RIN-m cells (ISCs) and/or human MSCs (hMSCs) were encapsulated within PEGDA hydrogel sheets by photopolymerizing cells suspended in a precursor solution formed by combining 0.1 g/mL 10 kDa PEGDA (10% w/v; Laysan Bio, Inc.) with (1.5% v/v) triethanolamine/HEPES buffered saline (pH 7.4), 37 mM 1-vinyl-2-pyrrolidinone, 0.1 mM eosin Y. The prepolymer solution was combined with cells (1 × 104 cells/μL), pipetted into 1 cm2 custom made molds and exposed to white light for 20 s to achieve 400 µm thick cell-laden hydrogel sheets containing both MSCs and ISCs, MSCs, or ISCs at 0.5 × 106 cells per 400 µm thick hydrogel sheets. Crosslinked hydrogels were gently lifted with blunt forceps and placed in Transwells® (0.4 mm pore polycarbonate membrane Transwell® inserts; Corning, Inc., Lowell, MA) in a 12-well plate with 3 mL culture medium. Hydrogels were maintained in a tissue culture incubator. For monolayer comparisons, 1 × 106 ISCs or hMSCs were plated on 6-well tissue culture plates.
Pegda
Manufactured by Laysan Bio
Sourced in United States
PEGDA is a synthetic polymer that is commonly used in the production of hydrogels for various biomedical applications. It is a water-soluble, biocompatible, and biodegradable material that can be chemically modified to meet the specific requirements of different applications.
Automatically generated - may contain errors
Lab products found in correlation
Mh 100 metal halide lamp, by Edmund Optics (2 mentions)
Peg dithiol, by Laysan Bio (2 mentions)
Rgds peptide, by Merck Group (1 mentions)
Pegda, by Merck Group (1 mentions)
2 hydroxy 1 4 hydroxyethoxy phenyl 2 methyl 1propanone irgacure 2959, by BASF (1 mentions)
Ultraviolet lamp, by Spectroline (1 mentions)
Irgacure 2959, by Novartis (1 mentions)
4 dimethylaminopyridine, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Tetrahydrofuran, by Merck Group (1 mentions)
Thioacetic acid, by Merck Group (1 mentions)
K2co3, by Merck Group (1 mentions)
N n diisopropylcarbodiimide, by Merck Group (1 mentions)
5 norbornene 2 carboxylic acid, by Merck Group (1 mentions)
8 arm peg thiol, by JenKem Technology (1 mentions)
Allyl bromide, by Merck Group (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Four arm peg, by JenKem Technology (1 mentions)
8 arm peg, by JenKem Technology (1 mentions)
14 protocols using pegda
1
Encapsulation of Stem Cells in PEGDA Hydrogels
2
Microencapsulation of hMSCs in PEGDA Hydrogel
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Cells were microencapsulated as previously described.39 (link) Briefly, hMSCs were harvested and combined at 104 cells/μL with a hydrogel precursor solution containing 0.1 g/mL 10 kDa PEGDA (10% w/v; Laysan Bio), 37 mM 1-vinyl-2-pyrrolidinone with hydrophilic photoinitiators (1.5% (v/v) triethanolamine and 0.1 mM eosin Y) in HEPES-buffered saline (pH 7.4). A hydrophobic photoinitiator solution (2,2-dimethoxy-2-phenyl acetophenone in 1-vinyl-2-pyrrolidinone; 300 mg/mL) was combined in mineral oil (3 μL/mL, sterile filtered) and then subjected to vortex (2 s) under white light (Edmund Optics MH-100 metal halide lamp, 20 s) to photopolymerize the resulting emulsion. Photopolymerized microspheres were isolated by two washes in complete culture medium followed by 5 min centrifugation at 300g and maintained in 25 cm2 flasks with complete culture medium in a humidified incubator at 37°C with 5% CO2.
