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.
Department of Chemical and Biological Engineering, University of Colorado, 3415 Colorado Avenue, Boulder, CO 80303, USA; BioFrontiers Institute, University of Colorado, 3415 Colorado Avenue, Boulder, CO 80303, USA. Electronic address: Luke.Amer@Colorado.EDU.
Luke D
LD
Amer
McEwen Centre for Regenerative Medicine, University Health Network, 8-601 TMDT 101 College St., Toronto, ON M5G 1L7, Canada. Electronic address: Aholtzin@uhnres.utoronto.ca.
Audrey
A
Holtzinger
McEwen Centre for Regenerative Medicine, University Health Network, 8-601 TMDT 101 College St., Toronto, ON M5G 1L7, Canada. Electronic address: gkeller@uhnresearch.ca.
Gordon
G
Keller
Department of Chemical and Biological Engineering, University of Colorado, 3415 Colorado Avenue, Boulder, CO 80303, USA; BioFrontiers Institute, University of Colorado, 3415 Colorado Avenue, Boulder, CO 80303, USA. Electronic address: Melissa.Mahoney@Colorado.EDU.
Melissa J
MJ
Mahoney
Department of Chemical and Biological Engineering, University of Colorado, 3415 Colorado Avenue, Boulder, CO 80303, USA; BioFrontiers Institute, University of Colorado, 3415 Colorado Avenue, Boulder, CO 80303, USA; Material Science and Engineering Program, University of Colorado, 3415 Colorado Avenue, Boulder, CO 80303, USA. Electronic address: Stephanie.Bryant@Colorado.EDU.
Stephanie J
SJ
Bryant
D000595:Amino Acid Sequence; D000818:Animals; D002449:Cell Aggregation; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D048429:Cell Size; D002470:Cell Survival; D004798:Enzymes / Q000378:metabolism*; D005434:Flow Cytometry; D000066449:Human Embryonic Stem Cells / Q000166:cytology*; D006801:Humans; D020100:Hydrogels / Q000737:chemistry*; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010179:Pancreas / Q000166:cytology*; D010455:Peptides / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry*; D058105:Polymerization; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism
D004798:Enzymes; D020100:Hydrogels; D010455:Peptides; D014157:Transcription Factors; D011092:Polyethylene Glycols
Controlled release; Human embryonic stem cells; Long-term culture platforms; Pancreatic precursor cells; Poly(ethylene glycol) hydrogels