Impedance-Based Monitoring of Mesenchymal Stromal Cell Three-Dimensional Proliferation Using Aerosol Jet Printed Sensors: A Tissue Engineering Application

Abstract

One of the main hurdles to improving scaffolds for regenerative medicine is the development of non-invasive methods to monitor cell proliferation within three-dimensional environments. Recently, an electrical impedance-based approach has been identified as promising for three-dimensional proliferation assays. A low-cost impedance-based solution, easily integrable with multi-well plates, is here presented. Sensors were developed using biocompatible carbon-based ink on foldable polyimide substrates by means of a novel aerosol jet printing technique. The setup was tested to monitor the proliferation of human mesenchymal stromal cells into previously validated gelatin-chitosan hybrid hydrogel scaffolds. Reliability of the methodology was assessed comparing variations of the electrical impedance parameters with the outcomes of enzymatic proliferation assay. Results obtained showed a magnitude increase and a phase angle decrease at 4 kHz (maximum of 2.5 kΩ and -9 degrees) and an exponential increase of the modeled resistance and capacitance components due to the cell proliferation (maximum of 1.5 kΩ and 200 nF). A statistically significant relationship with enzymatic assay outcomes could be detected for both phase angle and electric model parameters. Overall, these findings support the potentiality of this non-invasive approach for continuous monitoring of scaffold-based cultures, being also promising in the perspective of optimizing the scaffold-culture system.

Keywords: 3D monitoring; aerosol jet printing; impedance-based cell spectroscopy; mesenchymal stromal cells; tissue engineering.

Figure 1

Setup for impedance-based monitoring of human mesenchymal stromal cells (hMSCs) seeded in 3D hybrid hydrogel scaffold: (a) Hybrid hydrogel scaffold and aerosol-jet printed (AJP) sensors, (b) cap for sterile measurements, (c) final measurement setup.

Figure 2

Preliminary impedance-based assessment for choosing the optimal cell concentration in the scaffold.

Figure 3

Impedance-based monitoring including controls (a) magnitude and (b) phase angle versus frequency) and seeded scaffolds (c) magnitude and (d) phase angle vs frequency.

Figure 4

Cell index in terms of (a) magnitudeand (b) phase angle contribution to the overall impedance value. Results concerning 4 kHz frequency are specifically highlighted for both (c) magnitude and (d) phase angle.

Figure 5

Equivalent circuit modeling the (a) blank and (b) cell seeded conditions: measured and fitted spectra for both the (c) module and (d) phase are reported.

Figure 6

Evolution of the relevant parameters concerning the cell contribution during time (C_cells, R_cells). (a): Equivalent circuit with R_cells and C_cells highlighted, (b): Evolution of C_cells during culture, (c): Evolution of R_cells during culture.

Figure 7

CKK-8 staining in 3D culture: (a) section and (b) upper-view of CKK-8 macroscopic staining of scaffolds, control (cell-less scaffold), hMSC culture (scaffold with cells after 14 days of culture); (c) box-plot of absorbance measurements at 21 days of culture. Distribution of absorbance measurements for scaffolds exposed to 4 CKK-8 treatments (x = average = 0.499 ± 0.062) and scaffolds exposed to 1 CKK-8 treatment (x = average = 0.474 ± 0.098), p = 0.64.

Figure 8

Box-plot of 3D culture proliferation detected with CKK-8 assay along the 21-day-long culture. X indicates samples average at each day.

Figure 9

Fluorescent images of hMSCs on hybrid gelatin-chitosan hydrogel scaffolds at several days of culture. Thinly sliced portions of the central part of the scaffolds. Nuclei staining with DAPI (blue), 10× magnification. (a) 3 days of culture, (b) 7 days of culture, (c) 14 days of culture and (d) 21 days of culture.