A modified protocol for highly efficient EBV-mediated immortalization of human B lymphocytes from small volumes of peripheral blood serum

Abstract

BackgroundMany human molecular and genetic studies require the use of a renewable biological material. Although primary fibroblast cell lines can be used for this purpose, there are disadvantages associated with human biopsies including the limited number of cell divisions. Peripheral blood has the advantage of being easier to obtain but also the drawback that blood cells produce only short-term cultures. Epstein-Barr virus is capable of transforming human B lymphocytes into indefinitely proliferating cells that can be maintained in tissue culture. Here, we report a convenient method of B-lymphocyte immortalization using small volumes of freshly collected human blood serum saturated with nucleated blood cells. Aim of the studyThe aim of the present study is modification and improvement of the protocol for highly efficient immortalization of human B lymphocytes from small volume of blood samples. Material and methodsCell line B95-8 was used as Epstein-Barr virus source for viral stock preparation. Immortalizing medium contains RPMI-1640, viral stock and additives. No feeder layer was used. ResultsAs result we present high efficient method for B lymphocytes immortalization with start blood volume less than 5 ml. ConclusionThe method is applicable for immortalization of B lymphocytes from small blood samples and is essential for studies involving children or patients when large blood volume sampling is impossible.