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>
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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.

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 

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

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 ...

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 ...

Traction Force Microscopy to Study B Lymphocyte Activation

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements from an experimentally observed displacement field produced by a cell pulling on an elastic substrate. Here, we adapted TFM to investigate the spatial and temporal structure of the force field exerted by B cells when activated by antigen engagement of the B cell receptor. Gel rigidity, bead density, and protein functionalization must be optimized for the study of relatively small cells (~ 6 &#181;m) that interact with, and respond specifically to ligands for cell surface receptors.

Here we present a protocol used to perform traction force microscopy experiments on B cells. We describe the preparation of soft polyacrylamide gels and their functionalization, as well as data acquisition at the microscope and a summary of data analysis.

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements ...

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 ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

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Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue