Establishment of Permanent Lymphoblastoid Cell Lines from Human Blood
Permanent Lymphoblastoid cell lines are established by in vitro transformation of human lymphocytes by Epstein Barr Virus ( Anderson, M.A & Gusella, J.F.,1984, In vitro 20: 856-858 )
Blood collected in yellow- top Vacutainer tubes containing ACD solution is processed for Epstein Barr Virus cell transformation. The whole blood is centrifuged in Cell Preparation Tube and the mononuclear cells which form a buffy coat are removed and resuspended in 10mls of Hanks Balanced Salt Solution.The cells are pelleted by centrifugation and the pellet is washed with Hanks balanced salt solution.
After the final wash, the lymphocytes are resuspended in 5- 7 mls of Iscoves Modified Dulbeccos Medium containing 15% Fetal Bovine Serum (FBS), 100U of Penicillin/Streptomycin/ml, 2mM Glutamin.(Growth medium)
To the resuspended cells, 1ml of EBV and 0.5mls of CyclosporinA (10 microgram/ml) are added and the cells are grown at 37degrees Celsius with 5% Carbon dioxide in an incubator.
Cultures are incubated for 8-10 days before being fed with growth medium. After 4-8 weeks, for most cultures, the proliferating transformed lymphocytes produce visible clumps of cells that settle but do not attach to the bottom of the flask. Cells are then transferred to a bigger flask and grown for cryopreservation.
For cryopreservation, the lymphoblastoid cell lines should be in the logarithmic phase of growth.Cells are frozen in freezing medium which contains 10%DMSO in Growth Medium.
Cells are frozen at a density of 6-10 million cells per vial.
Frozen cells are stored in liquid nitrogen freezers for long term storage. Cell lines are thawed routinely to check cell viability after freezing.