Name: 3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation (Video) | JoVE Journal
Uploaded: 2023-06-30T12:20:00.000+00:00
Duration: 7 min 17 s
Description: Learn 3D bioprinting of phototunable hydrogels to study fibroblast activation and extracellular matrix stiffening in lung tissue models. This method enables precise control of mechanical properties for chronic lung disease research.

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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 Biorender.com, 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 Award W81XWH-20-1-0037 (CMM), the National Cancer Institute of the NIH under Award R21 CA252172 (CMM), the Ludeman Family Center for Women&#39;s Health Research at the University of Colorado Anschutz Medical Campus (DDH and CMM), the National Heart, Lung, and Blood Institute of the National Institutes of Health under Awards R01 HL080396 (CMM), R01 HL153096 (CMM), F31 HL151122 (DDH), and T32 HL072738 (DDH and AT).

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

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<li>Hu, C. J., Zhang, H., Laux, A., Pullamsetti, S. S., Stenmark, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+contributing+to+persistently+activated+cell+phenotypes+in+pulmonary+hypertension%5BTitle%5D)+AND+%22The+Journal+of+Physiology%22%5BJournal%5D)">Mechanisms contributing to persistently activated cell phenotypes in pulmonary hypertension.</a> The Journal of Physiology. 597 (4), 1103-1119 (2019).</li>
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<li>Caracena, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alveolar+epithelial+cells+and+microenvironmental+stiffness+synergistically+drive+fibroblast+activation+in+three-dimensional+hydrogel+lung+models%5BTitle%5D)+AND+%22Biomaterials+Science%22%5BJournal%5D)">Alveolar epithelial cells and microenvironmental stiffness synergistically drive fibroblast activation in three-dimensional hydrogel lung models.</a> Biomaterials Science. 10 (24), 7133-7148 (2022).</li>
<li>Ruskowitz, E. R., DeForest, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteome-wide+analysis+of+cellular+response+to+ultraviolet+light+for+biomaterial+synthesis+and+modification%5BTitle%5D)+AND+%22ACS+Biomaterials+Science+%26+Engineering%22%5BJournal%5D)">Proteome-wide analysis of cellular response to ultraviolet light for biomaterial synthesis and modification.</a> ACS Biomaterials Science & Engineering. 5 (5), 2111-2116 (2019).</li>
<li>Kruse, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+pH+on+cell+viability%2C+cell+migration%2C+cell+proliferation%2C+wound+closure%2C+and+wound+reepithelialization%3A+In+vitro+and+in+vivo+study%5BTitle%5D)+AND+%22Wound+Repair+and+Regeneration%22%5BJournal%5D)">The effect of pH on cell viability, cell migration, cell proliferation, wound closure, and wound reepithelialization: In vitro and in vivo study.</a> Wound Repair and Regeneration. 25 (2), 260-269 (2017).</li>
<li>Filippi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perfusable+biohybrid+designs+for+bioprinted+skeletal+muscle+tissue%5BTitle%5D)+AND+%22Advanced+Healthcare+Materials%22%5BJournal%5D)">Perfusable biohybrid designs for bioprinted skeletal muscle tissue.</a> Advanced Healthcare Materials. , e1500758(2023).</li>
<li>Matthiesen, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Astrocyte+3D+culture+and+bioprinting+using+peptide-functionalized+hyaluronan+hydrogels%5BTitle%5D)+AND+%22Science+and+Technology+of+Advanced+Materials%22%5BJournal%5D)">Astrocyte 3D culture and bioprinting using peptide-functionalized hyaluronan hydrogels.</a> Science and Technology of Advanced Materials. 24 (1), 2165871(2023).</li>
<li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioprinting+a+skin+patch+with+dual-crosslinked+gelatin+%28GelMA%29+and+silk+fibroin+%28SilMA%29%3A+An+approach+to+accelerating+cutaneous+wound+healing%5BTitle%5D)+AND+%22Materials+Today+Bio%22%5BJournal%5D)">Bioprinting a skin patch with dual-crosslinked gelatin (GelMA) and silk fibroin (SilMA): An approach to accelerating cutaneous wound healing.</a> Materials Today Bio. 18, 100550(2023).</li>
<li>Bliley, J. M., Shiwarski, D. J., Feinberg, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((3D-bioprinted+human+tissue+and+the+path+toward+clinical+translation%5BTitle%5D)+AND+%22Science+Translational+Medicine%22%5BJournal%5D)">3D-bioprinted human tissue and the path toward clinical translation.</a> Science Translational Medicine. 14 (666), eabo7047(2022).</li>
</ol>

