Cell culture: Know your cells for successful, reproducible results

Although in vivo experiments are prized because they allow the study your protein/RNA/biological phenomenon within the complexity of the organism, in vitro systems, such as cell culture, also have their merits. For assays where you need a significant amount of input, or sensitivity that is unobtainable from within the organism, cell culture is a preferable alternative. In many ways, cells in culture are a semi-synthetic system, with conditions that do not reflect those likely observed within the organism. However, when using a cell line that is representative of your disease or organ of choice, the intracellular dynamics are thought to be reminiscent of that which occurs in vivo and so are worth preserving through prudent cell culture practices.

Essentially, cell culture consists of growing your cells within a plate/dish/flask, splitting them when they become too abundant or “confluent” through dissociation from each other and the dish they are in, and re-plating a lesser amount to continue culturing or for an experiment. Most labs now practice cell culture, and there are variations on technique, but good cell culture lies in knowing your cell line well through careful handling, research, and observation. Here are some key questions to ask when getting started.

What should your cells look like?

A very basic and intuitive question, but I am surprised at how many students cannot answer it. For the most part, you can look up your cell line on ATCC or check the literature for bright-field pictures to get an idea of morphology. If pictures of your cell line are not available, check for something similar, as cell types, such as fibroblasts, tend to have similar morphology. Poor culturing can stress out your cells, and so it is also good to know how your cells look when they are ”happy” and growing as they should. Stressing the cells may alter cellular dynamics, such as signaling networks and RNA expression, or activate stress responses, so it is best to keep your cells properly cultured to ensure your experiment will be legitimate.

What do your cells look like when they are confluent?

Cells will continue to grow in the culture dish until you split them. Some cells do stop growing due to contact inhibition, but this is less commonly observed in standard cell culture. Once your cells become overly confluent, it is generally believed that the signaling networks are altered, potentially changing your cells and leading to aberrant and irreproducible results. So, when your cells become confluent or reach a maximum desired density, you should split them. For your cell line, what does confluency look like? For example, fibroblasts will spread out and cover the dish as much as they can, and so one could split the cells once 80% of the dish is covered, or at 80% confluency. Alternatively, cells such as NMuMGs tend to grow in a cluster, and can become confluent without covering the culture plate. Remember to also maintain the same confluency when setting up and performing experiments.

How quickly do your cells grow?

This is important for both splitting cells once they become confluent and knowing how many cells to plate when setting up experiments. Basically, you should know how many cells to plate today so that your dish will be at a desired confluency either the next day or at the end of the experiment. I know several people who like to split their cells harshly (i.e. put less cells back into the culture dish after dissociation), in order to avoid having to split their cells as often. However, some cells become stressed when they are too sparse, so you shouldn’t push your cells without knowing that they will survive a harsh split.

Be aware of the cell passage

Every time you split or “passage” your cells, not only does the passage number increase but also the time they have been in culture. Depending on your cell line and your interests, this may be good or bad. From my experience, cell signaling and proliferation rates change the longer you have cells in culture. It is also believed that random mutations accumulate the longer you culture cells. Accordingly, most people grow their cells until passage 10, at which point they toss them and take out a new frozen vial of cells to avoid these potential issues. On a similar note, once you do begin to culture cells, it is best to freeze a few vials the first time you split them to ensure you have low passage cells frozen for future needs.

Now that you have your cells growing nicely, the next trick in culturing is to avoid contamination.  Happy cells help make successful experiments—and happy scientists.


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Brittany Carson

Brittany Carson

I am a post doc at the Lunenfeld-Tanenbaum Institute in Toronto. I currently work on glioma, but have studied breast cancer, multiple myeloma and renal cancer, with a focus on cell-signaling pathways, translational regulation, the cell cycle and the cytoskeleton to develop novel biomarkers and therapeutic targets. My science journey has taken me through cell culture, flies, worms and mice and through many different techniques. I love photography (including microscopy), traveling and non-science reading.