Cell Culture Guide - Techniques and Protocols

*Please note that all cell culture must be undertaken in a laminar flow hood using aseptic techniques.

Cell culture remains one of the most widely used techniques in research today due to the consistency and reproducibility of results that can be obtained from using a batch of clonal cells.

The growth of cells from an animal or plant in a favorable environment is known as cell culture. Different cells have different culturing requirements necessary for optimum growth, which can be catered for via medium selection and supplementation. The medium used acts to supply the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, gases (O₂, CO₂) and regulates the physicochemical milieu (pH, osmotic pressure, temperature).

A primary culture refers to cells which have been cultured directly after tissue isolation. Once these cells become confluent i.e. occupy all available space in the flask they need to be passaged or split by transferring them to a new growth vessel which will provide the cells with more room for continued growth and expansion. Following the first passage the primary culture now becomes known as a cell line. Cell lines derived from primary cultures have a limited life span, meaning that they can only divide a limited number of times before becoming senescent.

As the cells are passaged cells with the highest growth capacity predominate, resulting in genotypic and phenotypic uniformity in the population. Immortal cell lines are widely used in cellular and molecular biology in order to bypass problems such as senescence. Cells can become transformed and thereby immortalized through a number of different means; spontaneously, chemically or virally. Once transformed these cells acquire the ability to divide indefinitely and become a continuous cell line.

Cell lines can either be:

1. Adherent (Anchorage Dependent) - Adherent cells must be cultured while attached to a solid or semi-solid substrate and will often require trypsinization for subculturing (see 2.6 for more details)

2. Suspension (Anchorage Independent) – Suspension cells do not require a solid growth substrate and can be grown floating in culture medium.

Below is a general guideline for the culturing of cell lines. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility, protective clothing must be worn throughout.

Good aseptic technique is designed to create a sterile environment and act as a barrier between microorganisms and the sterile cell culture hood. Key aspects of this technique include the use of sterile reagents, media. Sterile handling ensures that nothing is entered into the hood which hasn’t been sprayed with 70% Ethanol.

The cell culture hood provides as aseptic work area while ensuring the containment of any infectious splashes or aerosols generated by microbiological procedures. There are 3 kinds of cell culture hoods, designated as Class I, II and III, these have been developed to meet varying research and bio-safety needs. Choose the right one for your experimental needs.

Before starting ensure that you are working with the right media type and culture supplements for your cell line of interest. These must always be sterile and only opened inside the hood. Most cell lines can be grown using Dulbecco’s Modified Eagle Medium (DMEM) culture media or Roswell Park Memorial Institute (RPMI) culture media with 10% Foetal Bovine Serum (FBS), 2 mM glutamine and antibiotics can be added if required.

Certain endothelial cell lines require special collagen matrices to grow. These matrices ensure attachment, differentiation and growth of the cell line. Before starting your cell culture experiment it is important to carry out a literature review of your cell line of interest to ensure any additional growth requirements such as matrices are meet. Be aware of the type of flask you are using i.e. vented or non-vented. If using a non-vented/plug flask be sure to loosen the cap to allow gaseous exchange. If using a vented flask ensure that the filter does not get wet as this will affect the efficacy of gaseous exchange.

Cells should be checked daily under the microscope to ensure they are healthy and growing as expected. It is important to become familiar with what healthy cells look under the microscope as this will make it easier to detect quiescent or infected cells through changes in morphology.

Mammalian cells should be split/passaged when they reach confluence, which occurs when 70-80% of the flask is covered with cells. In adherent cells confluence can be noted under the microscope when there is no visible space available for cellular growth. For suspension cells confluence can be noted when the cells clump together and the medium appears turbid when the flask is swirled.

It is important not to let the cells become over confluent, with 70-80% confluency being the optimum amount, after such a point contact inhibition comes into effect and the cells require a longer recovery period following re-seeding. The speed at which your cell line grows will determine the split ratio used. For instance slow growing cell lines will not do well if split at a high ratio.

