Automatic analysis of growth onset, growth rate and colony size of individual bone marrow progenitors

Abstract

A method for automatic enumeration of proliferating bone marrow progenitors after single cell sorting is described. The system is based on regular inverse microscopy, recording with a video camera, and image analysis using dedicated software on an Apple computer. Single CD34+ progenitor cells were sorted in 96-well plates. Three times weekly phase-contrast video images of each well were stored and analyzed for the actual number of cells. From the subsequent counts growth curves were plotted for each individual progenitor. Enumeration by image analysis correlated very well with manual cell counting (r = 0.99, P < 0.0001). To show the capability of the method to analyze growth rate and growth delay, more differentiated (CD34+/CD13+/CD33+) progenitors were compared with more primitive (CD34+/CD13+/CD33g-) progenitors. Differences in the timing of colony outgrowth were shown to be based on delay in growth initiation. Initiation of growth was delayed 2.6-3.1 days in CD34+/CD13+/ CD33- fraction of 3 different donors (P < 0.0001). The growth rates of the progenitors in both fractions were not significantly different. The described method seems important to more accurately evaluate subpopulations of progenitors, the effect of growth promoting or inhibiting factors, and effects of cytotoxic drugs and irradiation.