PubMed2002
To increase the efficiency of in vitro transformation of human lymphocytes by Epstein-Barr virus (EBV) and establish permanent lymphoblastoid cell lines from patients with abnormal chromosome karyotype, B lymphoid cells were prepared from cryopreserved heparinized blood samples. The lymphoid cell pellet was resuspended with 0.5 ml medium of RPMI with 20% fetal calf serum(FCS), and added 2 ml virus-containing superatant of the EB virus-producing cell lines by filtrated, and mixed. Four 25 cm2 cell culture bottles were put upright. A total of 2.5 ml of RPMI with 20% FCS was put in each of them. The blood-virus mixture was distributed among the four cell culture bottles as follows: Bottle I, Bottle II, Bottle III and Bottle IV were added with 0.3 ml, 0.6 ml, 1.2 ml and the rest respectively. The cells culture bottles were put into the cell culture incubator in an upright position. After 3 days the cells were puting new medium with 20% FCS as follows: Bottle I 3 ml, Bottle II 4 ml, Bottle III 5 ml and Bottle IV 6 ml. After one week, the medium was changed again as described above. The medium change was conducted until the cells grew very fast. The right ratio between blood cells and virus titer can not be exactly determined for every blood sample, and therefore a dilution series with four different blood/virus ratios was set up. Due to the dilution series, addition of immune inhibitors like cyclosporine, was not necessary. Forty-seven permanent lymphoblastoid cell lines of patients with abnormal chromosome karyotype. Transformed cells were found in only one or two of the four cell culture bottles. The total successive rate increased up to 97.87%. Of the four cell culture bottles, Bottle I, Bottle II, Bottle III and Bottle IV, the successive rates were 6.39%, 61.70%, 31.91% and 8.51% respectively. This method can be used for preserving large number of lymphoblastoid cell lines, and also provide enough research materials for further studies.