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.

Multicellular Spheroid Formation for FLIM-Based Metabolic and Hypoxia Analysis

Nothing to disclose.

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 standardization71, these protocols are expected to help researchers choose the best spheroid formation method for the live quantitative fluorescence lifetime imaging microscopy experiments. The methods presented take advantage of cell staining nanoparticle sensors and intrinsic cellular autofluorescence, which can be easily combined with staining cell death.
The most critical steps are in spheroid formation protocol; thus, for the high throughput agarose-based method, it is essential to sufficiently equilibrate the agarose molds for the culturing media. Insufficient equilibration time will lead to the dilution of nutrients in the remaining PBS and bring additional factors for spheroid heterogeneity21,26,72,73. The freshly made agarose mold has to be also visually checked for the presence of air bubbles, micro-cracks and other defects.
Although efficient for imaging, nanoparticle suspension can display self-aggregation and cause the formation of irregularly shaped spheroids in low-attachment formation methods. This can often be mitigated by the addition of serum to the culturing media or by pre-staining the monolayer cell culture before spheroid formation. Frequently, during spheroid compactization, an addition of a small amount of nanoparticles (e.g., 10% of the initial staining concentration) can also help staining the proliferating zone of the spheroids. Lastly, it is important to use &#34;dust-free&#34; sterile pipette tips with all formation methods.
The potential impact of illumination on cell viability must always be considered during imaging experiments. Thus, it is important to know the risk factors negatively affecting each live imaging experiment. In general, photodamage involves several mechanisms: (1) high local rise of temperature, (2) mechanical stress, and (3) photochemical stress (oxidative and free-radicals reaction)74. Depending on the imaging modality (e.g., one-photon confocal vs. multiphoton imaging, continuous vs. pulse illumination modes, the sensitivity of detectors and power of the light source at different wavelengths if the excitation tuning is possible), cell and tissue type (e.g., tissue type-dependent accumulation of efficiently light absorbing pigments or other autofluorescence molecules, 3D vs. 2D organization, growth and imaging media composition), the origin of the fluorescent probe (e.g., photosensitizing properties) different combination of risk factors can provoke phototoxicity during imaging. In addition, even if no increase in cell death is observed after illumination, light power-dependent earlier pre-death effects on mitochondria and metabolism can potentially interfere with experiment analysis and interpretation55.
To exclude the phototoxic impact on experimental results the researcher should perform general monitoring of cell viability and changes in cell morphology in comparison to intact control or using cell death probes to estimate the increase of cell death rate after illumination. If the metabolic experiments are performed, the optimization of imaging parameters should be done accordingly in the pilot experiments to estimate and minimize the imaging impact on measured metabolic parameters. In the case of oxygen-sensitive probes, the light overdose can be easily tracked by a sudden increase of O2-sensitive dye phosphorescence lifetime and intensity (corresponding changes in the intensity ratio of reference to O2-sensitive dye) due to so-called &#39;photoinduced O2 consumption&#39; effect53,75.This can be easily done by kinetic curve analysis of time-lapse experiments with different light power parameters using, for example, the following published protocol25. In &#39;defense&#39; of the pulse-illumination (e.g., utilized in TCSPC-F(P)LIM microscopy), the recent studies demonstrated its minimal impact on ROS production and probe photobleaching in comparison to steady-state illumination measurements76,77, pointing to additional advantage of TCSPC-FLIM vs. conventional intensity-based imaging approach.
The main limitation of the presented spheroid formation methods lies in direct and long-term monitoring applications. The agarose molds require a long working distance objective (or additional upright microscope or fluorescence stereo microscope) due to the thick layer of agarose and the material of the 5D Spheriplate may interfere with measurements in red fluorescent channels (O2 probe). The spheroid size produced by high throughput methods is limited by microwell diameter and requires frequent media exchange to avoid media depletion and acidification. With all these limitations, we recommend the 3D Petri Dishes (MicroTissue) for experiments requiring high-throughput (e.g., for 3D bioprinting applications) and low-attachment methods for &#34;medium-throughput&#34; basic studies, also requiring lower cell numbers.
Ratiometric oxygenation monitoring and conventional live cell fluorescence dye-based microscopy experiments are affordable on widely available inverted microscopes if equipped with a near-infrared (NIR)-sensitive camera, light-emitting diode (LED)-based excitation, and appropriate objective25. For more appropriate optical sectioning and FLIM, off-the-shelf available commercial confocal microscope systems (such as described here) are recommended. While we also describe a method for detecting NAD(P)H autofluorescence, requiring a more costly two-photon FLIM microscope, there are many different approaches, compatible and useful for confocal FLIM systems, equipped ideally with white light laser and hybrid or SPAD detectors. These include assessment of cell metabolism, FRET-FLIM biosensor proteins, imaging cell and tissue mechanics, autofluorescence of specific cell and tissue types, and receptor-drug interactions visualized via FLIM-FRET31.
The vendor-provided FLIM acquisition and analysis software can affect the success of the experiment, as it is not always possible to export the IRF or the images in a format compatible with the downstream open-source analysis. Post-processing is an important part of the FLIM analysis workflow, and it is still not universally standardized or accepted. Curve fitting requires a higher number of photons and more computational power compared to the phasor approach. FLIMfit45 (global and pixel-by-pixel fitting) and Flimview78 are among the open software used for curve fitting analysis, while PAM79, FLUTE64, and AlliGator80 are used for phasor analysis. FLIMJ46 is a useful ImageJ plugin that works with both approaches. Altogether, these options help improve the post-processing routine and make it faster, more versatile, and user-friendly.

