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Materials

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Bioengineering

Generation and Recovery of β-cell Spheroids From Step-growth PEG-peptide Hydrogels

Published: December 6th, 2012

DOI:

10.3791/50081

1Department of Biomedical Engineering, Purdue School of Engineering and Technology, Indiana University - Purdue University at Indianapolis

The following protocol provides techniques for encapsulating pancreatic β-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

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.

Hydrogels are hydrophilic crosslinked polymers with exceptional potential as scaffolding materials for repairing and regenerating tissues.1-3 The high water content of hydrogels permits easy diffusion of oxygen and exchange of nutrients and cellular metabolic products, all of which are crucial to maintaining cell viability. In addition, hydrogels are excellent carriers for controlled release and cell delivery due their high tunability.2 Synthetic hydrogels such as those prepared from poly(ethylene glycol) (PEG) are increasingly used in tissue engineering applications, largely due to their cytocompatibility, tissue-like elasticity, and hig....

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A. Macromer and Peptide Synthesis

  1. Synthesize 4-arm PEG-norbornene (PEG4NB) and photoinitiator Lithium arylphosphanate (LAP) using established protocols.18,19
  2. Synthesize bis-cysteine containing chymotrypsin-sensitive peptide CGGY↓C (arrow indicates chymotrypsin cleavage site) using standard solid phase peptide synthesis in a microwave peptide synthesizer (CEM Discover SPS).
    1. Calculate the amount of resin (Rink-amide MBHA resin) needed based on the substitutio.......

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Figures 1-4 show representative results for encapsulation, survival, proliferation, spheroid formation, and spheroid recovery in thiol-ene hydrogels. Figure 1 shows the reaction schematic of (1) step-growth thiol-ene photopolymerization using PEG4NB and CGGYC, and (2) chymotrypsin mediated gel erosion which follows a surface erosion mechanism. Figures 2 and 3 present viability results obtained using Live/Dead staining and AlamarBlue assay. We observe tha.......

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The described protocol presents details on easy encapsulation of cells in thiol-ene hydrogels formed by step-growth photopolymerization. While a stoichiometric ratio of 1:1 of norbornene to thiol functional groups was used in this protocol, the ratio can be adjusted depending on the experiments. In addition to a correct formulation, it is important to maintain homogeneity in the pre-polymer solution. In particular, use gentle pipetting to ensure that cells are well distributed in the pre-polymer solution in order t.......

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This project was funded by NIH (R21EB013717) and IUPUI OVCR (RSFG). The author thanks Ms. Han Shih for her technical assistance.

....

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Name Company Catalog Number Comments
Name Company Catalog Number Comments
4-arm PEG (20kDa) Jenkem Technology USA 4ARM-PEG-20K
Fmoc-amino acids Anaspec
Live/Dead cell viability kit Invitrogen L3224 Includes Calcein AM and Ethidium homodimer-1
AlamarBlue reagent AbD Serotec BUF012
CellTiter Glo reagent Promega G7570
DPBS Lonza 17-512F Without Ca+2 and Mg+2
HBSS Lonza 10547F Without Ca+2 and Mg+2
High Glucose DMEM Hyclone SH30243.01
FBS Gibco 16000-044
Antibiotic-Antimycotic Invitrogen 15240-062
β-Mercaptoethanol Sigma-Aldrich M7522-100ML
Trypsin-EDTA Invitrogen 15400-054
Trypsin-free α-chymotrypsin Worthington Biochemical Corp LS001432
Mouse Inusin ELISA kit Mercodia 10-1247-01
1 ml disposable syringe BD biosciences

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