Enhancing all-in-one bioreactors by combining interstitial perfusion, electrical stimulation, on-line monitoring and testing within a single chamber for cardiac constructs

Abstract

Tissue engineering strategies have been extensively exploited to generate functional cardiac patches. To maintain cardiac functionality in vitro, bioreactors have been designed to provide perfusion and electrical stimulation, alone or combined. However, due to several design limitations the integration of optical systems to assess cardiac maturation level is still missing within these platforms. Here we present a bioreactor culture chamber that provides 3D cardiac constructs with a bidirectional interstitial perfusion and biomimetic electrical stimulation, allowing direct cellular optical monitoring and contractility test. The chamber design was optimized through finite element models to house an innovative scaffold anchoring system to hold and to release it for the evaluation of tissue maturation and functionality by contractility tests. Neonatal rat cardiac fibroblasts subjected to a combined perfusion and electrical stimulation showed positive cell viability over time. Neonatal rat cardiomyocytes were successfully monitored for the entire culture period to assess their functionality. The combination of perfusion and electrical stimulation enhanced patch maturation, as evidenced by the higher contractility, the enhanced beating properties and the increased level of cardiac protein expression. This new multifunctional bioreactor provides a relevant biomimetic environment allowing for independently culturing, real-time monitoring and testing up to 18 separated patches.

Figure 1

(a) Schematic representation of the bioreactor chamber including the scaffold holder and the MC connector linked by a silicone tube; medium perfusion through the scaffold is achieved by bidirectional oscillation (270° spanned angle) along the axis perpendicular to the culture chamber. (b) Detailed scheme of the scaffold holder composed by two complementary parts housing electrodes, two round arrays of pillars and glass windows. (c) Representation of the two functioning positions of the scaffold holder to hold the construct during culture (top) and to release it for testing (bottom). (d) The OPB bioreactor housing the PDMS culture chambers mounted on 3D printed supporting disks; the newly designed chamber allows the optical assessment of constructs during perfusion and electrical stimulation.

Figure 2

(a) Fluid dynamic FEM computational analyses: (i) model representing the medium and the scaffold domains with initial conditions (V = 100 µm/sec; P_out = 0 atm); (ii) fluid flow velocity profile evaluated at different quotes of the chamber; (iii) colorimetric maps highlighting the velocity profile at different quotes of the chamber and within the scaffold. (b) Electrical FEM computational analyses: (i) model representing the fluidic and the electrode domains with the applied electric field (V = 5 V/cm); colorimetric maps and streamlines (arrows) (ii) of the electric field and (iii) of the current density at different quotes within the chamber.

Figure 3

Electric current measurement within the culture chamber when (a) a biphasic stimulation of 5 V for 2 ms at 1 Hz, typical for cardiac cell culture is performed within the chamber and when a monophasic stimulation of 5 V for 4 ms at (b) 1 Hz or (c) 10 Hz, typically used for construct pacing, is provided.

Figure 4

(a) The OPB bioreactor housing the transparent culture chambers mounted on 3D printed support disks and integrating a digital microscope for the on-line monitoring of the constructs through the glass windows. (b) The OPB allows the release of a single chamber from the bioreactor frame. (c) Thanks to the presence of the two transparent glass windows, the construct can be observed using a fluorescence microscope for live imaging of labeled cells without removing the construct from the scaffold holder.

Figure 5

(a) Experimental plan: cells were embedded in Matrigel, seeded on a collagen scaffold, let adhere for two hours and placed in the culture chambers; constructs were electrically stimulated for 4 days after a static culture of 3 days (Elect), bidirectionally perfused for 7 days (Perf) or perfused for 7 days in combination with an electrical stimulation, which started after 3 days (Perf + Elect). (b) Cytocompatibility of the stimuli provided by the chamber was assessed on NRCF constructs by Live/Dead (scale bars: 500 µm) and MTT assays performed after 7 days of culture. Cardiac functionality was evaluated by: (c) measuring the percentage of fraction area change of the construct during its beating (scale bar 1 mm); (d) analyzing the ET and MCR parameters during the pacing test; (e) evaluating the transcriptional expression of cardiac markers, such as myosin heavy chains, troponins and gap junction proteins, in constructs cultured for 7 days.