Method to Disassemble Spheroids into Core and Rim for Downstream Applications Such as Flow Cytometry, Comet Assay, Transcriptomics, Proteomics, and Lipidomics

Advancements in 3D spheroid imaging: Optimised cryosectioning and immunostaining techniques

“…The methods presented in this protocol were tested using 21-day-old HepG2/C3A spheroids stained for alpha-smooth muscle actin (α-SMA) (Uniprot Accession Number P62736). 21-day-old HepG2/C3A spheroids are large, approximately 800 to 1000 µm pseudo tissue structures with a viable rim and necrotic core [ 3 , 8 ]. Such spheroids are dense and difficult to analyse using fluorescent light microscopy without sectioning.…”

“…This approach was originally developed for sectioning animal and plant tissues, and the existing protocols are not optimal for handling small and fragile spheroids and organoids. Typically, spheroids larger than 600 µm in diameter develop a distinctly structured necrotic core [3] . The necrotic core is located in the central part of the spheroids and consists of compacted cell debris and DNA fragments.…”