Development of a Laminin-511 Inspired 3D Zwitterionic Hydrogel for Human Pluripotent Stem Cell Culture

Human pluripotent stem cells (hPSCs) hold enormous potential for regenerative medicine and disease modeling applications. These cells, however, are commonly cultured on Matrigel, a 2D coating of mouse origin and significant complexity, hindering their clinical translation. These issues have motivated the development of (a) 2D natural alternatives, which can mimic the extracellular matrix niche, but result in cytoskeletal and epigenetic changes, insufficient cell-cell and cell-matrix interactions, lineage biases upon differentiation, and challenges with scale-up due to surface area constraints and (b) 3D synthetic alternatives, which can be xenogeneic-free but are afflicted with spontaneous differentiations, incompatibility with common media, and a lack of receptor-specific ligands. To overcome these limitations, we developed a fully synthetic, chemically defined, xenogeneic-free, highly biocompatible, ligand-incorporated, and cell-mediated degradable 3D culture system. We demonstrated a zwitterionic polycarboxybetaine (PCB) hydrogel inspired by laminin-511 in the early embryonic niche (PCB-LN511) can outperform other commercial substrates such as Matrigel and iMatrix-511, and we successfully performed 10 passages over 40 days, maintaining excellent pluripotency expression and retaining differentiation capabilities. The resulting PCB-LN511 provides a synthetic alternative to Matrigel for 3D hPSC culture, overcoming the current limitations with both 2D natural and 3D synthetic alternatives for stem cell culture.