Optical Clearing of Skeletal Muscle Bundles Engineered in 3-D Printed Templates.
Publication Year:
2021
PubMed ID:
32748107
Funding Grants:
Public Summary:
Scientists are creating 3D muscle tissues in the lab to better study muscle diseases and test treatments, but current methods are often slow and need special tools. This study used a regular 3D printer to quickly make templates that help grow these muscle bundles more easily. They also used new imaging techniques to see the entire 3D muscle structure clearly, instead of just thin slices. While the clearing process for imaging caused some tissue damage, these improvements make it faster and easier to study muscle tissues and could help with finding new treatments.
Scientific Abstract:
Many techniques for engineering and interrogating three-dimensional (3-D) muscle bundles from animal- or patient-derived myoblasts have recently been developed to overcome the limitations of existing in vitro and in vivo model systems. However, many approaches for engineering 3-D muscle bundles rely on specialized and time-consuming techniques, such as photolithography for fabrication and cryosectioning for histology. Cryosectioning also limits visualization to a single plane instead of the entire 3-D structure. To address these challenges, we first implemented a consumer-grade 3-D-printer to rapidly prototype multiple templates for engineering muscle bundles. We then employed our templates to engineer 3D muscle bundles and identify template geometries that promoted bundle survival over three weeks. Subsequently, we implemented tissue clearing, immunostaining, and confocal imaging to acquire z-stacks of intact muscle bundles labelled for myogenic markers. With this approach, we could select the imaging plane on-demand and visualize the intact 3-D structure of bundles. However, tissue clearing did cause some tissue degradation that should be considered. Together, these advances in muscle tissue engineering and imaging will accelerate the use of these 3-D tissue platforms for disease modeling and therapeutic discovery.