对于机械学研究,体外人B细胞分化和浆细胞的产生是非常有价值的技术。然而,T细胞依赖性(TD)和T细胞非依赖性(TI)刺激的异质性以及现有方案中使用的培养条件的差异使得结果的解释具有挑战性.本研究的目的是使用分离的CD19B细胞和外周血单核细胞(PBMC)培养物达到最佳的B细胞分化条件。我们解决了多种播种密度,不同的培养时间,以及包括B细胞受体(BCR)触发的TD和TI刺激的各种组合。B细胞扩增,扩散,并在6天和9天后通过测量B细胞增殖和扩增来分析分化,浆细胞和浆细胞形成,和免疫球蛋白(Ig)分泌。此外,使用冷冻保存的细胞外推这些条件,并比较分化潜能.
该研究表明,在使用CD40L和IL-21作为TD刺激的B细胞和PBMC培养物培养9天后,以及CpG和IL-2作为TI刺激的培养6天后,分化效率提高。我们达成了优化的协议,每个培养孔需要2,500和25,000个B细胞用于TD和TI测定,分别。PBMC培养物的结果与B细胞培养物具有高度可比性,这允许在培养之前解除额外的B细胞分离步骤。在这些优化的TD条件下,添加抗BCR对表型B细胞分化的影响很小;但是,它会干扰Ig分泌测量。将IL-4添加到TD刺激物显示出显著降低的Ig分泌。向优化的TI条件中添加BAFF显示B细胞分化和Ig分泌增强,而不是在PBMC培养物中。通过这种方法,当从新鲜或冷冻保存的样品开始时,实现有效的B细胞分化和Ig分泌。
我们的方法展示了优化的TD和TI刺激方案,用于更深入地分析原代人B细胞和PBMC培养物中的B细胞分化,同时需要少量的B细胞。使它们非常适合未来对来自不同队列的B细胞介导的疾病的患者样品的B细胞分化的临床和研究研究。
UNASSIGNED: For mechanistic studies, in-vitro human B-cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T-cell-dependent (TD) and T-cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols make the interpretation of results challenging. The aim of the present study was to achieve the most optimal B-cell differentiation conditions using isolated CD19+ B cells and peripheral blood mononuclear cell (PBMC) cultures. We addressed multiple seeding densities, different durations of culturing, and various combinations of TD and TI stimuli including B-cell receptor (BCR) triggering. B-cell expansion, proliferation, and differentiation were analyzed after 6 and 9 days by measuring B-cell proliferation and expansion, plasmablast and plasma cell formation, and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared.
UNASSIGNED: This study demonstrates improved differentiation efficiency after 9 days of culturing for both B-cells and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2,500 and 25,000 B-cells per culture well for the TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B-cell cultures, which allows dismissal of additional B-cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed a little effect on phenotypic B-cell differentiation; however, it interferes with Ig secretion measurements. The addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B-cell differentiation and Ig secretion in B-cell but not in PBMC cultures. With this approach, efficient B-cell differentiation and Ig secretion were accomplished when starting from fresh or cryopreserved samples.
UNASSIGNED: Our methodology demonstrates optimized TD and TI stimulation protocols for more in-depth analysis of B-cell differentiation in primary human B-cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B-cell differentiation of patient samples from different cohorts of B-cell-mediated diseases.