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Tissue Engineering

Tissue Engineering - Poster Session E

Poster BB13 - The effect of alginate bioink concentration on real-time monitoring of cell properties using dielectric impedance spectroscopy

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

Introduction:  Bioprinting uses the layer-by-layer deposition of bioink, a biocompatible and crosslinkable hydrogel, to produce personalized cell-laden constructs for tissue regeneration. To create bioprinted constructs that are GMP-compliant and FDA approved for human clinical trials, extensive testing is conducted to determine the cell bioactivity within the constructs after printing. These tests involve the use of destructive techniques such as biochemical assays, histology, and biomechanical testing. Destructive techniques necessitate the creation of multiple constructs per patient, increasing production time and cost. An alternative means of assessing cell bioactivity is through in-line, non-destructive testing, such as dielectric impedance spectroscopy (DIS). DIS uses an alternating current across a frequency sweep to temporarily charge a conducting solution and polarize cells. This polarization is then quantified using impedance (Z).

We previously incorporated DIS onto a syringe to monitor cell concentration and viability in saline solutions as a proof-of-concept [1][2]. Bio-inks are composed of charged, polymeric chains with branching charged groups that interact with a secondary solution to crosslink, forming a network that encapsulates cells. Additionally, bio-ink properties such as concentration and charge can be tuned to mimic native tissue structure. While previous studies have measured cell properties of bioprinted constructs, the effect of bio-ink properties on dielectric measurements has not been considered. The goals of our study were to (1) characterize the dielectric properties for alginate bio-ink at different concentrations and (2) determine the effect of alginate concentration on cell dielectric properties.

Materials and

Methods:  Alginate bio-ink samples were created by reconstituting sodium alginate powder (PRONOVA) in phosphate buffered saline. Samples were sterilized through syringe filtering, and final concentrations were 0.5, 1, and 3 wt/v%. Each bio-ink concentration was prepared with or without cells. Primary chondrocytes were isolated from neonatal bovine condyles, digested overnight, and added to the alginate samples at a concentration of 25×106 cells/mL [3]. Before printing, a portion of the cell-laden samples were stained with LIVE/DEAD assay to confirm the cell concentration and viability of the samples.

Alginate samples were loaded into a syringe with our device (referred to as the smart syringe), which uses antiparallel gold electrodes embedded into a luer lock and rests between the syringe barrel and syringe tip (Figure 1). Z was measured across frequency sweeps from 0.1-25,000 kHz. There was a total of 16-17 measurements per sample that were collected across 4 independent experiments with n=4-5 replicates each. The electrodes in the smart syringe were cleaned between each sample and each replicate.

Results, Conclusions, and Discussions: All alginate samples showed high impedance ranging from 130-1530 Ω across 0.1-4 kHz and comparatively lower impedance from 100-130 Ω across 4-25,000 kHz (Figure 2). No differences in impedance were found between alginate samples without cells. This finding indicates that changing the bioink concentration during a print will have minimal impact on the measured dielectric properties of the sample.
Next, primary chondrocytes were infused into the alginate bio-inks, and the data was normalized using a percent change calculation to highlight the cell response (Figure 3). Notably, impedance from all bio-ink concentrations showed an effect from the addition of cells, which is seen from the non-zero response. Cell-infused samples with 0.5 wt/v% and 3 wt/v% alginate showed increased normalized impedance. Additionally, 1 wt/v% alginate with cells displayed a range with both increased and decreased impedance. Comparing the alginate concentrations, cell-infused 3 wt/v% alginate showed the highest impedance on average at nearly all frequencies. Interestingly, from approximately 10-1,000 kHz, both 0.5 wt/v% and 1 wt/v% samples displayed nearly zero impedance on average, indicating that the cellular response was low compared to the contributions of the bio-ink at these frequencies.
For all samples, impedance measurements were particularly pronounced from 0.1-4 kHz, which correlates to the α-dispersion, a range of frequencies that result in ionic diffusion between cells and the surrounding substrate [4]. This trend suggests that the cell-alginate interface may contribute to the dielectric properties of the cells at low frequencies.

Conclusions: These studies show the dielectric measurements of alginate bio-ink at concentrations ranging from 0.5-3 wt/v% and show the resulting effect of alginate concentration on the cellular response. Alginate samples without cells showed similar impedance values from 0.1-25,000 kHz across all concentrations. The addition to cells to the bio-inks increased or decreased the sample impedance, particularly from 0.1-4 kHz. These results suggest that DIS may be used to monitor cell-laden bioink during printing, reducing the need for destructive testing post-fabrication.

Acknowledgements (Optional):  This work was funded in part by West Pharmaceutical Services.

References:
[1] 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.
[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] Chang SCN, Rowley JA, Tobias G, Genes NG, Roy AK, Mooney DJ, et al. Injection molding of chondrocyte/alginate constructs in the shape of facial implants. J Biomed Mater Res. 2001 Jun 15;55(4):503–11.
[4] Heileman K, Daoud J, Tabrizian M. Dielectric spectroscopy as a viable biosensing tool for cell and tissue characterization and analysis. Biosensors and Bioelectronics. 2013 Nov;49:348–59.