Name: Preparation of Phototunable Hydrogel Bioink
Uploaded: 2023-06-30T12:20:00
Description: Watch this Scientific Journal Video about Preparation of Phototunable Hydrogel Bioink at JoVE.com

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.

Research

JoVE Journal

Bioengineering

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...