Quantitative image analysis of connective tissue progenitors.
OBJECTIVE
To develop an image analysis system to automatically identify colony-forming units (CFUs) in in vitro cell cultures of connective tissue progenitors. This system was designed to quantitatively assess colony morphology and number of colonies in 4-cm(2) culture wells.


METHODS
Large field-of-view high-resolution fluorescence images of 4',6-diamidino-2-phenylindole (DAPI)- and alkaline phosphatase (AP)-stained bone marrow cell cultures were obtained using an epi-fluorescence microscope and automated scanning stage. Cell nuclei were identified in the DAPI-stained images after removal of fluorescent debris from the image. An Euclidean distance map (EDM) of the segmented cell nuclei was used to cluster cell nuclei into colonies. The automated system was evaluated using 40 tissue culture wells of bone marrow aspirate samples. The results of the automated analysis were compared to the manual tracings of colonies by 3 reviewers.


RESULTS
The automated method agreed with all 3 reviewers on average 87.5% of the time. Additionally, reviewers identified other colonies not outlined by the reviewers on average 2.7 times more than the automated method.


CONCLUSIONS
The automated method is a less biased method for identifying CFUs than individual reviewers, it provides more quantitative information about colony morphology than can be obtained manually and it is less time consuming.
Powell
Kimerly A
KA
Whitaker Biomedical Imaging Laboratory and Orthopaedic Research Center, Department of Biomedical Engineering, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio, USA. kim.powell@lsbio.com
Nakamoto
Chizu
C
Villarruel
Sandra
S
Boehm
Cynthia
C
Muschler
George
G
D001707:Biopsy, Needle; D001854:Bone Marrow Cells / Q000502:physiology; D001842:Bone and Bones; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D003239:Connective Tissue Cells / Q000502:physiology*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D010006:Osteoblasts / Q000502:physiology; D013234:Stem Cells / Q000502:physiology*