Enzymatically degradable poly(ethylene glycol) hydrogels for the 3D culture and release of human embryonic stem cell derived pancreatic precursor cell aggregates

Abstract

This study aimed to develop a three dimensional culture platform for aggregates of human embryonic stem cell (hESC)-derived pancreatic progenitors that enables long-term culture, maintains aggregate size and morphology, does not adversely affect differentiation and provides a means for aggregate recovery. A platform was developed with poly(ethylene glycol) hydrogels containing collagen type I, for cell-matrix interactions, and peptide crosslinkers, for facile recovery of aggregates. The platform was first demonstrated with RIN-m5F cells, showing encapsulation and subsequent release of single cells and aggregates without adversely affecting viability. Aggregates of hESC-derived pancreatic progenitors with an effective diameter of 82 (15) μm were either encapsulated in hydrogels or cultured in suspension for 28 days. At day 14, aggregate viability was maintained in the hydrogels, but significantly reduced (88%) in suspension culture. However by day 28, viability was reduced in both culture conditions. Aggregate size was maintained in the hydrogels, but in suspension was significantly higher (~2-fold) by day 28. The ability to release aggregates followed by a second enzyme treatment to achieve single cells enabled assessment by flow cytometry. Prior to encapsulation, there were 39% Pdx1+/Nkx6.1+ cells, key endocrine markers required for β-cell maturation. The fraction of doubly positive cells was not affected in hydrogels but was slightly and significantly lower in suspension culture by 28 days. In conclusion, we demonstrate that a MMP-sensitive PEG hydrogel containing collagen type I is a promising platform for hESC-derived pancreatic progenitors that maintains viable aggregates, aggregate size, and progenitor state and offers facile recovery of aggregates.

On-demand dissolution of modular, synthetic extracellular matrix reveals local epithelial-stromal communication networks

“…A barrier to productive use of 3D in vitro models to parse epithelial-stromal communication is access to the local cell-cell communication networks, as standard proteolytic digestion methods also degrade many target proteins (Table 1), and methods to deconstruct synthetic ECMs have limitations (14–23, 63). Here, we used the example of stromal-epithelial communication in the human endometrium to illustrate the design and implementation of a MSD-ECM that is made from readily-available or easily-synthesized reagents, can be tuned to support differentiated function in stromal-epithelial cultures, and can be dissolved rapidly on demand to release cells and proteins largely intact, before or after fixation or lysis, for further analysis by multiplex methods to yield insights into local cell-cell communication networks.…”

“…Methods to avoid degradation of proteins and other macromolecules are desirable, not only to preserve cell surface receptors and soluble signaling molecules for analysis and quantification, but also because proteolytic cleavage of cell surface growth factors and receptors triggers near-instantaneous changes in signaling networks, altering the parameters under investigation (14–17). Previously, synthetic ECM breakdown strategies using thermal (18), chemical (19), ionic shifts (20), photodegradation (21, 22), and proteolytic degradation (23) have all been deployed to release cells, but these approaches are either relatively slow, have variable success in minimizing cell damage, or are limited in application to relatively thin tissues.…”

Tuning Reaction and Diffusion Mediated Degradation of Enzyme‐Sensitive Hydrogels

“…[6,7] We investigated a hydrogel platform based on a thiol-norbornene PEG hydrogel system with peptide crosslinks comprised of the collagenase-sensitive peptide VPLS-LYSG (Scheme 1B). [8] The enzyme that was used in this study was a commercially available collagenase blend. We first calibrated and validated our model with a simple one-dimensional (1D) experimental system (Scheme 1C) and then applied it to a more complex three-dimensional (3D) experimental hydrogel system mimicking enzyme-releasing cells (Scheme 1D).…”

Strategies for Bioengineered Scaffolds that Support Adipose Stem Cells in Regenerative Therapies

“…The use of vitronectin-derived attachment peptides promoted the development of larger lipid vacuoles, further suggesting that the interaction of the ASCs with their scaffolding may have a significance impact on the desired differentiation and health of implanted cells [90,91]. Biomimetic PEG hydrogels may prove to be superior synthetic scaffolds for use in tissue reconstruction [92][93][94].…”