title: Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells.
abstract: Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.
authors: Institute of Biomaterials and Biomedical Engineering, University of Toronto.
authors: Celine L
authors: CL
authors: Bauwens
authors: Department of Comparative Biology and Experimental Medicine, University of Calgary.
authors: Derek
authors: D
authors: Toms
authors: Department of Comparative Biology and Experimental Medicine, University of Calgary; mdungrin@ucalgary.ca.
authors: Mark
authors: M
authors: Ungrin
mesh_terms: D018929:Cell Culture Techniques; D002454:Cell Differentiation; D053595:Embryonic Stem Cells; D006801:Humans; D032383:Myocytes, Cardiac; D039904:Pluripotent Stem Cells; D013535:Suspensions
chemical_list: D013535:Suspensions