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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)?m 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 under 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 ?-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.

SUBMITTER: Amer LD

PROVIDER: S-EPMC4503244 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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

Amer Luke D LD Holtzinger Audrey A Keller Gordon G Mahoney Melissa J MJ Bryant Stephanie J SJ

Acta biomaterialia 20150422

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 ...[more]

PMID: 25913222

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Project description:Radiation therapy for head and neck cancers leads to permanent xerostomia due to the loss of secretory acinar cells in the salivary glands. Regenerative treatments utilizing primary submandibular gland (SMG) cells show modest improvements in salivary secretory function, but there is limited evidence of salivary gland regeneration. We have recently shown that poly(ethylene glycol) (PEG) hydrogels can support the survival and proliferation of SMG cells as multicellular spheres in vitro. To further develop this approach for cell-based salivary gland regeneration, we have investigated how different modes of PEG hydrogel degradation affect the proliferation, cell-specific gene expression, and epithelial morphology within encapsulated salivary gland spheres. Comparison of non-degradable, hydrolytically-degradable, matrix metalloproteinase (MMP)-degradable, and mixed mode-degradable hydrogels showed that hydrogel degradation by any mechanism is required for significant proliferation of encapsulated cells. The expression of acinar phenotypic markers Aqp5 and Nkcc1 was increased in hydrogels that are MMP-degradable compared with other hydrogel compositions. However, expression of secretory acinar proteins Mist1 and Pip was not maintained to the same extent as phenotypic markers, suggesting changes in cell function upon encapsulation. Nevertheless, MMP- and mixed mode-degradability promoted organization of polarized cell types forming tight junctions and expression of the basement membrane proteins laminin and collagen IV within encapsulated SMG spheres. This work demonstrates that cellularly remodeled hydrogels can promote proliferation and gland-like organization by encapsulated salivary gland cells as well as maintenance of acinar cell characteristics required for regenerative approaches. Investigation is required to identify approaches to further enhance acinar secretory properties.Statement of significanceRegenerative strategies to replace damaged salivary glands require the function and organization of acinar cells. Hydrogel-based approaches have shown promise to control cell function and phenotype. However, little is known about how specific parameters, such as the mechanism of hydrogel degradation (e.g., hydrolytic or enzymatic), influence the viability, proliferation, organization, and phenotype of salivary gland cells. In this work, it is shown that hydrogel-encapsulated primary salivary gland cell proliferation is dependent upon hydrogel degradation. Hydrogels crosslinked with enzymatically degradable peptides promoted the expression of critical acinar cell markers, which are typically downregulated in primary cultures. Furthermore, salivary gland cells encapsulated in enzymatically- but not hydrolytically-degradable hydrogels displayed highly organized and polarized salivary gland cell markers, which mimics characteristics found in native gland tissue. In sum, results indicate that salivary gland cells respond to cellularly remodeled hydrogels, resulting in self-assembly and organization akin to acini substructures of the salivary gland.

Dataset Information

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

Publications

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

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