Assay Methods for 3D Cultures
How to assay spheroids in culture is a continuing challenge. 3D cultures are dense in cells and ECM, and thus fluorescent signals can be of limited use (6) . Furthermore, 3D cell cultures can be delicate, and it can be difficult to add or remove solutions without disrupting spheroids. We tested two luminescent cell viability assays—CellTiter-Glo® and RealTime-Glo™ —to evaluate their performance with bioprinted 3D cultures. The CellTiter-Glo® Assay is an endpoint viability assay that quantifies the amount of ATP in metabolically active cells. Cells are lysed and luminescence is read from the media. The RealTime-Glo™ Assay is a non-lytic assay that allows real-time tracking of viability continuously during culture. In the RealTime-Glo™ Assay, a cell-permeant prosubstrate is reduced to a NanoLuc® luciferase substrate by viable cells. The NanoLuc® substrate diffuses out into the culture medium, where it is converted to a luminescent signal by NanoLuc® luciferase. The cells remain alive in the presence of the assay reagent and constantly produce luminescent signal for ~ 72 hours. In both of these assays, viability is measured with single additive steps, avoiding excessive handling issues with delicate spheroid cultures.
Materials and Methods
Cell Culture: HepG2 human hepatocellular carcinoma cells (ATCC, Manassas, VA) and PC3 human prostate cancer cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were maintained in a humidified environment (37ºC, 5% CO2).
Magnetic 3D Bioprinting: At 70-80% confluence, NanoShuttle™ (Nano3D Biosciences) was added to cells (1 µl/10,000 cells) and incubated overnight. The next day, the magnetized cells were enzymatically detached, counted, and resuspended in media. At the same time, a cell-repellent 384-well plate was placed on top of a 384-well spheroid drive (Nano3D Biosciences; Figure 2). The cells were distributed into the 384-well plate at concentrations of 50, 200, and 2,000 cells/well (50 µl/well), where they were instantly aggregated into spheroids. Spheroids were left to form on the magnet for 1 hour, after which time the plate was removed to culture in an incubator.
CellTiter-Glo® Cell Viability Assay: At 72 hours, media was exchanged with 50 µl of a 1:1 solution of media and CellTiter-Glo® Reagent. A 384-well spheroid drive was used to hold down spheroids while adding the solution, maintaining spheroid integrity. After the CellTiter-Glo® Reagent was added, the 384-well plate was removed from the spheroid drive and luminescence read on a luminometer (Synergy 4, Biotek, Winooski, VT). We also studied the effect of acetaminophen (Sigma-Aldrich, St. Louis, MO) on viability. Varying amounts of acetaminophen were added to HepG2 spheroids (2,000 cells/spheroid) and incubated for 72 hours. At 72 hours, the CellTiter-Glo® Assay was performed.
RealTime-Glo® MT Cell Viability Assay: Immediately after printing spheroids, the media was exchanged with 50 µl of media and 2X RealTime-Glo® Reagent at a 1:1 ratio. A spheroid drive was used to hold down spheroids while adding the solution, maintaining the spheroid integrity. After addition of the reagent, the 384-well plate was removed from the spheroid drive and left to culture in an incubator. Luminescence was measured at specified time points up to 72 hours using a luminometer (Synergy 4).
Live/Dead Staining: After 72 hours, spheroids were stained to detect viable cells using a live/dead staining assay (Biotium, Hayward, CA) to confirm the results of the CellTiter-Glo® and RealTime-Glo™ Assays. Media was replaced with a solution containing 2 µM calcein-AM and 4 µM ethidium homodimer-1. After a 10-minute incubation, the spheroids were imaged under a fluorescence microscope.