There are over one hundred trillion cells in the human body. These tidy parcels collaborate in exquisite synchrony—dividing, differentiating, morphing and stretching, beating, and reaching. Like something akin to magical realism these tiny building blocks of self-contained life stack on top of each other giving way to an unimaginably complex machine: the human body. And yet, inside of each cell is a vast universe complete with a nuclear sun at its center.
For well over one hundred years, scientists have sought to peek inside of these cells, to coax out well-guarded secrets, to unearth the mechanisms that yield life (and death). But how can one understand the cell when it comes attached to an organ within a body?
Cell culture got its start as tissue culture. Scientists lopped off bits of animals and bits of organs and tried their luck at growing them for a few days inside of a dish. The earliest tissue culture consisted of chicken embryos floating in warm saline. This technique was pioneered by an embryologist named Wilhelm Roux in the late 19th century. Others soon followed suit. The 20th century brought a flurry of advances that eventually allowed for cells, not tissue, to be separated and kept alive in a soup of nutrients. The 1940s brought the first mouse lymphocyte cell line; the 1950s brought Eagle’s famous media recipe and the establishment of the now controversial HeLa line.
Today, the basic principles remain the same, but the techniques evolve at breakneck speed. Cells now live indefinitely inside of incubators. Stem cells transform into cardiomyocytes that beat in plates, or neurons with long, winding axonal tendrils. Fibroblasts are reprogrammed as stem cells. Forget two dimensions! Cancer cells are grown in three dimensions (or four, if you add the component of time) mimicking tumors in the body. Scientists edge closer and closer to therapeutic frontiers, and the progress is staggering.
Lee Ligon is associate professor and associate department head in the department of biological sciences at Rensselaer Polytechnic Institute. She’s worked with cells for nearly 25 years and remembers the excitement cell culture evoked in her early days at the bench. “The first time I actually saw fluorescent mitochondria moving in axons and dendrites—that sounds so tame now, but at the time, it was awe-inspiring!”
Members of Lee Ligon's Lab
These days, her lab works to make biomimetic tissue culture models, especially of the tumor microenvironment. “We have developed ways to co-culture multiple cell types, to use different 3D ECM (extracellular matrix) models with varying mechanical properties, and various combinations of these features. We're now working to develop a high(er) throughput method to look at the biomechanical properties of the tumor microenvironment.”
More sophisticated set-ups, same old scoundrels
Yet, despite the innumerable advances, there are the old foes that plague experiments and hinder findings. These are the microbial saboteurs that boldly set up shop inside of cell culture experiments. These are the ill-trained staff that don’t fully grasp aseptic technique, cut corners, or ignore protocol. These, according to scientists and experts, are the things new researchers need to get a handle on if they want to be successful and competitive.
Yet, despite the innumerable advances, there are the old foes that plague experiments and hinder findings.
Sokol Todi, associate professor of pharmacology at Wayne State University, stressed how critical it is to train staff properly: “Contamination has always been an issue and will always be an issue. You can have all the bells and whistles, but as soon as you introduce humans into the equation you will have problems. Train your staff yourself and if you are new to cell culture, make sure you find someone who has lots of experience or an online journal and learn from them.” Consider reading Geraghty RJ, et al.’s article to get started.1
Beware of surprising sources of contamination, too. Fluorescent lights, for example, can photoactivate HEPES buffer, riboflavin, and tryptophan, leading to the production of hydrogen peroxide and free radicals. Sera supplements are a common source of both biological and chemical contamination. For this reason, it is best to stick with a particular source and lot of serum that has been successful in the past.
Melanoma masquerading as breast cancer—Why cell-line validation is a priority
To date, there are 488 cell lines that are cross-contaminated or misidentified. The database documenting these cells is maintained by the International Cell Line Authentication Committee.
One of the most notorious is the MDA-435 (or MDA-MB-435) line. These cells emerged in lab research in 1975. Since then, the cells made appearances in more than a thousand scientific articles as breast cancer cells. This turned out to be a big problem, however. When scientists examined the DNA of these cells, they were stunned to discover that MDA-435 was a melanoma line, not a breast cancer line.
Journals now require, or strongly encourage, scientists to authenticate their cell lines. A Nature commentary from 2015 called on scientists to be more proactive in this regard, yet acknowledged common obstacles: “If we exclude ignorance and indifference, cost and simplicity of assays appear to be the biggest roadblocks to universal use of cell authentication.”2
STR (short-tandem repeat) and SNP (single nucleotide polymorphism) analyses represent viable methods for verifying cell-line identity. STR involves analyzing repetitive sequence elements, 3–7 base pairs long, that are scattered throughout the genome. SNPs, which are genetic variations between members of the same species, are conserved within a specific locus and can be used to confirm identity. Commercial kits for SNP assessment are available, whereas scientists can send cells to companies such as Promega or ATCC for STR analysis.
The nonprofit, ATCC, claims that all cell lines are STR-validated, on their website. In addition, ATCC touts a database that scientists can use to compare their results against profiles of documented human cell lines.
Profiling has limitations, too. While these methods are useful in establishing identity, they do not assess cell purity, i.e. absence of interspecies cross contamination or contaminants, such as viruses and microbes, phenotype, morphology, or cell ploidy.
