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3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Published: June 30th, 2023



1Department of Bioengineering, University of Colorado Denver | Anschutz Medical Campus, 2Department of Pediatrics, University of Colorado Anschutz Medical Campus, 3Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado Anschutz Medical Campus

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEGαMA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

3D bioprinting is a transformative technology that enables researchers to precisely deposit cells and biomaterials within 3D volumes and recreate the complex hierarchical structure of biological tissues. Over the past decade, advances in 3D bioprinting have created beating human cardiac tissues1, functional models of kidney tissues2, models of gas exchange within the lung3, and tumor models for cancer research4. The invention of embedded 3D bioprinting techniques, such as Freeform Reversible Embedding of Suspended Hydrogel (FRESH) bioprinting, has made it possible to reprod....

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1. PEGαMA synthesis and characterization

NOTE: Poly(ethylene glycol)-alpha methacrylate (PEGαMA) synthesis was adapted from Hewawasam et al. and performed under moisture-free conditions9.

  1. Weigh the reactants.
    NOTE: For example, weigh out 5 g 10 kg/mol 8-arm PEG-hydroxyl (PEG-OH) and 0.38 g sodium hydride (NaH) (see Table of Materials).
  2. Add a stir bar to 250 mL Schlenk flask and purge with .......

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This protocol describes how to 3D bioprint phototunable hydrogels within a support bath to create constructs capable of dynamic and temporal stiffening for studying fibroblast activation in geometries that mimic human tissues. First, the protocol explained how to synthesize PEGαMA, the backbone of this phototunable polymer system. Nuclear magnetic resonance (NMR) spectroscopy measurements showed successful PEGαMA functionalization at 96.5% (Figure 1). Functionalization values of 90.......

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Dual-stage polymerization reactions in response to controlled light exposure can stiffen biomaterials with spatial and temporal control. Several studies have harnessed this technique to evaluate cell-matrix interactions in various platforms5,8,9,10,11,21,22,23

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The authors would like to acknowledge Dr. Adam Feinberg (Carnegie Mellon University) and those who hosted the 3D Bioprinting Open-Source Workshop. These individuals made it possible to learn the techniques of FRESH bioprinting and build the 3D bioprinter used for these studies. Additionally, the authors would like to acknowledge, which was used to produce figures in this manuscript. This work was supported by multiple groups or funding sources including the Rose Community Foundation (DDH and CMM), a Colorado Pulmonary Vascular Disease Research Award (DDH and CMM), the National Science Foundation under Award 1941401 (CMM), the Department of the Army under....

