Characterization of heterogeneous primary human cartilage-derived cell population using non-invasive live-cell phase-contrast time-lapse imaging.

Reliable and reproducible cell therapy strategies to treat osteoarthritis demand an improved characterization of the cell and heterogeneous cell population resident in native cartilage tissue. Using live-cell phase-contrast time-lapse imaging (PC-TLI), this study investigates the morphological attributes and biological performance of the three primary biological objects enzymatically isolated from primary human cartilage: connective tissue progenitors (CTPs), non-progenitors (NPs) and multi-cellular structures (MCSs). The authors' results demonstrated that CTPs were smaller in size in comparison to NPs (P < 0.001). NPs remained part of the adhered cell population throughout the cell culture period. Both NPs and CTP progeny on day 8 increased in size and decreased in circularity in comparison to their counterparts on day 1, although the percent change was considerably less in CTP progeny (P < 0.001). PC-TLI analyses indicated three colony types: single-CTP-derived (29%), multiple-CTP-derived (26%) and MCS-derived (45%), with large heterogeneity with respect to cell morphology, proliferation rate and cell density. On average, clonal (CL) (P = 0.009) and MCS (P = 0.001) colonies exhibited higher cell density (cells per colony area) than multi-clonal (MC) colonies; however, it is interesting to note that the behavior of CL (less cells per colony and less colony area) and MCS (high cells per colony and high colony area) colonies was quite different. Overall effective proliferation rate (EPR) of the CTPs that formed CL colonies was higher than the EPR of CTPs that formed MC colonies (P = 0.02), most likely due to CTPs with varying EPR that formed the MC colonies. Finally, the authors demonstrated that lag time before first cell division of a CTP (early attribute) could potentially help predict its proliferation rate long-term. Quantitative morphological characterization using non-invasive PC-TLI serves as a reliable and reproducible technique to understand cell heterogeneity. Size and circularity parameters can be used to distinguish CTP from NP populations. Morphological cell and colony features can also be used to reliably and reproducibly identify CTP subpopulations with preferred proliferation and differentiation potentials in an effort to improve cell manufacturing and therapeutic outcomes.