3D Cell Culture Assays

Three-dimensional (3D) cell culture models have become a well established in vitro experimental approach for cancer research, enabling more physiologically relevant cell-to-cell interactions compared to 2D cell culture systems. The use of clear U bottom ultra low attachment (ULA) microplates that promote spheroid formation and growth have become a standard 3D cell culture system for applications such as quantifying spheroid proliferation and viability.


Label-Free Spheroid Proliferation Assay

Brightfield imaging provides a convenient label-free method for quantifying spheroid size. In addition to simple endpoint evaluation, onboard environment control within the BioSpa Live Cell Analysis System enable detailed long-term kinetic analysis of spheroids proliferation.


Video 1. Label-free imaging and Gen5 image analysis tools automatically identify spheroids and report size for quantification and comparison across treatment conditions. HT-1080 cells were seeded in a 96-well ULA microplate (1000 cells/well) and incubated for 48 hours to allow for spheroid formation. Spheroid proliferation was monitored using the BioSpa Cell Analysis System with brightfield images acquired every 24 hours using a 4x objective. Gen5 object masks (yellow) were placed around each spheroid for analysis with Object Sum Area reported in real time.


High density microplate format for evaluating multiple treatment conditions with replicates

Spheroid Proliferation Assay is compatible with 96- and 384-well ULA microplates for higher throughput applications

Figure 1. Spheroid Proliferation Assay is compatible with 96- and 384-well ULA microplates for higher throughput applications. (A) Matrix-view of kinetic profiles of spheroid area for each well in 96-well format. (B) Mean profiles of spheroid area over time for each drug concentration with error bars (SD) displayed for comparison across treatments.


Fluorescence-based 3D Cell Model Proliferation and Viability Assay

Fluorescent labels and expressed tags enable detailed evaluation of spheroid size and cell viability. Fluorescence imaging protocols for the Cytation Imaging Readers and the Lionheart FX Automated Microscope and Gen5 image analysis tools enable quantitative analysis of spheroid size and viability using area and fluorescence intensity metrics. Additionally, the Cytation C10 Confocal Imaging Reader enables direct counts of labeled cells within spheroid samples for determination of relative cell number and percent apoptotic or necrotic cells.

3D cell model proliferation and viability assay.

Figure 2. 3D cell model proliferation and viability assay. (A) Schematic of the assay workflow combining imaging-compatible ULA microplates that promote uniform spheroid formation, and a broad range of available fluorescent cell viability markers. (B) Acquisition of individual optical slices or z-stacks using the Cytation C10 and Gen5 image analysis tools enable detailed quantitative analysis of spheroids.  


Direct cell counts within spheroids for evaluating treatment response

Direct cell counts provide a quantitative readout of drug treatment response

Figure 3.  Direct cell counts provide a quantitative readout of drug treatment response. (A) Relative total number of cells and (B) relative number of apoptotic or necrotic cells are determined using Gen5 image analysis tools (yellow and fuchsia masks, respectively) Dose response curves are generated using (C) relative total cell number or (D) ratio of cells positive for necrotic marker to total cell number. 

Increase assay throughput and performance

Automated, gentle media exchange for 3D cell cultures
MultiFlo FX

The BioTek MultiFlo FX with the Automated Media Exchange (AMX) module
Automates media exchange and washing for spheroids, tumoroids and other 3D cell structures
Helps protect 3D cell culture models and encourage cell structure growth



Application Notes:


Visual Abstract:

For Research Use Only. Not for use in diagnostic procedures.