title: Generation and recovery of β-cell spheroids from step-growth PEG-peptide hydrogels.
abstract: Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY↓C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 β-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.
authors.affiliation: Department of Biomedical Engineering, Purdue School of Engineering and Technology, Indiana University - Purdue University at Indianapolis.
authors.forename: Asad
authors.initials: A
authors.lastname: Raza
authors.forename: Chien-Chi
authors.initials: CC
authors.lastname: Lin
mesh_terms: D000818:Animals; D002918:Chymotrypsin / Q000737:chemistry; D020100:Hydrogels / Q000737:chemistry*; D050417:Insulin-Secreting Cells / Q000166:cytology*; D051379:Mice; D009636:Norbornanes / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry*; D018874:Spheroids, Cellular; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D020100:Hydrogels; D009636:Norbornanes; D011092:Polyethylene Glycols; D002918:Chymotrypsin