BEaTS-α an open access 3D printed device for in vitro electromechanical stimulation of human induced pluripotent stem cells

Abstract

3D printing was used to develop an open access device capable of simultaneous electrical and mechanical stimulation of human induced pluripotent stem cells in 6-well plates. The device was designed using Computer-Aided Design (CAD) and 3D printed with autoclavable, FDA-approved materials. The compact design of the device and materials selection allows for its use inside cell incubators working at high humidity without the risk of overheating or corrosion. Mechanical stimulation of cells was carried out through the cyclic deflection of flexible, translucent silicone membranes by means of a vacuum-controlled, open-access device. A rhythmic stimulation cycle was programmed to create a more physiologically relevant in vitro model. This mechanical stimulation was coupled and synchronized with in situ electrical stimuli. We assessed the capabilities of our device to support cardiac myocytes derived from human induced pluripotent stem cells, confirming that cells cultured under electromechanical stimulation presented a defined/mature cardiomyocyte phenotype. This 3D printed device provides a unique high-throughput in vitro system that combines both mechanical and electrical stimulation, and as such, we foresee it finding applications in the study of any electrically responsive tissue such as muscles and nerves.

Figure 1

BEaTS-α a 3D Printed device for mechanical and electrical stimulation of cells in vitro. (A) Parts and components of the 3D printed stimulation device; Standard Tessellation Language (.stl) files for these, and all other 3D printed parts are accessible https://figshare.com/s/504b0f7ccbdc7b893e3a. (B) The bottom of the wells of a 6-well plate were removed and replaced by flexible, translucent silicone sheets. Cells were cultured on the membranes and mechanically stimulated by means of a vacuum-driven system. In addition, to simultaneously stimulate cells electrically, a C-PACE EP system was placed over the 6-well plate. The electrodes of the C-PACE EP system were inserted in the media and a TTL-controlled signal was sent to create a physiologically relevant cyclic stimulation cycle. (C) Top view of a well with the modified well bottom and the electrodes of the C-PACE EP system. Once assembled, the cells were seeded in a growth area of 2.26 cm². (D) Finite Element Analysis (FEA) of the strain generated on the flexible membranes. A pressure of 55 kPa resulted in the deflection of the membranes with an average strain of 2.34%. FEA of the stress (Von Mises) and deflection (URES) profiles are shown in Fig. S1. (E) Actual image of the system assembled depicting the electrical impulse generator (1), mechanical stimulator (2), and Arduino UNO interface, red-boxed (3). All dimensions shown are in [mm].

Figure 2

Effect of stimulation on hiPSC-derived cardiomyocyte cultures. (A) Representative immunofluorescent images of hiPSC-derived cardiomyocytes cultured on flexible silicone membranes under either no stimulation (control), mechanical stimulation, electrical stimulation, or mechanical and electrical stimulation at different time points (4 and 7 days). Cells were stained for α-sarcomeric actin (α-SA; red) and connexin-43 (Cx43; green). Nuclei were stained using DAPI (blue). Scale bars = 50 µm. (B) Effect of stimulus on sarcomere length measured at 4 and 7 days (n = 2). Values calculated from the measurement of 100 random cells over 6 different fields of view. (C) Relative gene expression for TNNI1 and TNNI3 measured for hiPSC cells cultured for 4 and 7 days without and with the different stimulation regimes (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 calculated using One-way ANOVA with Tukey posthoc analysis.