IGEM:SWC/2009/Notebook/General Procedures for Cell Culture/Entry Base

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General Procedures for Cell Culture by Dan Gilliland

Purpose Cells can be cultured in the Lab and experimented on in a test tube environment. This environment can be controlled to experiment on and understand the exact reactions of cultured cells, tissues, or organs without hurting living beings. Unfortunately normal cells can't really survive in the Lab environment so the cells are manipulated to become a continuous cell line that can keep dividing instead of dieing out like normal cells. These continuous cells can be produced by adding viruses or carcinogenic chemicals. The advantage to this strategy is an unlimited supply of cells to experiment on. The disadvantage is the cells being tested are not typical cells and the test can only explain the specific action within a cell instead of how the experiment would effect a normal cell.


Materials & Methods

sterile environment optimal pH and temperature for growth. Minimum Essential Medium Eagle Carbohydrates (glucose or fructose) added as energy source inorganic salts (buffering capacity and osmotic balance) typical medium pH 7.2 - 7.4 fibroblasts pH 7.4 - 7.7 transformed cells 7.0 - 7.4 sodium bicarbonate (buffering) 5% CO2 HEPES growth serums (5% - 20%) pipette refrigerator autoclave dishwasher DMSO

General Safety: Antibodies, cells and other media are generally considered a bio-hazard. Lab coats are essential, you may be working with acids or other bio-hazards and you do not want to be contaminating yourself or others. Protective glasses need to be worn at all times. Eyes either can be damaged or can be an infection point if coming into contact with hazard.

No eating, drinking should be permitted. Contamination to and or from food can destroy a project or a life.

Wearing of gloves is strongly recommended.

Immunization and common vaccinations are recommended for those in contact with primary human material.

HEPA-filtered workstations are common to protect Lab employees

Disinfection procedures should always be in effect, but SOPs must be followed due to the corrosive nature of many disinfectants

Data, Results & Calculations

Cell culture should be manipulated in a clean clutter free area using sterile equipment.

Cell lines must come from reputable sources

Stock cultures of 2 cell lines should never be worked on at the same time because the risk of cross contamination is too great.

Before stored stock is used for large studies the stock needs to be a aliquoted to ensure against contamination.

The use of antibiotics continuously in culture medium will cause growth of antibiotic resistant strains.

Reagent quality control testing ensures reproducible growth characteristics of medium.


Results

To maintain cell cultures in optimum conditions keep the cells in Log phase as long as possible. Adherent cells when kept in the Stationary state too long will not adhere properly when being studied.

Suspension cells do not produce attachment factors and can be subcultured by dilution. If the amount of cells becomes to concentrated toxic by-products can accumulate.

Cell lines can be saved by cryopreservation of cells.


Discussion

"Minimum Essential Medium (MEM), developed by Harry Eagle, is one of the most widely used of all synthetic cell culture media. Early attempts to cultivate normal mammalian fibroblasts and certain subtypes of HeLa cells revealed that they had specific nutritional requirements that could not be met by Eagle's Basal Medium (BME). Subsequent studies using these and other cells in culture indicated that additions to BME could be made to aid growth of a wider variety of fastidious cells. MEM, which incorporates these modifications, includes higher concentrations of amino acids so that the medium more closely approximates the protein composition of mammalian cells. MEM has been used for cultivation of a wide variety of cells grown in mono-layers. Optional supplementation of non-essential amino acids to the formulations that incorporate either Hanks' or Eagles' salts has broadened the usefulness of this medium. The formulation has been further modified by optional elimination of calcium to permit the growth of cells in suspension." http://www.sigmaaldrich.com/life-science/cell-culture/classical-media-salts/mem-media.html

"HEPES is widely used in cell culture, largely because it is better at maintaining physiological pH despite changes in carbon dioxide concentration (produced by cellular respiration) when compared to bicarbonate buffers, which are also commonly used in cell culture."

http://en.wikipedia.org/wiki/HEPES

"DMSO, which is often used in cryopreservation, must be handled with care. The chemical is flammable, although high temperatures are required to generate a risk of explosion. DMSO readily permeates some types of gloves and the skin. In itself DMSO is not hugely toxic to the body; however, DMSO may carry other chemicals and allergens through the skin barrier."

Cell Biology - A Laboratory Handbook p.19 & (Freshney, 2000).

Conclusion

This is a general overview of lab procedures when handling cell cultures.



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