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

<ol>
	<li>Ahrens, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programming+cellular+alignment+in+engineered+cardiac+tissue+via+bioprinting+anisotropic+organ+building+blocks.">Programming cellular alignment in engineered cardiac tissue via bioprinting anisotropic organ building blocks.</a> Advanced Materials. 34 (26), e2200217 (2022).</li><li>Lin, N. Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+reabsorption+in+3D+vascularized+proximal+tubule+models.">Renal reabsorption in 3D vascularized proximal tubule models.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (12), 5399-5404 (2019).</li><li>Grigoryan, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivascular+networks+and+functional+intravascular+topologies+within+biocompatible+hydrogels.">Multivascular networks and functional intravascular topologies within biocompatible hydrogels.</a> Science. 364 (6439), 458-464 (2019).</li><li>Kang, Y., Datta, P., Shanmughapriya, S., Ozbolat, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+bioprinting+of+tumor+models+for+cancer+research.">3D bioprinting of tumor models for cancer research.</a> ACS Applied Biomaterials. 3 (9), 5552-5573 (2020).</li><li>Davis-Hall, D., Thomas, E., Pena, B., Magin, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-bioprinted,+phototunable+hydrogel+models+for+studying+adventitial+fibroblast+activation+in+pulmonary+arterial+hypertension.">3D-bioprinted, phototunable hydrogel models for studying adventitial fibroblast activation in pulmonary arterial hypertension.</a> Biofabrication. 15 (1), (2022).</li><li>Mirdamadi, E., Tashman, J. W., Shiwarski, D. J., Palchesko, R. N., Feinberg, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRESH+3D+bioprinting+of+a+full-size+model+of+the+human+heart.">FRESH 3D bioprinting of a full-size model of the human heart.</a> ACS Biomaterials Science &amp; Engineering. 6 (11), 6453-6459 (2020).</li><li>Shiwarski, D. J., Hudson, A. R., Tashman, J. W., Feinberg, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+FRESH+3D+printing+as+a+platform+for+advanced+tissue+biofabrication.">Emergence of FRESH 3D printing as a platform for advanced tissue biofabrication.</a> APL Bioengineering. 5 (1), 010904 (2021).</li><li>Petrou, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clickable+decellularized+extracellular+matrix+as+a+new+tool+for+building+hybrid+hydrogels+to+model+chronic+fibrotic+diseases+in+vitro.">Clickable decellularized extracellular matrix as a new tool for building hybrid hydrogels to model chronic fibrotic diseases in vitro.</a> Journal of Materials Chemistry B. 8 (31), 6814-6826 (2020).</li><li>Hewawasam, R. S., Blomberg, R., Serbedzija, P., Magin, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modification+of+human+decellularized+extracellular+matrix+for+incorporation+into+phototunable+hybrid+hydrogel+models+of+tissue+fibrosis.">Chemical modification of human decellularized extracellular matrix for incorporation into phototunable hybrid hydrogel models of tissue fibrosis.</a> ACS Applied Materials &amp; Interfaces. 15 (12), 15071-15083 (2023).</li><li>Saleh, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+hybrid+hydrogels+comprised+healthy+or+diseased+decellularized+extracellular+matrix+to+study+pulmonary+fibrosis.">Engineering hybrid hydrogels comprised healthy or diseased decellularized extracellular matrix to study pulmonary fibrosis.</a> Cellular and Molecular Bioengineering. 15 (5), 505-519 (2022).</li><li>Guvendiren, M., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiffening+hydrogels+to+probe+short-+and+long-term+cellular+responses+to+dynamic+mechanics.">Stiffening hydrogels to probe short- and long-term cellular responses to dynamic mechanics.</a> Nature Communications. 3, 792 (2012).</li><li>Rosales, A. M., Vega, S. L., DelRio, F. W., Burdick, J. A., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+with+reversible+mechanics+to+probe+dynamic+cell+microenvironments.">Hydrogels with reversible mechanics to probe dynamic cell microenvironments.</a> Angewandte Chemie International Edition English. 56 (40), 12132-12136 (2017).</li><li>Wynn, T. A., Ramalingam, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+fibrosis:+therapeutic+translation+for+fibrotic+disease.">Mechanisms of fibrosis: therapeutic translation for fibrotic disease.</a> Nature Medicine. 18 (7), 1028-1040 (2012).</li><li>Huertas, A., Tu, L., Humbert, M., Guignabert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+inflammation+within+the+vascular+wall+in+pulmonary+arterial+hypertension:+more+than+a+spectator.">Chronic inflammation within the vascular wall in pulmonary arterial hypertension: more than a spectator.</a> Cardiovascular Research. 116 (5), 885-893 (2020).</li><li>Kendall, R. T., Feghali-Bostwick, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+in+fibrosis:+novel+roles+and+mediators.">Fibroblasts in fibrosis: novel roles and mediators.