Organ-on-chips (OoCs) are microfluidic cell culture devices with two independently addressable, parallel channels separated by a porous membrane, enabling the co-culture of different cell types and creating complex tissue-tissue interfaces. While OoCs offer a powerful alternative to traditional in vitro and animal models, their use is labor-intensive and requires expertise in both microfluidics and cell culturing. To improve throughput and facilitate commercial applications like drug screening and personalized medicine, multiplexed OoCs are being developed. Existing systems like the Mimetas OrganoPlate®, while offering multiple wells, require extensive pipetting and have small culture areas. Other designs with shared inlets and parallel outlets still necessitate considerable manual handling. Microfluidic systems with integrated valves offer automation potential, reducing the need for manual liquid handling. Normally open valves, particularly Quake-style valves, are popular due to their ease of fabrication and small footprint. However, the standard fabrication process for Quake-style valves, relying on reflow photoresist, limits channel height to tens of micrometers, making them unsuitable for the hundreds-of-micrometer dimensions commonly found in OoCs and 3D cell culture systems. Furthermore, the low flow rates (0.05-0.15 µL/min) achievable with these valves are inadequate for the higher flow rates (0.5-3.3 µL/min) often needed in OoCs. While alternative fabrication methods like photolithography and 3D printing have been explored, they present challenges such as extra fabrication steps, material compatibility issues, and limitations in resolution and speed. Micromilling offers advantages over these methods: it's a fast, versatile, cleanroom-free, and cost-effective technique capable of creating complex 3D geometries. This paper presents a cleanroom-free fabrication method using micromilled direct positive molds to create Quake-style PDMS ‘macrovalves’ with dimensions suitable for OoC applications. The use of a direct positive mold simplifies the fabrication process and minimizes errors from PDMS shrinkage compared to double casting methods. This approach facilitates the commercialization of molds and the multiplexing of PDMS OoCs.

The authors reviewed existing organ-on-chip (OoC) technologies, highlighting the limitations of current systems in terms of throughput and automation. They discussed existing microfluidic valve technologies, focusing on the commonly used Quake-style valves fabricated using reflow photoresist. The limitations of this method for OoC applications, specifically the restrictions on channel height and flow rate, were emphasized. The authors then examined alternative fabrication methods, such as photolithography and 3D printing, outlining their respective advantages and disadvantages concerning OoC integration and scalability. The advantages of micromilling as a cleanroom-free and cost-effective rapid prototyping technique were highlighted, contrasting it with previous methods using micromilled molds for PDMS macrovalves which involved negative molds and double or multi-step casting processes.

The researchers developed a cleanroom-free fabrication method for PDMS macrovalves using micromilling to create direct positive molds. The devices consisted of three layers: a glass slide, a thin PDMS control layer with control channels, and a PDMS flow layer with rounded flow channels. The micromilled molds were used to cast both layers. To improve valve sealing, a chloroform treatment was explored to reduce the surface roughness of the micromilled PMMA molds. The effectiveness of the chloroform treatment was evaluated using scanning electron microscopy (SEM) and profilometry. A systematic characterization was performed to determine the relationship between valve closure, flow channel dimensions (width and height), control channel width, and actuation pressure. Four characterization chips, each with a different fixed flow channel width (250, 500, 750, or 1000 µm), were fabricated. Each chip contained 25 cross-sections with varying flow channel heights and control channel widths. The valves' ability to close off channels was assessed under different actuation pressures (1, 1.25, 1.5, and 1.75 bar). Based on the characterization results, two valve designs were selected for further experiments: one for a peristaltic pump and a mixing/metering device, and another for a biocompatibility test with endothelial cells under continuous perfusion. The peristaltic pump and mixing device performance were evaluated in terms of pumping rate and mixing efficiency. The biocompatibility test assessed the long-term cell viability and functionality in the device.

The study demonstrated a successful cleanroom-free fabrication method for PDMS macrovalves using micromilled direct positive molds. The macrovalves effectively closed off rounded channels up to 700 µm high and 1000 µm wide. The systematic characterization revealed a strong correlation between valve closure, flow channel dimensions, control channel width, and actuation pressure. The researchers found that even without chloroform treatment for surface smoothing, the valves were leak-free when closed. The developed peristaltic pump achieved a pumping rate of up to 48 µL/min, suitable for OoC applications. The mixing and metering device demonstrated complete mixing of a 6.4 µL volume in 17 s. The biocompatibility test using endothelial cells showed successful cell culture over multiple days under continuous perfusion and automated medium refreshment, indicating the suitability of the macrovalves for long-term cell culture experiments in OoC systems. The results suggest that the macrovalves can be successfully integrated into OoC devices without compromising cell viability.

This research successfully addresses the limitations of current microfluidic valve technologies for organ-on-chip applications. The developed cleanroom-free fabrication method using micromilled molds provides a scalable and cost-effective approach for creating macrovalves with dimensions suitable for OoCs. The high flow rates and efficient mixing capabilities demonstrated by the integrated peristaltic pump and mixing device significantly enhance the functionality of OoC systems. The successful endothelial cell culture experiment confirms the biocompatibility of the system and showcases its potential for long-term, automated cell culture. The findings contribute to advancing OoC technology by enabling higher throughput and reducing reliance on manual handling, thus facilitating more complex and physiologically relevant in vitro models. The simplified fabrication process and the system's biocompatibility make it suitable for widespread adoption in OoC research and development.

This study introduces a novel, cleanroom-free fabrication method for PDMS macrovalves using micromilling, enabling the creation of valves suitable for organ-on-chip applications. The macrovalves demonstrated high flow rates, efficient mixing, and biocompatibility, paving the way for automated and higher-throughput OoC experiments. Future research could explore the integration of these macrovalves into more complex OoC systems and investigate their application in various cell types and organ models. Further optimization of the micromilling process and exploration of different materials could further improve the performance and versatility of these macrovalves.

While the study demonstrated successful fabrication and functionality of the macrovalves, further investigation is needed to fully assess the long-term stability and reliability of the valves under continuous operation. The current characterization was limited to a specific set of dimensions and pressures; a more comprehensive characterization across a wider range of parameters would enhance the design guidelines. The biocompatibility study was conducted with one cell type; further studies are needed to validate biocompatibility with other cell types relevant to OoC applications. The scalability of the micromilling process for mass production requires further evaluation.