Comparison of flow cytometry and laser scanning cytometry for the analysis of CD34+ hematopoietic stem cells.
BACKGROUND
Characterization of hematopoietic stem cells (HSCs) by laser scanning cytometry (LSC) was compared with conventional flow cytometry (FCM). The method was evaluated for application in the development of advanced cell culture substrates that were supposed to support the ex vivo expansion of HSC. For this purpose, adherent HSCs were grown in culture on thin polymer films coated with reconstituted collagen I fibrils and subsequently analyzed by LSC.


METHODS
CD34+ HSCs were isolated from cord blood by immunomagnetic separation and cultivated on polymer films coated with reconstituted collagen I fibrils. Cell surface antigens (CD34, CD29) were stained with antibodies, and nuclei were labeled with a DNA stain (TO-PRO-3 iodide) that does not interfere with the fluorochromes of the antibodies. Fluorescence intensity of the adherent cells was measured by means of LSC. Before and after in vitro expansion for time periods of up to 7 days, suspension cells were analyzed with LSC and FCM.


RESULTS
LSC-based analysis enabled reliable quantification of CD34+ cells with bright antigen expression before cell culture. At this stage, LSC and FCM data for CD34 expression at given HSC samples largely coincided. After in vitro expansion, LSC data deviated from FCM data for cells with dim CD34 antigen expression, whereas the fluorescence intensity of the CD29 antigen remained comparable. The deviation between LSC and FCM data for CD34dim was attributed to the better resolution of weak fluorescence by FCM. Based on the preceding evaluation of the method, LSC analysis could be applied to characterize HSCs cultivated on collagen I-coated polymer films without detachment of the cells from the substrate.


CONCLUSIONS
LSC-based analysis allows for the automated evaluation of adherent HSCs. Although resolution of weakly expressed antigens can be achieved more precisely with FCM, the method provides a valuable tool to study interactions of HSCs with bioartificial substrates.
Oswald
Joachim
J
Institute of Polymer Research Dresden, Department of Biocompatible Materials, and Max Bergmann Center of Biomaterials Dresden, Dresden, Germany.
Jørgensen
Birgitte
B
Pompe
Tilo
T
Kobe
Fritz
F
Salchert
Katrin
K
Bornhäuser
Martin
M
Ehninger
Gerhard
G
Werner
Carsten
C
D018952:Antigens, CD34 / Q000032:analysis*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000737:chemistry*; D006801:Humans; D019044:Image Cytometry / Q000379:methods; D007231:Infant, Newborn; D019012:Integrin beta1 / Q000378:metabolism; D007834:Lasers; D011247:Pregnancy
D018952:Antigens, CD34; D019012:Integrin beta1; D016650:Fluorescein-5-isothiocyanate