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

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

To begin, fix the spheroids in a fume hood on day one as outlined in the text protocol. On day two, place the agarose gel into a water-filled beaker in a microwave oven to heat it, and keep the gel warm on a benchtop heating plate set to 60 degrees Celsius until needed.
Wash the spheroids twice in the fume hood using 1 milliliter of ice-cold 1X PBS per wash. Then, aspirate most of the PBS. Prepare a 20-microliter pipette tip by cutting it at an incline to obtain a pointier tip with a larger hole.
After this, make an agarose gel drop on a microscope slide. Place the slide on a warm heating block to prevent the agarose from solidifying. Using the modified pipette tip, catch as many spheroids as possible in a volume of 15 to 20 microliters.
Carefully inject the spheroids into the center of the agarose gel drop, making sure to not touch the microscope slide. Incubate at room temperature or at 4 degrees for 5 to 10 minutes to let the agarose gel drop harden. When the gel drop has solidified somewhat but is still rather soft, use a scalpel to carefully push it from the microscope slide into a plastic tissue cassette. Transfer the tissue cassette to a beaker filled with ethanol and store at room temperature.

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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>

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...

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

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