</a> Frontiers in Pharmacology. 5, 123 (2014).</li><li>Parker, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrotic+extracellular+matrix+activates+a+profibrotic+positive+feedback+loop.">Fibrotic extracellular matrix activates a profibrotic positive feedback loop.</a> The Journal of Clinical Investigation. 124 (4), 1622-1635 (2014).</li><li>Habiel, D. M., Hogaboam, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+fibroblast+proliferation+and+survival+in+idiopathic+pulmonary+fibrosis.">Heterogeneity in fibroblast proliferation and survival in idiopathic pulmonary fibrosis.</a> Frontiers in Pharmacology. 5, 2 (2014).</li><li>Hu, C. J., Zhang, H., Laux, A., Pullamsetti, S. S., Stenmark, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+contributing+to+persistently+activated+cell+phenotypes+in+pulmonary+hypertension.">Mechanisms contributing to persistently activated cell phenotypes in pulmonary hypertension.</a> The Journal of Physiology. 597 (4), 1103-1119 (2019).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+fibroblasts+with+a+proinflammatory+epigenetically+altered+phenotype+in+severe+hypoxic+pulmonary+hypertension.">Emergence of fibroblasts with a proinflammatory epigenetically altered phenotype in severe hypoxic pulmonary hypertension.</a> The Journal of Immunology. 187 (5), 2711-2722 (2011).</li><li>Hinton, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+printing+of+complex+biological+structures+by+freeform-reversible+embedding+of+suspended+hydrogels.">Three-dimensional printing of complex biological structures by freeform-reversible embedding of suspended hydrogels.</a> Science Advances. 1 (9), e1500758 (2015).</li><li>Brown, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+photocrosslinking+of+click+hydrogels+to+probe+myoblast+mechanotransduction+in+three+dimensions.">Secondary photocrosslinking of click hydrogels to probe myoblast mechanotransduction in three dimensions.</a> Journal of the American Chemical Society. 140 (37), 11585-11588 (2018).</li><li>Ondeck, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamically+stiffened+matrix+promotes+malignant+transformation+of+mammary+epithelial+cells+via+collective+mechanical+signaling.">Dynamically stiffened matrix promotes malignant transformation of mammary epithelial cells via collective mechanical signaling.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (9), 3502-3507 (2019).</li><li>Caliari, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiffening+hydrogels+for+investigating+the+dynamics+of+hepatic+stellate+cell+mechanotransduction+during+myofibroblast+activation.">Stiffening hydrogels for investigating the dynamics of hepatic stellate cell mechanotransduction during myofibroblast activation.</a> Scientific Reports. 6, 21387 (2016).</li><li>Liu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedback+amplification+of+fibrosis+through+matrix+stiffening+and+COX-2+suppression.">Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression.</a> Journal of Cell Biology. 190 (4), 693-706 (2010).</li><li>Tschumperlin, D. J., Ligresti, G., Hilscher, M. B., Shah, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensing+and+fibrosis.">Mechanosensing and fibrosis.</a> The Journal of Clinical Investigation. 128 (1), 74-84 (2018).</li><li>Chelladurai, P., Seeger, W., Pullamsetti, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinases+and+their+inhibitors+in+pulmonary+hypertension.">Matrix metalloproteinases and their inhibitors in pulmonary hypertension.</a> European Respiratory Journal. 40 (3), 766-782 (2012).</li><li>Caracena, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+epithelial+cells+and+microenvironmental+stiffness+synergistically+drive+fibroblast+activation+in+three-dimensional+hydrogel+lung+models.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome-wide+analysis+of+cellular+response+to+ultraviolet+light+for+biomaterial+synthesis+and+modification.">Proteome-wide analysis of cellular response to ultraviolet light for biomaterial synthesis and modification.</a> ACS Biomaterials Science &amp; Engineering. 5 (5), 2111-2116 (2019).</li><li>Kruse, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+pH+on+cell+viability,+cell+migration,+cell+proliferation,+wound+closure,+and+wound+reepithelialization:+In+vitro+and+in+vivo+study.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusable+biohybrid+designs+for+bioprinted+skeletal+muscle+tissue.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+3D+culture+and+bioprinting+using+peptide-functionalized+hyaluronan+hydrogels.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprinting+a+skin+patch+with+dual-crosslinked+gelatin+(GelMA)+and+silk+fibroin+(SilMA):+An+approach+to+accelerating+cutaneous+wound+healing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-bioprinted+human+tissue+and+the+path+toward+clinical+translation.">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

