The capacity to monitor cell proliferation is pivotal to many research studies. Yet the process is often more complicated than simply evaluating whether there are now two cells where there used to be one. Often researchers want additional detail, requiring that they measure DNA synthesis, analyze cellular metabolism, study key proliferation markers, or identify specific mitotic phases. Additionally, it is important to understand that cell proliferation in vitro does not necessarily represent normal physiologic behavior. To address this discrepancy, advanced substrates and cell culture systems are being developed with the aim of more closely mimicking the in vivo environment.
Understand your cell line
“It’s important to recognize that proliferation characteristics are a cellular phenotype,” says Fang Tian, Ph.D., lead scientist at ATCC. “Different cell lines will proliferate in a different manner and, irrespective of the method used to monitor proliferation, it is essential to understand these features for a given cell line and to perform routine monitoring to avoid any unintended changes. Generation of a growth curve is relatively straightforward and can be useful for establishing optimal seeding density, subculture ratio and frequency, and culture conditions.”
Adding that cells typically grow more slowly, or stop proliferating, when they reach high density or confluence, Tian notes that allowing this to occur in vitro can have a significant impact on downstream studies. “When a culture is allowed to proliferate beyond a healthy confluence, the cells are forced to dysregulate certain signaling pathways to attenuate contact inhibition,” she says. “This may lead to changes in the gene-expression profile, poor cell growth after subculture, or unintended selection of cells with malignant transformation. Genotypic or phenotypic drift is also a distinct possibility.”
Physiomimetic cell substrates can regulate proliferation
“For the majority of cell types in the human body, continuous proliferation is not an activity associated with healthy cell function under normal conditions,” notes John O’Neill, CSO of East River BioSolutions. “Although some cell types turnover repeatedly, others proliferate rapidly only upon activation by an external stimulus. Researchers should be aware that rapid cell growth in vitro may not represent normal cellular behavior in vivo.”
O’Neill elaborates that cells cultured in vitro on rigid, 2D surfaces often proliferate extensively, including those cell types that are not proliferative in a normal state of homeostasis. “Gene-expression analysis by RNA sequencing has shown that human cells cultured on 2D plasticware significantly upregulate their expression of cell cycle and stress genes, suggesting that under these artificial conditions the normal cell program is sufficiently perturbed or dysregulated to alter gene expression. This calls into question the validity and relevance to human physiology of results obtained through cell culture systems supporting rapid, extensive, cell proliferation.”
To promote slower, more regulated, proliferation, East River BioSolutions has developed a range of physiomimetic 2D and 3D extracellular matrix (ECM) cell substrates. “Many cell types proliferate less or at slower rates in the presence of their tissue-specific ECM, indicating an important regulatory role of the ECM on the cell cycle,” says O’Neill. “Our highly optimized tissue-specific ECM cell culture substrates, including hydrogels and scaffolds, recreate the in vivo environment to regulate proliferation more consistent with native conditions. These substrates may also function to protect cells from stress during media changes.”
Consider the impact of environmental fluctuations
Also strongly focused on preventing cellular stress, BioSpherix has developed a modular technology designed to protect the entire cell-based process from fluctuating environmental conditions. Alicia Henn, CSO, explains that while a multitude of factors can influence cell proliferation, minimizing variation in critical parameters such as temperature, CO2 concentration, or pH affords more consistent, physiologically relevant results. “One should never under-estimate the effects of a highly variable environment on cellular proliferation,” she says. “For example, a recent publication in Cell clearly demonstrated that even a brief exposure to room air had a rapid, deleterious effect on numbers and repopulating potential of hematopoietic stem cells. The ability to maintain an optimal cellular environment is therefore pivotal to the generation of robust, reproducible cellular data.”
Henn adds that by considering the needs of the cells first, BioSpherix’ Cytocentric Xvivo Systems are designed to provide full-time optimal conditions to ensure proliferation more similar to that which occurs in vivo. “Cells within the body reside in a highly regulated environment that is continually refreshed,” she says. “In contrast, prevention of nutrient depletion in vitro requires frequent media changes, which can stress cells when performed in room air conditions. By facilitating these media changes under constant physiologic conditions, this stress can be avoided.”
Evolving tools to monitor cell proliferation
With so many different methods available to monitor cell proliferation, researchers may find themselves spoiled for choice when it comes to selecting suitable reagents. “Encompassed within our suite of cell proliferation products are reagents enabling the most popular methods for cell growth analysis,” reports Brenda Karim, product manager for cell biology reagents at Bio-Rad Laboratories. “These include CytoTrack™ cell proliferation dyes, alamarBlue®, BrdU, cell cycle antibodies, and dyes that bind DNA in a stochiometric manner. The selection of a suitable reagent for monitoring cell proliferation often comes down to researcher preference, however we’ve covered most eventualities.”
Bio-Rad’s CytoTrack dyes, launched in 2014, have already been widely cited, including a recent publication in Scientific Reports where the authors demonstrated the anti-proliferative properties of Climacostol (a protozoal toxin with antitumor potential) in melanoma cells. Karim explains that these specialized reagents function by reacting with primary amines to become retained intracellularly. “Upon entering the cell, esterases cleave a blocker from the dye to achieve fluorescence,” she says. “As cell division occurs, the dye is successively halved, allowing up to 10 generations to be resolved based on consistent reduction in fluorescence intensity. CytoTrack dyes can be added directly to the culture media, allowing for efficient staining of live, dividing cells, and with several excitation and emission profiles available, these reagents fit seamlessly into existing workflows.”
Image: CytoTrack dyes to monitor cell proliferation. Human PBLs were stained with CytoTrack red and stimulated with PHA. Proliferating cells show a reduction in the amount of dye with each cell division. (Red—stimulated; green—unstimulated; blue—unlabeled.) Image courtesy of Bio-Rad.
The requirement to understand and monitor the complex and highly regulated process of cell proliferation is fundamental to every area of cell-based research. An abundance of tools is available to facilitate its study, while specialized technologies are continually being developed to promote more physiologically relevant cell growth. As awareness grows that common manipulations such as viral infection or the overexpression of transgenes can impact on cell proliferation, and that these changes may be detrimental to cell health, it is more important than ever that cell proliferation is given due consideration.