Fast growing cell lines may require a high split ratio as it will take them a shorter amount of time to reach confluency in comparison to slower growing cell lines. As a general rule cells should not be split more than 1:10 as this is too low for the cells to survive. Varying the seeding density of your cultures will ensure that your cells are ready for an experiment on a particular day.

As a general guide, from a confluent flask of cells: 1:2 split should be 70-80% confluent and ready for an experiment in 1 to 2 days. 1:5 split should be 70-80% confluent and ready for an experiment in 2 to 4 days. 1:10 split should be 70-80% confluent and ready for sub-culturing or plating in 4 to 6 days. However this may vary depending on the growth rates of your cell line.

* Trypsinization can be toxic to some cells. It can also induce temporary internalization of some membrane proteins, this should be taken into consideration when designed experiments. Other methods such as gentle cell scraping, or use of detergent can often be used as a substitute in these circumstances.

If your cells have been growing for a few days but are not confluent enough to be split the media will have to be changed to replenish nutrients and maintain pH.

The passage number is the number of sub-cultures the cells have gone through. Passage number should be recorded and not get too high. Cell lines with passage numbers of greater than 30 are more likely to acquire genetic abnormalities compared to lower passage cells (Esquenet et al., 1997; Lin et al., 2003; O’Driscoll et al., 2006).

P30 is generally accepted as the upper limit of passage number for certain cells in culture, as further culturing may result in substantial variation of molecular profiles, limiting their in vivo applications and reproducibility (Calles et al., 2006; O’Driscoll et al., 2006; Wenger et al., 2004).

Freezing down cell lines is essential component of cell culture as replacing cell lines is expensive and time consuming, it also ensures that if your cells become infected you have back up stocks and can start again. As soon as surplus cells become available, preferably at a low passage number to limit genetic drift, they should be frozen as seed stocks. Working stocks can then be prepared from the seed stock.

The best and most widely used method for cryopreserving cells is storing them in liquid nitrogen in media with a cryoprotective agent such as dimethylsulfoxide (DMSO). Cyroprotective agents such as DMSO reduce the freezing point of the medium and enable a slower cooling rate, which greatly reduces the risk of ice crystal formation, known to cause cell death and damage.

*Freeze your cultured cells at as a high concentration and as low a passage number as possible. Ensure that at least 90% of the cells are viable before freezing them down. Always use sterile cryovials for storing frozen cells. Always use proper sterile technique and work in a laminar flow hood.

Safety Notes: Caution should be exerted when using DMSO as it is known to facilitate the entry of organic molecules into tissues. Use gloves and wear appropriate protective clothing when handling this reagent. Dispose of the reagents in compliance with local regulations.

Thawing cells is extremely stressful to the frozen cells and therefore unlike freezing down cells which should be done slowly, thawing cells should be done as rapidly as possible, to ensure a high survival rate of cells.

One of the most common contaminants in cell culture laboratories is mycoplasma. Mycoplasma is a basic bacterium lacking a cell wall, considered to be the smallest self-replicating organism. Mycoplasma are very difficult to detect as a result of their small size approximately > 1 micrometer, it is not until they achieve extremely high densities and cause the cell culture to deteriorate that their presence becomes known.

Many slow growing mycoplasma can persist in culture undetected without causing cell death however, they can alter the behaviour and metabolism of the host cell in culture. Chronic mycoplasma infections may also lead to a decreased rate of cell proliferation, reduced saturation density, and agglutination in suspension cultures. The only way to detect such mycoplasma infections is by periodically testing the cell cultures using; fluorescent staining (e.g., Hoechst), ELISA, PCR, immunostaining, autoradiography, or microbiological assays.

Antibiotics in cell culture should be used sparingly. Continuous use of antibiotics encourages antibiotic resistance, allows low-level contamination to exist and can mask a variety of contaminants. Some antibiotics can also cross react with the cells and interfere with the cellular processes under investigation.

Cell culture laboratories can vary greatly depending on research institute and area of investigation. All cell culture laboratories do however have some essential common equipment and requirements, the most important of which being the maintenance of an aseptic environment free from pathogens. Below is a list of the common equipment and materials you can expect in more cell culture laboratories. This list is not all inclusive, deviations from which can be noted depending on research area.