Morphological characterization of heterogeneous progenitor and non-progenitor populations resident in human articular cartilage using time-lapse imaging- implications for cartilage cellular therapy approaches

Abstract

Background & Aim Adult connective tissues contain heterogeneous connective tissue progenitors (CTPs) with the capability to proliferate and differentiate to one or more connective tissue phenotypes. The aims of this study were: 1) Define morphological features that can distinguish CTPs from the non-CTPs in day 1 in vitro culture, and 2) Characterize the relationship between attributes of colony founding CTPs and their progeny (resulting colonies). Methods, Results & Conclusion Human articular cartilage (Grade 1-2) obtained from five knee arthroplasty patients were digested to isolate cells for 2-D cell culture assay. Large field of view phase contrast (LFOVPC) images were captured every one hour over 10 days. This time-lapse video allowed characterization of colony founding CTPs (n=174) and non-CTPs (n=100). Day 10 PC images were assessed to characterize CTP-derived colonies that were formed by one and only one founding CTP (n=66). Colonies formed by more than one CTP and colonies that grew into one another were excluded. The non-CTPs (median: 19µm2, range: 1.5 – 195) were smaller than the CTPs (median: 38µm2, range: 11 – 227), (p Non-invasive LFOVPC time-lapse microscopy demonstrate that colony founding CTPs are larger than non-CTPs on Day 1. Colonies with greater density and faster growth contained smaller cells. LFOVPC imaging can be a powerful technique for real time identification and characterization of colony founding cells and associated colony formation kinetics and outcome metrics. These data may be useful in guiding cell sourcing and processing decisions to enhance the quality, repeatability and reproducibility of systems for cell fabrication.