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    Knowledge database

    A guide to cell culture media

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    Cell culture media

    What is cell culture? 

    Cell or tissue culture refers to the growth and maintenance of cells derived from tissues of an animal, from insect to human, in an artificial environment often referred to as in vitro.

    There are two ways to grow in vitro cell cultures;

    1. Adherent cell culture - Cells are grown as monolayers in plastic dishes or flasks. Some cell types can be grown on gel-like substrates as multicellular aggregates to better mimic a physiological environmental stiffness.

    2. Suspension cell culture – Cells are grown without adhesion contact to the substrates suspended in cell media. Generally, to ensure optimum cell growth, the suspension is agitated regularly.

    Adherent cell culture is the most commonly used method of growing cells as it is able to be used across a wide variety of cell culture types. Suspension cell culture is useful as it can be easily scaled up for protein purification.

    What is the difference between primary and secondary cell culture? 

    A primary cell culture refers to cells that have been removed from a live donor/specimen and have been established in an in vitro environment.

    Secondary cell culture refers to cell lines that have been immortalized, usually by overexpressing an enzyme called human telomerase reverse transcriptase (hTERT), and can divide indefinitely. Many tissue types are not amenable to immortalisation, so primary cell culture may be the only option in some circumstances.

    Both primary and secondary cell cultures have advantages and disadvantages:

    1. Using primary cell cultures will provide biological results in specific cells of interest, that are more accurate to those seen in the host tissue. However, there will be a finite number of cells with a finite lifespan for you to use.
    2. In contrast, secondary cell cultures will be easier to maintain and have a more uniform and long-lasting cell population, but as the cultures have grown and been passaged in artificial cell culture media, they will provide biological responses different from cells within the original host tissue. Additionally, these cells may have genetic drifting, changes or mutations acquired during repeated passaging and culture maintenance.

    To overcome some of the limitations of secondary cell cultures, some researchers prefer to use human plasma-like cell culture media such as PlasmaxTM, rather than standard media.

    How many types of cell culture media are there?

    There are many types of cell culture media as the growth and metabolic requirements and biological environments of cultured cells are myriad. However, all cell culture media can be divided into two main groups – natural and artificial:

    1. Natural media are derived from natural tissues, such as blood or serum.  Various tissues, in physiological or modified form, can be used as cell culture media. These include foetal bovine serum (FBS), and umbilical cord serum. Natural media is very hard to reproduce as the exact composition of these media is undefined and subject to batch-to-batch variability.

    2. Artificial media are chemically defined and synthetically manufactured with different ratios of organic and inorganic compounds such as vitamins, amino acids, nutrients and salts. These cell culture media can be supplied in either a liquid or powder form. Artificial media are often supplemented with FBS or some other type of natural sera to add essential natural nutrients and growth factors for cell proliferation.

    Cell culture media (both natural and artificial) have been produced to a generalised formula of nutrients needed for cell culture growth. Researchers often need to amend the cell culture media to accommodate the optimum growth requirements of a specific cell type. This can include altering serum concentrations, and/or adjusting the concentrations/ratios of other supplements, such as additional amino acids, vitamins, cytokines and/or selection compounds (e.g. puromycin).

    Some cell types require very specific amounts of multiple supplements and selecting the correct combination of supplements is time consuming, laborious and difficult to reproduce across cell culture media batches. Recently, some new physiologically-relevant cell culture media were developed aiming to resolve some of these issues. One in particular, PlasmaxTM, is the only ready-to-use cell culture medium containing growth enhancing components at physiological levels as well as essential trace elements.

    What are the advantages/ disadvantages of including serum in media?

    As serum is derived from natural tissues, adding serum to your cell culture growth media, provides your cell culture with some physiological elements: 

    • Presence of growth enhancing nutrients, leading to increased cell culture growth rates. 
    • Presence of basic nutrients and minerals found within the in vivo environment, e.g. binding proteins, ensuring your cell cultures better resemble in vivo cell cultures.  
    • Presence of inhibitors protecting cells from a variety of conditions such as proteolysis. 

    However, there are also disadvantages to using serum, particularly as the exact composition of serum is undefined: 

    • Difficulty in ensuring batch-to-batch consistency, leading to increased testing taking place at the start of each new batch. 
    • Less control over the cell culture’s physiological response 
    • Increased costs of purchasing both sera and cell culture media.

    New cell culture media are trying to replicate the advantages of including serum within cell culture media, without the disadvantages. One example of these new physiologically-relevant cell culture media is PlasmaxTM, which is the only ready-to-use cell culture medium containing growth-enhancing components at physiological levels as well as essential trace elements.

