A non-invasive method for in situ quantification of subpopulation behaviour in mixed cell culture.
Ongoing advances in quantitative molecular- and cellular-biology highlight the need for correspondingly quantitative methods in tissue-biology, in which the presence and activity of specific cell-subpopulations can be assessed in situ. However, many experimental techniques disturb the natural tissue balance, making it difficult to draw realistic conclusions concerning in situ cell behaviour. In this study, we present a widely applicable and minimally invasive method which combines fluorescence cell labelling, retrospective image analysis and mathematical data processing to detect the presence and activity of cell subpopulations, using adhesion patterns in STRO-1 immunoselected human mesenchymal populations and the homogeneous osteoblast-like MG63 continuous cell line as an illustration. Adhesion is considered on tissue culture plastic and fibronectin surfaces, using cell area as a readily obtainable and individual cell specific measure of spreading. The underlying statistical distributions of cell areas are investigated and mappings between distributions are examined using a combination of graphical and non-parametric statistical methods. We show that activity can be quantified in subpopulations as small as 1% by cell number, and outline behaviour of significant subpopulations in both STRO-1+/- fractions. This method has considerable potential to understand in situ cell behaviour and thus has wide applicability, for example in developmental biology and tissue engineering.
MacArthur
Ben D
BD
University of Southampton, Bone and Joint Research Group, Developmental Origins of Health and Disease, Southampton General Hospital, Southampton SO16 6YD, UK. bdm@soton.ac.uk
Tare
Rahul S
RS
Please
Colin P
CP
Prescott
Philip
P
Oreffo
Richard O C
RO
D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D018920:Coculture Techniques / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008856:Microscopy, Fluorescence / Q000379:methods*; D010006:Osteoblasts