Dipartimento di Patologia e Oncologia Sperimentali dell'Universitá degli Studi di Firenze e Istituto Toscano Tumori, viale G.B. Morgagni 50, 50134 Firenze, Italy.
Curr Pharm Des. 2013;19(30):5374-83. doi: 10.2174/1381612811319300006.
The Culture-Repopulating Ability (CRA) assays is a method to measure in vitro the bone marrow-repopulating potential of haematopoietic cells. The method was developed in our laboratory in the course of studies based on the use of growth factorsupplemented liquid cultures to study haematopoietic stem/progenitor cell resistance to, and selection at, low oxygen tensions in the incubation atmosphere. These studies led us to put forward the first hypothesis of the existence in vivo of haematopoietic stem cell niches where oxygen tension is physiologically lower than in other bone marrow areas. The CRA assays and incubation in low oxygen were later adapted to the study of leukaemias. Stabilized leukaemia cell lines, ensuring genetically homogeneous cells and enhancing repeatability of results, were found nevertheless phenotypically heterogeneous, comprising cell subsets exhibiting functional phenotypes of stem or progenitor cells. These subsets can be assayed separately, provided an experimental system capable to select one from another (such as different criteria for incubation in low oxygen) is established. On this basis, a two-step procedure was designed, including a primary culture of leukaemia cells in low oxygen for different times, where drug treatment is applied, followed by the transfer of residual cell population (CRA assay) to a drug-free secondary culture incubated at standard oxygen tension, where the expansion of population is allowed. The CRA assays, applied to cell lines first and then to primary cells, represent a simple and relatively rapid, yet accurate and reliable, method for the pre-screening of drugs potentially active on leukaemias which in our opinion could be adopted systematically before they are tested in vivo.
造血细胞重建成活力（CRA）测定是一种测量造血细胞体外骨髓重建潜能的方法。该方法是在我们实验室的研究过程中开发的，研究过程中使用了添加生长因子的液体培养来研究造血干细胞/祖细胞在孵育大气中低氧张力下的抵抗和选择。这些研究使我们提出了体内存在造血干细胞龛的第一个假说，其中氧张力比其他骨髓区域的生理氧张力低。CRA 测定和低氧孵育后来被适用于白血病的研究。然而，稳定的白血病细胞系确保了遗传上同质的细胞并增强了结果的可重复性，但表现出表型异质性，包括具有干细胞或祖细胞功能表型的细胞亚群。这些亚群可以单独进行测定，只要建立了能够从另一个亚群中选择一个亚群的实验系统（例如，低氧孵育的不同标准）。在此基础上，设计了两步程序，包括在低氧条件下对白血病细胞进行不同时间的初级培养，在此期间进行药物治疗，然后将剩余细胞群（CRA 测定）转移到标准氧张力下无药物的次级培养中，允许细胞群扩增。CRA 测定首先应用于细胞系，然后应用于原代细胞，是一种简单、相对快速、准确和可靠的方法，用于对潜在作用于白血病的药物进行初步筛选，我们认为在体内测试之前可以系统地采用这种方法。
