Preparation of Cancer Spheroid Blocks by Flash Freezing: A Procedure To Preserve Cancer Spheroids in Frozen Matrix Blocks

Abstract

Source: Wang, Y. et al. 3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer. J. Vis. Exp. (2018)

This video demonstrates the preparation of cancer spheroid blocks by flash freezing, a technique in which a tissue sample of interest is frozen to an ultracold temperature, restraining the spheroids inside the matrix.

Protocol

1. Preparation of Sample Block Containing Cancer Spheroids for Frozen Tissue Sectioning

1. Carefully lift the block of collagen gel and spheroids from the chamber slides by using small forceps. Place the collagen gel block in a plastic histology mold.
2. Rinse the collagen gel with 1x PBS briefly and then fix it with 4% paraformaldehyde (PFA) in PBS for 30 min at room temperature.
   NOTE: PFA is a potential carcinogen and should be handled with care.
3. Wash the collagen gel with 1.5 mL of 1x PBS on a shaker for 15 min. Replace the PBS and repeat the previous washing step 3x.
4. Apply a thin layer (about 3 mm) of optimal cutting temperature (OCT) compound to cover the bottom of a new plastic histology mold. Place the fixed and washed collagen gel on top of the OCT compound, then carefully embed the whole gel by filling the mold with OCT compound while avoiding the formation of any air bubbles in the OCT.
5. Leave the mold at 4 °C for 1 h.
6. Place the mold containing the sample and OCT compound on a 100 mm Petri dish floating on liquid nitrogen. Allow the sample to flash-freeze completely.
7. Store the frozen sample block at -80 °C for future use.

Materials

DMEM cell culture medium	Thermo Fisher Scientific	11995065
Fetal bovine serum	Thermo Fisher Scientific	26140079
Antibiotic-Antimycotic (100x)	Thermo Fisher Scientific	15240062
Bovine serum albumin (BSA)	Sigma-Aldrich	A3803
Phosphate-buffered saline (PBS), pH 7.4	Thermo Fisher Scientific	10010023
Collagen type 1 from rat tail, high concentration	Corning	354249
Nunc Lab-Tek II Chambered Coverglass	Thermo Fisher Scientific	155382
Paraformaldehyde solution	Electron Microscopy Sciences	15710
O.C.T compound	Thermo Fisher Scientific	23730571