3
Microencapsulation of Immortalized Mesenchymal Stem Cells
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hTERT-MSCs were microencapsulated as previously described [8 (link),9 ]. Briefly, hydrogel prepolymer solution was prepared by combining 10 % w/v 10 kDa PEGDA (Laysan Bio, Arab, AL, USA), 37 mM 1-vinyl-2-pyrrolidinone, 1.5% v/v triethanolamine, 0.1 mM eosin Y, and 1% w/v Pluronic Acid F68 in HEPES buffered saline (pH 7.4). hTERT-MSCs were collected following incubation with trypsin, centrifuged at 300 g for 5 min, then resuspended in hydrogel prepolymer solution at 1 × 104 cells/µL. A hydrophobic photoinitiator solution was prepared by combining 300 mg 2,2-dimethoxy-2-phenyl acetophenone in 1 mL 1-vinyl-2-pyrrolidinone. The hydrophobic photoinitiator solution was combined with sterile filtered mineral oil at 3 µL/mL. Prepolymer containing cells was injected into mineral oil solutions, vortexed for 2 s under white light (MH-100 metal halide lamp, Edmund Optics, Barrington, NJ, USA) and exposed to the white light for an additional 20 s to photopolymerize resulting emulsion droplets. Photopolymerized microspheres were washed twice with complete culture medium, centrifuged at 300 g for 5 min, and maintained in 6-well plates in complete basal culture medium in a humidified incubator at 37 °C with 5% CO2.
4
Hierarchical Tissue Engineering Scaffold
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Poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA) were used to fabricate the inner cell-laden pattern and outer cell-laden pattern of the hierarchical microstructure. PEGDA (Mw = 3400 Da) was purchased from Laysan Bio. Inc. (USA). 2-Hydroxy-1-(4-(hydroxyethoxy)phenyl)−2-methyl-1-propanone (Irgacure 2959) as a photoinitiator (PI) was purchased from BASFSE (Germany). For cell culture, PEGDA was modified with RGDS peptides (Sigma-Aldrich, USA) [29 (link)]. A PEGDA solution with 20% (w/v) PEGDA, 0.5% (w/v) PI, 5 mM RGDS-modified PEGDA, and 1 × 106 mL−1 HepG2 cells in DMDM (HYCLONE, USA) was used to fabricate the inner radial pattern with cell encapsulation through photopatterning.
GelMA was prepared by synthesizing 10% (w/v) gelatin and 20% (w/v) methacrylic anhydride according to a previous report [30 (link)]. Lithium phenyl-2,4,6-trimethyl-benzoylphosphinate (LAP) was purchased from Allevi, USA. To obtain the GelMA solution, 10% (w/v) GelMA, 0.5% (w/v) LAP, and DMEM with 1 × 106 mL−1 HUVECs were mixed at 37°C.
GelMA was prepared by synthesizing 10% (w/v) gelatin and 20% (w/v) methacrylic anhydride according to a previous report [30 (link)]. Lithium phenyl-2,4,6-trimethyl-benzoylphosphinate (LAP) was purchased from Allevi, USA. To obtain the GelMA solution, 10% (w/v) GelMA, 0.5% (w/v) LAP, and DMEM with 1 × 106 mL−1 HUVECs were mixed at 37°C.
5
PEGDA Hydrogel Scaffold Fabrication
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Hydrogel scaffolds were prepared using a polymer solution containing one of four different molecular weight PEGDA (Laysan Bio, Arab, AL) in HEPES‐buffered saline (10 mM N‐[2‐hydroxyethyl]piperazine‐N0‐[2‐ethanesulfonic acid] and NaCl in ultra pure water), with 10 µL/mL photoinitiator solution (2,2‐dimethoxy‐2‐phenyl‐acetophenone 300 mg/mL in N‐vinylpyrrolidone). The four molecular weights of PEGDA used were 3.4, 5, 10, and 20 kDa. The concentrations used for 10 and 20 kDa PEGDA were 10 and 20%. The concentrations for 3.4 and 5 kDa were 20 and 40%. We define the lower concentrations as 1× and the higher concentrations as 2×. The concentrations of PEGDA used are summarized in Table 1 . The scaffolds were fabricated using molds constructed of two 25 × 75 mm2 precleaned glass microscope slides separated by a 500‐μm Teflon spacer. The molds were disinfected with 70% ethanol and exposed to UV light (B‐200SP UV lamp, UVP, 365 nm 10 mW2/cm2) for further sterilization for at least an hour prior to use. The prepolymer PEGDA solutions were injected into the molds through a 0.2‐µm polyethersulfone syringe filter, then the molds were exposed to the UV light for 3 min. After polymerization, rectangular‐shaped hydrogel scaffolds were removed from the molds with tweezers and fully immersed in 5 mL phosphate buffered saline (PBS) within petri dishes and allowed to swell for 24 h.
