Direct Production of Human Cardiac Tissues by Pluripotent Stem Cell Encapsulation in Gelatin Methacryloyl

Abstract

Direct stem cell encapsulation and cardiac differentiation within supporting biomaterial scaffolds are critical for reproducible and scalable production of the functional human tissues needed in regenerative medicine and drug-testing applications. Producing cardiac tissues directly from pluripotent stem cells rather than assembling tissues using pre-differentiated cells can eliminate multiple cell-handling steps that otherwise limit the potential for process automation and production scale-up. Here we asked whether our process for forming 3D developing human engineered cardiac tissues using poly­(ethylene glycol)-fibrinogen hydrogels can be extended to widely used and printable gelatin methacryloyl (GelMA) hydrogels. We demonstrate that low-density GelMA hydrogels can be formed rapidly using visible light (<1 min) and successfully employed to encapsulate human induced pluripotent stem cells while maintaining high cell viability. Resulting constructs had an initial stiffness of approximately 220 Pa, supported tissue growth and dynamic remodeling, and facilitated high-efficiency cardiac differentiation (>70%) to produce spontaneously contracting GelMA human engineered cardiac tissues (GEhECTs). GEhECTs initiated spontaneous contractions on day 8 of differentiation, with synchronicity, frequency, and velocity of contraction increasing over time, and displayed developmentally appropriate temporal changes in cardiac gene expression. GEhECT-dissociated cardiomyocytes displayed well-defined and aligned sarcomeres spaced at 1.85 ± 0.1 μm and responded appropriately to drug treatments, including the β-adrenergic agonist isoproterenol and antagonist propranolol, as well as to outside pacing up to 3.0 Hz. Overall results demonstrate that GelMA is a suitable biomaterial for the production of developing cardiac tissues and has the potential to be employed in scale-up production and bioprinting of GEhECTs.

Synthesis and Properties of Gelatin Methacryloyl (GelMA) Hydrogels and Their Recent Applications in Load-Bearing Tissue

“…3D scaffold of GelMA hydrogels can guide the formation of a desired tissue due to their attachment sites and signaling cues. A variety of applications of GelMA hydrogels had been reported in tissue engineering, such as bones [44,45,46,47,48,49,50,51,52,53,54,55,56,57], endochondral bone [58,59], skin [60,61,62,63,64,65], myocardium [66], cardiac tissues [67,68,69,70,71,72], cartilage [42,73,74,75,76,77,78,79,80,81], vascular networks [82,83,84,85,86,87,88,89], skeletal muscle [90,91,92,93], cornea [94,95], interface [96] and so on. Ovsianikov A et al [44] prepared 3D CAD scaffolds for tissue engineering applications using two-photon polymerization (2PP).…”

Rapid Production of Cell‐Laden Microspheres Using a Flexible Microfluidic Encapsulation Platform

“…Using the established microfluidic encapsulation platform, microspheres have been produced using multiple photocrosslinkable polymers that have been widely employed for various tissue engineering applications . Here, we examined the photocrosslinkable hydrogel materials PF, GelMA, and PEGDA; these materials have been used for cancer tissue engineering, cardiac tissue engineering, and bone regeneration . These polymers were selected to demonstrate the compatibility of this setup with various polymers.…”

Mechanical Characterization for Cellular Mechanobiology: Current Trends and Future Prospects

“…For instance, using fluorescent bead density as a readout for stiffness gradients in gels instead of using expensive techniques like AFM (Barber-Pérez et al, 2020). Recent technology also allows assessment of mechanical properties of materials with extremely precise force (resolution down to 10 nN claimed by the manufacturer) through micro-scale compression testing, particularly for soft biomaterials (Kerscher et al, 2017).…”