Aerosol Jet® Printing of Poly(3,4-Ethylenedioxythiophene): Poly(Styrenesulfonate) onto Micropatterned Substrates for Neural Cells In Vitro Stimulation

Abstract

In neural tissue engineering (NTE), topographical, electrical, mechanical and/or biochemical stimulations are established methods to regulate neural cell activities in in vitro cultures. Aerosol Jet^® Printing is here proposed as enabling technology to develop NTE integrated devices for electrically combined stimulations. The printability of a poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT: PSS) commercial ink onto a reference substrate was firstly investigated and the effect of the process parameters on the quality of printed lines was analyzed. The study was then extended for printing thick electrodes and interconnects; the print strategy was finally transferred to a silicon-based wafer with patterned microchannels of proven cellular adhesion and topographical guidance. The results showed values of electrical resistance equal to ~16 Ω for printed electrodes which are ~33 μm thick and ~2 mm wide. The electrical impedance of the final circuit in saline solution was detected in the range of 1 - 2 kΩ at 1 kHz, which is in line with the expectations for bioelectronic neural interfaces. However, cells viability assays on the commercial PEDOT: PSS ink demonstrated a dose dependent cytotoxic behavior. The potential cause is associated with the presence of a cytotoxic co-solvent in the ink's formulation, which is released in the medium culture, even after a post-sintering process on the printed electrodes. This work is a first step to develop innovative in vitro NTE devices via a printed electronic approach. It also sheds new insights the transfer of AJ^® print strategies across different substrates, and biocompatibility of commercial PEDOT: PSS inks.

Keywords: Aerosol Jet® printing; Biomedical; Conductive polymers; Neural tissue engineering; Printing of electronics.

Figure 1

Optical profilometer analysis on Si-channels substrates before and after Parylene-C coating. The figure shows a profilometer extract (A) before and (B) after coating. (C) Values of mean thickness t and width w of Si-channels in both cases. (D) Representative optical image of the homogenous Parylene-C coating.

Figure 2

Quantitative image detection analysis. (A) Representative figure of a printed line, with emphasis on the terms w_l, w_o (w₀ = w_o1+w_o2), and the total line with (w_l + w_o). (B) Original microscopic line image. (C) Line image with enhanced contrast. (D) Line length division (in red color) for analysis purposes. (E) Image size in μm plotted against the grayscale levels shows the printed line profile, w_l and w_o (w₀ = w_o1+w_o2).

Figure 3

Aerosol Jet^® Printing (AJ^®P) process on the NTE substrate. (A) Schematic design of the bioelectrical integrated device for electrical and morphological stimulation, with the printed pattern (light blue color) around the micro-patterned channels (black color) on the NTE substrate. (B) Representation of the entire fabrication procedure of the device: (i) Fabrication of the microchannels via photolithography, including spin coating and ultraviolet exposure of the SU-8 photoresistor; (ii) pyrolysis treatment of the micropatterned substrate to obtain glassy carbon microchannels; (iii) Parylene-C coating and subsequent oxygen plasma treatment of the substrate; (iv) AJ^®P of conductive patterns using a commercial inkjet PEDOT: PSS ink on the preheated substrate; (v) the AJ^®P printed circuit before and (iv) after annealing in a thermal oven at 140°C for 1 h. (C) Schematic figure of the AJ^®P on the Parylene-C-coated NTE substrate. Firstly, the PEDOT: PSS ink is atomized from liquid to mist in a glass vial positioned in the ultrasonic bath. Secondly, the mist is transported by a carrier gas into a transportation PTFE tube, and consequently focused by an annular sheath gas in the print head. The focused aerosolized beam is then printed according to the design pattern on the NTE substrate, located on the printing platform at a selected platen temperature. Figure adapted from Degryse et al., International Conference on Biofabrication, 2021[33].

Figure 4

(A-C) Contour plot of line quality, q, from 1 (worst) to 5 (ideal) at T = (25, 40, 60) °C for R_f = (1, 1.5, 2) with PEDOT: PSS ink and glass slides.

Figure 5

R_avg and t_avg of AJ^®P PEDOT: PSS interconnects (single printed line) and electrodes on glass slides (A and B) and NTE substrates (C) at 40°C.

Figure 6

Immunofluorescence assays of HFs on (A) NTE substrates ante plasma, showing a non-adhesion of HFs, with (B) the presence of agglomerates in round-like shapes, detectable as an unhealthy cellular condition, and on (C) NTE substrates post plasma, representing good cellular adhesion of HFs, with (D) HFs aligned in the micro-channels, demonstrating the validity of the plasma treatment and the applied topographical cue. Immunofluorescence assays of NSCs on Parylene-C-coated NTE substrates post plasma at 24 h, magnification ×10 (E) and ×20 (F), and at 120 h, magnification ×10 (G) and ×20 (H), showing good cellular adhesion and morphological alignment, with cellular network formation across the channels. Immunofluorescence assays of NSCs on plastic control and PEDOT: PSS ink: (E) NSCs cultured on Matrigel-coated plastic, showing good adhesion, (F) NSCs cultured on Matrigel-coated AJ^®P PEDOT: PSS samples, showing an unhealthy cellular state.

Figure 7

Cell viability assay of NSCs on plastic control and PEDOT: PSS samples. (A) Representative image of the ink’s hydrophobicity. (B) Optical image with focus on the presence of NSCs agglomerates near the well edges detected after 48 h. (C) NSCs rATP direct viability assay at 3 time points (24 h, 48 h and 96 h) on printed PEDOT: PSS samples, revealing an increasing cytotoxic behavior of the PEDOT: PSS ink. (D) NSCs rATP undirect viability assay at 3 time points (24 h, 48 h and 120 h) on printed PEDOT: PSS samples, confirming a high cytotoxic behavior of the PEDOT: PSS ink already after 24 h. (E) NSCs rATP undirect viability assay at 2 time points (24 h and 48 h) on spin-coated PEDOT: PSS samples, cured at six different conditions (150°C for 8, 60, and 120 min, and 200°C for 8, 60, and 120 min), showing good biocompatibility. (F) NSCs rATP undirect viability assay at 3 time points (24 h, 72 h, and 120 h) on spin-coated PEDOT: PSS samples, cured at 150°C for 120 min, confirming a dose-dependent behavior of the PEDOT: PSS ink.

Figure 8

Printing of the device: on (A) glass substrate and (B) NTE substrate.

Figure 9

Characterization of the device: (A) Contour profile of printed electrodes at different printing layers n = (5, 20, 30, 40, 50), showing the potentiality of reaching ca. 30 μm with n 40; (B) Electrical impedance of the printed circuit (40 layers) on Parylene-C NTE substrate. Impedance (Ω) and phase (°) are plotted versus frequency (Hz).