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10 Tips for Successful Development of Cell Culture Assays

10 Tips for Successful Development of Cell Culture Assays content piece image

Cell culture was first developed in the early 1900s.1,2 It was developed to visualize and investigate cells in simplistic studies, isolated from the multitude of other cells and biochemical factors present in the body. It is now ubiquitous in biomedical laboratories and is rapidly evolving to meet growing pharmaceutical demand. Traditionally, cell culture has been used to produce biotherapeutics such as antibodies.3 Now, cell culture has also become a key procedure both in regenerative medicine4 and in drug discovery.5 Briefly, cells are grown on dishes either to replace damaged cells in patients, or to recreate cellular models that mimic the organs of patients to test drug efficacy and safety. While technological advances are reshaping the cell culture field, many basic steps are still to this day performed manually in laboratories. This means that the protocols are far from being standardized and are often prone to variability and human error. Here, we offer what we think are essential assay tips, designed for anyone starting out with cell culture, which can be complemented with these tips for 3D cellular assays.

Primary or immortalized cells?

Your choice of cell source is crucial: it defines the physiological relevance of your cellular model. Scientists have a choice between primary or immortalized cells, also called cell lines. Immortalized cells keep dividing due to a mutation which causes them to evade their normal cellular senescence. In contrast, primary cells have limited expansion capacity and can therefore only be passaged a certain number of times, limiting the number of cells one can obtain. Using primary cells might be more expensive and cumbersome, as you need access to animal or human tissue or to obtain them repeatedly from a vendor. However, they are more physiologically relevant, as they are directly extracted from the tissue, have undergone less population doubling and represent a distinct and unique donor.

Cell lines are very practical because you can passage them many times, and there are numerous well characterized cell lines available. However, they are more homogeneous genetically, seeing that they come from the same original donor. Note that homogeneity can sometimes minimize noise and thus facilitate obtaining statistically significant results.

Finally, although it requires specialized expertise, one can also immortalize cells after isolation6 from patients or animals. In the current race towards personalized medicine, one could say that primary cells have an edge, but think carefully whether you have the appropriate budget and facilities!

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Mechanical techniques

You would be surprised at how much of a problem this is in the biomedical field, even to this day. Up to 30,000 studies have been identified that use the “wrong” cell line.7 Upon obtaining your cells for the first time, try to double check that they are the ones you want. You can for example stain them for a marker they are known to express. After that, make sure you do not contaminate them with any other cell types, and be very careful with your labeling!

Carefully prepare your stock of cells

The cell stock is made of numerous vials filled with cells that all come from the same original sample, which were expanded together and passaged a similar amount of times. Be cautious to not make any mistakes while preparing your cell stock as all your subsequent experiments will be affected! For example, make sure you label your tubes appropriately with the cell’s name, their passage number and the date the cell stock was made. Additionally, make sure to not contaminate your cell stock with other cell types or bacteria: in other words, handle your cell stock with care to be confident that it can be trusted for all your subsequent experiments.

To make the stock, cells are typically resuspended in a solution of the cell’s media with up to 10% dimethyl sulfoxide (DMSO) which prevents the formation of ice crystals during the freezing process. You can also add varying concentrations of serum to maximize your cell’s recovery. DMSO can however be toxic to cells, so make sure that once your cells are resuspended in the solution containing DMSO, you are quick to transfer the vials to the -80°C freezer. This means that you should have labeled your vials prior to this step so that they are ready to be filled. Little but important detail: use commercially available labels that can stand up the extreme cold, otherwise they might not stay attached to the tube after their long-term storage! Ideally, first place the vials in a freezing box, this optimizes the cooling rate upon transfer to the -80 °C freezer. After that, make sure to transfer your vials to the liquid nitrogen the next day to ensure your cell’s viability.

Consistently use the same stock of cells

When you make your stock of cells, make sure to prepare enough vials to complete your entire set of experiments. There is often a lot of variability between cell stocks due to many user or environment dependent factors, or even between different passages due to genetic drift. Using one cell stock will therefore minimize noise in your results. However, it is always wise to generalize your experiments to other batches of cells, or cell types further down the road!

Thaw your cells gently!

Thawing cells is a catch-22: you can’t thaw them too quickly otherwise the temperature shock for the cells is too brutal, but at the same time you can’t be too slow because the DMSO in the freezing solution will damage your cells.

Do not thaw your vial for too long and add progressively, and quickly, new warm media to your cells to neutralize the DMSO!

Some protocols recommend immediate centrifugation to plate cells with fresh new media devoid of DMSO. Others suggest skipping the centrifugation which can be harsh to cells and directly placing cells in a flask while diluting the DMSO content by adding a large volume of fresh media. Neither procedure is perfect, and your choice will most likely vary with the cell type you are handling. Check the vendor’s protocol to see what they recommend.

Minimize exposition to trypsin

Generally, cells adhere tightly to surface cultures and must be dissociated using chemical agents such as trypsin. However, these reagents can be toxic to cells. Minimize exposure to trypsin and, once cells are dissociated, neutralize the trypsin quickly with a volume of media that is much larger than that of trypsin.

Coat surfaces

Often, surfaces for cell culture are coated with extracellular matrix proteins to improve cell growth, survival8 or differentiation.9 For example, collagen I can be deposited on flasks to improve cells proliferation by filling the flasks with a layer of collagen solution for at least several hours. Make sure to wash the coating solution well with water before culturing cells as some components of the solution might be harmful to cells upon direct exposure.

Optimize the media you are going to use

Media solutions are often like black boxes. They are made of a basal solution, complemented with growth factors and serum. The added growth factors can mask your results if you are interested in their endogenous secretion in your assay. In addition, different batches of media can be highly variable as they often contain serum (e.g. fetal bovine serum (FBS)) that are isolated from animals.10 Thus, when possible, use the same batch of media for your set of experiments. Go serum-free if you can: researchers have shown that genetically modified endothelial cells can survive without serum.11

Control your cell’s confluency

Confluency is a measure of the cell culture area covered by cells. The more cells there are on a flask, the more confluent they are. Some cells, upon increased confluency can have an altered morphology, proliferation, gene expression and viability.12,13 Many experimental procedures call for a confluency from 60–90% to ensure cell viability and success. Therefore, make sure you do not let your growing cells become too confluent by checking them regularly under the microscope. If you are not ready for your experiment when your cells are already confluent, simply passage your cells and keep a fraction in a new flask (what is called sub-culturing). Note that the original density of cells plated also matters: if the number of cells placed in the flask is too low they might not grow, or they will but very slowly. This means you should not dilute the cells too much upon sub-culturing. But remember to be consistent. Always passage your cells the same number of times for a given set of experiments.

Check for contamination

Cells are prone to becoming contaminated during their in vitro culture due to either chemical or biological contamination. While this is a vast and complicated subject of its own,14 some contaminations are easier to detect than others. For example, you can check for the presence of moving bacteria in your culture under the microscope (to not be confused with floating cellular debris!). Mycoplasma however must be tested for using kits available commercially. In any case, make sure that you minimize the probability of contamination by using aseptic techniques!

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