Cellular tissue culture has come a long way since the beginning of the 20th century. Published papers in the early 1900+s report a theory and a procedure, although harsh and crude, to keep cells alive in media, and those cells+ behavior in vitro would be similar cells in vivo. The advantages of these cell cultures soon came into perspective, medical research could soar from this. Certain difficulties were soon overcome and a serum free media was produced, allowing a huge repertoire of cell lines to be produced. These cell lines created a diversity, allowing scientist to look at how different species of cells could respond to different things.
To produce a cell line worth studying, the first thing that has to be considered is contamination. Cell and tissue culture work requires that cultures are free of contamination microorganisms. The harvest site is sterilized, and sterile instruments are used throughout any procedure when working with the tissue. Next, the overall health of the cells being taken is examined. Tissue culture also requires that the procedure starts with healthy cells, or the experiment will probably not work.
Once the tissue is collected, it undergoes a mincing process to break apart the cells, i.e. mechanical dissociation. After mincing, the chunks of tissue are exposed to proteolytic enzymes and mechanically agitated. A number of chemicals can be used for tissue dissociation to liberate individual cells, but trypsin and collagenase are most often used. When the desired amount of cells are attained, the cells are washed, rinsed, and put into fresh medium. Cells suited for growth in the culture vessels undergo rounds of growth and mitotic reproduction. The primary culture, when it becomes too crowded can be subcultured by making a one-to-one or one-to-four split thereby terminating the primary culture and having the first |passageX initiate secondary cultures. As the cell lines proliferate and are subcultured, a selection process may occur which narrows the range of variation within the line until only one cell type remains. Such a culture may be designated as a cell strain. However, cell strains are often established by the cloning of a culture, insuring that all the descendents have a common in vitro origin. After dissociation is complete, the cells are then rinsed of trypsin or old media and resuspended in fresh medium and inoculated into culture vessels. The whole process can be done over and over again to produce gigantic amounts of cells.
A characteristic of most normal diploid cell lines is that they are attachment dependent, they respond to contact inhibition signals, they grow as monolayers, and they have a finite in vitro lifespan. On occasion, the cell will show spontaneous changes in morphology and/or growth behavior. Also, it is not uncommon for cells to alter the phenotypic expression then removed for the regulated environment of the whole organism. But steps have be taken to make medium and create conditions where phenotypic expression is under some control.
In order to insure the cells+ survival and to get any kind of positive results, the environment conditions must be satisfied. Most important are controlled temperature, most cells lines require 37 C; pH, 7.2-7.4; osmotic pressure, 260-320 mOsm/kg; a surface suitable for attachment, adequate respiratory gas exchange, and a non-toxic culture medium which satisfies several roles. The culture medium, prepared from non-toxic chemicals and purified water, ordinarily consists of a mixture of essential and non-essential amino acids, a carbon source such as glucose, a variety of vitamins, and a mixture of inorganic salts.
An important goal of the in vitro methodology has been to maximize reproducibility and control of the cellular environment. It was soon realized that |naturalX supplement were making the goal very difficult to achieve. The serums were often fluctuating depending on season, diet, etc. The ingredients for the medium were often then synthesized to produce more dependable results.
Mass culture technology as described as come a long way, and it has provided new opportunities for enhanced product yield as well as cell yield. Much success as come out of culturing and some of that success was because of cooperation between many people and agencies to achieve the goal of successful culturing technique. Now the we know how to culture, where will the future lead us from here? Mass food production, the revival of extinct species, who knows???