    How do I select the right cell culture media?

    Cell culture media and their associated supplements have been targeted to specific cell culture types and have been tailored to provide a variety of different formulation options, so there are a vast range to choose from. Identifying your research objectives, and selecting the most appropriate cell type, are therefore crucial first steps in selecting the right cell culture media.  

    Once these decisions have been made, the choice of cell culture media will have been substantially narrowed. E.g. Primary cell cultures (fibroblasts, mesenchymal stem cells, etc.) generally require specific growth factors to be supplemented to artificial media, whereas established cell lines such as HeLa or RPE1 require minimal supplements (FBS, L-Glutamine, optional antibacterial/fungal), beyond the basal media. 

    Much of the cell culture media and different supplements necessary for cell growth and survival are empirically determined. These can be found in databases such as ATCC, ECACC, or other cell repositories, providing a first reference point for identifying the necessary media, supplement types and concentrations for your chosen cells. 

    However, your research objectives will also need to be considered. If you are investigating cellular metabolism or the impact of specific nutrients on cellular processes, a more physiologically-relevant cell culture medium might be the best option to obtain more relevant results. These types of cell culture media have only recently been developed, with one of the first being PlasmaxTM, a ready-to-use medium, that mimics the metabolic and physiological profile of human plasma. 

    Cell culture tips and tricks

    • Bring the temperature of the cell culture media to 37oC in a clean water bath, preferably dedicated to cell culture use only, to decrease chances of contamination.

    • Keep the biosafety cabinet (the cell culture hood) empty, except when it is in use and stay clear of the vents to promote proper air circulation. This decreases the chances of contamination. Wipe down the work surface with 70% ethanol before and after using the biosafety cabinet to maintain a sterile/ an aseptic environment.

    • Spray and wipe down everything (pipets, media bottles, other conical tubes, tube racks, etc) that enters the biosafety cabinet with 70% ethanol, to reduce chances of contamination.

    • Refrain from placing caps, pipets, or other equipment down on the sterilized surfaces. If a lid needs to be opened, be swift in keeping bottles and flasks open, and quickly close them to reduce the time the bottle stays open to potential contaminants. If caps and lids must be placed onto the work surface, briefly place them open-side up, and ensure that nothing touches the inside of the cap/lid.

    • If you suspect a pipet tip may have touched a surface (even if it has been sterilised), dispose of the pipet tip immediately and start with a fresh tip. The contact, albeit brief, could introduce contaminants (e.g. bacteria, fungi) that could exist on surfaces (the biosafety cabinet walls, pipets, cell culture dishes/flasks) into the cell culture medium, the cell stock itself, or any other supplements and addition stocks.

    • Unopened cell culture media can be stored in the cold room or a 4 oC fridge, and should be used close to, or before the expiration date. Once supplemented with FBS and/or other additives, the media should be used within 6 weeks of opening.  The supplemented media can be stored at 4oC during this time, and warmed to 37oC before using.

    How often should you change your cell culture media?

    In general, media should be changed every 2-3 days. However, this will depend on the cell culture type cell density and volume of medium used per unit of surface. Allowing the medium to turn yellow as a consequence of the acidic pH generated by cell metabolic activity should be avoided as it can lead to cell death and phenotypic changes in the culture. Moreover not all the media will turn yellow before being exhausted of nutrients.

    Usually (though not always, depending on the cell culture type), the timings of when you change your cell culture media coincide with what is called passaging or split. In general, this involves the transfer of either adherent or suspension cell cultures once they have grown to confluency, to ensure continued cell culture growth:

    • Adherent cells are usually passaged by treating the cell culture with an enzyme that helps to lift adherent cells from the surface, before a fraction of this suspension is reseeded to a clean, unused plate/flask and additional media is added. The plate/flask manufacturer usually has information on the media volume for each dish/flask for proper gas exchange. Cells should be seeded at a density that is most optimal for proliferation for the specific cell type and should form a monolayer.

    • In suspended cells, a proportion of the cells suspended in the cell culture medium are removed from the container and placed into a fresh flask/dish to maintain the correct seeding density for that particular cell type in a given flask/dish.

    How long does cell culture media last?

    In general, cell culture media lasts for 3-4 weeks once it has been opened and supplemented, though this will vary across cell culture media and the rate of usage – PlasmaxTM, for example, lasts for 6 weeks once it has been opened. You should receive instructions on storage and shelf life with your cell culture media order, however databases such as ATCC also have information on shelf life and storage, in the unlikely event you can’t find the relevant information on the manufacturer’s website.

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