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).

1. PEG&#945;MA synthesis and characterization
NOTE: Poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) synthesis was adapted from Hewawasam et al. and performed under moisture-free conditions9.
<ol>
	<li>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).</li>
	<li>Add a stir bar to 250 mL Schlenk flask and purge with ...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#8217;,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)&#160;</td>
			<td >Sigma-Aldrich</td>
			<td>10197777001</td>
			<td>Bioink component</td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td >Cytiva</td>
			<td >SH30271.FS</td>
			<td></td>
		</tr>
		<tr >
			<td >Ethyl 2-(Bromomethyl)Acrylate (EBrMA)</td>
			<td >Ambeed Inc.</td>
			<td >A918087-25g</td>
			<td>Used during PEGaMA synthesis</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&#34; 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&#34; 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&#160;</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&#174; 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&#160;</td>
			<td >LulzBot</td>
			<td >N/A</td>
			<td>Bioprinter adapted</td>
		</tr>
		<tr >
			<td >Methacryloxyethyl Thiocarbamoyl Rhodamine B&#160;</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 &#945;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 >&#160;Lumen Dynamics</td>
			<td >S2000-XLA</td>
			<td>UV light source used for photostiffening</td>
		</tr>
		<tr >
			<td >Paraformaldehyde (PFA)&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Used to fix samples</td>
		</tr>
		<tr >
			<td >pH Meter</td>
			<td>Mettler Toledo&#160;</td>
			<td>FP20&#160;</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 &#181;L Pipette Tips</td>
			<td>USA Scientific</td>
			<td>1120-3710</td>
			<td></td>
		</tr>
		<tr >
			<td >20 &#181;L Pipette Tips</td>
			<td>USA Scientific</td>
			<td>1183-1510</td>
			<td></td>
		</tr>
		<tr >
			<td >200 &#181;L Pipette Tips</td>
			<td>USA Scientific</td>
			<td>1111-0700</td>
			<td></td>
		</tr>
		<tr >
			<td >1000 &#181;L Pipette Tips</td>
			<td>USA Scientific</td>
			<td>1111-2721</td>
			<td></td>
		</tr>
		<tr >
			<td >Poly(Ethylene Glycol)-Alpha Methacrylate (PEG&#945;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 &#181;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&#160;</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&#160;</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&#160;</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.

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

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

We engineered 3D models of lung tissue to help the world breathe easier. Our mission is to use 3D biomaterials and techniques like 3D printing to build platforms that help us understand what is causing chronic lung diseases and how to better treat these conditions. One experimental challenge in our research is the difficulty of 3D printing soft materials into complex shapes.We precisely deposit cells by printing our bioink and tissue thinning support bath that helps maintain their shape during the printing process. Our biomaterials provide control over sample mechanical properties throughout the entire course of the experiment. This formulation allows researchers to grow cells in a hydrogel that matches the stiffness of both healthy and diseased lung tissue.Users can decide when to stiffen the samples and measure cellular responses. The photo-tunable biomaterials used in this protocol allow for a softened model to be created, which can later be stiffened to explore cellular responses to microenvironmental stiffening. Similar techniques that use a static biomaterial instead of a photo-tunable biomaterial result in a model that cannot be stiffened after printing.Results from other studies in our lab suggests sex and age play critical roles in lung disease progression. Future models will study these variables and include more complexity, like additional cell-specific layers. Coupling these efforts will result in more realistic bioprints and lead to new ways of treating fibrotic diseases.

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

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

Preparation of Phototunable Hydrogel Bioink

Begin the preparation of the hydrogel bioink aseptically inside a biosafety cabinet by first preparing a 0.25 milligrams per milliliter stock solution of PEG alpha methacrylate in sterile cell culture media without FBS. Also, using 20-millimolar sterile TCEP, prepare 250-millimolar stock solutions of DTT, MMP2 degradable crosslinker, and the RGD peptide. Finally, prepare a 15 weight percent stock solution of PEO using distilled water.Combine the required amounts of the prepared reagent stock solutions and low-serum cell culture media with the fibroblasts in a 15-milliliter conical tube. Mix the resultant bioink thoroughly using a positive displacement pipette to ensure the cells are single. Verify that the final precursor solution has a pH of 6.2.Remove the print head from the bioprinter to access the glass syringe. To load the bioink into the glass syringe, remove the syringe plunger. Then, collect the bioink from the centrifuge tube using a separate syringe fitted to a 1.5-inch-long, 15-gauge, blunt-tip needle.Replace the 15-gauge needle with a 30-gauge, 0.5-inch-long, blunt-tip needle and transfer it to the glass syringe, avoiding air bubble formation. Finally, place the glass syringe within the print head and attach the print head components while ensuring a firm assembly for printing.

3D Bioprinting, Culturing, and Photostiffening of Phototunable Hydrogel

To begin 3D printing of the phototunable hydrogel constructs, place the glass syringe with the hydrogel bio-ink containing fibroblasts within the printhead and attach the printhead components while ensuring a firm assembly for printing. Then using the directional arrows in the Pronterface software, manually and carefully adjust the extrusion needle's position within the support bath slurry in the center of the well of the well plate. Leave at least one millimeter of slurry below the needle tip.Once the needle tip is correctly placed, press the print button in the Pronterface software and wait for the printing to complete. Repeat the previously demonstrated steps until the desired number of bioprinted constructs are obtained. Following printing, leave the well plate with the constructs covered at room temperature in the biosafety cabinet for one hour.This allows for base catalyzed polymerization to occur with the phototunable hydrogel bio-ink. Then place the well plate with the 3D bioprinted constructs in a 37 degree Celsius sterile incubator for 12 to 18 hours to melt the support bath slurry. Next, inside a biosafety cabinet, change the media surrounding the bioprinted constructs.To do so, manually remove the media and the melted gelatin support bath from the wells without disturbing the constructs. Then add an appropriate volume of low-serum media to each well. Again, 24 hours prior to the desired stiffening time point, replace media in the wells with low-serum media supplemented with 2.2 millimolar sterile LAP.At the desired stiffening time point, remove half of the media from the wells to be stiffened and place the plate without the lid under the ultraviolet or UV light of an OmniCure. Turn on the UV light with a 365 nanometer band-pass filter to stiffen the constructs for five minutes. Once stiffened, remove the remaining media from these wells before adding fresh low-serum media to each well.Return the plate to the incubator until performing the fibroblast activation study at the desired time point, The combination of an acidic bio-ink and basic support bath slurry facilitated the polymerization of the 3D bioprinted constructs and maintained the cylindrical structures. Microscopy of fluorescently-labeled hydrogel showed pores within the hydrogel induced by gelatin microparticles in the support bath. Confocal microscopy showed spatial control over-stiffening in 3D.Fibroblast viability assays revealed that constructs with 300 micron wall thickness, and having 4 million cells per milliliter outperformed all other conditions at every time point. Viability peaked on day seven, with about 91%of the cells staining live, and by day 14, 85%were still viable.

962 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. 

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

Summary