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Name Company Catalog Number Comments
AccuMax Radiometer/Photometer Kit Spectronics Corporation XPR-3000 To measure light intensity, used for photostiffening
Acetic Acid  Fisher Scientific BP2401-500 Used during PEGaMA synthesis
Acetone Fisher Scientific A184 Used with the cryosections
ActinGreen 488 ReadyProbes Fisher Scientific R37110 Used for staining
Aluminum Foil Reynolds F28028
Anhydrous Tetrahydrofuran (THF) Sigma-Aldrich 401757-1L Used during PEGaMA synthesis
Argon Compressed Gas Airgas AR R300 Used during PEGaMA synthesis
8 Arm Poly(ethylene glycol)-hydroxyl (PEG-OH) JenKem Technology 8ARM-PEG-10K Used during PEGaMA synthesis
365 nm Bandpass Filter Edmund Optics 65-191 Used for photostiffening
Bovine Serum Albumin (BSA) Fisher Scientific BP9700-100 Used during staining process
Buchner Funnel Quark Glass QFN-8-14 Used during PEGaMA synthesis
Calcein AM Invitrogen 65-0853-39 Used during staining process
Celite 545 (Filtration Aid) EMD Millipore CX0574-1 Used during PEGaMA synthesis
Charged Microscope Slides Globe Scientific 1358W
Chloroform-d Sigma-Aldrich 151823-10X0.75ML Used to characterize PEGaMA
Click-iT Plus EdU Cell Proliferation Kit Invitrogen C10637 Used for staining
50 mL Conical Tubes CELLTREAT 667050B
Cryogenic Safety Kit Cole-Parmer EW-25000-85
Cryostat Leica CM 1850-3-1
Dialysis Tubing Repligen 132105
4’,6-Diamidino-2-Phylindole (DAPI) Sigma-Aldrich D9542-1MG Used for staining
Diethyl Ether Fisher Scientific E1384 Used during PEGaMA synthesis
1,4-Dithiothreitol (DTT)  Sigma-Aldrich 10197777001 Bioink component
Dulbecco's Modified Eagle's Medium (DMEM) Cytiva SH30271.FS
Ethyl 2-(Bromomethyl)Acrylate (EBrMA) Ambeed Inc. A918087-25g Used during PEGaMA synthesis
Filter Paper Whatman 1001-090 Used during PEGaMA synthesis
Freezone 2.5L Freeze Dry System Labconco LA-2.5LR Lyophilizer
Fusion 360 Autodesk N/A Software download
2.5 mL Gastight Syringe Hamilton 81420 Used for bioprinting
15 Gauge 1.5" IT Series Tip Jensen Global JG15-1.5X Used for bioprinting
30 Gauge 0.5" HP Series Tip Jensen Global JG30-0.5HPX Used for bioprinting
Goat Anti-Mouse Alexa Fluor 555 Antibody Fisher Scientific A21422 Used for staining
Glycine Fisher Scientific C2H5NO2 Used during staining process
Hemocytometer Fisher Scientific 1461
Hoechst Thermo Scientific 62249 Used during staining process
Human Pulmonary Artery Adventitial Fibroblasts (HPAAFs) AcceGen ABC-TC3773  From a 2-year-old male patient
Hydrochloric Acid (HCl) Fisher Scientific A144-500 Used to pH adjust solutions
ImageJ National Institutes of Health (NIH) N/A Free software download
ImmEdge® Pen Vector Laboratories H-4000 Used during staining process
Incubator VWR VWR51014991
LifeSupport Gelatin Microparticle Slurry (Gelatin Slurry) Advanced Biomatrix 5244-10GM Used for bioprinting
Light Microscope Olympus CKX53 Inverted light microscope
Lithium Phenyl-2,4,6-Trimethylbenzoylphosphinate (LAP) Sigma-Aldrich 900889-5G Photoinitiator used for photostiffening
Liquid Nitrogen N/A N/A
LulzBot Mini 2  LulzBot N/A Bioprinter adapted
Methacryloxyethyl Thiocarbamoyl Rhodamine B  Polysciences Inc. 669775-30-8
2-Methylbutane Sigma-Aldrich M32631-4L
Microman Capillary Pistons CP1000 VWR 76178-166 Positive displacement pipette tips
MMP2 Degradable Crosslinker (KCGGPQGIWGQGCK) GL Biochem N/A Bioink component
Mouse Anti-Human αSMA Monoclonal Antibody Fisher Scientific MA5-11547 Used for staining
OmniCure Series 2000  Lumen Dynamics S2000-XLA UV light source used for photostiffening
Paraformaldehyde (PFA)  Electron Microscopy Sciences 15710 Used to fix samples
pH Meter Mettler Toledo  FP20 
pH Strips Cytiva 10362010
Phosphate Buffered Saline (PBS) Hyclone Laboratories, Inc. Cytiva SH30256.FS
Pipette Set Fisher Scientific 14-388-100
10 µL Pipette Tips USA Scientific 1120-3710
20 µL Pipette Tips USA Scientific 1183-1510
200 µL Pipette Tips USA Scientific 1111-0700
1000 µL Pipette Tips USA Scientific 1111-2721
Poly(Ethylene Glycol)-Alpha Methacrylate (PEGαMA) N/A N/A Refer to manuscript for synthesis steps
Poly(Ethylene Oxide) (PEO) Sigma-Aldrich 372773-250G Bioink component
Positive Displacement Pipette Fisher Scientific FD10004G 100-1000 µL
Potassium Hydroxide (KOH) Sigma-Aldrich 221473-500G Used to pH adjust solutions
ProLong Gold Antifade Reagent Invitrogen P36930 Used during staining process
Pronterface All3DP N/A Software download
Propidium Iodide Sigma-Aldrich P4864-10ML Used for staining
RGD Peptide (CGRGDS) GL Biochem N/A Bioink component
Rocker VWR 10127-876
Rotary Evaporator  Thomas Scientific 11100V2022 Used during PEGaMA synthesis
Rubber Band Staples 808659
Schlenk Flask  Kemtech America F902450 Used during PEGaMA synthesis
Slic3r Slic3r N/A Software download
Smooth Muscle Cell Growth Medium-2 (SmGM-2) BulletKit Lonza CC-3182 Kit contains CC-3181 and CC-4149 components
Sodium Hydride  Sigma-Aldrich 223441-50G Used during PEGaMA synthesis
Sorvall ST 40R Centrifuge Fisher Scientific 75-004-525
Stir Bar VWR 58948-091
Syringe Filter VWR 28145-483 Used to sterile filter solutions
T-75 Tissue-Cultured Treated Flask VWR 82050-856 Used for cell culture work
Tissue-Tek Cyromold Sakura 4557
Tissue-Tek O.C.T Compound (OCT) Sakura 4583
Tris(2-Carboxyethyl) Phosphine (TCEP) Sigma-Aldrich C4706-2G
Triton X-100 Fisher Bioreagents C34H622O11 Used during staining process
Trypan Blue Sigma-Aldrich T8154-20ML Used for cell culture work
0.05% Trypsin-EDTA Gibco 25-300-062 Used for cell culture work
Tween 20 Fisher Bioreagents C58H114O26 Used during staining process
Upright Microscope Olympus BX63F Fluorescent microscope capabilities
Water Bath PolyScience WBE20A11B
24-Well Tissue Culture Plates Corning 3527

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