Self-folding 3D microfluidics for tissue engineering

Introduction: 3D chemical patterns, including gradients, control information flow in the biology of broad relevance to tissue engineering, microphysiological systems, and developmental biology. While researchers have created a variety of microfluidic systems for tissue engineering, they are predominantly applicable to 2D form factors. However, recent innovations in tissue engineering demand 3D platforms to interface organoids and assembloids. For instance, controlled 3D chemical interaction with brain organoids is critical for investigating neurodevelopment, mainly because the spatiotemporal regulation of signaling molecules, such as bone morphogenic proteins, Wnt, fibroblast sonic hedgehog, and retinoic acid, drives morphogenesis during in-utero brain development. Moreover, advancements in assembloids and morphogenesis studies bring about spatial asymmetry in organoid's chemical signature, requiring spatial chemical monitoring systems. Here, we report the development of a 3D self-folding microfluidic shell that can distribute chemical sources and sinks around the contour of a sphere to create a controlled microenvironment and continuous chemical monitoring. Guided by computational fluid dynamics (CFD) simulations, we show how complex gradients can be formed in gels and tissue constructs using this 3D self-folding microfluidic system.

Materials and

Methods: We used wafer-scale, batch-manufacturable, self-folding 3D shell microfluidics for bidirectional chemical interaction with millimeter-scale 3D biological constructs such as organoids. We achieved this feat by employing a photolithography fabrication method involving six photomasks. The fabrication mainly involves (i) a sacrificial layer patterning for selectively detaching the microfluidic shell petals that encapsulate the organoids, (ii) channel wall fabrication, (iii) temporary filling of the channel with a sacrificial polymer, (iv) removal of the sacrificial polymer for microchannel formation, and (v) selective differential crosslinking to induce self-folding, as shown in Fig 1a. 3D organic objects are placed inside the shell microfluidics system once the self-folding is triggered to achieve encapsulation of these objects. A CFD analysis that considers a combination of laminar flow in the channel, convective and diffusive spreading of the transport species from the openings into the media, and diffusive transport into a porous media is used to predict the gradient profile.

Results, Conclusions, and Discussions: We successfully fabricated a 3D self-folding shell microfluidic system that can inject liquid around 3D millimeter and submillimeter objects, as illustrated in Fig.1 b. We demonstrate the selective fluid infusion and extraction capabilities of this self-folding 3D shell microfluidic using alginate beads and brain organoids. Unlike 3D printed microfluidics, the presented microfluidic shell can self-fold to wrap around the 3D object, as illustrated in Fig. 1 c. CFD analysis using COMSOL demonstrates strategies for generating global and local chemical gradients using this 3D microfluidic platform. The results are being validated by employing fluorescent dyes and alginate beads. The platform can also extract chemical-containing solutions from various spatial locations around the bead/organoid through the surrounding shell ports for chemical monitoring. For the demonstration, we will use a composite of conductive polymer containing Poly(3,4-ethylenedioxythiophene) polystyrene sulfonate and carbon nanotube to detect dopamine. Our calibration studies show that the sensor can accurately monitor dopamine using this platform at a detection limit of 75 nM.

In conclusion, we show spatiotemporally controlled, CFD-backed fluid delivery and extraction with a 3D self-folding microfluidic shell, presenting a unique platform for controlled 3D gradient generation and spatial metabolomics. Our ongoing research will translate the gradient generation and monitoring efforts on alginate beads to live organoids using these biocompatible microfluidic shells. Combined with our previous work on a shell microelectrode array1, the presented microfluidic system can be a multimodal interrogation and stimulation platform relevant to various research studies related to organoids, including the developing field of organoid intelligence.

References
1. Huang, Q. et al. Shell microelectrode arrays (MEAs) for brain organoids. Sci Adv 8, eabq5031 (2022).

Acknowledgements (Optional): We acknowledge funding from the JHU Surpass Program and the NSF EFMA-2318093.