Cell Culture

Cell culture is a process of growing cells under controlled conditions outside of their natural environment. This is useful for cell biologists to study specific pathophysiological, disease or injury, or phenotypic, particular cellular genetics, responses in a controlled environment. Different cell cultures exist from traditional 2D cultures such as adherent cell cultures to 3D cell cultures and organ-on-a-chip models. The use of cell culture might be dependent on the cell type used. Typically, cancerous compared to non-cancerous cell lines require less stringent media and growth factors due to the “cancerous” aspect where they expand exponentially due to less growth-regulating factors.

This video and blog post is exploring suspension cell cultures using Jurkat T-cells. The Jurkat T-cells were primarily cultured for the Optimising Me Manufacturing Systems (OMMS) project. Briefly, my part of OMMS was to produce T-Cells for the leukapheresis, separation and enrichment of white blood cells, as well as supporting the transfection using electroporation -on-a-chip. Read more about the microfluidic inertial separation here.

Media Used

The media used in cell culture is somewhat specific to the cell used. For example, we are using Jurkat T-cells so it is recommended we use RPMI 1640 with glutamine, a building block for many proteins. RPMI 1640 has been used with HeLa (cervical cancer cells), astrocytes (a central nervous system cell), and carcinomas (cancerous skin cells) just to name a few.

In this cell culture, Foetal Bovine Serum (FBS) and antibiotics were added to the solution. The FBS solution is a universal growth supplement of cell and tissue culture media whilst the antibiotic solution of penicillin and streptomycin aids in avoiding contamination. Contamination in cell biology is the culturing of anything but the intended cell or cell types in the cell culture flask. Contamination is more common in shared laboratories due to the requirement of each individual to keep a high level of aseptic techniques and the traffic through the cell culture hoods. Without good aseptic techniques, contamination can be common wasting time, money, and effort. In particular, the cost increases very fast in cell culture.

If everyone has good aseptic techniques, antibiotics are not required. Most cell biologists, from personal experience, use antibiotics as a “safety net” if contamination were to happen. However, this mindset of using antibiotics can cover up poor aseptic techniques and some cell lines must be cultured without antibiotics as they can interfere with the development and cell functionality.

Keeping contamination-free

Having no contamination is ideal but in reality, it requires patience, skill, and time to achieve.  The most basic technique starts with spraying everything with 70% Industrial Methylated Spirit (IMS) or 70% ethanol. This ranges from cleaning the bottom of the cell culture hood with IMS to everything and anything that enters the cell culture hood.

The next major part is cell culture skills. This is picked up over time but some of the basic skills are holding lids in one hand with the sample, opening and attaching strippettes, and most importantly keeping a clean, clear, and tidy cell culture hood. I talk about these techniques in the video so make sure to watch!

Last but not least is time. Not rushing and taking time to prepare and label samples is fundamental. Unlabelled samples are useless to everyone and can result in work being prematurely disposed of when they could be critical to the work. Labeling cell cultures are important to tell others what your sample is whereas suspension cell cultures can look like contaminated adherent cell cultures that no one wants in their incubators.

Difference between suspension and adherent

Comparison between suspension cell culture and adherent cell culture. Suspension cell culture has cells freely in suspension. Adherent cell culture has cells attached to the bottom of the flask. Adherent cells require Trypsin to unbind from the bottom of cell flasks.

Suspension cell cultures are treated very similarly to adherent cell cultures except for how cells are initially suspended. As the name suggests, suspension cell cultures are already in suspension. Adherent cell cultures have cells adhered to the bottom of the flask. The cells adhere to the flask with membrane-binding proteins expressed. The adherent cells require an enzyme, trypsin, to digest the membrane-binding proteins to release cells into suspension. Adherent cells generally do not survive for long periods in suspension but are fine for short periods such as freezing and thawing. Once adherent cells are in suspension, the procedure is very similar to suspension cell cultures. However, the beauty of adherent cell cultures is that they adhere to the flask therefore media can be changed simply by emptying and replacing the media without disturbing the cells.

With the cells in suspension, they need to be transferred to a falcon tube to undergo centrifugation. The purpose of the centrifugation is to create a pellet, a ball of cells, at the bottom of the falcon tube. Depending on the cells used, different centrifugal maximum forces can be used. The lower the spin force, the longer it can take to deposit the cells out of suspension. Once a pellet has been formed, we can remove the supernatant and resuspend the cells in fresh media.

Why we change the media

The media should be changed regularly to remove waste produced by the cells. When thawing cells, the media change is important to remove Dimethyl sulfoxide (DMSO), a preservative agent which penetrates membranes and stops the formation of ice, as well as providing the cells with fresh nutrients and oxygen.

How often the media needs to be changed is dependent on the cell culture metabolism and cell population. It is difficult to predict how often media changes and it differs from culture to culture. Often cell cultures need to change when the media changes colour which indicates there is more cellular waste than fresh nutrients in the culture.

Conclusion

Cell culture is a key skill for cell biologists as it produces a controllable study of a specific cell type or population of cells. The skills around cell culture can be overlooked but are pivotal to contamination-free, repeatable cell culture. Hopefully, the video and this blog highlight some of the key skills used in cell biology and will help you in the near future!

If you have any key skills, share them in the comments here or on YouTube!

Funded by EPRAT – Keep your face toward the PRAT, shadows will wall behind you
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