June 10, 1990
Rosalie Veile
Purpose:
To grow lymphoblastoid cells for permanent storage and for DNA extraction.
Safety Considerations:
All cultured animal and human cells have the potential for carrying viruses, latent viral genomes, and other infectious agents. Cell cultures should be handled very carefully by trained persons under laboratory conditions which afford adequate biohazard containment. A Biological Safety Cabinet must be used when passaging cell lines. Use bleach in a suctioning apparatus to kill unused virus. All material used in passaging the cell lines must be autoclaved. Gloves are always worn to protect hands from contamination . A laboratory coat should be worn to protect clothes from contamination. Doors of the tissure culture room should remain closed to decrease the amount of airborn contaminants entering the incubators and the room. Equipment (incubators, centrifuges, microscopes, tabletops, etc.) should be cleaned routinely to help maintain a sterile work environment.
Time required:
3-4 weeks to grow a cell line from a frozen stock, or to grow an established cell line arriving in a T-25cm2 flask to 1 x 100 million cells.
Allow 6-8 weeks establish and grow a lymphoblastoid cell line from whole blood to 1 x 108 cells.
Procedure:
Maintaining lymphoblastoid cultures is fairly simple if two important characteristics are taken into consideration: 1) the cell cycle (primary culture and established cell line) and 2) the cell concentration.
Cell cycle:
Every lymphoblastoid culture is unique and should be treated accordingly. For example, some cultures will grow very rapidly, while others may require twice the amount of time. Cultures which require the media to be changed every other day are rapidly dividing and will form many clumps. Cell lines which grow slowly will change the color of the media every 3-4 days, and may require the use of cyclosporin A and less media with each feeding.
Lymphobast cultures grow in clumps and do best if periodically shaken up to break up the clumps. The cells will usually settle to the bottom of the flask, but do not attachunless the culture is in the primary stage of transformation or is lacking in nutrients. Cultures growing well will turn the media acidic within 12-24 hours after being fed. The color is a good indication of cell growth and concentration (yellow: growing well; orange or pink: not growing well).
Lymphoblasts can be grown in T-25cm2 flasks or T-75cm2 flasks. Occasionally it is necessary to use a 24 or 96 well plate if a culture is not growing well. Primary cultures are set up in a 25cm2 flask and maintained until a volume of 15-20 ml is reached. The culture is then transferred to a larger flask to continue growth.
Cell concentration:
The cell concentration of the suspension is important. A cell count above a certain number means decreased viability (the dead cells stain blue with Trypan blue, see cell counting procedure). When the cell count is too low, cultures will show little growth. The absolute lowest cell concentration for any cell line should be 1.5-2.0 X 100 thousand viable cells/ml. Cultures can be split when the cell count is 2.0 x million viable cells/ml.
Cultures are grown upright in T-flasks. They are maintained with RPMI-1640 (supplemented with 1% of a 200 mM L-glutamine solution) plus 15% fetal bovine serum (heat inactivated) plus an antibiotic, such as gentamicin reagent or penicillin/streptomicin. Incubation conditions are 37 degrees C and 5% CO2. Cultures are fed every 3 to 4 days. If a cell line is not fed frequently enough, the majority of the cells will not be in the logarithmic phase of growth; therefore the optimum growth of the cell line is never reached. Cultures are fed by removing half of the media from the flask and replacing it with a slightly increased volume of new media. If a culture is not growing well, half of the media is removed, and the volume of added media is decreased slightly.
References:
Human Genetic Mutant Cell Repository, Coriell Institute for Medical Research, Camden,New Jersey, Assurance Form, and Optimum method for passing lymphocyte cultures.
Dr. Ray White, Maintaining and Freezing Lymphoblasts Procedure, 3/25/85.
首页
