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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 high tunability in material physical and mechanical properties.4-6
Although a commonly used hydrogel platform, studies have shown that PEG diacrylate (PEGDA) hydrogels formed by chain-growth photopolymerizations have a tendency to damage encapsulated cells during network crosslinking and in situ cell encapsulation.7 The cellular damage was largely attributed to radical species generated by the photoinitiator molecules, which propagate through the vinyl groups on PEGDA to crosslink polymer chains into hydrogels. Unfortunately, these radical species also cause stresses and cellular damage during cell encapsulation, especially for radical-sensitive cells such as pancreatic β-cells.8-10 In order to obtain a higher mesh size for better diffusion and cell survival, higher molecular weights PEGDA are often used for cell encapsulation. This, however, compromises polymerization kinetics and causes sub-optimal gel biophysical properties.7,11,12 In addition to the above mentioned disadvantages, it is very difficult to recover cell structures from PEGDA hydrogels due to the heterogeneity and non-degradable nature of the crosslinked networks. While protease-sensitive peptides can be incorporated into PEG macromer backbone to render the otherwise inert PEGDA hydrogels sensitive to enzymatic cleavage, the conjugation often uses expensive reagents and the resulting networks still contain high degree of heterogeneity due to the nature of chain-growth polymerization.13-15
Recently, PEG-peptide hydrogels formed via step-growth thiol-ene photopolymerization have been shown to exhibit preferential properties for cell encapsulation over hydrogels formed by chain-growth photopolymerization.7 The superior gelation kinetics of thiol-ene hydrogels is attributed to the 'click' nature of reaction between thiol and ene functionalities. Compared to chain-growth polymerization of PEGDA, thiol-ene reaction is less oxygen inhibited which results in faster gelation rate.16,17 Thiol-ene hydrogels also have higher polymerization efficiency and better gel biophysical properties compared to chain-growth PEGDA hydrogels,7,18 which results in limited cellular damage caused by radical species during photopolymerization.
Previously, thiol-ene hydrogels formed by 4-arm PEG-norbornene (PEG4NB) macromer and bis-cysteine containing peptide crosslinkers, such as protease-sensitive peptides have been utilized for cell encapsulation.7,18 High tunability of PEG hydrogel networks offers a flexible and controllable 3D microenvironment for investigating cell survival and activity, while the use of protease-sensitive peptide sequence provides a mild way for recovery of cell constructs formed naturally within hydrogels. In this protocol we utilize step-growth photopolymerized thiol-ene hydrogels fabricated using 4-arm PEG-norbornene (PEG4NB) and a chymotrypsin-sensitive peptide crosslinker (CGGY↓C) for the encapsulation of MIN6 β-cells. This protocol systematically elaborates techniques for studying the survival, proliferation and spheroid formation of MIN6 β-cells in thiol-ene hydrogels. We further provide method for β-cell spheroid recovery and biological characterization of recovered spheroids.
A. Macromer and Peptide Synthesis
Synthesis scale | 0.1 mmole | 0.2 mmole |
Power | 20 W | 50 W |
Temperature | 75 °C | 75 °C |
Time | 3 min | 3 min |
Synthesis scale | 0.1 mmole | 0.2 mmole |
Power | 20 W | 50 W |
Temperature* | 75 °C | 75 °C |
Time* | 5 min | 5 min |
B. Material Preparation and Sterilization
C. Cell Preparation
D. Hydrogel Fabrication and Cell Encapsulation
E. Cell Viability Assay
Encapsulated cell morphology can be observed using light microscope (since the synthesized hydrogels are transparent). Cell viability can be visualized and measured qualitatively using Live/Dead staining and quantitatively using AlamarBlue reagent.
E.1. Live/Dead staining
E.2. AlamarBlue Assay
F. Chymotrypsin Mediated Gel Erosion and Spheroid Recovery
G. Functional Assay of Recovered Spheroids
G.1. Glucose stimulated insulin secretion from the recovered cell spheroids
G.2. CellTiter Glo Assay
G.3. Determine insulin secretion
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...
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...
No conflicts of interest declared.
This project was funded by NIH (R21EB013717) and IUPUI OVCR (RSFG). The author thanks Ms. Han Shih for her technical assistance.
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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