>90 &#181;m</td>
			<td>400 &#181;m</td>
			<td>800 &#181;m</td>
			<td>1 mm</td>
		</tr>
		<tr>
			<td>Culture volume</td>
			<td>1 mL</td>
			<td>5 mL</td>
			<td>1 mL</td>
			<td>200 &#181;L</td>
		</tr>
		<tr>
			<td>Other consumables</td>
			<td>/</td>
			<td>7 mL of 3% agarose</td>
			<td>500 &#181;L of 2% agarose</td>
			<td>/ *</td>
		</tr>
		<tr>
			<td>Preparation time</td>
			<td>10 min</td>
			<td>2 h + 3 days media adaptation</td>
			<td>0.5 h + 15 min media adaptation</td>
			<td>10&#8211;30 min + 1 h drying</td>
		</tr>
		<tr>
			<td>Monitoring</td>
			<td>Yes</td>
			<td>No**</td>
			<td>Yes</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Autofluorescent</td>
			<td>Yes</td>
			<td>No</td>
			<td>No</td>
			<td>No</td>
		</tr>
		<tr>
			<td>Reusable</td>
			<td>No</td>
			<td>Yes</td>
			<td>Yes</td>
			<td>No**</td>
		</tr>
		<tr>
			<td rowspan="2">Cost</td>
			<td rowspan="2">&#8364;&#8364;&#160;</td>
			<td rowspan="2">&#8364;</td>
			<td rowspan="2">&#8364;&#8364;&#8364;&#8364;</td>
			<td>&#8364;&#8364;&#8364;&#8364;: Coating and Matrigel</td>
		</tr>
		<tr>
			<td>&#8364;&#8364;: Commercial 96-well plate</td>
		</tr>
		<tr>
			<td colspan="3">*Some cell lines need addition of&#160; ECM (i.e. 2%&#8211;5% Matrigel) to form compact spheroids.&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="5">**The coating is reusable until depleted. However, each plate will consume a small amount of media and dust can accumulate over time. Filter sterilization is regularly needed.&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of multiple spheroid formation methods29. &#34;Monitoring&#34;: the ability to monitor spheroid without the need for transfer to a microscopy dish. &#8364;: 0-50&#8364;, &#8364;&#8364;: 50-150&#8364; , &#8364;&#8364;&#8364;: 150-500&#8364; , &#8364;&#8364;&#8364;&#8364;: &#62;500&#8364;
Fluorescence microscopy enables direct monitoring of the key biological aspects within spheroids, including cell death, viability, proliferation, metabolism, viscosity, and even mechanical properties30. Fluorescence lifetime imaging microscopy (FLIM) provides an additional quantitative dimension for studying fluorescent probe interactions within their (micro)environment31,32,33,34, allowing resolving the overlapping emission spectra according to different emission lifetimes35,36 and probing cell metabolism based on intrinsic cellular autofluorescence. Thus, such widespread cellular autofluorescent compounds as nicotinamide adenine dinucleotide phosphate (NAD(P)H), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), protoporphyrin IX, and others can be measured with one- and two-photon FLIM and serve as intrinsic &#39;sensors&#39; of glucose catabolism, oxidative phosphorylation (OxPhos) and provide a general overview of the cell redox state. NAD(P)H exists in free cytoplasmic, or in protein-bound mitochondrial forms37,38. Similarly, the oxidized state of FAD is fluorescent with a longer lifetime of the free form. NAD(P)H and FAD microscopies usually involve two-photon excited FLIM, aiming at preventing sample photodamage39. Frequently, &#39;optical metabolic imaging&#39; FLIM can be combined with the use of dye-based probes, genetically encoded biosensors, phosphorescence lifetime imaging microscopy (PLIM), and ratiometric intensity-based measurements in order to provide a more complete picture of spheroid or organoid metabolism, oxygenation, proliferation and cell viability29,30,31. In addition, FLIM can also be combined with F&#246;rster resonance energy transfer (FRET) method to measure the lifetime variation of the donor fluorophore when in close contact with the acceptor to investigate the binding of a drug with its target domain33,40,41.
The acquired FLIM images are typically analyzed to calculate the lifetime pixel-by-pixel. Currently, there are at least 3 common strategies used to obtain fluorescence lifetime: semi-quantitative &#39;fast FLIM&#39;42 (sometimes referred to as &#39;tau sense&#39;43,44), decay curve fitting, using one-, two- or three-exponential fitting, and &#39;fitting-free&#39; approach with phasor transformation and phasor plot analysis. Depending on the vendor, either provided (LAS X, Symphotime, SPCImage, etc.) or open-source software (e.g., FLIMfit45, FLIMJ46, or others47) can be used to handle measured FLIM data. Typically, vendor-provided software is useful for preliminary data analysis, while open-source solutions can provide for more accurate studies using, e.g., phasor plots and 3D visualization.
Despite the usefulness and attractiveness of FLIM as a method for studying spheroids, very few experimental protocols are available, and there is a general lack of knowledge in choosing the most appropriate formation method for successful live multiparametric microscopy experiments involving FLIM. Here, a detailed comparison of commonly used spheroid formation protocols is presented based on their morphology, viability, and oxygenation with the recently validated and characterized far-red and near-infra-red (NIR) oxygen-sensing nanosensor (MMIR1). The cationic nanoparticle is impregnated with two reporter dyes, the reference O2-insensitive aza-BODIPY (excitation 650 nm, emission 675 nm) and the NIR O2-sensitive metalloporphyrin, PtTPTBPF (excitation 620 nm, emission 760 nm). The MMIR1 enables real-time analysis of oxygen gradients on a conventional fluorescence microscope (using ratiometric analysis) or phosphorescence lifetime microscope (PLIM) without introducing cellular toxicity and allowing for stable signals, long-term monitoring, and multiplexing25,29. Depending on the need to stain with dyes or nanosensors, spheroid throughput, or cell type, the most appropriate formation protocol can be chosen. Since the studies of spheroids viability and oxygenation are relevant for studies of cancer and stem cell-derived spheroids, the presented protocols also include examples and expected typical results of NAD(P)H-FLIM and&#160;FAD-FLIM with these models. The presented imaging and analysis pipelines target the most popular time-correlated single photon counting-based FLIM microscopy platforms.

