Name: Video: Embedding 3D Spheroids in Agarose Gel Drops: A Technique to Preserve Spheroids for Downstream Analysis - Concept
Uploaded: 2023-04-30T13:41:01.000+00:00
Duration: 1 min 34 s
Description: 2.7K Views. Source: Rolver, M. G. et al. Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells. J. Vis. Exp. (2019) This video describes the technique to embed 3D spheroids in agarose gel drops for preservation. The embedded 3D spheroids can be further used for downstream analysis.

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1. Embedding of 3D Spheroids
<ol>
	<li>Prepare the agarose gel into which the spheroids are embedded (only necessary first time performing the protocol)
	<ol>
		<li>Mix 1 g of bacto-agar in 50 mL of ddH&#8322;O.</li>
		<li>Heat slowly in the microwave oven until the bacto-agar has dissolved and a homogeneous gel has formed. Do not allow the gel to boil.</li>
		<li>Keep the bacto-agar warm in a water bath at 60 &#176;C.</li>
		<li>Keep at 4 &#176;C between experiments.</li>
	</ol>
	</li>
	<li>Embedding of spheroids
	<ol>
		<li>On day 1, for each condition, pool a minimum of 12 spheroids in a 1.5 mL tube.</li>
		<li>Wash once with 1 mL of ice-cold 1X PBS.</li>
		<li>To fix the spheroids, add 1 mL of 4% paraformaldehyde. 
		NOTE: Handling of paraformaldehyde should be performed in a fume hood.</li>
		<li>Let them incubate for 24 h at RT.</li>
		<li>On day 2, heat the agarose gel carefully by placing it in a water-filled beaker in a microwave oven. Ensure that the gel does not boil. Keep warm on a benchtop heating plate, at 60 &#176;C until use.</li>
		<li>Wash spheroids twice with 1 mL of ice-cold 1X PBS.</li>
		<li>Aspirate most of the 1X PBS (leaving approximately 100 &#181;L at this point is practical for handling the spheroids).</li>
		<li>Prepare a 20 &#181;L pipette by cutting the pipette tip at an incline to obtain a pointier tip with a larger hole (Figure 1). 
		NOTE: The next part has to be done quickly to ensure optimal spheroid transfer and to avoid solidification of gel drop. If no heating block is available, it is recommended to first catch the spheroids and then make the agarose drop (i.e., switching the order of points 1.2.9 and 1.2.10).</li>
		<li>Make an agarose gel drop on a microscope slide. Place the slide on a warm heating block to prevent the agarose from solidifying.</li>
		<li>Using the modified pipette tip (step 1.2.8), catch as many spheroids as possible in a volume of 15&#8211;20 &#181;L.</li>
		<li>Carefully inject the 15&#8211;20 &#181;L spheroid-containing 1X PBS into the center of the agarose gel drop without touching the microscope slide. 
		NOTE: This is a slightly difficult point. The spheroids will be lost if the pipette tip touches the microscope slide when injecting the spheroids into the gel drop. It is advisable to practice the whole process of making the agarose drop and injecting the spheroids by injecting a colored liquid into the drop. This will allow visualization of a potential penetration through the drop, as the colored liquid will be leaking out onto the slide.</li>
		<li>Let the agarose gel drop harden by incubating for 5&#8211;10 min at RT or at 4 &#176;C. Once the gel drop has solidified somewhat (but is still rather soft), carefully push the gel drop from the microscope slide into a plastic tissue cassette with a scalpel.</li>
		<li>Cover the plastic tissue cassettes in 70% ethanol. 
		NOTE: At this point, the spheroids can be used directly or stored for months.</li>
		<li>Embed the agarose-embedded spheroid in paraffin, section into 2&#8211;3 &#181;m thick layer slides, and stain with hematoxylin and eosin or subject to immuno-histological staining.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>Bactoagar</td><td>BD Bioscience</td><td>#214010</td><td>Used for agarose gel preparation</td></tr><tr><td>Formaldehyde</td><td>VWR Chemicals</td><td>#9713.1000</td><td>Used for cell fixation</td></tr><tr><td>Medim Uni-safe casette</td><td>Medim Histotechnologie</td><td>10-0114</td><td>Used for storage of embedded spheroids</td></tr><tr><td>Superfrost Ultra-Plus Adhesion slide</td><td>Menzel-Gl&amp;auml;ser</td><td>#J3800AMNZ</td><td>Microscope glass slide used for embedding</td></tr><tr><td>CellTiter-Glo 3D Cell Viability Assay</td><td>Promega</td><td>#G9681</td><td>Used for the cell viability assay</td></tr></tbody></table>

embedding 3d spheroids in agarose gel drops: a technique to preserve spheroids for downstream analysis

Source: Rolver, M. G. et al. <a href="https://www.jove.com/video/59714" target="_blank">Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells.</a> J. Vis. Exp. (2019)
This video describes the technique to embed 3D spheroids in agarose gel drops for preservation. The embedded 3D spheroids can be further used for downstream analysis.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20481/20481fig1.jpg" /> 
Figure 1: Fixing, embedding and immunohistochemistry analysis of spheroids. (A) Schematic representation of the protocol for embedding of spheroids. Individual steps are marked as (i-vii). (B) Image of embedded MDA-MB-231 spheroid. Scale bar: 50 &#181;m. (C) Representative image of chemotherapy-treated MDA-MB-231 sphe...

Embedding 3D Spheroids in Agarose Gel Drops: A Technique to Preserve Spheroids for Downstream Analysis

Source: Rolver, M. G. et al. Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells. J. Vis. Exp. (2019)
This video describes the ...

Spheroids are three-dimensional in vitro spherical cellular aggregates that mimic the in vivo tumor microenvironment and help study tumor progression. To preserve these spheroids by embedding, begin by taking a sterile tube containing spheroids in a desired buffer.
Treat the spheroids with paraformaldehyde. Paraformaldehyde crosslinks cellular proteins, resulting in the stabilization and preservation of cell structure. Centrifuge the spheroids and remove the paraformaldehyde-containing supernatant. Resuspend the spheroid pellet in buffer.
Next, prepare the desired concentration of molten agarose gel solution. Make an agarose gel drop on a microscope slide. Maintain the slide on a heating block to avoid the solidification of agarose.
Using a micropipette with a wide orifice tip, collect the spheroids from the tube. Carefully pipette the spheroids into the center of the agarose gel drop without touching the microscope slide. Subsequently, incubate the slide at a suitable temperature to allow the ECM to solidify and entrap the spheroids within.
Gently transfer the semi-solid drop from the microscope slide into a tissue cassette. Store the embedded spheroids in ethanol until further downstream analysis.

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2.7K Views. Source: Rolver, M. G. et al. Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells. J. Vis. Exp. (2019) This video describes the technique to embed 3D spheroids in agarose gel drops for preservation. The embedded 3D spheroids can be further used for downstream analysis. 

Video: Embedding 3D Spheroids in Agarose Gel Drops: A Technique to Preserve Spheroids for Downstream Analysis - Concept

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Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

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Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

Embedding 3D Spheroids in Agarose Gel Drops: A Technique to Preserve Spheroids for Downstream Analysis

Transcript

Embedding 3D Spheroids in Agarose Gel Drops: A Technique to Preserve Spheroids for Downstream Analysis

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

A 3D Spheroid Model for Glioblastoma

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production