Professor of Mechanical Engineering Seoul National University, United States
Introduction: : We investigated the effects of freezing conditions on muscle fiber morphology using tissues sampled from store-bought beef, chicken, and salmon. To enable high-throughput assessment, we engineered a custom tissue mold inspired by tissue microarray (TMA) technology. Tissue samples were sectioned en bloc and stained using H&E stain. We observed increased loss of muscle fiber structure when samples were treated with multiple freeze-thaw cycles compared to a single freeze.
Materials and
Methods: : 3D printing: BioMorpho12 tissue mold was created using Autodesk Fusion360 for design and modeling, and Formlabs Form 4 resin printer was used for printing. Test prints in multiple orientations (face-up, face-down, and with varied support placements) were performed to minimize potential damage during printing or support removal. Histology: tissues were sampled using a 3mm biopsy punch and placed into the mold sample well. Each sample well was filled with optimal cutting compound (OCT), placed at -80°C for 5 min, then sectioned using a cryomicrotome at thicknesses 2, 5, 10, and 20 microns. A single section was placed on a glass slide, then placed at different freezing conditions: Condition A, -80°C for 5 days; Condition B, freeze-thaw cycle 3x (1 hour at -80°C, 1 hour at 25°C (RT)); Condition C, not frozen. H&E staining was performed following standard procedures.
Results, Conclusions, and Discussions:: The tissue microarray mold underwent multiple design iterations. Initially, we explored the idea of printing the mold using resin that can be sectioned together with the embedded sample. However, this approach caused tissue damage due to rapid dulling of the cryostat blade. In the final design, BioMorpho12 (Figure 1), we used a rigid resin, and we redesigned the mold so that frozen sample blocks can be ejected and sectioned without the mold. BioMorpho12 measures 22 mm (L) x 15 mm (W) x 8.25 mm (H) and is compatible with cryosectioning, allowing single-pass analysis of up to 12 samples.
Using BioMorpho12, we prepared muscle tissue from beef, chicken, and salmon using two freezing protocols: Condition A (single freeze) and Condition B (three freeze-thaw cycles) (Figure 2). Samples were stained with hematoxylin and eosin (H&E) and examined histologically. Condition A samples displayed longer muscle fibers and less tearing compared to Condition B. Salmon tissue from both conditions lacked distinct muscle fibers, likely because the store-bought specimen had already undergone multiple freeze–thaw cycles resulting in compromised structural integrity. Among all tested species, chicken muscle exhibited the most uniform staining.
In conclusion, we successfully engineered a tissue microarray mold and applied it to evaluate the effects of different freezing conditions on muscle tissue from multiple animal sources. Our results demonstrate that reducing freeze–thaw cycles improves the preservation of muscle morphology. This finding is consistent with previous food science research, which shows that repeated freeze–thaw cycles and slow freezing of meat induce exudate formation, or "drip", where intracellular proteins and water are released due to structural degradation of muscle tissue. Our results provide insight into optimal preservation strategies for musculoskeletal tissues.
Acknowledgements and/or References (Optional): : Acknowledgements: This work was supported by Seon Ki Kim and Professor Noo Li Jeon from the Department of Mechanical Engineering at Seoul National University, Seoul, South Korea.
References: 1) Leonard, K. C., Worden, N., Boettcher, M. L., Dickinson, E., & Hartstone-Rose, A. Anatomical Record (Hoboken), 2022, 305, 199–208. (Effects of Freezing and Short-Term Fixation on Muscle Mass, Volume, and Density) 2) Ko, J., Park, D., Lee, S., Gumuscu, B., & Jeon, N. L. (2022). Engineering Organ-on-a-Chip to Accelerate Translational Research. Micromachines, 13(8), 1200. https://doi.org/10.3390/mi13081200
