An Optimized Method to Generate Human Active Osteoclasts From Peripheral Blood Monocytes.
An Optimized Method to Generate Human Active Osteoclasts From Peripheral Blood Monocytes.
复制标题
DOI：
10.3389/fimmu.2018.00632
复制
发表时间：
2018
影响因子：
7.3
通讯作者：
Velard F
中科院分区：
文献类型：
--
作者：
Abdallah D;Jourdain ML;Braux J;Guillaume C;Gangloff SC;Jacquot J;Velard F
破骨细胞(OCs)是骨吸收细胞，在骨骼发育和成人骨重建中起着关键作用。它们还参与了各种骨骼疾病的发病机制。在处理人类材料时，生成OCs的主要技术困难之一是实现从人外周血单核细胞(PBMC)中大量分化成熟OCs的能力。为了更好地分析人类OCS的作用，需要访问活动OCS的标准化来源。本研究的目的是开发一种从新鲜的人外周血单核细胞中提取活性和成熟的OCs的方法。因此，我们使用含有巨噬细胞集落刺激因子(M-CSF)和核因子kappa-B受体激活剂(RANKL)的未剥离和炭剥离的血清，在不同的细胞培养液中检测了PBMC向OCs分化的情况。我们还研究了维生素D3对PBMCs向OCs分化水平的影响。采用Phalloidin-AlexaFluor®488/DAPI荧光染色和扫描电子显微镜牙本质吸收分析方法，观察培养7~14~21天OCs的数量和大小、每细胞核数及吸收活性。这项研究报告了一种优化的方法，通过使用含有胎牛炭剥离血清的培养液，在有M-CSF和RANKL的情况下，在没有维生素D3的情况下，从低种植密度的PBMC中高效地生产人活性OCS。
Osteoclasts (OCs), the bone-resorbing cells, play a key role in skeletal development and adult bone remodeling. They also participate in the pathogenesis of various bone disorders. One of the major technical difficulties in the generation of OCs, when working on human material, is the ability to achieve large differentiation of mature OCs from human peripheral blood mononuclear cells (PBMCs). Access to a standardized source of active OCs is needed to better analyze the roles of human OCs. The aim of this study was to develop a procedure yielding active and mature OCs from fresh human PBMCs. We therefore examined the differentiation of PBMCs to OCs in different cell culture media, using non-stripped and charcoal-stripped sera in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). We also studied the effects of vitamin D3 in the differentiation level of PBMCs to OCs. Phalloidin-AlexaFluor®488/DAPI fluorescent stainings and dentin resorption analyses by scanning electron microscopy were used to identify the number and size of differentiated OCs, number of nuclei per cell and resorption activities of OCs for a 7–14–21-day culture period. This study reports an optimized method for an efficient production of human active OCs from a low seeding density of PBMCs, after a 14-day culture period by using a medium containing fetal bovine charcoal-stripped serum in the presence of M-CSF and RANKL, and in the absence of vitamin D3.