When combined with high-content confocal microscopy and commercially available viability reagents, Visikol® HISTO-M™ allows the interior environment of 3D cell cultures to be imaged, facilitating the detection and quantification of a necrotic core in large HepG2 spheroids. Spheroids larger than 200 µm in diameter present a necrotic core, indicated by higher frequencies of centralized, non-viable cells.
Given their low-cost and high-throughput nature, multicellular spheroid models have become a mainstay in high content screening approaches to drug discovery and development in various physiological and pathophysiological contexts. For example, multicellular spheroids have been generated from cancerous cells to screen potential chemotherapeutic agents, hepatocytes to screen for drug-related liver toxicity, and even isolated primary cells from the human cortex to study drug penetration into the blood brain barrier. Given the crucial nature of cell viability in assaying the cytotoxic effects of therapeutic candidates in these various contexts, a reliable determination of the frequency and distribution of non-viable cells is necessary for the establishment of a baseline in newly developed models and for determining the localization of cytotoxic effects in treated models.
Here, we employed the commonly used hepatocyte cell line, HepG2 to generate multi-cellular spheroids of varying sizes using Corning® Ultra-low Attachment Spheroid Micro-plates. Combined with ThermoFisher’s LIVE/DEAD™ Fixable Green Dead Cell Stain and spheroid clearing with Visikol HISTO-M we were able to determine the frequency and localization of non-viable cells.
HepG2 cell culture
HepG2 cells were maintained in Advanced DMEM, supplemented with 5% fetal bovine serum, 1x GlutaMAX and 1x antibiotic-antimitotic in a humidified, 37°C, 5% CO2 incubator and passaged via light trypsinization with 0.05% trypLE and 1 mM EDTA upon reaching 80% confluence. Trypsinized cells were resuspended in complete medium and 5 x 102 – 16 x 103 cells were plated in each of 96 wells of a Corning Round Bottom Ultra Low Attachment Spheroid Microplate. Spheroids were maintained under standard culture conditions for 4 days, exchanging half of the media volume with fresh complete media after 2 days.
Staining and fixing HepG2 spheroids
Spheroids were washed twice with 1X D-PBS, and a 1:1000 dilution of Thermo LIVE/DEAD Fixable Green Dead Cell Stain (reconstituted according to manufacturer’s instructions in 1X D-PBS) was added to each well. Following 45 minutes of room temperature incubation with the LIVE/DEAD staining solution, spheroids were washed twice with 1X D-PBS, fixed with 10% neutral buffered formalin and permeabilized with 0.2% Triton, each for 30 min at room temperature. Fixed spheroids were then stained with a 1:5000 dilution of DAPI in Visikol HISTO Antibody Buffer for 1 h at room temperature.
Clearing and high throughput imaging of spheroids
Stained spheroids were washed twice with Visikol HISTO washing buffer, once with deionized water, once with 50% methanol in deionized water, and once with 100% methanol. For clearing, as much methanol as possible was removed from the well, and Visikol HISTO-M was added to each well for subsequent imaging on a CX7 LZR High Content Confocal Imager. Z-stacks were collected for each spheroid, using 10 µm steps. Image analysis was performed using both custom ImageJ macros and CellProfiler.