(C-88) Chondrocytes seeded in different alginate bioink concentrations display frequency-dependent alteration of resistance signature

Introduction:: Subtrack: Bioprocess Monitoring and In-Line Sensing


With the advent of 3D bioprinted constructs in human clinical trials, the need for quality control of the printing process is becoming more apparent. Currently, bioprinted constructs are analyzed post-fabrication using destructive techniques.

However, dielectric spectroscopy (DIS) is a non-destructive technique used to detect cellular properties in cell suspensions [1,2,3]. This technique uses an electric current to polarize the ions in the cell cytoplasm and the substrate, leading to a frequency-dependent dielectric response. Low frequencies, referred to as the α-dispersion from 0.1-1 kHz, display the dependence of cell-substrate interactions. High frequencies, called the β-dispersion from 10-1000 kHz, isolate the behavior of ions polarizing across the cell membrane [2]. While the behavior of different cell types has been studied in suspensions, very few studies have investigated cell dielectric behavior in bioinks. Moreover, the effect of alginate, a low-cost and biocompatible hydrogel [2,5], bioink concentration has not been investigated using DIS.

To address the knowledge gap, the goal of the study is to measure the impact that two different alginate bioink concentrations have on the DS signature of model cells, chondrocytes (CHs). We propose that the higher concentration of alginate bioink will directly impact the dielectric property of resistance of the cells in the bioink sample. Furthermore, we anticipate that this impact will be more pronounced in the α-dispersion region than in the β-dispersion.




Materials and Methods:: To assess the effect of alginate bioink concentration on the DS signature of chondrocytes, four groups were prepared: 1 w/v% alginate with and without chondrocytes (n=19 each) and 3 w/v% alginates with and without chondrocytes (n=9 each). Chondrocytes were isolated from neonatal bovine joints, as previously published [6]. Chondrocytes were diluted to 25*10⁶ cells/mL and then included in either 1 w/v% or 3 w/v% alginate. Bioink samples were composed of Pronova® low viscosity sodium alginate mixed with phosphate-buffered saline using a three-way stopcock. Bioink pH was measured and adjusted using 1M HCl or 1M NaOH until it reached 7-7.5.

The samples were individually loaded into our custom device that uses DS onboard a syringe [7], and a Diligent Analog Discovery 2 device measured resistance using the software, Waveforms (100Hz to 1 MHz, 101 steps, 50mV Amp, 1 kOhm). The custom device was washed between samples.




Results, Conclusions, and Discussions:: Consistent with previous literature [4], resistance was higher in the α-dispersion region and then decreased to a plateau in the β-dispersion region (Fig. 1). Within the β-dispersion, resistance was higher for the average alginate 1% with no cells compared to alginate 1% cell group, however, 1% without cells also showed the highest standard error (Fig. 1). Alginate 3% with cells and alginate 3% with no cells followed in the order stated (Fig. 1) and all groups plateaued in the β-dispersion region.

To clearly visualize the effect of the different alginate bioink concentrations on the CHs resistance signal, the alginate resistance (Rs_alg) was subtracted from the resistance of alginate with CHs (Rs_{alg + CHs}). Upon subtracting, both samples displayed negative resistance (Fig. 2), indicating that cell-seeded alginate showed lower resistance than non-seeded bioink. This non-zero delta Rs indicates that the presence of bioink alters the resistance signature of CHs (Fig. 2). Furthermore, the unequal 3 w/v % alginate bioink resistance with that of 1 w/v % indicates that bioink concentration has an impact on resistance signature alteration. Alginate 3 w/v % was higher than alginate 1 w/v %, which suggests that increased bio-ink concentration leads to an increase in resistance of the cells (Fig. 2). In addition, at low frequencies (α-dispersion), 60 Hz noise was experienced and the delta Rs was a smaller fraction of the overall resistance (Fig.1). The largest effect on resistance was seen in the β-dispersion region (Fig. 2) above 10 kHz. This study also shows the potential for DIS as a tool with sensitivity to bio-ink composition, which is a measure critical to tissue regulation.




Acknowledgements (Optional): : Research supported by the Cornell University Louis Stokes Alliance for Minority Participation.

References (Optional): : [1] Kell D et al TrAC Trends in Analytical Chemistry 10.1016

[2] Haring AP et al 2020 Biofabrication 12(3):035026

[3] Lokesh Karthik Narayanan et al 2018 Biofabrication 10 035012

[4] Heileman K, Daoud J, Tabrizian M. Dielectric spectroscopy as a viable biosensing tool for cell and tissue characterization and analysis. Biosens Bioelectron. 2013 Nov 15;49:348-59. doi: 10.1016/j.bios.2013.04.017. Epub 2013 May 15. PMID: 23796534.

[5] Lee SM, Han N, Lee R, Choi IH, Park YB, Shin JS, Yoo KH. Real-time monitoring of 3D cell culture using a 3D capacitance biosensor. Biosens Bioelectron. 2016 Mar 15;77:56-61. doi: 10.1016/j.bios.2015.09.005. Epub 2015 Sep 6. PMID: 26386332.

[6] Genes, N. G., J. A. Rowley, D. J. Mooney, and L. J. Bonassar. Effect of substrate mechanics on chondrocyte adhesion to modified alginate surfaces. Arch. Biochem. Biophys. 422(2):161–167, 2004.

[7] Matthews J, Bonassar L; Cornell University. System and devices for cell-containing materials and methods of their use. United States patent US 2021/0096094 A1. 2021 April 1.