preparation of phototunable hydrogel bioink

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

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.

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.

Watch this Scientific Journal Video about Preparation of Phototunable Hydrogel Bioink at JoVE.com

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.

preparation-of-phototunable-hydrogel-bioink

Research

JoVE Journal

Bioengineering

262 Views.  University of Colorado Denver | Anschutz Medical Campus. 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...

Video: Preparation of Phototunable Hydrogel Bioink

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 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, 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.