Cells used for culturing are described by origin and manner of growth. Primary cells come directly from a living organism and they can originate from any organ. These cells are difficult to culture because they have limited ability to divide, making their life spans short. Finite cell lines also come directly from animal tissues and are difficult to propagate. These cells can be cultured over several generations and so they last longer than direct primary lines. Continuous cell lines come from an action called transformation whereby their genetics have spontaneously changed in culture. These cells are easy to propagate because they have an almost indefinite lifespan, however, they are not as representative of the original organism due to this change.
Primary cells must come directly from the living organism. For example, if you are studying mouse mammary glands, the mouse must be sacrificed, the mammary tissue isolated, and the cells separated and grown into culture dishes. This is expensive and time consuming but will give a more accurate indication of real life application. Most of the cells plated after isolation will die within 48 hours of culture so many mice are necessary to get a large number of healthy plates. If you do not have the animal needed to obtain cells, you will have to research another lab that is working on the same type of project and see if they can spare some cells for you. Cell lines can be ordered from biological supply companies or obtained from another lab that you work with.
The method of cell culture will be different for each cell type; however, there are some general rules to learn to be successful. Most cells are kept frozen so the first task is to thaw the cells out. If you have primary cells it is imperative that you learn how to culture them from another person who is very experienced in that particular cell line. Cell lines will usually come with a protocol to follow. Frozen cells must be thawed in a 37 degree C warm water bath quickly in order to prevent unnecessary cell death. In addition, the growth media that is used in the plates should also be warmed prior to use. After thawing, transfer the cells to a culture dish with the prescribed amount of nutrient media and incubate for 24 hours. This time period allows the cells to stick to the bottom of the dish so that the media can be removed the next day and replaced with fresh media.
Monitor your cells daily by looking at them under the microscope. Look for signs of failure, such as many floaters. Floaters are dead cells that will no longer adhere to the bottom of the plate. Use the suction tip in the tissue culture hood to evacuate the media and dead cells and then refresh with new media. Healthy cells should replicate and spread out as they mature in the incubator. When the bottom of the plate is almost fully covered with cells, it is time to split them. Each lab will have a specific protocol to follow but generally this is done by washing the cells with a buffer solution and then detaching the cells from the bottom of the plate using media and a pipette or scraping. If using media, then fill the pipette with media and then press it firmly down on the bottom of the plate and release the liquid. The flood of liquid from the pipette will dislodge the cells and suspend them in the media. If scraping, use the cell scraper indicated in the protocol to suspend the cells. Transfer this cell suspended media to new plates that have been previously prepared and incubate for 24 hours. If you find yourself with too many plates full of cells, they must be used or frozen.
Contamination is a serious problem in tissue culture. Many weeks of hard work can be ruined with the introduction of bacteria in the incubator. Signs of contamination in your tissue culture include cloudy media, observation of clumping cells under the microscope, unexplained death, or an unusual smell. Always perform your tissue culture under a laminar air flow hood designed especially for the task. Do not use a bottle of media for more than one cell line. Do not have two different cell types under the hood at the same time. Ideally, you should not even have more than one cell type in each incubator, but this can sometimes be impractical. If you must share, keeps plates as far away from each other as possible and always make sure your incubator is clean.