Lutz M B, Kukutsch N, Ogilvie A L, Rössner S, Koch F, Romani N, Schuler G
Department of Dermatology, University of Erlangen, Germany.
J Immunol Methods. 1999 Feb 1;223(1):77-92. doi: 10.1016/s0022-1759(98)00204-x.
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.
由于树突状细胞（DC）在所有器官中都是稀有群体，因此从造血前体大量生成DC已被证明对研究其生物学特性至关重要。用GM-CSF培养8天后，每只小鼠可从鼠骨髓中获得约5×10⁶个纯度为70%的细胞。我们改进了这一标准方法，通常可使产量提高50倍，即每只小鼠可获得1-3×10⁸个纯度为90-95%的未成熟和成熟DC。主要改进包括：（i）避免对骨髓细胞亚群进行任何主动清除以避免前体丢失，（ii）降低骨髓细胞的接种密度，（iii）延长培养期至10-12天，（iv）从第8天或第10天起降低GM-CSF剂量以减少粒细胞污染。第10-12天的最终非贴壁群体由未成熟和成熟DC组成。在最后24小时用高剂量的LPS或TNF-α可诱导DC进一步成熟，其中50-70%的非贴壁部分为具有高水平NLDC-145、CD86和CD40的成熟DC。该方法通过简单的手段即可生成大量B细胞或粒细胞污染极低的鼠DC。这对于特别是在分子水平上研究DC生物学将是有价值的。