The authors do not have any conflicts of interest to disclose. Portions of this manuscript are reproduced with permission from &#169; IOP Publishing https://doi.org/10.1088/1758-5090/aca8cf.5&#160;All rights reserved.

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</s...

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 reproduce complex soft tissue structures such as pulmonary blood vessels5 and even human heart6 in 3D. FRESH 3D bioprinting facilitates layer-by-layer printing of soft and low-viscosity bioinks through extrusion into a shear-thinning support bath. The support bath consists of a material such as closely packed gelatin microparticles that acts as a Bingham plastic and maintains the intended shape and structure of the bioink after printing. Once the printed construct has solidified, the support bath can then be dissolved away by increasing the temperature to 37 &#176;C7.
A recent review article summarized the materials that have been 3D bioprinted in various publications using FRESH technique. These naturally derived materials range from collagen type I to methacrylated hyaluronic acid and represent several different gelation mechanisms7. Most research studies performed using this 3D bioprinting technique employ static biomaterials that do not change in response to external stimuli. Dynamic phototunable hydrogel biomaterials have been used by our lab and others8,9,10,11,12 to model a variety of fibrotic diseases. Unlike static biomaterials, phototunable bioinks allow for a softened model with lower elastic modulus value to be created and later stiffened to explore cellular responses to increases in microenvironmental stiffening.
Fibrotic diseases are characterized by an increase in the extracellular matrix production that can cause scarring and stiffening13. Tissue stiffening can initiate further injury and destruction of the impacted tissue, causing permanent organ damage and even death; fibrotic disorders are responsible for one-third of mortality worldwide. Fibroblasts produce excess and aberrant extracellular matrix in this disease state14,15.&#160;Increased fibroblast proliferation and extracellular matrix deposition further stiffen the tissue and activates a profibrotic positive feedback loop16,17,18,19.&#160;Studying fibroblast activation is vital to understanding fibrotic diseases. Here we present human pulmonary arterial hypertension (PAH) as an example of one fibrotic disorder in which it is important to mimic the 3D geometry of the blood vessel using 3D bioprinting and introduce the dynamic stiffening capabilities of phototunable hydrogels. PAH is a condition in which pressure in the main pulmonary arteries surpasses normal levels and applies strain to the heart, increasing human pulmonary artery adventitial fibroblast (HPAAF) activation and stiffens the blood vessel tissues16,17,18,19. A phototunable poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) bioink formulation allows for temporal stiffening in constructs and helps model both healthy tissue&#160;and disease progression5,8,9,10. Exploiting this unique feature enables&#160;the quantification of HPAAF activation and proliferation in response to microenvironmental stiffening in 3D and may provide valuable insight into the cellular mechanisms involved in this disease. The protocol described here will allow researchers to create 3D models that recapitulate changes in the extracellular microenvironment during disease progression or tissue repair and study fibroblast activation.