6
Cell-Laden UV-Crosslinked Hydrogel
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Hydrgel precursor solution was prepared by mixing PEGDA (10% w/v; Laysan Bio), 0.05% w/v photoinitiator Irgacure 2959 (Ciba), and EL4 cells (105 cells/ml). The precursor solution was transferred to a 60 mm culture dish, and exposed to an ultraviolet lamp (365 nm, 5 mW/cm2; Spectroline) for 15 min to induce gelation. The crosslinked hydrogel was placed in culture medium containing SYTO 62 to stain the encapsulated cells for 1 hour. The hydrogel was washed twice by replacing the culture medium.
7
Hydrogel Synthesis and Fabrication
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Acrylate-acrylate hydrogels were synthesized using polyethylene glycol diacrylate monomers (PEGDA, Laysan Bio, 3.4 kDa). Thiol-ene hydrogels were synthesized by a reaction between PEG-dithiol (Laysan Bio, 3.4 kDa) and 8-arm PEG norbornene (JenKem Tech, 20 KDa) at an eight to one molar ratio. Precursor solutions were created at a 10% weight/volume in phosphate buffer saline (PBS) with 2 mM of LAP, unless otherwise stated. Gel solutions were passed through a 0.22 μm filer for sterilization. Additionally, fibronectin (60 μg/ml) was added into the precursor solution to improve cell attachment to the hydrogel. After thoroughly mixed, solutions were pipetted into a 0.5 mm thick mold and exposed to UV light (365 nm, 3.4 mW/cm2) for 2 min, unless otherwise stated. Biopsy punches were used to cut hydrogels into appropriate sized samples and placed into well plates for imaging or cell seeding.
8
Acrylate-Thiol-ene Hydrogel Synthesis
9
Synthesis of Functionalized PEG Hydrogels
10
Functionalization of PEG-NHS Hydrogel Scaffolds
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A similar protocol to that described above was used to produce an N-hydroxysuccinimide (NHS) ester-functionalized ICC scaffold, with a pre-polymer solution containing 50% (w/v) PEG-DA, 10% (w/v) acryloyl-PEG-NHS (Laysan Bio, AL), and 0.05% photo initiator in deionized water. The prepared PEG-NHS ICC scaffold was coated with either collagen I or fibronectin by centrifugation, shaking, and incubation in 20 μg/ml collagen I (Merck Millipore, Germany) or fibronectin (Merck Millipore) solution in pH 7.4 PBS at 4 °C overnight. Excess collagen I or fibronectin was removed by repeated washing with PBS.
To characterize the ECM coating, the ICC scaffolds were fixed in 4% paraformaldehyde (Alfa Aesar, MA) then incubated with a mouse primary antibody against collagen I or fibronectin (Abcam, UK) at 4 °C overnight in the presence of 3% bovine serum albumin (BSA; Sigma-Aldrich) and labeled with an anti-mouse secondary antibody conjugated to a fluorophore. Green fluorescent Alexa Fluor 488 was used to label collagen I and red fluorescent Alexa Fluor 555 was used to label fibronectin. After PBS washing, the ECM-functionalized scaffolds were imaged using a Carl Zeiss LSM 710 confocal microscope.
To characterize the ECM coating, the ICC scaffolds were fixed in 4% paraformaldehyde (Alfa Aesar, MA) then incubated with a mouse primary antibody against collagen I or fibronectin (Abcam, UK) at 4 °C overnight in the presence of 3% bovine serum albumin (BSA; Sigma-Aldrich) and labeled with an anti-mouse secondary antibody conjugated to a fluorophore. Green fluorescent Alexa Fluor 488 was used to label collagen I and red fluorescent Alexa Fluor 555 was used to label fibronectin. After PBS washing, the ECM-functionalized scaffolds were imaged using a Carl Zeiss LSM 710 confocal microscope.
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