Stable establishment of the NIH3T3-CD40L-IL2-IL-4-IL21-BAFF cell line and its application to in vitro culture of B cells

[Abstract]  Objective: The aim of this study was to express CD40L, IL-2, IL-4, IL-21 and BAFF in NIH3T3 cells to further optimize the in vitro culture conditions of B cells. Methods: Lentiviral expression vectors containing CD40L, IL-2, IL-4, IL-21, and BAFF genes were constructed by molecular cloning, lentiviral infection of NIH3T3 cells was prepared, and the expression of CD40L and BAFF was detected by flow cytometry, the secretion levels of IL-2, IL-4, and IL-21 were detected by ELISA, and the lentiviral expression vector was used as a feeder cell and B cells were in co-cultured in vitro for 16 days, the proliferation of B cells was observed microscopically, and the secretion level of IgG was detected by ELISA. Results: In this study, we successfully constructed the NIH3T3-CD40L-IL-2-IL-4-IL-21-BAFF cell line, which was able to stably express CD40L, BAFF, and secrete high levels of IL-2, IL-4, and IL-21, and co-cultured with B cells in vitro for 16 days was able to support the efficient expansion of low-density B cells and induce the differentiation of B cells into plasma cells, and its antibody secretion was as high as 517 ng/mL.Conclusion: In this study, we successfully constructed feeder cell lines capable of supporting B cell proliferation and activation in vitro, which laid a good foundation for the preparation of monoclonal antibodies.

[摘要] 目的：本研究旨在于NIH3T3细胞中表达CD40L、IL-2、IL-4、IL-21及BAFF，以进一步优化B细胞的体外培养条件。方法：通过分子克隆技术构建携带CD40L、IL-2、IL-4、IL-21及BAFF基因的慢病毒表达载体，制备经慢病毒感染的NIH3T3细胞；采用流式细胞术检测CD40L与BAFF的表达水平，通过酶联免疫吸附试验（ELISA）检测IL-2、IL-4及IL-21的分泌水平；以该转染细胞作为饲养层细胞，与B细胞体外共培养16天，通过显微镜观察B细胞增殖情况，并采用ELISA检测免疫球蛋白G（IgG）的分泌水平。结果：本研究成功构建了NIH3T3-CD40L-IL-2-IL-4-IL-21-BAFF细胞株，该细胞株可稳定表达CD40L与BAFF，并能高水平分泌IL-2、IL-4及IL-21；将其与B细胞体外共培养16天，可支持低密度B细胞的高效扩增，并诱导B细胞分化为浆细胞，抗体分泌量可达517 ng/mL。结论：本研究成功构建了可在体外支持B细胞增殖与活化的饲养层细胞系，为单克隆抗体制备奠定了良好基础。

搜集汇总

背景与挑战

背景概述

该数据集描述了成功构建NIH3T3-CD40L-IL-2-IL-4-IL-21-BAFF细胞系的研究，该细胞系能稳定表达CD40L和BAFF并分泌高水平的IL-2、IL-4和IL-21。在体外与B细胞共培养16天后，该细胞系支持B细胞高效扩增和分化为浆细胞，抗体分泌量达517 ng/mL，为单克隆抗体的制备提供了重要基础。