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.remotexs.ntu.edu.sg/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><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&#039;s modified Eagle&#039;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&amp;nbsp;(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&#039;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&lt;br/&gt; 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 &amp;mu;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 &amp;micro;g/mL at 1h incubation</td></tr><tr><td>PVDF syringe filter 0.22 &amp;micro;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&amp;sup2; )</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&amp;sup2;)</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

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.

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 ...

production-multi-parameter-live-cell-fluorescence-lifetime-imaging

Nanyang Technological University

Research

JoVE Journal

Bioengineering

829 Views.  Ghent University. In dit werk presenteren en vergelijken we sferoïde vormingsmethoden die kunnen worden gebruikt voor analyse van celmetabolisme en zuurstofverdeling met behulp van levende celmicroscopie. In de afgelopen jaren is fluorescentiebeeldvormingsmicroscopie op grote schaal gebruikt om metabole biomarkers zoals NADPH en FAD in levende cellen te onderzoeken, en er zijn verschillende fluorescerende nanodeeltjes ontwikkeld om dergelijke metingen te multiplexen met beeld...

Video: Auteur in de schijnwerpers: standaardisatie van sferoïde vormingsmethoden voor metabole en oxygenatieanalyse met behulp van fluorescentiebeeldvormingsmicroscopie

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Cancer Research

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

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 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.

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major clinical challenge because no efficient method exist for their elimination once their dissemination is underway. A better understanding of the mechanisms regulating cancer cell invasion may lead to the development of novel potent therapies. Due to their physiological resemblance to tumors, spheroids embedded in collagen I have been extensively utilized by researchers to study the mechanisms governing cancer cell invasion into the extracellular matrix (ECM). However, this assay is limited by (1) a lack of control over the embedding of spheroids into the ECM; (2) high cost of collagen I and glass bottom dishes, (3) unreliable immunofluorescent labeling, due to the inefficient penetration of antibodies and fluorescent dyes and (4) time-consuming image processing and quantification of the data. To address these challenges, we optimized the three-dimensional (3D) spheroid protocol to image fluorescently labeled cancer cells embedded in collagen I, either using time-lapse videos or longitudinal imaging, and analyze cancer cell invasion. First, we describe the fabrication of a spheroid imaging device (SID) to embed spheroids reliably and in a minimal collagen I volume, reducing the assay cost. Next, we delineate the steps for robust fluorescence labeling of live and fixed spheroids. Finally, we offer an easy-to-use Fiji macro for image processing and data quantification. Altogether, this simple methodology provides a reliable and affordable platform to monitor cancer cell invasion in collagen I. Furthermore, this protocol can be easily modified to fit the users&#8217; needs.

Presented here is a protocol for the fabrication of a spheroid imaging device. This device enables dynamic or longitudinal fluorescence imaging of cancer cell spheroids. The protocol also offers a simple image processing procedure for the analysis of cancer cell invasion.

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major ...

Biology

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale quantitative phenotypic measurements from cells. This approach has proved powerful for deepening our understanding of a wide range of both fundamental and applied events in cell biology. To date, the majority of applications for this technology have relied on the use of cells grown in monolayers, although it is increasingly realized that such models do not recapitulate many of the interactions and processes that occur in tissues. As such, there has been an emergence in the development and use of 3-dimensional (3D) cell assemblies, such as spheroids and organoids. Although these 3D models are particularly powerful in the context of cancer biology and drug delivery studies, their production and analysis in a reproducible manner suitable for HCS and HCA present a number of challenges. The protocol detailed here describes a method for the generation of multicellular tumor spheroids (MCTS), and demonstrates that it can be applied to three different cell lines in a manner that is compatible with HCS and HCA. The method facilitates the production of several hundred spheroids per well, providing the specific advantage that when used in a screening regime, data can be obtained from several hundred structures per well, all treated in an identical manner. Examples are also provided, which detail how to process the spheroids for high-resolution fluorescence imaging and how HCA can extract quantitative features at both the spheroid level as well as from individual cells within each spheroid. This protocol could easily be applied to answer a wide range of important questions in cell biology.

This protocol details a method for the production of three different types of spheroids in a manner that makes them suitable for large-scale high-content screening and analysis. In addition, examples are presented showing how they can be analyzed at spheroid and individual cell levels.

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

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