Speaker
Description
Introduction.
The growing demand for sustainable meat alternatives has accelerated research on culture meat. However, scalable production of fully edible meat-like structured constructs remains a challenge. In this study, we use chaotic bioprinting, extrusive bioprinting enabled by chaotic advection, using an enzymatic crosslinking agent, transglutaminase (TGase), to fabricate compartmentalized gelatin constructs, which induced an accelerated cellular proliferation and differentiation.
Methods.
We developed a chaotic bioprinting strategy, enabled by static mixer-induced chaotic flows, to co-extrude three distinct inks: a hydroxyethyl cellulose (HEC)/TGase sacrificial ink to create hollow channels, a supprotivesupportive gelatin/TGase ink, and a cell-laden gelatin ink. Using this set of inks, with a 2in4e printhead we fabricated (1) a gelatin/TGase solid fiber, (2) a void channeled fiber with a gelatin ink and a HEC/TGase sacrificial ink, and with a 4in4e we fabricated (3) a supported channeled fiber including the support gelatin/TGase ink (Figure 1A). Structural assessment of printed fibers was performed with fluorescence microscopy. Additionally, biological compatibility was assessed in all printed constructs by loading C2C12 murine myoblasts in (1) one gelatin/TGase bioink, (2) a gelatin-based bioink, and (3) in two gelatin-based bioinks (Figure 1B). Cellular viability, proliferation and tissue maturation were evaluated with Live/Dead assays, Actin/DAPI staining, immunostaining, and RT-qPCR.
Results and discussion.
The use of TGase as crosslinking agent proved successful in the formation and maintenance of structural integrity (Figure 1C) in all the three evaluated conditions. Cell-laden bioprinted constructs showed high proliferation and tissue maturation (Figure 1D-E) while maintaining construct integrity and high cellular viability for up to 35 days.
Our results demonstrate that structural construct stability can be achieved when using TGase enzymatic activity as crosslinking agent for gelatin-based hydrogels when using chaotic bioprinting, amplifying the range of materials that can be used with this technique. Additionally, by maintaining the biocompatible properties of gelatin, the bioprinted constructs demonstrated that cellular proliferation can be achieved within the fiber, eliminating the need for extensive expansion of satellite cells prior to bioprinting. Moreover, the inclusion of hollow channels enhanced cell proliferation and tissue maturation, probably induced by improved diffusion within the fiber.
Conclusion.
This work introduces a scalable bioprinting technique that enables the use of highly biocompatible materials (gelatin), to biofabricate prevascularized microarchitected constructs, creating completely edible scaffolds suitable for cultured meat applications and effectively addressing current challenges in the field.
References:
Bolívar-Monsalve, E. J. et al. Continuous chaotic bioprinting of skeletal muscle-like constructs. Bioprinting 21, (2021).
Bolívar-Monsalve, E. J. et al. One-Step Bioprinting of Multi-Channel Hydrogel Filaments Using Chaotic Advection: Fabrication of Pre-Vascularized Muscle-Like Tissues. Adv Healthc Mater 11, (2022).
Ceballos-González, C. F. et al. Plug-and-Play Multimaterial Chaotic Printing/Bioprinting to Produce Radial and Axial Micropatterns in Hydrogel Filaments. Adv Mater Technol 8, (2023).
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