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Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation - Poster Session D

Poster F3 - Non-destructive cell detection with ultrasound on decellularized spinach scaffolds

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Advancements in cellular agriculture offer promising solutions to the world hunger crisis, such as the creation of cultured meat. Decellularized plants have been used as scaffolds for growing skeletal muscle cells, but this process is currently inefficient and difficult to do on a large scale. The use of bioreactors has been proposed to scale up the decellularization and cell growth processes. Monitoring the cell density is critical for scale-up. However, scaffolds must be destroyed in order to effectively track the progression of decellularization and recellularization. This study aims to demonstrate that ultrasound technology can be used to continuously monitor the entire cellular agriculture process in a non-destructive manner.

Materials and

Methods:  Spinach leaves were used to develop the use of ultrasound to detect cellular levels on plant scaffolds. A Clarius L20 ultrasound probe mounted 3.5 cm from the benchtop was submerged approximately 1 mm into a clear basin filled with 290 mL of PBS solution, 0.5 cm distant from the samples as seen in Figure 2B. Images were taken every 5 seconds. The samples were mounted with alligator clamps at a height approximately 2 cm from the benchtop. The leaves were cut into ten 1 cm x 3 cm rectangles, and ultrasound images were taken. The samples were then decellularized by placing samples in 1x SDS solution for 5 days, followed by 1% Tween-20 3% Bleach solution for 2 days. Partially decellularized leaves were imaged after 5 days in the SDS solution and fully decellularized samples were imaged after the full 7 day decellularization procedure. The scaffolds were then sterilized using 70% ethanol. NIH 3T3 cells were seeded on the sterilized scaffolds using 1 cm diameter cloning wells, which were filled with 5% penicillin 10% FBS Dulbecco’s Modified Eagle Medium cell culture media. 48 hours following cell seeding, ultrasound images were taken of the samples. Then, ultrasound images were analyzed based on mean pixel intensity using the Waikato Environment for Knowledge Analysis (Weka), a semi-automatic image segmentation software available through ImageJ.

Results, Conclusions, and Discussions: Analysis of the images comparing the fresh and decellularized leaves showed statistically different pixel intensities before and after the decellularization process (p < 0.001) . The fresh leaves had an average pixel intensity of 232.25 ± 3.57, and the decellularized leaves had an average intensity of 86.13 ± 16.63. Similar results were observed on the recellularized scaffolds. The cloning well cell clusters had statistically higher average pixel intensity of 121.46 ± 28.82 compared to the decellularized regions (81.95 ± 13.02), (p = 0.001).Preliminary visual analysis and mean pixel analysis indicate that ultrasound can be used to distinguish between fresh, decellularized and recellularized spinach scaffolds. This method can potentially be integrated into a bioreactor and provide continuous monitoring of the cell growth process. Further development is necessary to optimize the method for such application.

Acknowledgements (Optional):