Synthetic PEG-4MAL Hydrogels Support Patient-Derived Human Intestinal Enteroid Culture

Patient-derived intestinal enteroids are valuable models for studying gastrointestinal physiology and disease, but their dependence on Matrigel^™ limits reproducibility and clinical translation. Here, we developed a fully synthetic poly(ethylene glycol)-4-maleimide (PEG-4MAL) hydrogel platform to support human intestinal enteroid culture. Guided by enteroid transcriptomics revealing high expression of α2β1 integrin and matrix metalloproteinases, we systematically evaluated collagen-mimetic GFOGER and fibronectin-derived RGD (Arg-Gly-Asp) peptides combined with four MMP-sensitive crosslinkers. GFOGER-functionalized hydrogels significantly outperformed RGD formulations across all metrics. The optimized PEG-4MAL-GFOGER formulation demonstrated enteroid formation efficiency and viability approaching Matrigel^™ performance. Bulk RNA-sequencing across multiple patient-derived lines demonstrated transcriptomic similarity between synthetic and Matrigel^™ hydrogels, with preservation of stem cell, differentiation, and regional identity markers. This rationally designed synthetic platform overcomes key limitations of biological matrices while supporting robust enteroid growth and functionality comparable to standard biological matrices.