(413i) Development of Hydrolytically Stable, Functionalizable, Tunable, and Robust Zwitterionic Hydrogels for the 3D Culture of Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) hold broad capabilities for clinical applications as they can undergo multi-lineage differentiation into various cell types for disease modeling, patient-specific drug screening, and transplantation. The most widely used biomaterial for culturing hPSCs is Matrigel, a matrix harvested from mouse tumors that suffers from batch-to-batch variability, animal-derived contaminants, and nonspecific protein interactions. These issues severely limit the clinical translation of hPSC-derived therapies. This has motivated the pursuit of synthetic alternatives to Matrigel that can be chemically-defined, xenogenic-free, reproducible, and optimized for specific cell types and applications. We propose the use of zwitterionic materials, specifically poly(carboxybetaine) (PCB), a unique super-hydrophilic synthetic polymer that achieves extremely low protein adsorption as a replacement to Matrigel for 3D hPSC culture. PCB is an ideal material for generating a “noise-free,” clean background in which specific signals for study or differentiation can be incorporated. The development of the PCB hydrogel for hPSC culture and differentiation will revolutionize the biomaterial and stem cell fields and will impact not only hPSCs, but also hPSC-derived cells, organoids, and any cell-based therapy dependent on Matrigel for clinical translation.

We have developed a highly efficient method for synthesizing functional PCB, offering access to star-shaped zwitterionic polymers with superior hydrolytic stability, excellent functionality, and tunable stiffness under physiological conditions. Previous formulations of zwitterionic PCB hydrogels were limited by a low product yield of about 20-40%, moderate functionality of about 60-70%, a tedious synthetic and purification process of over 7 days, and hydrolytic stability issues due to the use of an ester core. Using a one-pot aqueous reaction, we have achieved over 95% yield, over 90% functionality, reduced the synthetic and purification process to 1-2 days, and replaced the ester core with a hydrolytically stable amide core. Moreover, by using DBCO chain-ends, the gelation time is set to an optimal 5-10 minutes ensuring that the gel forms before cells have time to settle. Due to the higher functionality, we can incorporate both MMP degradable peptides as well as cell adhesive peptides, and due to the stable formulation, cells can be cultured for extended time periods without the risk of uncontrolled hydrogel loss. Additionally, bioactive molecules like RGD can be efficiently incorporated into the polymer by direct copolymerization instead of post-polymerization modification, enabling the independent control of scaffold stiffness and bioactivity possible.

The developed PCB hydrogel system, incorporating an MMP-degradable crosslinker and cell-adhesive peptide, enables direct encapsulation and release of hPSCs in a user-friendly manner at physiological conditions. This new PCB formulation was validated for iPSC biocompatibility, pluripotency maintenance, and differentiation capacity. Results indicate PCB-cultured iPSCs had high viability of at least 80% over 7 days of culture, sustained cell growth reaching nearly 70 million cells per mL gel at 7 days, and a normal karyotype. Pluripotency analysis revealed high expression of critical pluripotency markers like NANOG and OCT4 by immunofluorescence and RT-qPCR. Additionally, germ layer differentiation results demonstrate that PCB-cultured iPSCs can successfully differentiate to the ectoderm, mesoderm, and endoderm germ layers. This cell culture system has several key advantages including (a) noise-free background, (b) flexibility to adjust the polymer and peptide formulations as needed, (c) ability to incorporate specific cell-adhesive ligands, (d) cell-mediated hydrogel degradation to allow for cell growth, and (e) easy and biocompatible user-desired release of encapsulated cells from the hydrogel.