Table 2 Overview of techniques for cell viability assessment in 2D and 3D cultures: applicability, limitations, and optimization strategies

MTT

No limitation;

Gold standard method;

Well-established and widely used;

Notes:

Formazan solubility: insoluble (requires solubilization);

Can involve mechanisms beyond mitochondrial activity, leading to overestimated results;

Lipidic compounds and surfactants may interact with formazan, causing over- or underestimation of cell viability.

Very limited: Penetration issues;

Optimization:

Low penetration impacts results in dense 3D structures;

As alternative, use soluble tetrazolium-based assays (e.g., WST-1, WST-8).

Spectrophotometry

Angius and Floris 2015; Stepanenko and Dmitrenko (2015); Adan et al. (2016); Carvalho et al. (2017); Ghasemi et al. (2021)

MTS

No limitation;

Notes:

Formazan solubility: soluble;

Can be reduced by non-mitochondrial pathways.

Limited: Diffusion in 3D is better than MTT but may underestimate inner regions;

Optimization: Increases incubation time or fragment structure;

Pre-select 3D structures based on size and shape to reduce variability.

Spectrophotometry

Riss et al. (2013); Khalef et al. (2024)

WST-1/WST-8

No limitation;

Notes:

Formazan solubility: highly soluble;

Can be reduced by non-mitochondrial pathways.

Limited: Better diffusion and reactivity than MTS;

Optimization: Increases reaction time or fragment highly dense structure;

Pre-select 3D structures based on size and shape to reduce variability.

Spectrophotometry

Riss et al. (2013); Oner et al. (2023); Khalef et al. (2024)

Resazurin

No limitation;

Notes:

Little to no interaction with NPs;

Reproducibility depends on resazurin concentration and incubation time.

Limited: Better diffusion than formazan-based reagents, but may still require optimization;

Optimization:

Extend incubation time or fragment highly dense structures;

Pre-select 3D structures based on size and shape to reduce variability.

Spectrophotometry or fluorescence

Bonnier et al. (2015); Adan et al. (2016); Gong et al. (2020)

ATP levels

No imitation;

Quick and easy method;

Does not rely on reagent penetration but on cell lysis.

Limited: Depends on homogeneous lysis of the 3D structure;

Variability may arise in large or dense spheroids if lysis is incomplete;

Optimization:

Adjust lysis protocol for 3D models;

Use detergent-based lysis buffers designed for 3D models;

Extend incubation time to ensure ATP extraction;

Pre-select 3D structures based on size and shape to reduce variability.

Luminometry

Kijanska and Kelm (2016); Dominijanni et al. (2021)

SRB

No limitation;

Suitable for long-term studies;

High sensitivity;

Can also evaluate cell proliferation;

Note:

Cannot differentiate between live and dead cells.

Limited: depends on homogeneous lysis of the structure;

Optimization:

Adjust lysis protocol;

Pre-select 3D structures based on size and shape to reduce variability;

Use complementary viability assays to confirm results.

Spectrophotometry

Vichai and Kirtikara (2006); Adan et al. (2016)

Neutral Red

No limitation;

Simple and sensitive method;

Notes:

Not suitable for long-term studies;

Interference with silver NPs.

Limited: depends on homogeneous lysis of the structure;

Optimization:

Adjust lysis protocol;

Pre-select 3D structures based on size and shape to minimize variability;

Use complementary viability assays to confirm results.

Spectrophotometry

Repetto et al. (2008); Perez et al. (2017); Mello et al. (2020)

LDH

No limitation;

Notes:

Results may be altered by culture conditions (e.g., growth/death rates, background LDH release in controls);

Cooper-based compounds may hinder LDH detection.

Limited: requires uniform collection of culture medium;

Optimization:

Strict volume control and thorough mixing of medium before sampling;

Pre-select 3D structures based on size and shape to minimize variability;

Use complementary viability assays to confirm results.

Spectrophotometry, fluorescence or luminometry

Kong et al. (2011); Cox et al. (2021); Bromma et al. (2023)

Live/Dead Assay

No limitation;

Quick and easy method;

Provides visual results to support quantitative methods;

Note:

Propidium iodide may enter viable cells via NP endocytosis, producing false positives.

Limited: allows in-depth analysis with confocal microscopy but may underestimate inner regions;

Optimization:

Increase reaction time;

Adjust reagent concentration;

Pre-select 3D structures based on size and shape to reduce variability;

Use confocal microscopy with Z-stack imaging to assess inner layers.

Fluorescence or confocal microscopy

Kong et al. (2011); Dominijanni et al. (2021); Khalef et al. (2024)

Trypan Blue/Erytrosine B

No limitation;

Note:

Automated analysis is recommended to reduce errors compared to manual readings.

Limited: 3D structure requires lysis prior to analysis;

Optimization:

Adjust lysis protocol to ensure viable cells post-dissociation;

Pre-select 3D structures based on size and shape to reduce variability;

Use complementary viability assays to confirm results.

Optical microscopy

Piccinini et al. (2017); Khalef et al. (2024)