<table><tbody><tr><td>AccuMax Radiometer/Photometer Kit</td><td>Spectronics Corporation</td><td>XPR-3000</td><td>To measure light intensity, used for photostiffening</td></tr><tr><td>Acetic Acid&amp;nbsp;</td><td>Fisher Scientific</td><td>BP2401-500</td><td>Used during PEGaMA synthesis</td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A184</td><td>Used with the cryosections</td></tr><tr><td>ActinGreen 488 ReadyProbes</td><td>Fisher Scientific</td><td>R37110</td><td>Used for staining</td></tr><tr><td>Aluminum Foil</td><td>Reynolds</td><td>F28028</td><td></td></tr><tr><td>Anhydrous Tetrahydrofuran (THF)</td><td>Sigma-Aldrich</td><td>401757-1L</td><td>Used during PEGaMA synthesis</td></tr><tr><td>Argon Compressed Gas</td><td>Airgas</td><td>AR R300</td><td>Used during PEGaMA synthesis</td></tr><tr><td>8 Arm Poly(ethylene glycol)-hydroxyl (PEG-OH)</td><td>JenKem Technology</td><td>8ARM-PEG-10K</td><td>Used during PEGaMA synthesis</td></tr><tr><td>365 nm Bandpass Filter</td><td>Edmund Optics</td><td>65-191</td><td>Used for photostiffening</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Fisher Scientific</td><td>BP9700-100</td><td>Used during staining process</td></tr><tr><td>Buchner Funnel</td><td>Quark Glass</td><td>QFN-8-14</td><td>Used during PEGaMA synthesis</td></tr><tr><td>Calcein AM</td><td>Invitrogen</td><td>65-0853-39</td><td>Used during staining process</td></tr><tr><td>Celite 545 (Filtration Aid)</td><td>EMD Millipore</td><td>CX0574-1</td><td>Used during PEGaMA synthesis</td></tr><tr><td>Charged Microscope Slides</td><td>Globe Scientific</td><td>1358W</td><td></td></tr><tr><td>Chloroform-d</td><td>Sigma-Aldrich</td><td>151823-10X0.75ML</td><td>Used to characterize PEGaMA</td></tr><tr><td>Click-iT Plus EdU Cell Proliferation Kit</td><td>Invitrogen</td><td>C10637</td><td>Used for staining</td></tr><tr><td>50 mL Conical Tubes</td><td>CELLTREAT</td><td>667050B</td><td></td></tr><tr><td>Cryogenic Safety Kit</td><td>Cole-Parmer</td><td>EW-25000-85</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM 1850-3-1</td><td></td></tr><tr><td>Dialysis Tubing</td><td>Repligen</td><td>132105</td><td></td></tr><tr><td>4&amp;rsquo;,6-Diamidino-2-Phylindole (DAPI)</td><td>Sigma-Aldrich</td><td>D9542-1MG</td><td>Used for staining</td></tr><tr><td>Diethyl Ether</td><td>Fisher Scientific</td><td>E1384</td><td>Used during PEGaMA synthesis</td></tr><tr><td>1,4-Dithiothreitol (DTT)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>10197777001</td><td>Bioink component</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Cytiva</td><td>SH30271.FS</td><td></td></tr><tr><td>Filter Paper</td><td>Whatman</td><td>1001-090</td><td>Used during PEGaMA synthesis</td></tr><tr><td>Freezone 2.5L Freeze Dry System</td><td>Labconco</td><td>LA-2.5LR</td><td>Lyophilizer</td></tr><tr><td>Fusion 360</td><td>Autodesk</td><td>N/A</td><td>Software download</td></tr><tr><td>2.5 mL Gastight Syringe</td><td>Hamilton</td><td>81420</td><td>Used for bioprinting</td></tr><tr><td>15 Gauge 1.5&quot; IT Series Tip</td><td>Jensen Global</td><td>JG15-1.5X</td><td>Used for bioprinting</td></tr><tr><td>30 Gauge 0.5&quot; HP Series Tip</td><td>Jensen Global</td><td>JG30-0.5HPX</td><td>Used for bioprinting</td></tr><tr><td>Goat Anti-Mouse Alexa Fluor 555 Antibody</td><td>Fisher Scientific</td><td>A21422</td><td>Used for staining</td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>C2H5NO2</td><td>Used during staining process</td></tr><tr><td>Hemocytometer</td><td>Fisher Scientific</td><td>1461</td><td></td></tr><tr><td>Hoechst</td><td>Thermo Scientific</td><td>62249</td><td>Used during staining process</td></tr><tr><td>Human Pulmonary Artery Adventitial Fibroblasts (HPAAFs)</td><td>AcceGen</td><td>ABC-TC3773&amp;nbsp;</td><td>From a 2-year-old male patient</td></tr><tr><td>Hydrochloric Acid (HCl)</td><td>Fisher Scientific</td><td>A144-500</td><td>Used to pH adjust solutions</td></tr><tr><td>ImageJ</td><td>National Institutes of Health (NIH)</td><td>N/A</td><td>Free software download</td></tr><tr><td>ImmEdge&amp;reg; Pen</td><td>Vector Laboratories</td><td>H-4000</td><td>Used during staining process</td></tr><tr><td>Incubator</td><td>VWR</td><td>VWR51014991</td><td></td></tr><tr><td>LifeSupport Gelatin Microparticle Slurry (Gelatin Slurry)</td><td>Advanced Biomatrix</td><td>5244-10GM</td><td>Used for bioprinting</td></tr><tr><td>Light Microscope</td><td>Olympus</td><td>CKX53</td><td>Inverted light microscope</td></tr><tr><td>Lithium Phenyl-2,4,6-Trimethylbenzoylphosphinate (LAP)</td><td>Sigma-Aldrich</td><td>900889-5G</td><td>Photoinitiator used for photostiffening</td></tr><tr><td>Liquid Nitrogen</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>LulzBot Mini 2&amp;nbsp;</td><td>LulzBot</td><td>N/A</td><td>Bioprinter adapted</td></tr><tr><td>Methacryloxyethyl Thiocarbamoyl Rhodamine B&amp;nbsp;</td><td>Polysciences Inc.