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Tissue Engineering

Tissue Engineering - Poster Session E

Poster L19 - Using Dielectric Impedance Spectroscopy to Monitor Bioinks in Real-Time During 3D Bioprinting

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  3D bioprinting is a rapidly advancing field with the potential to help thousands. Before any 3D bioprinted objects can be implanted into patients, it is critical to understand the various cellular properties of the 3D bioprinted construct. Currently, there is no non-destructive way to assess these cellular characteristics, causing multiple constructs to be created and destroyed, wasting time and money [1]. The smart syringe, a syringe attachment consisting of two electrodes, offers a solution to this issue by providing a non-destructive, real-time analysis of bioinks during the bioprinting process through dielectric impedance spectroscopy (DIS) (Figure 1) [2].

Our device, the smart syringe, has undergone numerous proof-of-concept studies to monitor varying cell concentrations and viabilities of cells in phosphate-buffered saline. However, until now, all experiments with the smart syringe have yet to be conducted during a 3D bioprinting process with bioink. The smart syringe analyzes bioprints through impedance, a measurement sensitive to cell concentration, viability, cell type, and size. However, the effect of movement, such as travel paths during bioprinting, on impedance measurements has not been assessed. This project aims first to assess the potential impacts of motion from the 3D bioprinting process on impedance collection over time and, secondly, to determine whether real-time impedance measurements can monitor the presence of cells in bioink samples during the bioprinting process

Materials and

Methods:  This study used 1wt/v% calcium alginate bioink that was resuspended from powder (high molecular weight PRONOVA®) in DIH2O. The alginate solution was crosslinked with 30mM CaCl2 in a 7:3 ratio. The smart syringe was attached to a Digilent impedance analyzer, which emitted a constant frequency of 10kHz with 1kΩ and 50mV.

To determine if motion from the bioprinting process affects impedance readings from the smart syringe, calcium alginate bioink was bioprinted to form various shapes: straight lines and squares. Impedance was collected and normalized for each print. A mixed model was applied to all bioprinted shapes with cell and no cell containing bioinks to determine potential differences amongst impedance measurements of various prints. Two bioinks were prepared to analyze the ability of the smart syringe to detect changes in cell densities. In addition, n=1 calcium alginate bioink was seeded with chondrocytes harvested from neonatal bovines (1-3 days old), producing the 7:3 ratio of alginate to CaCl2 with a final cell concentration of 25x106 cells/mL. Fluorescein isothiocyanate isomer, a yellow dye, was added to the cell-laden bioink for improved image analysis. The no-cell and cell-laden bioinks were loaded together into one syringe and were bioprinted such that the bioink would change from cell-laden bioink to no-cell-laden bioink during the print. Impedance was collected and normalized for the duration of each print. A Student’s t-test was then performed using each print's start and end impedance values to determine potential differences in measurements from the two bioinks.

Results, Conclusions, and Discussions: The impedance of various-shaped prints stayed almost constant throughout the duration of each bioprint, regardless of the direction and velocity of the nozzle print movements. This consistency in impedance was seen in all 3D bioprinted shapes, with and without corners, along with bioinks with and without cells (p > 0.05, Figure 2). This is important because knowing that movement does not affect impedance measurements means that the smart syringe can be used accurately to monitor bioinks during the 3D bioprinting process and while printing more complicated shapes. When analyzing the variation of cell-based bioinks (Bioink 1) to no cell-based bioinks (Bioink 2), there was a decrease a decrease in impedance measurements after transitioning to the no cell-based bioink (Bioink 2, p < 0.05, Figure 3). This is expected because cells will polarize in an alternating current, resulting in higher impedance. As a result, bioink without cells displays reduced resistance to current compared to bioink with cells because cells are not present to obstruct the electrical signal [3]. These results show that bioinks with and without cells can be monitored in real-time during impedance bioprinting.

Studies show that the motion of the syringe in the 3D bioprinting process does not affect impedance measurements during bioprinting regardless of the type of movement: straight lines or sharp corners. It is also seen that during the bioprinting process, the smart syringe can detect differences in impedance between cells and no cell-based alginate bioinks. This suggests that the smart syringe can serve as a viable solution, offering real-time bioink monitoring during bioprinting, reducing the need for destructive 3D bioprinting testing to determine cellular characteristics.

Acknowledgements (Optional):  This work was funded in part by West Pharmaceuticals Services and Cornell University's Engineering Learning Initiatives.

References:
[1] D. Stanco, P. Urbán, S. Tirendi, G. Ciardelli, J. Barrero. 2020 Sep 23. 3D bioprinting for orthopedic applications: Current advances, challenges and regulatory considerations. Elsevier. https://www.sciencedirect.com/science/article/pii/S2405886620300300.
[2] Jared Matthews and Lawrence Bonassar. (2024). System and devices for monitoring cell-containing materials and methods of their use (US Patent No.12019040). U.S, Patent and Trademark Office.
[3] Matavosian, A., Griffin, A., Bhuiyan, D., Lyness, A., Bonassar, L. (April 2023). Real-Time Measurement of Cell Concentration and Viability in Bioinks During Bioprinting Using Dielectric Impedance Spectroscopy [Poster Presentation]. TERMIS, Boston, MA, United States.