Abstract
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.
Keywords: Hypoxia, stem cell cycling, stem cell niche, leukaemia stem cells, minimal residual disease, chronic myeloid leukaemia, BCR/Abl, resistance to Imatinib.
Current Pharmaceutical Design
Title:The Culture-Repopulating Ability Assays and Incubation in Low Oxygen: A Simple Way to Test Drugs on Leukaemia Stem or Progenitor Cells
Volume: 19 Issue: 30
Author(s): Maria Grazia Cipolleschi, Elisabetta Rovida and Persio Dello Sbarba
Affiliation:
Keywords: Hypoxia, stem cell cycling, stem cell niche, leukaemia stem cells, minimal residual disease, chronic myeloid leukaemia, BCR/Abl, resistance to Imatinib.
Abstract: 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.
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Cipolleschi Grazia Maria, Rovida Elisabetta and Sbarba Dello Persio, The Culture-Repopulating Ability Assays and Incubation in Low Oxygen: A Simple Way to Test Drugs on Leukaemia Stem or Progenitor Cells, Current Pharmaceutical Design 2013; 19 (30) . https://dx.doi.org/10.2174/1381612811319300006
DOI https://dx.doi.org/10.2174/1381612811319300006 |
Print ISSN 1381-6128 |
Publisher Name Bentham Science Publisher |
Online ISSN 1873-4286 |
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