Enzymatically degradable poly(ethylene glycol) hydrogels for the 3D culture and release of human embryonic stem cell derived pancreatic precursor cell aggregates.
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 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 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.
['Amer|Luke D|LD|', 'Holtzinger|Audrey|A|', 'Keller|Gordon|G|', 'Mahoney|Melissa J|MJ|', 'Bryant|Stephanie J|SJ|']
D000595:Amino Acid Sequence D000818:Animals D002449:Cell Aggregation D018929:Cell Culture Techniques D045744:Cell Line, Tumor D048429:Cell Size D002470:Cell Survival D004798:Enzymes D005434:Flow Cytometry D000066449:Human Embryonic Stem Cells D006801:Humans D020100:Hydrogels D051379:Mice D018613:Microscopy, Confocal D008969:Molecular Sequence Data D010179:Pancreas D010455:Peptides D011092:Polyethylene Glycols D058105:Polymerization D051381:Rats D014157:Transcription Factors
Poly(ethylene glycol) hydrogels Human embryonic stem cells Pancreatic precursor cells Controlled release Long-term culture platforms