Dynamic gelatin-based hydrogels promote the proliferation and self-renewal of embryonic stem cells in long-term 3D culture

Long-term maintenance of embryonic stem cells (ESCs) in the undifferentiated state is still challenging. Compared with traditional 2D culture methods, 3D culture in biomaterials such as hydrogels is expected to better support the long-term self-renewal of ESCs by emulating the biophysical and biochemical properties of the extracellular matrix (ECM). Although prior studies showed that soft and degradable hydrogels favor the 3D growth of ESCs, few studies have examined the impact of the structural dynamics of the hydrogel matrix on ESC behaviors. Herein, we report a gelatin-based structurally dynamic hydrogel (GelCD hydrogel) that emulates the intrinsic structural dynamics of the ECM. Compared with covalently crosslinked gelatin hydrogels (GelMA hydrogels) with similar stiffness and biodegradability, GelCD hydrogels significantly promote the clonal expansion and viability of encapsulated mouse ESCs (mESCs) independent of MMP-mediated hydrogel degradation. Furthermore, GelCD hydrogels better maintain the pluripotency of encapsulated mESCs than do traditional 2D culture methods that use MEF feeder cells or medium supplementation with GSK3β and MEK 1/2 inhibitors (2i). When cultured in GelCD hydrogels for an extended period (over 2 months) with cell passaging every 7 days, mESCs preserve their normal morphology and maintain their pluripotency and full differentiation capability. Our findings highlight the critical role of the structural dynamics of the hydrogel matrix in accommodating the volume expansion that occurs during clonal ESC growth, and we believe that our dynamic hydrogels represent a valuable tool to support the long-term 3D culture of ESCs.