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

Abstract

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.

Protocol

1. Embedding of 3D Spheroids

1. Prepare the agarose gel into which the spheroids are embedded (only necessary first time performing the protocol)
   1. Mix 1 g of bacto-agar in 50 mL of ddH₂O.
   2. 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.
   3. Keep the bacto-agar warm in a water bath at 60 °C.
   4. Keep at 4 °C between experiments.
2. Embedding of spheroids
   1. On day 1, for each condition, pool a minimum of 12 spheroids in a 1.5 mL tube.
   2. Wash once with 1 mL of ice-cold 1X ....

Results

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bactoagar	BD Bioscience	#214010	Used for agarose gel preparation
Formaldehyde	VWR Chemicals	#9713.1000	Used for cell fixation
Medim Uni-safe casette	Medim Histotechnologie	10-0114	Used for storage of embedded spheroids
Superfrost Ultra-Plus Adhesion slide	Menzel-Gläser	#J3800AMNZ	Microscope glass slide used for embedding
CellTiter-Glo 3D Cell Viability Assay	Promega	#G9681	Used for the cell viability assay