Standard Procedures for Culture of Mammalian Cells

This article is about the standard procedures for culturing mammalian cells.An overview of the cell line as a whole and the L8 cell line specifically is discussed in this article. This article is to familiarize students in culturing, which is a common procedure in biology.Culturing cell is not easy. It needs careful study and the exact procedure for culturing to succeed.

There are three major factors to be considered in culturing cells. These are the following: Knowing and controlling the density at which cell are grown. The cells grow best when “plated” and on the surface of culture plastic. Growths of cells are few and very slow because things added by the cell in the medium affects their own growth. When the cells are too many cells will run out the medium and may contaminate the medium with their waste products. Another thing to be considered is the fusion of L8 to form myotubes. Once the cells fuse, they stop division. In order to continue the growth of the cell, put the cells onto plastic dishes and let it grow where they nearly cover the plastic and remove them and let them seed in unto another dish.

Another thing to be considered in cell culture is to keep the cells uncontaminated by bacteria and molds. The use of devices and procedure are required like: a) inclusion of antibiotics in the culture fluid. If one is cautious in handling culture media there is no need to use antibiotics because cells preferably grow with out antibiotic. b) Use sterile media and sterile plastic container. Culture media is sensitive with other foreign materials they easily react to other factors. So the materials used in culturing must be sterilized to avoid contamination. c) Mixing and transferring culture medium from one container to another must be done in a sterile place. This procedure is called laminar flow hood, which sterile air is continuously blowing in the work area to prevent the entry of air borne germs.Last to be considered in cell culturing is that cell need moist and warm environment to grow. The use of an incubator and buffering of the culture fluid is needed.

A method in to prolonging the shelf life of a culture media is by means of freezing. The freezing temperature required is usually at 80 degree centigrade. A special ingredient called Dimethyl Sulfide is mixed in the cells which prohibit the development of crystal in the cells as they freeze.The article discusses in detail on how to grow a cell. You need to have a vial of frozen cells dissolved in a bucket with water at 37 degrees centigrade. A small cell suspension is added and put to a plastic culture dish. They will seat onto the plastic and grow in the plastic surface and undergo repeated cycles of cell division. In order to grow the cell correctly it should be grown at a certain density. The chosen density is 3000 cells per square cm.of surface area.

If you have a small dish you need to add a certain amount of cell to be equal in the surface area to have an exact number of cells per dish. Monitoring the growth of the cell in the flask is obtainable by placing the sealed culture dish on the stage of a special microscope which is called tissue culture microscope; this microscope is an inverted version. The object is found beneath the stage and its illuminator is above the stage of the tissue culture microscope. The set up of tissue culture microscope are arranged so that the cells are imaged through the plastic on the bottom of the culture dish.