An Overview of the Current State of Cell Viability Assessment Methods Using OECD Classification

Abstract

Over the past century, numerous methods for assessing cell viability have been developed, and there are many different ways to categorize these methods accordingly. We have chosen to use the Organisation for Economic Co-operation and Development (OECD) classification due to its regulatory importance. The OECD categorizes these methods into four groups: non-invasive cell structure damage, invasive cell structure damage, cell growth, and cellular metabolism. Despite the variety of cell viability methods available, they can all be categorized within these four groups, except for two novel methods based on the cell membrane potential, which we added to the list. Each method operates on different principles and has its own advantages and disadvantages, making it essential for researchers to choose the method that best fits their experimental design. This review aims to assist researchers in making this decision by describing these methods regarding their potential use and providing direct references to the cell viability assessment methods. Additionally, we use the OECD classification to facilitate potential regulatory use and to highlight the need for adding a new category to their list.

Keywords: OECD cell viability classification; cell viability; cell-based methods; in vitro toxicology.

Figure 1

Graphical scheme of the cell viability methods based on structural cell damage (non-invasive). (a) Cell morphology changes between two samples with an inverted microscope. The left image shows viable cells, while the right image displays cells treated with 12.5 mg/L of Arsenic V. (b) Measurement of leaking molecules from viable and non-viable cells. The provided example uses the LDH enzyme, which leaks in greater amounts from non-viable cells due to membrane disruption. The supernatant (extracellular fluid) is collected and measured with a spectrophotometer, where higher values indicate non-viable cells.

Figure 2

Graphical scheme of the cell viability methods based on structural cell damage (invasive). (a) In non-viable cells, a molecule like PI is transported within the cell. While it can also be transported in viable cells, it occurs to a much lesser extent. Once PI binds to RNA or DNA, it emits fluorescence upon excitation. (b). Lipophilic dyes pass through the cell membrane and are cleaved by esterases within viable cells, occurring more frequently in viable cells. After cleavage, they emit fluorescence upon excitation. (c) Antibodies pass through permeabilized cells and attach to specific molecules related to cell death pathways, such as caspase. If the target molecule is present, the antibodies bind to them, allowing visualization or quantification using fluorescence measurement instruments.

Figure 3

Graphical scheme of the cell viability methods based on cell growth. (a) The cell count of a sample is assessed before and after treatment using an inverted microscope or, alternatively, a flow cytometer. (b) BrdU or EdU are integrated into the cell DNA, making them visible in daughter cells and allowing for the precise quantification of replicated cells. (c) The absorbance of Sulforhodamine B, which binds to cellular proteins, can be measured with a spectrophotometer. Thus, this measurement enables the quantification of the total cell protein content.

Figure 4

Graphical scheme of the cell viability methods based on cellular metabolism. (a) Molecules are metabolized in the mitochondria at varying rates depending on the cell’s metabolism (viability). The viable cell on the left has a higher number of reduced molecules compared to the non-viable cell on the right, resulting in lower absorbance or fluorescence intensity in the latter. (b) The mitochondrial membrane potential is measured using a dye that emits different intensities and bandwidths based on the mitochondrial membrane potential. (c) Neutral red is transported within the cell by lysosomes. In viable cells, this transport is unaltered, while in non-viable cells, it is disrupted. The amount of neutral red within the cell is then measured, with higher levels found in viable cells. (d) Viable cells produce more ATP than non-viable cells. After cell lysis, ATP content is measured using luminometric assays.

Figure 5

Graphical scheme of the cell viability methods based on cell membrane potential. (a) The intensity of dye fluorescence is proportional to the membrane potential. Previous studies utilized Fluovolt™ (FV) (Thermo Fisher Scientific, Waltham, MA, USA) for this purpose. (b) Cell viability is assessed by measuring both the cell membrane potential and DNA content using the dye Vybrant™. Viable cells and cells in cell cycle arrest exhibit stable FV intensity, while apoptotic and dividing cells show higher intensity. Necrotic cells have the lowest fluorescence emission due to dye leakage. Dividing cells and cells in cycle arrest emit higher fluorescence intensity corresponding to 2n DNA.

Figure 6

Suggested decision tree for the desired method.