Effect of liquid flow by pipetting during medium change on deformation of hiPSC aggregates.

INTRODUCTION: Maintaining the pluripotency and homogeneity of human induced pluripotent stem cells (hiPSCs) requires stable culture conditions with consistent medium change. In this study, we evaluated the performance of medium change by machine vs. medium change performed manually in terms of their impact on the aggregate shape of hiPSCs. METHODS: Aggregates of two hiPSC lines (1383D2 and Tic) were cultured, and the medium change was conducted either manually or with a machine. The populational homogeneity in aggregate shape was determined based on the projected aggregate area for size expansion as well as the circularity for spherical morphology. RESULTS: In the case of manually performed medium changes, the size of 1383D2 aggregates expanded homogeneously, maintaining its spherical morphology as culture duration increased, while spherical morphology was deformed in Tic aggregates, which had a heterogeneous population in terms of shape. In the case of medium change performed by a machine under a low flux of liquid flow, cultures of both aggregates showed homogeneous populations without deformation, although a high flux led to a heterogeneous population. The heterogeneous population observed in manually performed medium change was caused by the low stability of motion. In addition, time-lapse observation revealed that the Tic aggregates underwent tardive deformation with cellular protrusions from the aggregate surface after medium change with high flux. Histological analysis revealed a spatial heterogeneity of collagen type I inside 1383D2 aggregates, which had a shell structure with strong formation of collagen type I at the periphery of the aggregates, while Tic aggregates did not have a shell structure, suggesting that the shell structure prevented aggregate deformation. CONCLUSION: Medium change by a machine led to a homogeneous population of aggregate shapes. Liquid flow caused tardive deformation of aggregates, but the shell structure of collagen type I in aggregates maintained its spherical shape.