<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>10 mL serological pipets</td>
			<td>VWR</td>
			<td>612-3700</td>
			<td>Similar products are also available from Sarstedt, Corning, VWR and other companies</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>3D Petri Dish micromolds</td>
			<td>Microtissue</td>
			<td>Z764000-6EA</td>
			<td></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>70% ethanol</td>
			<td>ChemLab</td>
			<td>CL02.0537.5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Biofloat</td>
			<td>Sarstedt</td>
			<td>83.3925.400</td>
			<td>Commercial available coated 96-well plate for spheroid formation</td>
		</tr>
		<tr>
			<td>Calcein Green-AM</td>
			<td>Tebubio</td>
			<td>AS-89201</td>
			<td>Apply in dilution 1:1000</td>
		</tr>
		<tr>
			<td>CellSens Dimension software</td>
			<td>Olympus</td>
			<td>version 3</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen from human placenta, type IV</td>
			<td>Sigma</td>
			<td>C5533</td>
			<td>For the preparation of 0.07 mg/mL Collagen and 0.03 mg/mL Poly-D-lysine coated microscopy dishes</td>
		</tr>
		<tr>
			<td>Confocal FLIM Microscope</td>
			<td>Leica Microsystems</td>
			<td>N/A</td>
			<td>Stellaris 8 Falcon inverted microscope with white-light laser, HyD X detectors, climate / T control chamber (OkoLab), 25x/0.95 W objective</td>
		</tr>
		<tr>
			<td>D(+)-Glucose</td>
			<td>Merck</td>
			<td>8342</td>
			<td>Prepare 1 M stock solution, 1:100 for preparation of imaging medium (final concentration 10 mM)</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>Fetal Bovine Serum&#160;(FBS)</td>
			<td>Gibco</td>
			<td>10270-098</td>
			<td>Also available from Sigma. Needs to be heat-inactivated before use.</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 10 mM)</td>
		</tr>
		<tr>
			<td>Human colon cancer cells HCT116</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td>version 1.54f</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite X (LAS X)</td>
			<td>Leica Microsystems</td>
			<td>version 4.6.1.27508</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030</td>
			<td>Also available from Sigma. Apply in dilution 1:100.</td>
		</tr>
		<tr>
			<td>Lipidure-CM5206</td>
			<td>Amsbio</td>
			<td>AMS.52000034GB1G</td>
			<td></td>
		</tr>
		<tr>
			<td>McCoy&#39;s 5A, need addition of 1 mM Sodium Pyruvate and 10 mM HEPES</td>
			<td>VWR</td>
			<td>392-0420</td>
			<td>Standard growth medium for HCT116 cells</td>
		</tr>
		<tr>
			<td>micro-patterned 3D-printed PDMS stamps</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Provided by the Centre for Microsystems Technology, Professor Dr. Jan Vanfleteren, Ghent University</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Chemlab</td>
			<td>CL00.1429.100</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer couting chamber</td>
			<td>Fisher Scientific</td>
			<td>15980396</td>
			<td></td>
		</tr>
		<tr>
			<td>O2 probes: MMIR1</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Full characterization, validation and some applications can be found at: https://www.biorxiv.org/content/10.1101/2023.12.11.571110 
			v1</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher scientific</td>
			<td>Gibco18912014</td>
			<td>Dissolve PBS tablet in 500 mL of distilled water.</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>Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P6407-5mg</td>
			<td>For the preparation of 0.07 mg/mL Collagen and 0.03 mg/mL Poly-D-lysine coated microscopy dishes</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Sigma-Aldrich</td>
			<td>25535-16-4</td>
			<td>Cell death staining, use 1 &#181;g/mL at 1h incubation</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>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>SphericalPlate 5D 24-well</td>
			<td>Kugelmeiers</td>
			<td>SP5D-24W</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile petridish</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>Tissue culture flask (25 cm&#178; )</td>
			<td>VWR</td>
			<td>734-2311</td>
			<td>Similar products are also available from Sarstedt, Corning, VWR and other companies</td>
		</tr>
		<tr>
			<td>Tissue culture flask (75 cm&#178;)</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>U-bottom 96-well plate</td>
			<td>VWR</td>
			<td>10062-900</td>
			<td>Similar products are also available from Sarstedt, Corning, Greiner Bio-one 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>

