Flow cytometry is an analytical method that allows cells to be quickly analyzed and categorized by lasers as they pass in single-file. The way in which the laser light is scattered can be used to infer a degree of information regarding the cell, such as size, internal complexity, and cell granularity, while more specific structure identification can be achieved by incorporating fluorophores and fluorescent microscopy into the process.
Other characterization methods such as mass spectrometry may be incorporated directly into flow cytometry equipment to allow more refined characterization to be undertaken in situ.
Image Credit: paulista/Shutterstock.com
Flow cytometry in target identification
Flow cytometry has been utilized at each stage of the drug discovery process, from target identification to lead development. Valid drug targets in cells encompass almost the entire plethora of biomolecular structures available: the cell membrane, any protein involved in mechanistic function or signaling, DNA down to the level of the gene, and other smaller molecules such as mRNA.
Characterization of large and varied cell populations in a high throughput manner is useful in initial target identification, allowing dysfunctional (diseased) cells to be separated for deeper analysis by other techniques. This process can be further optimized by tagging molecular features within the cell suspected to be involved in disease with fluorescent probes, and in this way, structural features of the cell such as the abundance of a particular protein or the integrity of the cell membrane can be quickly assessed.
Following target identification and the generation of a drug lead the efficacy and toxicity of the drug can be investigated by flow cytometry. It is possible to quickly infer these outcomes by high-throughput cell counting, though cells are often then sorted based on the stage of apoptosis.
Among several other methods that mark out the activation of particular cellular mechanisms, apoptosis can be monitored using the protein fluorophore annexin V, which binds to phospholipid phosphatidylserine. This component of the cell membrane comes to the cell surface in high concentration during the early stages of apoptosis, allowing cells experiencing toxicity from the therapy under investigation to be identified.
Flow cytometry in lead development
Similarly, cells undergoing late apoptosis can be identified by their leaky membrane structure, and thus a variety of fluorophores that struggle to penetrate the intact membrane but bond with internal cell structures such as DNA are often used to mark out these cells. Propidium iodide is one such DNA-binding dye, which can also be used to differentiate between the stage of the cell cycle the cell is currently in.
Cells in the G2 or M phases have only one copy of DNA, while cells undergoing mitosis in the G0 or G1 phases bear two copies, and thus the intensity of fluorescence can be used to gauge the stage. A pause in the cell cycle can be indicative of drug efficacy or toxicity, depending on whether the cell being analyzed is considered the target of the therapy.
The aim of a drug compound is often to disrupt or augment bonding to a specific target or between specific molecules, and thus the intensity of fluorescence detected from the cell correlates well with interactions between the tagged compound of interest and the specific target within the cell.
Broad-spectrum in vitro testing of a variety of cells under a range of conditions and analysis by flow cytometry also helps researchers to identify the optimum in vivo tests to be performed later, paying particular attention to toxicity by identifying cells undergoing apoptosis or cell cycle arrest.
Future applications
Flow cytometry is also used to study vaccine efficacy and safety in a number of capacities, such as quantification of viral load, identification of IgG and IgM antibodies, and measurement of T- and B- cell responses.
In an example of the latter case, the CD4+ and CD8+ T-cell response of vaccinated individuals against SARS-CoV-2 spike peptides was assessed in vaccinated individuals by Ewer et al. (2020) using flow cytometry, where cells were firstly stained with a cocktail of anti-human T-cell surface protein antibodies and anti-human cytokines.
The presence of proteins involved in T-cell activation and the secretion of inflammatory cytokines is evidence of vaccine efficacy, and the high-throughput nature of flow cytometry has proven highly beneficial for such applications since the outbreak of the COVID-19 pandemic.
Modern high-end flow cytometers utilized in the pharmaceutical industry are capable of processing tens of thousands of cells per second in an entirely automated manner, though when undertaking higher degrees of characterization analysis is dramatically slower.
Continuously running bioreactors generate cells for testing that can then be exposed to a variety of conditions and later categorized by flow cytometry, including the use of specialized tubing that replicates the shear forces found in blood vessels.
Increased automation, cell processing speed, and a variety of characterization techniques incorporated directly into flow cytometry equipment will likely be observed going forward. Besides improving efficiency and therefore accuracy, as a greater number of cells can be tested and analyzed in detail, this will also allow more robust safety profiles to be developed for new compounds, with a greater variety of cells tested in relevant conditions.
References:
- Mckinnon, K. M. (2021) Flow Cytometry: An Overview. Current protocols in immunology.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5939936/#:~:text=Flow%20cytometry%20is%20a%20technology%20that%20rapidly%20analyzes%20single%20cells,one%20or%20multiple%20fluorescence%20parameters.
- Ewer, K. J., et al. (2020) T cell and antibody responses induced by a single dose of ChAdOx1 nCoV-19 (AZD1222) vaccine in a phase 1/2 clinical trial. Nature medicine.
https://www.nature.com/articles/s41591-020-01194-5
- Egia-Mendikute, L., et al. (2021) Sensitive detection of SARS-CoV-2 seroconversion by flow cytometry reveals the presence of nucleoprotein-reactive antibodies in unexposed individuals. Communications biology.
https://www.nature.com/articles/s42003-021-02011-6
- Boraldi, F. et al. (2016) Innovative Flow Cytometry Allows Accurate Identification of Rare Circulating Cells Involved in Endothelial Dysfunction. PLoS one.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160153
- Hughes, J. P. et al. (2011) Principles of early drug discovery. British journal of pharmacology.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3058157/
- Edwards, B. S. & Sklar, L. A. (2015) Flow Cytometry: Impact On Early Drug Discovery. Journal of biomolecular screening.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4606936/
Further Reading
How immune cells shape our earliest breaths