</td><td>669775-30-8</td><td></td></tr><tr><td>2-Methylbutane</td><td>Sigma-Aldrich</td><td>M32631-4L</td><td></td></tr><tr><td>Microman Capillary Pistons CP1000</td><td>VWR</td><td>76178-166</td><td>Positive displacement pipette tips</td></tr><tr><td>MMP2 Degradable Crosslinker (KCGGPQGIWGQGCK)</td><td>GL Biochem</td><td>N/A</td><td>Bioink component</td></tr><tr><td>Mouse Anti-Human &amp;alpha;SMA Monoclonal Antibody</td><td>Fisher Scientific</td><td>MA5-11547</td><td>Used for staining</td></tr><tr><td>OmniCure Series 2000</td><td>&amp;nbsp;Lumen Dynamics</td><td>S2000-XLA</td><td>UV light source used for photostiffening</td></tr><tr><td>Paraformaldehyde (PFA)&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Used to fix samples</td></tr><tr><td>pH Meter</td><td>Mettler Toledo&amp;nbsp;</td><td>FP20&amp;nbsp;</td><td></td></tr><tr><td>pH Strips</td><td>Cytiva</td><td>10362010</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Hyclone Laboratories, Inc.</td><td>Cytiva SH30256.FS</td><td></td></tr><tr><td>Pipette Set</td><td>Fisher Scientific</td><td>14-388-100</td><td></td></tr><tr><td>10 &amp;micro;L Pipette Tips</td><td>USA Scientific</td><td>1120-3710</td><td></td></tr><tr><td>20 &amp;micro;L Pipette Tips</td><td>USA Scientific</td><td>1183-1510</td><td></td></tr><tr><td>200 &amp;micro;L Pipette Tips</td><td>USA Scientific</td><td>1111-0700</td><td></td></tr><tr><td>1000 &amp;micro;L Pipette Tips</td><td>USA Scientific</td><td>1111-2721</td><td></td></tr><tr><td>Poly(Ethylene Glycol)-Alpha Methacrylate (PEG&amp;alpha;MA)</td><td>N/A</td><td>N/A</td><td>Refer to manuscript for synthesis steps</td></tr><tr><td>Poly(Ethylene Oxide) (PEO)</td><td>Sigma-Aldrich</td><td>372773-250G</td><td>Bioink component</td></tr><tr><td>Positive Displacement Pipette</td><td>Fisher Scientific</td><td>FD10004G</td><td>100-1000 &amp;micro;L</td></tr><tr><td>Potassium Hydroxide (KOH)</td><td>Sigma-Aldrich</td><td>221473-500G</td><td>Used to pH adjust solutions</td></tr><tr><td>ProLong Gold Antifade Reagent</td><td>Invitrogen</td><td>P36930</td><td>Used during staining process</td></tr><tr><td>Pronterface</td><td>All3DP</td><td>N/A</td><td>Software download</td></tr><tr><td>Propidium Iodide</td><td>Sigma-Aldrich</td><td>P4864-10ML</td><td>Used for staining</td></tr><tr><td>RGD Peptide (CGRGDS)</td><td>GL Biochem</td><td>N/A</td><td>Bioink component</td></tr><tr><td>Rocker</td><td>VWR</td><td>10127-876</td><td></td></tr><tr><td>Rotary Evaporator&amp;nbsp;</td><td>Thomas Scientific</td><td>11100V2022</td><td>Used during PEGaMA synthesis</td></tr><tr><td>Rubber Band</td><td>Staples</td><td>808659</td><td></td></tr><tr><td>Schlenk Flask&amp;nbsp;</td><td>Kemtech America</td><td>F902450</td><td>Used during PEGaMA synthesis</td></tr><tr><td>Slic3r</td><td>Slic3r</td><td>N/A</td><td>Software download</td></tr><tr><td>Smooth Muscle Cell Growth Medium-2 (SmGM-2) BulletKit</td><td>Lonza</td><td>CC-3182</td><td>Kit contains CC-3181 and CC-4149 components</td></tr><tr><td>Sodium Hydride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>223441-50G</td><td>Used during PEGaMA synthesis</td></tr><tr><td>Sorvall ST 40R Centrifuge</td><td>Fisher Scientific</td><td>75-004-525</td><td></td></tr><tr><td>Stir Bar</td><td>VWR</td><td>58948-091</td><td></td></tr><tr><td>Syringe Filter</td><td>VWR</td><td>28145-483</td><td>Used to sterile filter solutions</td></tr><tr><td>T-75 Tissue-Cultured Treated Flask</td><td>VWR</td><td>82050-856</td><td>Used for cell culture work</td></tr><tr><td>Tissue-Tek Cyromold</td><td>Sakura</td><td>4557</td><td></td></tr><tr><td>Tissue-Tek O.C.T Compound (OCT)</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>Tris(2-Carboxyethyl) Phosphine (TCEP)</td><td>Sigma-Aldrich</td><td>C4706-2G</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher Bioreagents</td><td>C34H622O11</td><td>Used during staining process</td></tr><tr><td>Trypan Blue</td><td>Sigma-Aldrich</td><td>T8154-20ML</td><td>Used for cell culture work</td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Gibco</td><td>25-300-062</td><td>Used for cell culture work</td></tr><tr><td>Tween 20</td><td>Fisher Bioreagents</td><td>C58H114O26</td><td>Used during staining process</td></tr><tr><td>Upright Microscope</td><td>Olympus</td><td>BX63F</td><td>Fluorescent microscope capabilities</td></tr><tr><td>Water Bath</td><td>PolyScience</td><td>WBE20A11B</td><td></td></tr><tr><td>24-Well Tissue Culture Plates</td><td>Corning</td><td>3527</td><td></td></tr></tbody></table>

