high-throughput generation of multicellular spheroids from human cancer cell line for live 3d imaging

Multicellular Spheroid Formation for FLIM-Based Metabolic and Hypoxia Analysis

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.

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

In this work, we present and compare spheroid formation methods that can be used for analysis of cell metabolism and oxygen distribution using live cell microscopy. In recent years, fluorescence lifetime imaging microscopy has been extensively used to investigate metabolic biomarkers like NADPH and FAD in live cells, and several fluorescent nanoparticles have been developed to multiplex such measurements with imaging of cell and tissue oxygenation. Multicellular spheroids, organoids, and organ-on-a-chip can replicate a complex, in-vivo-like microenvironment, minimizing the need in animal research.For spheroid production, we show different formation methods, can span from low to high throughput. They also highlight their optical accessibility, compatibility with fluorescence lifetime imaging microscope, and the possibility of including extracellular matrix components. So, even though 3D in vitro models provide better context in comparison to 2D cultures, their high variability, low reproducibility, and incomplete experimental reporting remain a problem.Parameters such as the spheroid size, the nutrient composition, the extracellular viscosity, and even spheroid formation methods can all lead to increased cellular heterogeneity. With this protocol, we aim to harmonize and standardize the spheroid production methods, highlighting key aspects important for life-continuous and multiparametric analysis of spheroids using FLIM microscopy.

High-Throughput Generation of Multicellular Spheroids from Human Cancer Cell Line for Live 3D Imaging

To cast 3D Petri dishes, rinse the micromolds in distilled water and place them in an autoclavable container for autoclaving. Add 0.9%sterile saline to the autoclave agarose powder in a 200 milliliter glass bottle. Screw on the lid loosely to avoid pressure buildup and swirl the bottle to mix the agarose powder.Boil the agarose powder in a microwave oven with intermittent shaking. Once the agarose solution is cooled down, pipette the molten agarose into the micro mold and wait until it solidifies. Afterward, carefully flex the micro mold to remove the 3D Petri dish and transfer it to a 12-well tissue culture plate.To equilibrate the 3D Petri dish, add 2.5 milliliters of McCoy's 5A medium per well, and incubate for 15 minutes at 37 degrees Celsius and 5%carbon dioxide. After the final equilibration, completely remove all culture medium surrounding the 3D Petri dish and inside of the dish. Dropwise, seed 190 microliters of cell suspension containing 40, 500 cells into the seeding chamber and allow the cells to settle for 10 minutes.Add two milliliters of McCoy's 5A medium to the outside of the 3D Petri dish, and incubate at 37 degrees Celsius and 5%carbon dioxide. After four days, the spheroids are compact enough for microscopy. Add 200 microliters of the coating solution to each well of a 96-well U-bottom culture plate, and incubate for one minute.Remove the excess coating solution and allow the plate to dry for one hour. See 200 microliters of cell suspension containing 500 cells to each well and incubate at 37 degrees Celsius and 5%carbon dioxide for at least four days.

high-throughput-generation-multicellular-spheroids-from-human-cancer

Research

JoVE Journal

Bioengineering

23 Views. Per fondere le piastre di Petri 3D, sciacquare i microstampi in acqua distillata e metterli in un contenitore autoclavabile per la sterilizzazione in autoclave. Aggiungere lo 0,9% di soluzione fisiologica sterile alla polvere di agarosio dell'autoclave in un flacone di vetro da 200 millilitri. Avvitare il coperchio senza stringere per evitare l'accumulo di pressione e agitare il flacone per mescolare la polvere di agarosio.Far bollire la polvere di aga...