Department of Chemistry G. Ciamician, Via Selmi 2, I-40126 Bologna, Italy.
J Chromatogr A. 2009 Dec 25;1216(52):9081-7. doi: 10.1016/j.chroma.2009.07.024. Epub 2009 Jul 17.
New cell sorting methodologies, which are simple, fast, non-invasive, and able to isolate homogeneous cell populations, are needed for applications ranging from gene expression analysis to cell-based therapy. In particular, in the forefront of stem cell isolation, progenitor cells have to be separated under mild experimental conditions from complex heterogeneous mixtures prepared from human tissues. Most of the methodologies now employed make use of immunological markers. However, it is widely acknowledged that specific markers for pluripotent stem cells are not as yet available, and cell labelling may interfere with the differentiation process. This work presents for the first time gravitational field-flow fractionation (GrFFF), as a tool for tag-less, direct selection of human hematopoietic stem and progenitor cells from cell samples obtained by peripheral blood aphaeresis. These cells are responsible to repopulate the hemopoietic system and they are used in transplantation therapies. Blood aphaeresis sample were injected into a GrFFF system and collected fractions were characterized by flow cytometry for CD34 and CD45 expression, and then tested for viability and multi-differentiation potential. The developed GrFFF method allowed obtaining high enrichment levels of viable, multi-potent hematopoietic stem cells in specific fraction and it showed to fulfil major requirements of analytical performance, such as selectivity and reproducibility of the fractionation process and high sample recovery.
需要新的细胞分选方法，这些方法应简单、快速、非侵入性，并且能够分离同质细胞群体，适用于从基因表达分析到基于细胞的治疗等各种应用。特别是在干细胞分离的前沿，祖细胞必须在温和的实验条件下从复杂的异质混合物中分离出来，这些混合物是用人组织制备的。现在使用的大多数方法都利用免疫标记物。然而，人们普遍认识到，多能干细胞的特异性标记物尚不可用，细胞标记可能会干扰分化过程。本工作首次提出将重力场流分离（GrFFF）作为一种工具，用于从通过外周血单采术获得的细胞样本中无标记、直接选择人造血干细胞和祖细胞。这些细胞负责重新填充造血系统，用于移植治疗。将血液单采术样本注入 GrFFF 系统，收集的级分通过流式细胞术检测 CD34 和 CD45 的表达，然后测试其活力和多向分化潜能。所开发的 GrFFF 方法允许在特定级分中获得高活力、多能造血干细胞的高富集水平，并且显示出满足分析性能的主要要求，例如分馏过程的选择性和重现性以及高样品回收率。
