To visualize micropellet organization, we labeled cell populations with Vybrant DiI and DiO cell membrane dyes (5 μl/1*106 million cells) (Life Technologies, Carlsbad, CA). The micropellets were imaged using inverted epifluoresent microscopes (Zeiss Axiovert 200M running SlideBook software and Leica DMi8 running LAS X).
The co-culture micropellets contain two different cell types that might vary in cohesivity, which could affect their adhesion-forming behavior. To quantify the intracellular cohesivity, we allowed 100% NPC and 100% MSC populations to interact overnight in agarose microwells and analyzed the contours of the resulting 100% NPC or 100% MSC micropellets. We measured circularity of the contours using FIJI’s built-in circularity measurement tool as previously described.30 (link) Briefly, circularity is a measure of the ratio of a micropellet’s area to the square of its perimeter, where C = 4π*area/perimeter2 (link). Higher circularity scores are correlated with smoother micropellet contours, which result from higher intracellular cohesivity.
The co-culture micropellets contain two different cell types that might vary in cohesivity, which could affect their adhesion-forming behavior. To quantify the intracellular cohesivity, we allowed 100% NPC and 100% MSC populations to interact overnight in agarose microwells and analyzed the contours of the resulting 100% NPC or 100% MSC micropellets. We measured circularity of the contours using FIJI’s built-in circularity measurement tool as previously described.30 (link) Briefly, circularity is a measure of the ratio of a micropellet’s area to the square of its perimeter, where C = 4π*area/perimeter2 (link). Higher circularity scores are correlated with smoother micropellet contours, which result from higher intracellular cohesivity.