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...

Watch this Scientific Journal Video about Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels at JoVE.com

Author Spotlight: Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels 

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

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

Research

JoVE Journal

Bioengineering

1.6K Views.  University of Colorado Denver | Anschutz Medical Campus. This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation. 

Video: Author Spotlight: Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels 

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

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

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.

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

Immunology and Infection

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

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 has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

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

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

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 is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Advancing 3D Bioprinting with &#954;-Carrageenan Sub-Microgel Medium

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

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic scaffolds. However, engineering a suitable gel suspension medium that balances precise bioink deposition with cell viability and function presents multiple challenges, particularly in achieving the desired viscoelastic properties. Here, a novel &#954;-carrageenan gel supporting bath is fabricated through an easy-to-operate mechanical grinding process, producing homogeneous sub-microscale particles. These sub-microgels exhibit typical Bingham flow behavior with small yield stress and rapid shear-thinning properties, which facilitate the smooth deposition of bioinks. Moreover, the reversible gel-sol transition and self-healing capabilities of the &#954;-carrageenan microgel network ensure the structural integrity of printed constructs, enabling the creation of complex, multi-layered tissue structures with defined architectural features. Post-printing, the &#954;-carrageenan sub-microgels can be easily removed by a simple phosphate-buffered saline wash. Further bioprinting with cell-laden bioinks demonstrates that cells within the biomimetic constructs have a high viability of 92% and quickly extend pseudopodia, as well as maintain robust proliferation, indicating the potential of this bioprinting strategy for tissue and organ fabrication. In summary, this novel &#954;-carrageenan sub-microgel medium emerges as a promising avenue for embedded bioprinting of exceptional quality, bearing profound implications for the in vitro development of engineered tissues and organs.

Author Spotlight: Development of Homogeneous &#954;-Carrageenan Sub-Microgel Baths for High-Resolution 3D Bioprinting

This study introduces a novel &#954;-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 three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic ...

Engineering

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

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

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

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.

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

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

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

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

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

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

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

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.

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster 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.

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Summary

Explore More Videos

Chapters in this video

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

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

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

Embedded Bioprinting of Tissue-like Structures Using κ-Carrageenan Sub-Microgel Medium

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

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

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

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

FRET Imaging in Three-dimensional Hydrogels