Berger Thomas G, Strasser Erwin, Smith Richard, Carste Curt, Schuler-Thurner Beatrice, Kaempgen Eckhart, Schuler Gerold
Department of Dermatology, University Hospital of Erlangen, Hartmannstr. 14, D-91052 Erlangen, Germany.
J Immunol Methods. 2005 Mar;298(1-2):61-72. doi: 10.1016/j.jim.2005.01.005.
Dendritic cells (DC) are promising tools for the immunotherapy of cancer. The induction of tumor-specific T cells and clinical regressions have already been observed in early phase I/II vaccination trials. As DC vaccination is now facing trials with larger patient collectives it becomes increasingly important to obtain large numbers of cells suitable for therapeutic applications under labor- and cost-effective conditions. We describe here a procedure that uses a novel cell separator (Elutra, Gambro BCT) to enrich monocytes from an entire apheresis product within one hour. Cells are separated on the basis of size and to a lesser extent density, by elutriation in a 40-ml conical chamber. The total monocyte recovery following elutriation (n = 6) was 98.53% (+/-8.07%), the recovery in the monocyte-rich fraction 75.45% (+/-11.31%), and the mean purity 82.95% (+/-6.01%). These monocytes can be cultured either in conventional culture dishes or in closed cell culture bags and differentiated, by using GM-CSF+IL-4 followed by a maturation cocktail composed of IL-1beta+IL-6+TNF-alpha+PGE2, into fully mature DC. The Elutra separator allows for fast and easy enrichment of monocytes within a closed system. Subsequently, elutriated monocytes can be successfully cultured into phenotypically and functionally mature DC for immunotherapeutic approaches. The method neither requires a density gradient step to enrich PBMC from leucapheresis products nor does it apply (xenogeneic) antibodies to target monocytes. Isolation of monocytes with Elutra may greatly facilitate future DC-based vaccination approaches.
树突状细胞（DC）是癌症免疫治疗中很有前景的工具。在早期的I/II期疫苗接种试验中已经观察到肿瘤特异性T细胞的诱导和临床缓解。由于DC疫苗接种目前正面临着针对更大患者群体的试验，在节省劳力和成本效益高的条件下获得大量适合治疗应用的细胞变得越来越重要。我们在此描述一种程序，该程序使用一种新型细胞分离器（Elutra，甘布罗BCT公司）在一小时内从整个单采血液成分中富集单核细胞。细胞在一个40毫升的锥形腔室中通过淘析法根据大小并在较小程度上根据密度进行分离。淘析后的单核细胞总回收率（n = 6）为98.53%（±8.07%），富含单核细胞部分的回收率为75.45%（±11.31%），平均纯度为82.95%（±6.01%）。这些单核细胞可以在传统培养皿或封闭细胞培养袋中培养，并通过使用GM-CSF + IL-4，随后使用由IL-1β + IL-6 + TNF-α + PGE2组成的成熟鸡尾酒，分化为完全成熟的DC。Elutra分离器允许在封闭系统中快速简便地富集单核细胞。随后，淘析得到的单核细胞可以成功培养成表型和功能成熟的DC用于免疫治疗方法。该方法既不需要密度梯度步骤从白细胞分离产物中富集PBMC，也不应用（异种）抗体靶向单核细胞。使用Elutra分离单核细胞可能会极大地促进未来基于DC的疫苗接种方法。
