Cell Culture

Cell Culture Definition

Cell culture is a method used to cultivate, propagate and grow a large amount of cells in a dish. The cells can be of a mixed, heterogeneous origin with different cell types growing, or they can be a singular cell type, sometimes clonal in origin. Cell culture allows one to grow cells outside of their natural environment and control the conditions in which they grow.

Cells grow in a dish with particular growth media and factors to support optimal growth. Once the cells have consumed most of the media and occupied, by proliferation, all of the space in the culturing dish, they are considered ‘confluent.’ The cells are then ‘passaged’, which removes them from their confluent dish, dilutes them into new growth media and places them at a low cell density into a new culturing dish. This allows the cells to keep expanding and proliferating in optimal culturing conditions. Most cells have a finite amount of time during which they can grow in culture because of their genetically determined cell lifespan. Other common cell culture cells have been ‘transformed’, which makes them immortal; they will continue to propagate infinitely, given optimal culturing conditions. A common type of immortal cell lines are cancer cell lines such as HeLa cells.

Cell cultures can also be adherent or non-adherent in their culturing conditions. Adherent cell types like fibroblasts or epithelial cells are grown by attaching to the culture dish surface. These types of cells need to be physically detached from their surface in order to passage them. Non-adherent cell types, such as lymphoid cells, are grown in suspension in a cell culture flask – they usually do not attach to a surface in their native environment or in culturing conditions.

Cell Culture Applications

Cell culture is widely used in basic and clinical research. It serves as a tool for cellular and molecular biology research where researchers can test drugs and growth conditions, and perform genetic manipulations on the cultured cell populations. Cell culture can be used to create a scaled-up source of a limiting population of cells found in-vivo, such as stem cells. It can also be used to create a large-scale production of some cell product, such as an antibody or secreted protein.

Cell culture is often considered an in-vitro model (i.e., outside the living organism) for a native, biological cell study. For example, skin epithelial cells from an organism may be isolated and prepared for cell culture; these cells can then be transduced or transformed to overexpress or reduce an epithelial gene of interest. This can allow the researcher to test the effects of the gene of interest on the epithelial cell morphology, growth rate, behavior etc., while the same type of gene manipulation may not be possible in-vivo (i.e., within the living organism) or in such a controlled, cell-type specific method as cell culturing.

Cell Culture Protocol

Materials needed for cell culture:

• A source of cells


• Growth media with essential nutrients


• Growth factors


• Culture dishes appropriate for the cell type


• Gas and temperature regulated incubator

All steps of the protocol should be attempted in a cell culture hood that is maintained for aseptic technique to prevent contamination.

Step 1

Cells can be isolated from a primary source, such as an organ or tissue from a living organism, or from a secondary source, such as a frozen cell line.

Step 2

Growth media solution is prepared with appropriate growth factors for the type of cell (e.g., epidermal growth factors for epidermal cells, cytokines for immune cells). The media should be sterile filtered and usually contains antibiotics to prevent bacterial contamination of the cell culture.

Step 3

These cells are counted if the quantity is not already known. The known number of cells is pelleted and re-suspended in the prepared growth media. The volume of cell resuspension should be optimal to the culture dish and to the amount of cells per dish (i.e., cell density).

Step 4

The cell resuspension is transferred to the appropriate culture dish and gently shaken to create an even dispersion of cells onto the dish.

Step 5

The dish is placed in the incubator with CO₂ and O₂, and the temperature set appropriately for the cell line or type. The cells are allowed to attach (if adherent) and grow over the next couple days.

Step 6

Cells are allowed to grow optimally and to confluency over the next couple of days using fresh media changes regularly.

Step 7

When the cells have reached confluency, they are ready for passaging. This is done by removing the growth media and adding a dissociation media so the cells, if they are adherent to the dish, detach themselves. Once detached, the cells are transferred to a tube where they will be pelleted and washed several times. They are also counted and usually assessed for the amount cell death. The new quantity of cells is then once again re-suspended and diluted in freshly prepared growth media, as before, for the appropriate cell density. This suspension of cells is then plated onto new culture dishes and allowed to grow again to confluency.

The process can be continued until the desired quantity of cells or passages has been reached.

Cell Culture Contamination

Cell culture contamination happens when the cells become infected with bacteria, mycoplasma, yeast and/or mold. Using aseptic technique is important in preventing this type of contamination. It involves using a sterile work environment as much as possible, such as a designated cell culture fume hood that is disinfected after each use. The hood is often kept under UV light when not in use to prevent the growth of microorganisms and regularly wiped down with 70% Ethanol. Furthermore, the reagents used during cell culture, such as the media and growth factors, should be kept under sterile conditions and handled with sterile instruments and dispensers.

Alternatively, cell culture contamination can also happen by non-biological sources, such as chemicals, detergents or endotoxins, that appear in the media or materials used to culture the cells.

Quiz

1. Which of the following could be an application of cell culture?
A. Large-scale cell production
B. In-vivo genetic studies
C. To study physical organ characteristics
D. Drug production

Answer to Question #1

A is correct. The applications of cell culture can be wide and varied; however, of the listed possibilities, large-scale cell production is the best answer, because cell culture can expand a small starting cell population. Cell culture is also considered an in-vitro model, and not an in-vivo model. Cell culture does not reproduce a whole organ so cannot be used to study the physical organ character. Lastly, drugs are produced chemically and not by cells.

2. Which of the following is a biological contaminant of cell culture?
A. Endotoxins
B. Mycoplasma
C. Antibiotics
D. Detergent

Answer to Question #2

B is correct. Mycoplasma are a type of bacteria organism that are a biological source of cell culture contamination. All the other answers are examples of non-biological substances.

3. How do you know when a cell culture has reached confluency?
A. The cells are at very low density
B. There is contamination in the culture
C. The cells have occupied almost all culture dish space
D. The cells are proliferating

Answer to Question #3

C is correct. Confluency is reached when cells have expanded, by proliferation and growth media usage, to cover the cell culture dish. Generally, cells will slow down or stop proliferation when they’ve reached confluency because there is no more room left to grow. If cells are at low density, it means they are sparse and have lots of room between them to expand and grow.

References

• Gibco (2016). “Cell Culture Basics” Thermo Fisher Retrieved 2017-04-30 from https://www.thermofisher.com/cellculturebasics

Cell Culture