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Nonadhesive micro-Mold for Enhancing Cell Alignment of Myotubes


Significant efforts have been made to develop various molds for application in tissue engineering and biofabrication. A range of factors affect the behaviors of cells on a substrate, including surface chemistry, feature geometry, and elastic modulus. The ability to control the position of cells in an organized pattern on a substrate has become increasingly crucial for the development of tissue engineering. With the advent of microfabrication techniques, the effects of micro-scale grooved models on cell alignment, elongation, migration, morphology, and orientation have been studied. When seeded into nonadhesive micro-molds (MM), cells self-assemble via the action of cytoskeletal-mediated contraction and cell-cell adhesion. The size and shape of the tissue is a function of the cell type and the size, shape, and obstacles of the MMs. In this context, an approach known as modular tissue engineering focuses on biofabricating tissues using cells or spheroids as building blocks with specific microarchitectural features.

Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados

The objective of our study is to microfabricate microtissues with cellular alignment, homogenous size, and shape using MMs fabricated by 3D printing technology. Methodology: First of all, the molds were designed an array with micro patterns using Rhinoceros software generating a Stereolithographic (STL) file. The patterns of MMs are 600 μm high and 400 μm wide. After that, the MMs were materialized by additive manufacturing (AM) or also 3D printing. The STL file was carried out into the 3D printer machine. The AM machine is the Polyjet Objet Connex 350 by Stratasys. The 3D printer jets and instantly UV-cures tiny droplets of liquid photopolymer commercially named VeroClear. Results: The MMs were designed to be used in both 6, 12 and 24-wells culture plates. When the gelled agarose already as a positive mold is removed from the negative MM, it is transferred to the standard 6, 12 or 24 wells tissue culture dishes and equilibrated with cell culture medium for cell seeding. C2C12 cell line - a subclone of myoblasts - was used to seed the MMs, and we have observed the morphology, migration, spheroid formation and alignment. The results suggested that cells can contribute to understanding the myotube formation (myogenesis), among other biological processes.

Considerações Finais/Final considerations/Consideraciones finales

The MMs produced during this study was successful in creating an environment to promote the cell alignment and growth of 3D micro-tissues, in a relatively inexpensive, user-friendly, and efficient nonadhesive MM as compared to other methods. This method enables the inclusion of topographical cues into 2D or 3D cultures to generate relevant physiological models to study differentiation processes of cells.

Palavras-chave/Key words/Palabras clave

Biofabrication, Micro-molds, Tissue spheroids, 3D printing, Myotubes.




JANAINA DE ANDREA DERNOWSEK, Renata Aquino de Carvalho, André Capaldo Amaral, Jorge Vicente Lopes da Silva