Protocol for Culturing Transformed Lymphocytes
From Shipment of Frozen Cells
Frozen cells will be received in 1 ml aliquots on dry ice. Keep cells frozen until thawed for cell culture or place in liquid nitrogen storage if not starting cultures right away. However, it is recommended that if you are going to store the cells for later use, grow up fresh stocks and freeze multiple ampoules. The cells are cryopreserved in 45% RPMI-1640, 50% fetal calf serum, and 5% DMSO (tissue culture grade).
- Place 10 ml of RPMI-1640, 15% fetal calf serum (FCS), ±antibiotics in a T-25 flask.
- Allow the media to equilibrate in a 37°C humidified incubator with 5% CO 2in air for 30 minutes.
- Thaw each ampoule of frozen cells in a 37°C water bath or a beaker of lukewarm water, one at a time.
- Quickly clean the outside of the ampoule off with a sterile isopropanol prep pad, then wrap the prep pad around the ampoule to protect fingers and crack the seal on the ampoule inside a biological safety hood.
- Remove the 1 ml of frozen cells with a sterile glass Pasteur pipet and place in a T-25 flask with 10 ml of medium. Clearly label flask to identify each cell line.
- Place the cells in a 37°C humidified incubator with 5% CO 2in the air.
- After 24 hours remove 5 ml of medium from the top without disturbing lymphocytes settled on the bottom of the flask and replace with 5 ml of pre-warmed medium.
- Feed the cells with 5 – 6 ml of fresh medium every 3 – 4 days and split into new cultures as required.
From Shipment of Live Cultures
- Live lymphocyte cultures will be received in T-25 flasks filled to the top with RPMI-1640, 15% FCS, 1% Penicillin-Streptomycin (Gibco). The caps will be tight and sealed with parafilm.
- Remove parafilm.
- Place cultures in a 37°C humidified incubator with 5% CO2 in air and allow cells to settle out (20 – 30 minutes).
- Using a sterile pipet remove all but 10 – 15 ml of medium and discard.
- Place caps back on flasks loosened slightly to allow gas exchange (Check to make sure cap threads do not have media on them. If so you may want to transfer to a new flask.) Place the cultures back in the incubator with 5% CO 2in air. They should recover in a day or two.
- Feed the cells with fresh medium every 3 – 4 days by resuspending the cell aggregates and removing 50 -60% of the volume and replacing with fresh media. Cells removed from the flask can be used to initiate new cultures as needed.
For Further Information Please Contact:
Leslie B. Gordon, MD, PhD
Professor of Pediatrics Research Warren Alpert Medical School of Brown University and Department of Pediatrics, Hasbro Children’s Hospital, Providence, RI Department of Anesthesia, Children’s Hospital Boston and Harvard Medical School, Boston, MA Medical Director, The Progeria Research Foundation