An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow
An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow
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DOI：
10.1016/s0022-1759(98)00204-x
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发表时间：
1999-02-01
影响因子：
2.2
通讯作者：
Schuler, G
中科院分区：
文献类型：
--
作者：
Lutz, MB;Kukutsch, N;Schuler, G
由于树突状细胞（DC）在所有器官中都是罕见的群体，它们从大量造血前体产生对于研究其生物学至关重要。用GM-CSF培养8天后，每只小鼠从鼠骨髓中获得约5 × 10（6）个纯度为70%的细胞。我们已经改进了这种标准方法，并常规地实现了50倍的高产率，即，1-3每只小鼠X 10（8）个未成熟和成熟DC，纯度为90-95%。主要修改如下：（i）避免骨髓细胞亚群的任何主动消耗以避免前体的损失，（ii）骨髓细胞的较低平板接种密度，（iii）10-12天的延长培养期，（iv）从第8天或第10天开始减少GM-CSF剂量以减少粒细胞污染。第10-12天的最终非粘附群体构成未成熟和成熟DC的混合物。在最后24小时内，高剂量的LPS或TNF-α可以诱导DC的进一步成熟，其中50-70%的非粘附部分代表具有高水平的NLDC-145、CD 86和CD 40的成熟DC。该方法允许通过简单的手段产生具有非常低的B细胞或粒细胞污染的大量鼠DC。在分子水平上研究DC的生物学特性具有重要意义。(C)1999 Elsevier Science B. V.保留所有权利。
As dendritic cells (DC) are rare populations in all organs, their generation from hematopoietic precursors in large quantities has proven critical to study their biology. From murine bone marrow about 5 X 10(6) cells at 70% purity are obtained per mouse after 8 days of culture with GM-CSF. We have improved this standard method and routinely achieve a 50-fold higher yield, i.e., 1-3 X 10(8) immature and mature DC per mouse at 90-95% purity. The major modifications were: (i) the avoidance of any active depletion of bone marrow cell subpopulations to circumvent loss of precursors, (ii) a lower plating density of bone marrow cells, (iii) a prolonged culture period of 10-12 days, (iv) the reduction of the GM-CSF dose from day 8 or 10 onwards to reduce granulocyte contaminations. The final non-adherent population at day 10-12 constitutes a mixture of immature and mature DC. Further maturation of DC could be induced by high doses of LPS or TNF-alpha for the last 24 h, where 50-70% of the non-adherent fraction represented mature DC with high levels of NLDC-145, CD86 and CD40. This method allows by simple means the generation of high numbers of murine DC with very low B cell or granulocyte contaminations. It will be valuable to study DC biology notably at the molecular level. (C) 1999 Elsevier Science B.V. All rights reserved.