JoVE Logo
Faculty Resource Center

Sign In





Representative Results






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

Published: December 6th, 2012



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....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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


Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

  1. Tibbitt, M. W., Anseth, K. S. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnology and bioengineering. 103, 655-663 (2009).
  2. Lin, C. C., Anseth, K. S. PEG hydrogels for the controlled release of biomolecules in regenerative medicine. Pharmaceutical research. 26, 631-643 (2009).
  3. Lin, C. C., Metters, A. T. Hydrogels in controlled release formulations: network design and mathematical modeling. Advanced drug delivery reviews. 58, 1379-1408 (2006).
  4. Khetan, S., Burdick, J. A. Patterning hydrogels in three dimensions towards controlling cellular interactions. Soft Matter. 7, 830-838 (2011).
  5. Aimetti, A. A., Tibbitt, M. W., Anseth, K. S. Human neutrophil elastase responsive delivery from poly(ethylene glycol) hydrogels. Biomacromolecules. 10, 1484-1489 (2009).
  6. Weber, L. M., He, J., Bradley, B., Haskins, K., Anseth, K. S. PEG-based hydrogels as an in vitro encapsulation platform for testing controlled beta-cell microenvironments. Acta biomaterialia. 2, 1-8 (2006).
  7. Lin, C. C., Raza, A., Shih, H. PEG hydrogels formed by thiol-ene photo-click chemistry and their effect on the formation and recovery of insulin-secreting cell spheroids. Biomaterials. 32, 9685-9695 (2011).
  8. Lin, C. C., Anseth, K. S. Glucagon-like peptide-1 functionalized PEG hydrogels promote survival and function of encapsulated pancreatic beta-cells. Biomacromolecules. 10, 2460-2467 (2009).
  9. Lin, C. C., Anseth, K. S. Cell-cell communication mimicry with poly(ethylene glycol) hydrogels for enhancing beta-cell function. Proceedings of the National Academy of Sciences of the United States of America. 108, 6380-6385 (2011).
  10. Hui, H., Nourparvar, A., Zhao, X., Perfetti, R. Glucagon-like peptide-1 inhibits apoptosis of insulin-secreting cells via a cyclic 5'-adenosine monophosphate-dependent protein kinase A- and a phosphatidylinositol 3-kinase-dependent pathway. Endocrinology. 144, 1444-1445 (2003).
  11. Weber, L. M., Lopez, C. G., Anseth, K. S. Effects of PEG hydrogel crosslinking density on protein diffusion and encapsulated islet survival and function. Journal of biomedical materials research. Part A. 90, 720-729 (2009).
  12. Weber, L. M., Hayda, K. N., Haskins, K., Anseth, K. S. The effects of cell-matrix interactions on encapsulated beta-cell function within hydrogels functionalized with matrix-derived adhesive peptides. Biomaterials. 28, 3004-3011 (2007).
  13. Hsu, C. W., Olabisi, R. M., Olmsted-Davis, E. A., Davis, A. R., West, J. L. Cathepsin K-sensitive poly(ethylene glycol) hydrogels for degradation in response to bone resorption. Journal of biomedical materials research. Part A. 98, 53-62 (2011).
  14. Leslie-Barbick, J. E., Moon, J. J., West, J. L. Covalently-immobilized vascular endothelial growth factor promotes endothelial cell tubulogenesis in poly(ethylene glycol) diacrylate hydrogels. Journal of biomaterials science. Polymer. 20, 1763-1779 (2009).
  15. Moon, J. J., Hahn, M. S., Kim, I., Nsiah, B. A., West, J. L. Micropatterning of poly(ethylene glycol) diacrylate hydrogels with biomolecules to regulate and guide endothelial morphogenesis. Tissue engineering. Part A. 15, 579-585 (2009).
  16. Hoyle, C. E., Bowman, C. N. Thiol-ene click chemistry. Angew. Chem. Int. Ed. Engl. 49, 1540-1573 (2010).
  17. Hoyle, C. E., Lowe, A. B., Bowman, C. N. Thiol-click chemistry: a multifaceted toolbox for small molecule and polymer synthesis. Chemical Society reviews. 39, 1355-1387 (2010).
  18. Fairbanks, B. D., et al. A Versatile Synthetic Extracellular Matrix Mimic via Thiol-Norbornene Photopolymerization. Adv. Mater. 21, 5005 (2009).
  19. Fairbanks, B. D., Schwartz, M. P., Bowman, C. N., Anseth, K. S. Photoinitiated polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility. Biomaterials. 30, 6702-6707 (2009).
  20. Zustiak, S. P., Leach, J. B. Characterization of protein release from hydrolytically degradable poly(ethylene glycol) hydrogels. Biotechnology and bioengineering. 108, 197-206 (2011).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved