Common Viability Assays for 3D Culture

Compared to 2D cultures, 3D cell cultures represent a significant step forward towards reflecting tumor biology in vivo. It’s important to be able to properly assess the cellular health and functional performance of these novel 3D bioprinted structures. The following common viability assays for 3D culture are optimized to ensure that you are able to properly analyze the health of your 3D structures.

Live/Dead Molecular Probes Assay

live/dead molecular probe assay 3d cell culture
Fig. 1: The molecules used in the Live/Dead molecular probes assay. Calcein AM (left) is readily absorbed by cells and remains in the cytosol of viable cells with intact membranes, fluorescing green. Ethidium Homodimer (right) binds to DNA in dead cells, fluorescing red.

Type: Fluorescent

Live/Dead imaging with molecular probes is by far the most common method for viability analysis of 3D cultures. This assay can be analyzed through fluorescent imaging or flow cytometry. This assay is highly effective at permeating many cell-encapsulating biomaterials used in bioprinting and can be used with minimal adjustments to the manufacturer’s protocol on many 3D cultures.

The biggest drawback of this assay is that samples must be sacrificed at each timepoint and cannot be reused.


AlamarBlue Assay

alamar blue assay 3d culture
Figure 2: AlamarBlue Assay utilizes the compound reazurin, which is reduced to the fluorescent resorufin through reduction reactions in metabolically active cells.  The fluorescence produced is proportional to the number of living cells.

Type: Fluorescent

The AlamarBlue Assay, available from ThermoScientific, is a popular colorimetric assay for 3D cultures. The most notable feature of this colorimetric viability assay is the lack of cell lysis, which allows for time-course experiments with the same sample. This assay is also very low cost.

The drawbacks of this assay are that it does not permeate all hydrogels effectively. While some hydrogels like gelatin and fibrin are penetrated easily by the AlamarBlue solution, the assay is less effective with others such as PEGDA.

ThermoFisher Scientific protocol calls for incubation times of 1-4 hours, however, up to 24 hours of incubation time has been used with 3D cultures. For new materials and 3D cellular cultures, users should test a variety of incubation times as well as concentrations to determine which is most accurate for that particular culture.


Promega 3D ATP Viability Assay

promega 3D ATP viability assay for 3d cell culture
Figure 3: Luciferin in the ATP assay. The homogeneous “add-mix-measure” format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present

Type: Luminescent

This assay is specifically designed for 3D cultures, with improved lytic activity compared to similar 2D assays. However, the assay is not designed for 3D cultures containing hydrogels or any other biomaterials. Therefore the protocol must be adjusted and optimized based on the microtissue.

Some 3D cultures are able to be penetrated by the assay solution with increased incubation time (up to 2 hours compared to the suggested 30 minutes in the protocol) and by placing the samples on a shaker during incubation, to further break up the tissue. Additionally, for some protein-based hydrogels, using an enzyme to break up the tissue before running the assay may also improve results. As this assay lyses cells, samples may not be reused after analysis.


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