High‐potentiality preliminary selection criteria and transformation time‐dependent factors analysis for establishing Epstein–Barr virus transformed human lymphoblastoid cell lines

Abstract

Infection of freshly isolated and cryopreserved lymphocytes with Epstein-Barr virus (EBV) leads to the establishment of human B lymphoblastoid cell lines (LCLs). Techniques for optimal infection of the lymphocytes are vital for the establishment of a human biobank. The present study found that more than half (58-86%) of such established LCLs had transport times of less than 48 h, cell densities exceeding 10(6) cells/ml and cell viabilities greater than 90%. After EBV infection, 3306 freshly isolated lymphocytes required 30.0 +/- 0.1 days to become LCLs. Conversely, 1210 cryopreserved lymphocytes required 36.2 +/- 0.4 days. Cell density and viability of the culture affected transformation time in freshly isolated lymphocytes. On the other hand, blood transport time, cryopreservation time and initial cell viability were major factors in cryopreserved specimens. These results contribute to general information concerning the establishment of a human biobank for EBV infected cells.

Assessment of the effect of TLR7/8, TLR9 agonists and CD40 ligand on the transformation efficiency of Epstein-Barr virus in human B lymphocytes by limiting dilution assay

“…Following 3 weeks, the frequency of transformation was evaluated based on the number of wells containing growing transformed B cells. Using the standard EBV transformation protocol in normal peripheral PBMCs, we and others (Chang et al 2006) have already shown that transformed B cells are established within 3-5 weeks after EBV infection. In the present study we employed an improved transformation protocol using irradiated fetal fibroblast feeder cells and tenfold concentrated EBV stock which accelerates the transformation process (data not presented).…”

Viable Mononuclear Cell Stability Study for Implementation in a Proficiency Testing Program: Impact of Shipment Conditions

“…12 Frozen viable PBMCs are fit-for-purpose, not only for immunomagnetic sorting of purified monocyte and lymphocyte populations, following cryopreservation, 13 but also for functional studies, 14 immunophenotyping, 15 establishment of lymphoblastoid cell lines (LCL) by Epstein Barr virus (EBV) transformation, 16 and purification of CD34 + cells. 17 The surrogate QC assay for either EBV transformation success 18 or immunophenotyping and proliferation assays 14 has shown cell viability, with a qualification cut-off at around 70% viability. Therefore, implementation of a PT scheme on cell viability for repositories, which process and cryopreserve mononuclear cells for all the above-mentioned end-uses, is of critical importance.…”

“…The HV suspension corresponded to the Jurkat cells incubated according to manufacturer's instructions (cell passage [1][2][3][4][5][6][7][8] or freshly isolated cells from whole blood for PBMCs; the IV suspension was obtained, following incubation of HV suspension of Jurkat cells (cell passage 1-8) or fresh isolated PBMCs for one week at + 4°C; the LV suspension was obtained, following incubation of Jurkat cells (cell passage [10][11][12][13][14][15][16][17][18] for one week at + 4°C.…”

Capacity of robot handling for Epstein‐Barr virus transformation

“…Epstein-Barr virus transformation was performed based on our previous study (6). Briefly, the cell pellet containing lymphocytes (cell number is in the range of 10 6 -10 7 cells) was infected with 1 mL EBV at 37 ° C for 1 h. Then, the sample was centrifuged at 270 g for 10 min to remove the EBV.…”

“…It has well accepted that growth of LCLs could be enhanced by mitogen such as PHA (4,11,12) and utilization of PHA in EBV transformation has been as standard EBV transformation protocol (12). Dosages of PHA that had been employed in EBV transformation were varied (10% PHA in Beck et al (4); 1% PHA in Chang et al (6)). In purpose of material saving, EBV transformation in this experiment was performed in the presence or absence of 1% PHA.…”