Method for large scale isolation, culture and cryopreservation of human monocytes suitable for chemotaxis, cellular adhesion assays, macrophage and dendritic cell differentiation
Method for large scale isolation, culture and cryopreservation of human monocytes suitable for chemotaxis, cellular adhesion assays, macrophage and dendritic cell differentiation
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DOI：
10.1016/j.jim.2004.03.003
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发表时间：
2004-05-01
影响因子：
2.2
通讯作者：
Berliner, J
中科院分区：
文献类型：
--
作者：
Danciger, JS;Lutz, M;Berliner, J
本文提出了一种使用标准实验室离心机大量高纯度分离、培养和冷冻保存人单核细胞的改进方法。使用双密度技术，使用 Ficoll Isopaque 和 46% 等渗 Percoll，从 300 至 360 ml 肝素化人血中分离单核细胞。单核细胞的产量为 75 至 2.05 亿个（300 至 360 毫升血液），平均纯度为 90.6%。在氧化 L-α-1-棕榈酰-2-花生四烯酸-sn-甘油-3-磷酸胆碱 (oxPAPC) 或脂多糖 (LPS) 存在下，新鲜或冷冻单核细胞粘附内皮细胞的能力没有差异，并且观察到对趋化刺激剂 N-甲酰基-L-甲硫氨酰-L-亮氨酰-苯丙氨酸 (FMLP) 的反应没有显着差异。我们定义了一种有用的方法，用于培养和分化通过该方法分离的新鲜或冷冻单核细胞，根据形态、巨噬细胞标记物 SRA-1 的表达以及炎症基因 TNF-α、IL-6 和 COX-2 的诱导来判断。此外，根据标志性表面蛋白 CD1a 和 DC-sign 的表达以及不存在 CD14 的情况判断，新鲜或冷冻的双密度分离细胞在 GM-CSF 和 IL-4 存在的情况下可以成功分化为树突状细胞。该方法还产生纯淋巴细胞群。 (C) 2004 Elsevier B.V. 保留所有权利。
This paper presents an improved method of isolating, culturing and cryopreserving human monocytes in large quantity with high purity using standard laboratory centrifuges. Monocytes were isolated from 300 to 360 ml of heparinized human blood using a Double Density technique employing Ficoll Isopaque and 46% iso-osmotic Percoll. Yields of monocytes ranged from 75 to 205 million (from 300 to 360 ml of blood) with an average purity of 90.6%. The ability of fresh or frozen monocytes to adhere to endothelial cells in the presence of oxidized L-alpha-1-palmitoyl-2-arachidonosyl-sn-glycero-3-phosphocholine (oxPAPC) or lipopolysaccharide (LPS) did not differ and no significant difference in response to the chemotactic stimulant N-formyl-L-methionyl-L-leucyl-phenylalanine (FMLP) was observed. We define a useful method for the culture and differentiation of fresh or frozen monocytes isolated by this method, into macrophages as judged by morphology, expression of the macrophage marker SRA-1 and induction of inflammatory genes TNF-alpha, IL-6 and COX-2. Also, fresh or frozen Double Density isolated cells can be successfully differentiated into dendritic cells in the presence of GM-CSF and IL-4 as judged by the expression of the hallmark surface proteins CD1a and DC-sign and the absence of CD14. This method also yields a pure population of lymphocytes. (C) 2004 Elsevier B.V. All rights reserved.