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

Cancer spheroids are three-dimensional cellular aggregates of tumor cells. These spheroids can be embedded in the collagen matrix, resulting in the formation of spheroid blocks. To flash-freeze a spheroid block, begin by placing a collagen gel spheroid block in the center of a histology mold.

Rinse the block with a suitable buffer and supplement the mold with a fixative solution. Incubate to allow this solution to penetrate the spheroids and cross-link the cellular proteins. This step helps maintain the spheroid structure for the subsequent procedure. Next, aspirate the fixative solution.

Place the spheroid gel block into a fresh histology mold containing a thin layer of embedding medium. Overlay it with more embedding medium to completely submerge the spheroid block. Finally, submerge the mold in a liquid nitrogen bath for a short duration.

Rapid freezing prevents the formation of bigger ice crystals within spheroids. It helps in retaining spheroid integrity without causing mechanical damage. Once the embedding medium layer solidifies around the spheroid block, remove the frozen block from the mold. Store the frozen spheroid block at low temperatures for further histological analysis.

First, use small forceps to lift the block of collagen gel and spheroids from the chamber slide. Then, place the collagen gel block in a plastic histology mold. Rinse the collagen gel block with 1X PBS briefly and fix it with 4% PFA in PBS for 30 minutes at room temperature. After this, wash the collagen gel block with 1.5 milliliters of 1X PBS on a shaker for 15 minutes.

Apply a thin layer of OCT compound to the bottom of a new plastic histology mold. Then, place the fixed and washed collagen gel block on top of the OCT compound before carefully filling the rest of the mold with OCT compound. Keep the mold at 4 degrees Celsius for 1 hour. After this, flash-freeze the mold on a 100-millimeter Petri dish floating on liquid nitrogen, and store the samples at minus 80 degrees Celsius.