This is it! The complete guide to performing cell culture!
Whether you are new to working with mammalian cells or you have wealth of experience with cells and just want to compare methods, you are so welcome to keep on reading. Cell culture is done in a very straightforward principle. When we grow them in the lab, most of the time it is on 2D flat surface of culture plates, which represent an in vitro setting. This serves as a platform for having a miniaturized biological system where we can subject the cells to many treatments, chemically and physically. This leads to discovery of many characteristics of mammalian cells, their behavior when subjected to certain treatment. As the field progresses, more culture systems have been developed to somehow simulate a closer physiological environment for the cells, such as spheroid culture done in a round bottomed wells, and so on. Overall, these are still accounted as in vitro culture and they have been reliable in translating cell or tissue responses to treatments, and contributed to discoveries of therapies.
- Basal medium = this usually contains the base medium, it can be Basal Eagle Medium (BME), Dulbecco's Modified Eagle Medium), or (RPMI), and there are plenty kinds of basal medium depending on the type of cells you grow. It is best to check with the cell bank, ATCC, or the supplier where you obtain the cell.
- Standard Culture Medium = this is usually made of basal medium, 1% penicillin -streptomycin, 1% L-glutamine, and 10% Fetal Bovine Serum (FBS)
- Confluent (adj) / confluency (noun) = the culture is confluent when cells have grown on or occupied all the available surface area due to proliferation
- Passaging = the activity of transferring cells from one flask/plate to another, typically when a culture has reached >90% confluency. It can also be referred as "Subculture".
- Passage number = the number of times the culture has been passaged. Many cells lose their proliferative and stemness behaviour after certain passage, so the earlier passage number, the better.
- Splitting = the activity of dividing a culture into multiple cultures. This shares the same idea with passaging. For example, from 1 confluent flask you may split it into 2, 3, 4.......and so on. as a result, each flask will have one half, one third, one fourth,...... of the original flask.
- The "hood" = which most of the time refers to the biosafety cabinet
- Seeding = placing cells in a well plate or flask at a certain density
- Contamination = when bacterial or fungal growth is found in the cell culture. But it can also mean when the growth of unwanted cells is found in a culture
- Cross contamination = when you are handling different cell lines at a time and it is possible that you mix them due to possible use of same pipette, same tips,
- Contact inhibition = it is the behavior of the cell that in the culture condition, they
- Adherent = Cells culture in a standard plastic culture flask will adhere to the surface after few hours of seeding
- Primary culture = The very first initiation of cell culture where tissue (taken from a donor or a animal) is digested and grown on the plates to allow cells to egress and grow further
- Cell line = the cells cultured after the successful initiation from primary culture
It is the step of collecting cells for the purpose of passaging, seeding, or extracting constituents from cells.
Passaging is the action of transferring cells that are growing rapidly in a flask or plate that confluency is reached. A 100% confluent culture flask means that the cells have grown on all the available surface area of the flask. Ideally, when confluency is about 80-90%, it means that passaging is required so that cells can have a new available surface to grow. Too confluent culture can result in cell death and loss of important function that may affect the result of your experiment.
This is an important step to seed the cells at a desired number or density required for experiment. Counting using hemocytometer or the Neubauer's chamber is the most common procedure in cell culture.
1. Prior to freezing cell, prepare an esky with dry ice, or pre cool the Mr. Frosty / Freezing Jar containing isopropanol. Label all your cryovials with the name of cells, date, passage number, GMO details, and owner initials. The more detailed info you put, the more thankful you will be later on for keeping good record.
2. To freeze cells for future use, harvest cells following the steps above. After centrifugation, remove the supernatant.
3. From a confluent T75 flask, you can usually split this to freeze into 3-4 vials. 1 vial contains 1 - 1.5 ml.
4. Prepare freezing medium, which contains standard culture medium + 10% DMSO (dimethyl sulfoxide + 30% FBS (fetal bovine serum). The amount of FBS also varied form cells to cells, but a concentration of 30-40% is usually preferred.
5. Re-suspend the pellet with the desired volume of the freezing medium.
6. Aliquot these to each cryovials. Place cryovials directly in dry ice or Mr. Frosty jar.
7. Place the vials directly in -80 C for at least 4 -6 hours. Cells in this freezer can last up to 1 year.
8. Transfer the vials into the vapor phase or liquid nitrogen storage for long term storage.
I hope you find this cell culture guide helpful. Reach out to me anytime via the comments section below for questions or troubleshooting in any cell related work. I am more than happy to hear from your experience and to discuss the A-Z of cell culture! 👍👍😉