Human Induced Pluripotent Spheroids' Growth Is Driven by Viscoelastic Properties and Macrostructure of 3D Hydrogel...

Stem cells, particularly human iPSCs, constitute a powerful tool for tissue engineering, notably through spheroid and organoid models. While the sensitivity of stem cells to the viscoelastic properties of their direct microenvironment is well-described, stem cell differentiation still relies on biochemical factors. Our aim is to investigate the role of the viscoelastic properties of hiPSC spheroids' direct environment on their fate. To ensure that cell growth is driven only by mechanical interaction, bioprintable alginate-gelatin hydrogels with significantly different viscoelastic properties were utilized in differentiation factor-free culture medium. Alginate-gelatin hydrogels of varying concentrations were developed to provide 3D environments of significantly different mechanical properties, ranging from 1 to 100 kPa, while allowing printability. hiPSC spheroids from two different cell lines were prepared by aggregation (⌀ = 100 µm, n > 1 × 10⁴), included and cultured in the different hydrogels for 14 days. While spheroids within dense hydrogels exhibited limited growth, irrespective of formulation, porous hydrogels prepared with a liquid-liquid emulsion method displayed significant variations of spheroid morphology and growth as a function of hydrogel mechanical properties. Transversal culture (adjacent spheroids-laden alginate-gelatin hydrogels) clearly confirmed the separate effect of each hydrogel environment on hiPSC spheroid behavior. This study is the first to demonstrate that a mechanically modulated microenvironment induces diverse hiPSC spheroid behavior without the influence of other factors. It allows one to envision the combination of multiple formulations to create a complex object, where the fate of hiPSCs will be independently controlled by their direct microenvironment.