P182 - Performance-Based Clonal Expansion Using High Throughput Automation for Cartilage Cellular Therapies
Safe, effective and reliable cellular therapies demand better characterization of the starting cell population being injected for cartilage repair. Primary human cartilage tissue contains a heterogeneous mixture of stem and progenitor cells. In this mixture, only a subsets of the populations possess the biological potential to repair the tissue. The goal of this study is to identify this subset of stem and progenitor cells by automated performance-based clonal expansion strategies.
Methods and Materials
Articular cartilage (Outerbridge grade 1-2) obtained from six knee arthroplasty patient’s for 2-D cell culture assay. The progenitors and their progeny were characterized using ASTM-based automated image analysis to define and quantify CQAs of the stem/progenitor cells. Based on the CQAs, 24 clonal populations from each patient were picked using Cell XTM robotic device in rapid, precise, repeatable and rigorously documented manner. Of the 24 clonal colonies, twelve fastest growing clones were expanded to 20 doublings (~passage 4) for trilineage differentiation assay and RNA sequencing.
Preliminary assessment indicated wide variation in morphological and biological characteristics of progenitor and their progeny: circularity (median: 0.665; range:0.15-0.93), area (median: 116.4µm2; range:51.9-204.2µm2), doubling time (median:32.9h; range:26.9-41.2h) and colony density (median:6.5%;range:2.3-20.7%). Differences seen between two representative clonal populations with respect to the trilineage differentiation potential and gene expression seen at 20 doublings (passage4) is shown in Figure 1 and 2 respectively. A complete detailed assessment of all the clonal populations and their culture-expanded population (n=72) will help us identify the CQAs that will aid in selection of quality clones.
An improved understanding of the heterogeneity of progenitor cells resident in adult cartilage will improve the rigor of cell sourcing and targeting decisions for pharmacological and cellular therapies. The automated methods for precision analysis and management of cell populations using Cell X™ represent an important advancement in the tools available for stem cell research and manufacturing.