What information do they provide for the development of biologics?
The development of biological medicines requires an understanding of how cells respond when exposed to a specific substance. Identifying its chemical composition or verifying its stability is not enough; it is also necessary to study its effects on living systems.
In this context, cell viability is one of the key parameters used to evaluate how cell cultures respond to new molecules and biotechnological products. Its analysis, together with proliferation and cytotoxicity assays, provides valuable information about cellular behaviour during the early stages of research.
Although these three concepts are related, each measures different biological phenomena. Assessing them together helps identify potential adverse effects, characterise biological activity and select candidates with greater potential to progress through development.
Cell viability: assessing cellular responses
Cell viability is used to evaluate the proportion of living and functional cells present in a sample. It is one of the most widely used indicators for studying how cell cultures respond to new molecules, compounds and biological products.
Common techniques include MTT, MTS and resazurin assays, which use indicators associated with cellular metabolic activity. Methods based on ATP quantification are also widely used, as ATP is an essential molecule for cell function.
These procedures make it possible to compare cultures treated with different product concentrations against control samples and identify potential changes in cellular activity.
For example, if one culture shows a viability signal equivalent to 90% of the control, while another reaches only 50%, there is a significant difference in the measured response. However, these percentages do not necessarily demonstrate that 10% or 50% of the cells have died.
A reduction in metabolic activity can occur without cell death. For this reason, it is important to complement cell viability assay results with other techniques that provide a more comprehensive characterisation of the biological response.
Cell proliferation: measuring growth capacity
Cell proliferation studies the ability of cells to divide and increase in number over a given period.
This parameter is particularly important in the development of monoclonal antibodies, growth factors, immunotherapies and other products capable of modifying cellular processes.
Tools commonly used to assess proliferation include EdU incorporation, which makes it possible to identify cells undergoing DNA synthesis, and Ki-67 detection, which identifies a protein associated with different phases of the cell cycle.
Cell counting using automated systems and flow cytometry techniques can also be performed.
A substance may maintain high cell viability while significantly reducing proliferation. This behaviour may correspond to a cytostatic effect.
This distinction is important because stopping cell multiplication does not necessarily mean causing cell death.
In certain cancer treatments, inhibiting tumour cell growth is precisely one of the desired therapeutic effects.
Cell cytotoxicity: identifying potential damage
Cell cytotoxicity assays determine whether a substance causes cellular damage or triggers cell death mechanisms.
One commonly used method involves measuring the release of lactate dehydrogenase (LDH), an enzyme that enters the extracellular medium when plasma membrane integrity is compromised.
Techniques involving annexin V and propidium iodide staining are also used to investigate changes associated with apoptosis and membrane alterations.
Results can be expressed using concentration-response curves. Under specific experimental conditions, it is possible to calculate the median cytotoxic concentration, known as CC50.
This value represents the concentration that produces the defined cytotoxic effect at 50% of the maximum response established by the assay.
Combining these techniques complements cell viability data and provides a more accurate understanding of the potential harmful effects of a substance on cells.
Differences between cell viability, proliferation and cytotoxicity
Although all three parameters are used to study cell culture responses, they do not provide the same information.
- Cell viability: evaluates indicators associated with the presence and functionality of living cells.
- Cell proliferation: measures the ability of cells to divide and increase in number.
- Cell cytotoxicity: identifies cellular damage and effects associated with cell death.
For example, a product may reduce proliferation without significantly affecting viability. Another may alter cellular metabolism before causing detectable structural damage.
Therefore, analysing these parameters together provides a more comprehensive picture of the biological response and prevents results from a single assay from being interpreted in isolation.
Why is it important to combine cell viability and cytotoxicity assays?
Isolated results can lead to incomplete interpretations of a substance’s effects.
Combining cell viability, proliferation and cytotoxicity assays makes it possible to build a more comprehensive profile of cell culture responses and identify potential differences between metabolic effects, growth and cellular damage.
It is also essential to include positive and negative controls, perform experimental replicates and assess potential interference from the substances being analysed with the detection methods used.
The international ICH Q6B guideline highlights the importance of characterising the biological activity of medicinal products obtained through biotechnological processes. Furthermore, assessing the potency of these products may require several complementary assays, depending on their characteristics and mechanisms of action.
Applications of cell viability in biological medicine development
Cell viability assays and other functional tests are used at different stages of research, from initial candidate selection to the characterisation of more advanced products.
Their main applications include:
- Immunotherapy: enable the study of cellular responses associated with the activity of specific treatments.
- Monoclonal antibodies: help characterise their effects on cell populations and specific biological processes.
- Regenerative medicine: contribute to evaluating the behaviour of cells used in therapeutic research.
- Formulation development: make it possible to investigate potential changes in cellular responses associated with different compositions.
- Manufacturing processes: provide information for studying the biological activity and certain functional characteristics of products.
However, cell-based assays do not replace the preclinical and clinical studies required to demonstrate the safety and efficacy of medicinal products.
Their main contribution is to provide reproducible experimental data that helps improve understanding of mechanisms of action, detect potential risks and support decision-making throughout the development of new biological medicines.
Frequently asked questions
- What is cell viability? Cell viability is a parameter used to evaluate the presence and functionality of living cells in a sample. It is used to study how cell cultures respond to different substances, compounds and biological products.
- How is cell viability measured? Cell viability can be measured using different techniques, such as MTT, MTS and resazurin assays, as well as ATP quantification methods. Each procedure uses specific indicators to obtain information about the condition of the cells.
- What is the difference between cell viability and cytotoxicity? Cell viability evaluates indicators related to the presence and activity of living cells, whereas cytotoxicity examines the harmful effects of a substance on cells, including mechanisms of cellular damage and death.
- What is a cell proliferation assay? It is an experimental test used to study the ability of cells to divide and increase in number over a given period. Common techniques include EdU incorporation and Ki-67 detection.
- What role does the EMA play in the development of biological medicines? The European Medicines Agency (EMA) evaluates biological medicines and establishes guidelines to ensure their quality, safety and efficacy throughout their development.
- What are in vitro cell viability assays? These are laboratory tests performed using cell cultures to study how cells respond to a specific substance. They are widely used in pharmaceutical and biotechnological research.