3d bioprinting phototunable hydrogels to study fibroblast activation

3D Bioprinting Phototunable Hydrogels to Study 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 (PEG&#945;MA) 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.

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 PEG&#945;MA, the backbone of this phototunable polymer system. Nuclear magnetic resonance (NMR) spectroscopy measurements showed successful PEG&#945;MA functionalization at 96.5% (Figure 1). Functionalization values of 90...

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 ...

3d-bioprinting-phototunable-hydrogels-to-study-fibroblast-activation

Research

JoVE Journal

Bioengineering

A bioprinter was used to create patterned hydrogels based on a sacrificial mold.  The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Förster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET Imaging in Three-dimensional Hydrogels

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

This study introduces a novel κ-carrageenan sub-microgel suspension bath, displaying remarkable reversible jamming-unjamming transition properties. These attributes contribute to the construction of biomimetic tissues and organs in embedded 3D bioprinting. The successful printing of heart/esophageal-like tissues with high resolution and cell growth demonstrates high-quality bioprinting and tissue engineering applications.

Embedded Bioprinting of Tissue-like Structures Using &#954;-Carrageenan Sub-Microgel Medium

Learn 3D bioprinting of phototunable hydrogels to study fibroblast activation and extracellular matrix stiffening in lung tissue models. This method enables precise control of mechanical properties for chronic lung disease research.

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

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