production and multi-parameter live cell fluorescence lifetime imaging microscopy (flim) of multicellular spheroids

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug therapies&#160;and using bio- and nanosensors in a 3D context. Their ease of production, predictable size, growth, and observed nutrient and metabolite gradients are important to recapitulate the 3D niche-like&#160;cell microenvironment. However, spheroid heterogeneity and variability of their production methods can influence overall cell metabolism, viability, and drug response. This makes it difficult to choose the most appropriate&#160;methodology, considering the requirements in size, variability, needs of biofabrication, and use as in vitro 3D tissue models in stem and cancer cell biology. In particular, spheroid production can influence their compatibility with quantitative live microscopies, such as optical metabolic imaging, fluorescence lifetime imaging microscopy (FLIM), monitoring of spheroid&#160;hypoxia with nanosensors, or viability. Here, a number of conventional spheroid formation protocols are presented, highlighting their compatibility with the live widefield, confocal, and two-photon microscopies. The follow-up imaging to analysis&#160;pipeline with multiplexed autofluorescence FLIM and, using various types of cancer and stem cell spheroids, is also presented.

Author Spotlight: Standardizing Spheroid Formation Methods for Metabolic and Oxygenation Analysis Using Fluorescence Lifetime Imaging Microscopy

Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids

Choosing the appropriate spheroid formation method 
The selected spheroid formation method can greatly influence spheroids&#39; size, shape, cell density, viability, and drug sensitivity (Figure 2). Previously, the effects of multiple high-throughput (SphericalPlate 5D, lab-made micromolds, and MicroTissue molds) and the &#39;medium throughput&#39; low attachment (Biofloat and Lipidure-coated 96-well plates) methods were compared on spheroids viability and oxygenation<s...

Here, we describe different multicellular spheroid formation methods to perform follow-up multi-parameter live cell microscopy. Using fluorescence lifetime imaging microscopy (FLIM), cellular autofluorescence, staining dyes, and nanoparticles, the approach for analysis of cell metabolism, hypoxia, and cell death in live three-dimensional (3D) cancer and stem cell-derived spheroids is demonstrated.

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug ...