Laser-Etched Designs for Molding Hydrogel-Based Engineered Tissues

Abstract

Rapid prototyping and fabrication of elastomeric molds for sterile culture of engineered tissues allow for the development of tissue geometries that can be tailored to different in vitro applications and customized as implantable scaffolds for regenerative medicine. Commercially available molds offer minimal capabilities for adaptation to unique conditions or applications versus those for which they are specifically designed. Here we describe a replica molding method for the design and fabrication of poly(dimethylsiloxane) (PDMS) molds from laser-etched acrylic negative masters with ∼0.2 mm resolution. Examples of the variety of mold shapes, sizes, and patterns obtained from laser-etched designs are provided. We use the patterned PDMS molds for producing and culturing engineered cardiac tissues with cardiomyocytes derived from human-induced pluripotent stem cells. We demonstrate that tight control over tissue morphology and anisotropy results in modulation of cell alignment and tissue-level conduction properties, including the appearance and elimination of reentrant arrhythmias, or circular electrical activation patterns. Techniques for handling engineered cardiac tissues during implantation in vivo in a rat model of myocardial infarction have been developed and are presented herein to facilitate development and adoption of surgical techniques for use with hydrogel-based engineered tissues. In summary, the method presented herein for engineered tissue mold generation is straightforward and low cost, enabling rapid design iteration and adaptation to a variety of applications in tissue engineering. Furthermore, the burden of equipment and expertise is low, allowing the technique to be accessible to all.

Practical adoption of state-of-the-art hiPSC-cardiomyocyte differentiation techniques

“…Thirdly, because ECT electromechanical function is closely linked to cardiomyocyte purity [19,33], the purity of control and lactate purified ECTs, which fall on either side of the ideal cardiomyocyte purity of roughly 50-60% [19], may mask changes resulting from cardiomyocyte bioenergetic phenotype. Finally, after lactate purification, tissues were returned to a glucose-based medium and lacked fatty acid supplementation, which has recently been shown to support electromechanical maturation [16,34]. Our findings suggest that though lactate purification changes hiPSC-cardiomyocyte metabolic phenotype, a corresponding matured electromechanical phenotype is not present after short culture periods (1 week) in standard glucosecontaining medium (RPMI/B27).…”

“…[15] Briefly, hiPSCcardiomyocytes were harvested at day 24 of differentiation and combined with 1.25 mg/mL rat-tail collagen-1 at 1x10 6 cells per tissue + 5% hCFs. ECTs were cultured under static stress [16] in RPMI/B27 with 1% penicillin-streptomycin and field-stimulated at a 1 Hz (IonOptix C-pace EP). ECTs were cultured for one week before further experiments.…”

IGF1 and NRG1 Enhance Proliferation, Metabolic Maturity, and the Force-Frequency Response in hESC-Derived Engineered Cardiac Tissues

“…Custom acrylic and polydimethylsiloxane (PDMS) molds were produced for ECT culture as previously reported [ 13 ]. Negative templates for tissue molds were created using a 100 W CO 2 laser to etch a pattern generated in Adobe Illustrator into 1/4” acrylic (TAP Plastics).…”

Digital Design and Automated Fabrication of Bespoke Collagen Microfiber Scaffolds

“…While often effective at inducing alignment, these methods either necessitate tissue fenestrations (reducing the efficacy and efficiency of tissues with function related to mechanics and structure) or prescribe high-aspect-ratio tissues with limited utility as replacement tissue patches. [2][3][4][5] Furthermore, this approach does not enable the design of the mechanical material anisotropy that is found in these native tissues and extracellular matrices (ECMs, often a defining feature of these tissues and materials), which plays an important role in cell and tissue development. 6,7 In recent years, a number of approaches to emulating ECM structural and mechanical cues in 3D engineered tissues have been used, including both aligned and random (unorganized) nanofiber mats, [8][9][10][11] aligned pores through directional freezing, [12][13][14][15] sphere-and rod-templated scaffolds, 16,17 and 3D printed scaffolds, [18][19][20][21] in a wide array of shapes.…”