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Three-dimensional (3D) printing has emerged as a transformative technology for fabricating complex microfluidic devices, enabling features that were previously unattainable with traditional layer-by-layer soft lithography. One key challenge in advancing 3D-printed microfluidics is the integration of functional microvalves across multiple spatial orientations. This study explores the design, simulation, and experimental realization of novel microvalve configurations to overcome the limitations of conventional, single-plane valves. We hypothesize that non-traditional valve orientations, such as those with vertically printed membranes or perpendicular control channels, present unique fabrication and operational challenges, including membrane delamination and stress-induced failure. To address these issues, we developed optimized geometries and fabrication techniques, supported by computational fluid dynamics (CFD) simulations to predict and mitigate stress concentrations. Our results demonstrate successful implementation of previously unreported valve configurations, validated through pressure testing and flow control experiments. These advancements expand the versatility of 3D-printed microfluidic systems, paving the way for more robust and adaptable devices in biomedical, chemical, and environmental applications. 

Recently, there has been an increasing effort in developing new fabrication methods for rapid prototyping of microfluidic chips using laser cutting and 3D printing. However, although these approaches can readily generate rigid parts of the devices, it is not trivial to integrate flexible components (e.g. on-chip valve and/or pump membranes) within the same build. This has led to the recent adoption of thermoplastic polyurethane (TPU) membranes sandwiched between the rigid layers to introduce the necessary flexibility to the chips. Despite its utility, TPU is not without its challenges—it is relatively expensive and surprisingly difficult to source. To overcome these difficulties, our study introduces the use of common food wrapping film as a cost-effective and readily available alternative to TPU, demonstrating its compatibility in fabricating essential microfluidic components such as on-chip valves and peristaltic pumps. Our findings show that this alternative maintains the performance standards required for sophisticated microfluidic applications while significantly alleviating logistical and financial constraints. The results show high cyclability of the membrane, up to 850,000 in continuous testing conditions, at 1 Hz, while also can block the fluid flow at as low as 250 kPa. Regarding the micropumps, it was shown that adequate flow rate of around 4 μL/min can be achieved. 

Tissue regeneration-promoting and drug-eluting biomaterials are commonly implanted into animals as a part of late-stage testing before committing to human trials required by the government. Because the trials are very expensive (e.g., they can cost over a billion U.S. dollars), it is critical for companies to have the best possible characterization of the materials' safety and efficacy before it goes into a human. However, the conventional approaches to biomaterial evaluation necessitate sacrificial analysis (i.e., euthanizing a different animal for measuring each time point and retrieving the implant for histological analysis), due to the inability to monitor how the host tissues respond to the presence of the material in situ. This is expensive, inaccurate, discontinuous, and unethical. In contrast, our manuscript presents a novel microfluidic platform potentially capable of performing non-disruptive fluid manipulations within the spatial constraints of an 8 mm diameter critical calvarial defect—a “gold standard” model for testing engineered bone tissue scaffolds in living animals. In particular, here, addressable microfluidic plumbing is specifically adapted for the in vivo implantation into a simulated rat's skull, and is integrated with a combinatorial multiplexer for a better scaling of many time points and/or biological signal measurements. The collected samples (modeled as food dyes for proof of concept) are then transported, stored, and analyzed ex vivo, which adds previously-unavailable ease and flexibility. Furthermore, care is taken to maintain a fluid equilibrium in the simulated animal's head during the sampling to avoid damage to the host and to the implant. Ultimately, future implantation protocols and technology improvements are envisioned toward the end of the manuscript. Although the bone tissue engineering application was chosen as a proof of concept, with further work, the technology is potentially versatile enough for other in vivo sampling applications. Hence, the successful outcomes of its advancement should benefit companies developing, testing, and producing vaccines and drugs by accelerating the translation of advanced cell culturing tech to the clinical market. Moreover, the nondestructive monitoring of the in vivo environment can lower animal experiment costs and provide data-gathering continuity superior to the conventional destructive analysis. Lastly, the reduction of sacrifices stemming from the use of this technology would make future animal experiments more ethical.