If you’re doing biological research on eukaryotic systems, there’s a good chance you’ve got cells in culture.
Logging time in the cell-culture room is a constant in the life of biomedical researchers. It’s what allows scientists to build up the raw materials—cells, or the biomolecules they produce—necessary for their experiments. But with countless cell lines and cell types in use in today’s laboratories, each with its own unique growth characteristics, how can researchers be sure those cultures are actually healthy?
Tool developers have created a wide array of tools for monitoring cell cultures. Some are specialized assays designed to monitor cellular changes in, for instance, drug-screening applications. But many are intended for use as part of day-to-day cell culturing. Here, we review some of those options.
Healthy cells impact results
It’s not just an academic question. Cultured cells, whether they are immortalized cell lines or primary cells derived from patients or model organisms, are the key ingredients of much of today’s biomedical research, says Del Trezise, director and European site head at Essen Bioscience. “In order for [those measurements] to be relevant, then it’s more than preferable to ensure the cells from which you make those measurements are healthy. Otherwise, it’s likely you will have artifacts and nonphysiological outcomes.”
Working with healthy cells—and knowing quantitatively what that means—also impacts reproducibility, making it easier to compare results from experiment to experiment and lab to lab, says Kamala Tyagarajan, R&D director for flow cytometry applications and assays at MilliporeSigma. “We do these experiments not just on a single day but also week to week, month to month, year to year. And we need to be sure that when comparing these results … there’s reproducibility in terms of the base material we are making these observations on.”
And if nothing else, Tyagarajan adds, having a good handle on cellular health provides researchers with a solid foundation for optimizing culture conditions.
Flow-based metrics
There is no one definition of cell “health”—the term can encompass everything from cell density and doubling time to oxidative stress and apoptotic state, says Tyagarajan.
Ultimately, the most straightforward indicator of health is cell viability.
Traditionally, viability has been assessed using a hemocytometer and trypan blue. Trypan blue is a membrane-impermeable dye, meaning it is excluded from healthy cells with intact membranes and can only enter and stain cells whose membranes are compromised. Researchers can thus simultaneously obtain a cell count and assess how many are alive simply by mixing the dye with an aliquot of cells and pipetting the mixture onto a hemocytometer, a special microscope slide for counting cells. That process can be automated, for instance with Bio-Rad Laboratories’ TC20™ Automated Cell Counter. But it’s also possible to abandon trypan blue in favor of flow cytometry-based methods.
MilliporeSigma’s Muse® Count & Viability Assay, for instance, uses a mixture of two fluorescent dyes, one that stains all nucleated cells and another for nonviable cells. By analyzing stained cells on the company’s Muse Cell Analyzer, researchers can obtain both the number of cells in the sample and the fraction of live cells.
According to Tyagarajan, MilliporeSigma has developed more than 25 assays for the Muse Cell Analyzer, at least 10 of which can be used to assess cell health, plus many additional assays for its other flow cytometers, the Guava easyCyte systems. These include assays that monitor indicators of apoptosis, oxidative stress and mitochondrial health and cell-cycle status.
Bio-Rad’s CytoTrack™ assay, which monitors proliferation, also is based on flow cytometry. According to Chris Linnevers, the company’s global product manager for cell biology, CytoTrack provides a measure of cell proliferation based on the abundance of a specific dye. In a CytoTrack assay, cells are loaded with dye, and as they divide, the amount of dye per cell decreases by half with each doubling. By passing stained cells through a flow cytometer (such as Bio-Rad’s S3e™ cell sorter),researchers can calculate how many times the cells divided since the dye was added. “You can watch these cells double up to 10 times,” Linnevers says.
Imaging assays
Several companies, including Bio-Rad, Essen Bioscience and GE Healthcare Life Sciences, have developed imaging platforms—capable of capturing morphologic changes, among other measures—for monitoring cultures.
Bio-Rad’s ZOE™ Fluorescent Cell Imager, for instance, is a benchtop manual microscope coupling “a simple tablet-like interface with an inverted microscope and digital fluorescence-imaging capabilities,” Linnevers says. With one brightfield and three fluorescent channels, ZOE is “a great tool for routine monitoring of cell health, especially in cultures expressing fluorescent proteins.” That said, researchers can also use the company’s VivaFix™ reagent, a single dye (available in multiple colors) that binds primary amines, to image and count both live and dead cells, using staining intensity to distinguish between the two. (VivaFix also works with flow cytometry, Linnevers notes.)
The Cytell™ Cell Imaging System, from GE Healthcare’s Life Sciences business, is a compact imager that fits (albeit sideways) within a standard tissue-culture hood, according to product manager William Yang. Controlled by a smartphone-like touchscreen interface and compatible with an array of culture vessels, Cytell can perform cell counting, viability assays and much more, says Yang. Users can create Cytell BioApps to run their own assays using an application wizard. They could, for instance, create a custom assay that counts live and dead cells and monitors mitochondrial health using three dyes. Yang explains: “You select the [fluorescent] channels, you select the [cellular] targets, and you set gating, for instance to count only live cells, and then you are done.”
Essen Biosciences’ IncuCyte ZOOM platform is a long-term cell imager that resides within a cell-culture incubator. According to Trezise, the platform is completely noninvasive and nonperturbing—cells are imaged constantly, using either label-free phase-contrast microscopy or innocuous mix-and-read reporter dyes that fluoresce only when cells are, for instance, dying.
“You don’t have to take the cells from their ‘happy place,’” Trezise says, referring to the incubator. “The moment you remove them from that environment, you make them subhealthy, so we don’t do that. We make measurements in situ.”
Basically, says Trezise, the IncuCyte records time-lapse movies of each desired location over days, weeks or even months, and then analyzes those data to quantify, for instance, doubling time, morphologic changes or motility. That's in contrast to most cellular assays, he notes, which make “endpoint” measurements, destroying a sample to analyze it. “If you want to learn about cells and how they behave, the best thing to do is to take movies of them, and then you will truly understand their dynamic nature.”
Coulter counting
One final monitoring option is the relatively old-school counting method called Coulter counting. Commercialized by Beckman Coulter (among others), this approach counts cells by passing them through a narrow pore; as they pass, the cells disrupt an electrical field, producing a measurable signal. Similarly, the CASY system from OMNI Life Science is based on “electric-current exclusion,” says marketing and communications director Ralf Ketterlinus. The system does not simply count objects, he notes; it also records their volume. “Thus, we can discriminate between cell debris and cell aggregates, and we can also discriminate between viable and dead cells in a fast, single, nonstaining measurement.”
Monitoring regimen
Though needs vary from lab to lab and situation to situation, it’s generally a good idea to assess the health of your cultures routinely—at least every time cells are split, or weekly.
That way, researchers can determine whether growth parameters are changing. Tyagarajan also recommends testing extensively after thawing new cells, for instance to assess their viability and health characteristics, or when assessing new growth conditions, such as with serum-free media.
What users should do if cell behavior seems to be changing, though, can only be determined on a case-by-case basis, Tyagarajan says. With immortalized cell lines, your best bet may be to thaw a new vial and start over. But precious primary cells, for instance, may be tougher to simply throw away. “This is why it’s important to understand the line’s health and growth characteristics so well,” she says. “Characteristics may change after multiple passages, and this change may not be ideal for your experiment.”
Image: Dreamstime