Mark Rothenberg, Ph.D., manager of scientific training and education at Corning, warns that a strong mycoplasma testing program should be in place. “Mycoplasma can stay viable 6–8 days in a drop of liquid in a hood, and can easily contaminate the whole facility,” he explains. Additionally, although it runs counter to protocols established in most labs, he says that you should consider not using antibiotics in your media, unless establishing primary cultures. “Antibiotics can hide the presence of bacteria and increase the risk of contaminating other cultures.” He strongly recommends a reference article written by Corning about how to manage contamination.3
Curbing contamination—Using technology and common sense
No matter the sophistication of the space or the technique, good old common sense can go a long way. Brian Langenderfer, a senior sales representative, has worked with DAI Scientific for 22 years. He says one of the biggest mistakes he sees is the set-up of cell culture space in high traffic areas such as next to a doorway.
Natalie Swartz is a lab designer with SmithGroupJJR in Detroit who works with universities and scientists to design spaces that match their needs: “We try to design for flexibility; this is the biggest innovation that we see on our end.” Flexibility means benches on wheels with adjustable heights. Ergonomics are built into these modular components so scientists can stand or sit depending on the task.
When possible, she suggests that researchers focus on a “triangle design” meaning that the biosafety cabinet, refrigerator, and incubator are installed strategically and in close proximity, while taking certain precautions. For example, “flow hoods should not be set up across from each other because the airflow could contaminate your samples,” she says. “Similarly, you want equipment three or four feet from biosafety cabinet so you can pull out your samples safely.”
Rothenberg adds that “incubators should be placed in a location with minimal potential for vibration from heavy equipment and elevators.”
All of the experts mentioned that when setting up your cell culture space you should consider what supplies you will use while at the biosafety cabinet. You should be able to reach most of these items without leaving your seat. The less you disturb the air around your cells the better.
According to Swartz most labs were designed in the 1970s and then remodeled in the early 2000s. Typically older labs have equipment spread out with the refrigerator, incubator, and hood in different rooms. But all that transport is not good and provides myriad opportunities for compromising a sterile space.
If you are one of the many scientists working with limited resources in an older space, there are plenty of steps you can take to design a successful cell culture space.
Swartz advises that you consider three main aspects when setting up a working space: organization, consolidation, and the above-mentioned triangle configuration. “First, organize the space. Try to consolidate all the things you are going to use by task. You can create task zones where all of the materials you need are in one place. Use wheels to move things to the biosafety cabinet,” she explains. Also, whenever possible you should follow the NIH guidelines.4
..Consider three main aspects when setting up a working space: organization, consolidation, and the…triangle configuration.
Rothenberg echoes Swartz and explains that you can work within the confines of the facility. Some things can’t be avoided. For example, if there are windows, make sure they are sealed.
Innovation only as good as the people who use it
Some cell culture mishaps can also be prevented with the proper equipment. Langenderfer believes that technology with certain features can prevent a lot of headaches, if it is used properly. Consider the case of the super pure deionized water; just because it is super clean doesn’t mean it should be used for every task. “For years, labs have been using deionized water in incubators, which is not recommended, because the water will pull the ions from the metal and over time create rust pitting inside,” he cautions.
“On the hood [side of things], if lab personnel use the UV light to substitute for good aseptic technique then we, as [sales] reps, cannot control this. However, there are features like a UV timer, and one-piece stainless steel interior that will help with making the cleaning process faster and easier,” says Langenderfer.
Biocompare’s Cell Culture Search Tool
Find, compare and review cell
culture products from different suppliers Search
He continues, “Some incubators do not have good controllers, hence may not set off an alarm if a lab tech leaves a door open or forgets to change the water in the humidity pan. Most mishaps that occur in a lab are related to training of the lab techs by the researcher with regards to cell culture process in general. We do all the free in-service training on all our equipment. We also cover proper use and setting of all the controllers. In most cases, yes, the equipment is great if the lab personnel use and maintain the equipment properly.”
Newer equipment takes into consideration the packed and strenuous day of scientists. Features include alarms, adjustable heights and wheels for chairs, hoods, and benches, as well as improved antimicrobial surfaces. And while an entire article could be written on it, both Swartz and Langenderfer stressed the importance of utilizing energy efficient devices whenever possible. If part of your grant is earmarked for operational costs, this is definitely something you’ll want to consider. That old incubator or hood you inherited for free might not be worth it in the long run. And do your legwork; “Don’t just buy the same old incubator your advisor had,” says Ligon.
Swartz warns that one fume hood consumes as much energy as 3.5 households per day, which is a massive amount. Whenever possible choose a variable air hood over a constant volume hood. Also, “Lower the sash as much as possible,” she advises. “If you do use a lower sash or a low flow velocity hood you can save up to 40% of your operating budget.”
Langenderfer offers some parting advice: “When setting up a cell culture lab or any research lab don’t just purchase the major equipment from a catalog; contact an equipment expert. In the end this will save you time and precious research dollars, as well as get you the exact equipment to match your research application.”
References
1. Geraghty, RJ, et al., “Guidelines for the use of cell lines in biomedical research,” British Journal of Cancer, 111(6), 1021–1046, 2014. [PMID: 25117809]
2. Freedman, LP, et al., “Reproducibility: changing the policies and culture of cell line authentication,” Nat Methods, 12(6):493-7, 2015. [PMID: 26020501]
3. Ryan, J, Understanding and Managing Cell Culture Contamination, Technical Bulletin, Corning Life Sciences.
4. NIH Design Policy and Guidelines, Office of Research Facilities.
Image: Shutterstock Images & Lee Ligon's Lab