title: The Culture-Repopulating Ability assays and incubation in low oxygen: a simple way to test drugs on leukaemia stem or progenitor cells.
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.
authors: Dipartimento di Patologia e Oncologia Sperimentali dell'Universitá degli Studi di Firenze e Istituto Toscano Tumori, viale G.B. Morgagni 50, 50134 Firenze, Italy.
authors: Maria Grazia
authors: MG
authors: Cipolleschi
authors: Elisabetta
authors: E
authors: Rovida
authors: Persio
authors: P
authors: Dello Sbarba
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D003470:Culture Media; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007938:Leukemia; D014411:Neoplastic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D010100:Oxygen / Q000494:pharmacology*
chemical_list: D003470:Culture Media; D010100:Oxygen
references: PLoS One. 2011 Feb 11;6(2):e17008
references: 21347297
references: Transfusion. 2000 Dec;40(12):1482-8
references: 11134568
references: Blood Rev. 1993 Sep;7(3):190-7
references: 7902155
references: Br J Haematol. 1972 Aug;23(2):221-34
references: 4672231
references: Cell Stem Cell. 2011 May 6;8(5):486-98
references: 21549325
references: Blood Cells. 1978;4(1-2):7-25
references: 747780
references: Leukemia. 2000 Apr;14(4):735-9
references: 10764163
references: Stem Cells. 1997;15 Suppl 1:185-95
references: 9368340
references: Nature. 2001 Nov 1;414(6859):105-11
references: 11689955
references: Blood Cells. 1994;20(2-3):468-79; discussion 479-81
references: 7538352
references: Clin Lymphoma Myeloma Leuk. 2011 Aug;11(4):355-60
references: 21816374
references: Cancer Metastasis Rev. 2007 Jun;26(2):225-39
references: 17440684
references: J Cell Comp Physiol. 1963 Dec;62:327-36
references: 14086156
references: Exp Hematol. 2002 Jan;30(1):67-73
references: 11823039
references: J Cell Physiol. 2012 Jun;227(6):2750-8
references: 21913190
references: Br J Haematol. 2000 Feb;108(2):424-9
references: 10691876
references: Stem Cells. 2004;22(5):716-24
references: 15342936
references: Aust J Exp Biol Med Sci. 1968 Oct;46(5):607-18
references: 5696493
references: J Clin Invest. 2003 Jul;112(1):126-35
references: 12840067
references: Clin Cancer Res. 1997 Sep;3(9):1571-8
references: 9815845
references: Exp Hematol. 1987 Feb;15(2):137-42
references: 3817047
references: Exp Cell Res. 1966 Oct;43(3):553-63
references: 5957746
references: J Cell Physiol. 2007 Jul;212(1):68-75
references: 17311290
references: Blood. 1991 Jul 1;78(1):1-19
references: 2070044
references: Aust J Exp Biol Med Sci. 1966 Jun;44(3):287-99
references: 4164182
references: Blood. 1993 Oct 1;82(7):2031-7
references: 8104535
references: Haematologica. 2011 Feb;96(2):204-12
references: 21071498
references: Stem Cells. 2002;20(4):347-54
references: 12110704
references: Exp Hematol. 1989 Mar;17(3):263-6
references: 2563690
references: Radiat Res. 1961 Feb;14:213-22
references: 13776896
references: Exp Hematol. 1997 Oct;25(11):1187-94
references: 9328456
references: Differentiation. 1976 Oct 7;6(3):191-4
references: 791737
references: Stem Cells. 2007 May;25(5):1119-25
references: 17255519
references: Exp Hematol. 2010 Oct;38(10):847-51
references: 20547202
references: J Cell Physiol. 2010 Jan;222(1):17-22
references: 19725055
references: J Cell Physiol. 2009 May;219(2):271-5
references: 19160417
references: Exp Hematol. 1996 Feb;24(2):299-306
references: 8641356
references: Int J Hematol. 2012 May;95(5):478-83
references: 22539363
references: Lymphat Res Biol. 2008;6(3-4):149-54
references: 19093787
references: Leukemia. 2006 Jul;20(7):1291-3
references: 16710305
references: Blood. 2002 Nov 15;100(10):3767-75
references: 12393646
references: Blood. 2002 Dec 15;100(13):4266-71
references: 12393432