Epstein–Barr virus‐induced transformation of B cells for the diagnosis of genetic metabolic disorders – enumerative conditions for cryopreservation

Abstract

Epstein-Barr virus (EBV) infection in vitro causes transformation of B cells and generates B lymphoblastoid cell lines (LCLs). These LCLs have been widely used for the diagnostic of several genetic metabolic disorders. However, up to now, efficiency of LCL generation has been based on misleading subjective analysis. In this study, quantitative analyses have been performed to indicate efficiency of B-cell transformation to measuring human lysosomal acid hydrolases associated with: GM1-gangliosidosis type I, Gaucher disease and mucopolysaccharidosis type I. Peripheral blood mononuclear cells were isolated from 13 subjects, and LCLs were produced by culturing them with EBV for 12 days. Activities of the enzymes beta-galactosidase, beta-glucosidase and alpha-iduronidase were measured before and after cryopreservation in liquid nitrogen for 30 days. Efficiency of the B-cell transformation was screened every 4 days by the enumeration of cell proliferation, cell counts and changes in granularity estimated by flow cytometry. We observed the generation of 13 LCLs. Cell transformation was confirmed by the gradual increase of cellular clusters, cell size and granularity. In addition, we determined that the activity of the enzymes mentioned above did not change following cryopreservation. These data suggest that our enumerative approach for screening of EBV-LCLs is efficient for the enzymatic determination of human lysosomal acid hydrolases and may thus replace misleading subjective analyses.

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

“…2003; Hur et al . 2005; Mello et al . 2006) or agents that can stimulate growth of LCLs (Penno et al .…”

Peripheral Biomarkers Revisited: Integrative Profiling of Peripheral Samples for Psychiatric Research

“…Thus, they can serve as an unlimited source of biomaterial and can be stored in liquid nitrogen. After re-establishment in culture (23), cells have been repeatedly used for multiple assays, including cellular functional assays (24). For instance, Sei et al .…”

Cytokine associated neopterin response of peripheral blood mononuclear cells to in vitro Epstein–Barr virus transformation process of B lymphocytes

“…In some cultures, where T lymphocytes have been depleted or physically inactivated, B cell transformation occurs more efficiently (16). In a previous study, where transformation was evaluated with LCL proliferation, transformed cells showed clumping, with increasing diameters and rising granularity (15). In our study, the cells attached to the flask bottom after 12 h (Figure 1) and transformed cells exhibited distinctively more clumping than others ( Figure 2).…”

“…Zerbini et al (13) showed that EBV can transform most of the B lymphocytes in cord blood, as shown in adult B lymphocytes (14). In vitro infection of B lymphocytes with EBV may cause the development of B lymphoblastoid cell lines (LCLs) (15). In some cultures, where T lymphocytes have been depleted or physically inactivated, B cell transformation occurs more efficiently (16).…”