Systematic characterization of cleanroom-free fabricated macrovalves, demonstrating pumps and mixers for automated fluid handling tuned for organ-on-chip applications

The study addresses the challenge of integrating automated fluid handling into organ-on-chip (OoC) devices, which typically use channels hundreds of micrometers in size—larger than dimensions compatible with conventional Quake-style PDMS microvalves fabricated via reflow photoresist. Conventional valves are limited to tens of micrometers in height and provide low stroke volumes, resulting in flow rates (approximately 0.05–0.15 µL/min) that are insufficient for many OoC applications requiring roughly 0.5–3.3 µL/min or higher. The research goal is to develop and systematically characterize cleanroom-free, PDMS-compatible “macrovalves” capable of sealing and actuating over OoC-relevant channel dimensions and achieving higher flow rates suitable for automated pumping and mixing. The work aims to enable higher-throughput, automated OoC operation, reduce manual handling, and facilitate translation of mLSI capabilities to larger-scale OoC platforms using an accessible, low-cost fabrication approach (micromilling of direct positive molds).

The introduction surveys OoC platforms and throughput challenges, highlighting existing multiplexed systems (e.g., Mimetas OrganoPlate) that still require extensive manual pipetting and have small culture areas. Integrated, normally open PDMS valves (Quake-style) are widely used for automation in microfluidics (mixing, pumping, multiplexing) but are constrained by fabrication via reflow photoresist, limiting channel heights to tens of micrometers and yielding low flow rates. Alternative fabrication methods include photolithography approaches capable of ~250 µm height/400 µm width valves (Freitas et al.), albeit with complex additional steps, and fully 3D-printed valves (Lee et al., Glick et al.) that are not directly PDMS-integrated. 3D-printed molds for PDMS face challenges with material compatibility, surface finish, resolution, repeatability, and photocrosslinker interference. Micromilling is positioned as a cleanroom-free, rapid, versatile method producing robust molds with complex 3D geometries and less labor than reflow-photoresist lithography. Prior PDMS macrovalve work using micromilled molds required negative molds and double casting or PET casting, and was limited by tool geometry. The gap identified is a direct positive micromilled mold method enabling Quake-style PDMS macrovalves at hundreds of micrometers dimensions without these limitations.

Devices comprised three layers: a glass substrate, a PDMS control layer with rectangular control channels covered by a thin flexible PDMS membrane, and a PDMS flow layer containing rounded flow channels. Direct positive molds were fabricated by micromilling polymethylmethacrylate (PMMA) to create upstanding, rounded structures for the flow channels and rectangular features for the control channels. PDMS formulations: flow layer cast using PDMS mixed at 7:1 (base:curing agent) on the micromilled mold; control layer spin-coated using PDMS mixed at 20:1 to form a thin membrane above the control channels. After precuring both layers, they were aligned and bonded (per Unger et al.) to assemble the final device with the control layer bonded to glass and the flow layer on top. Surface roughness of micromilled rounded features exhibited a staircase pattern (10 µm vertical steps) observable in SEM; optional smoothing of PMMA molds was performed by a 5-minute chloroform solvent treatment (per Ogilvie et al.), which reduced roughness as evidenced by SEM and profilometry. For systematic valve characterization, four chip versions were designed, each with a fixed rounded flow channel width (250, 500, 750, or 1000 µm). For each, the flow channel height was varied as a percentage of the width (from 10% to 70%). Five control channels with varying widths (as percentages of the flow channel width; including 10–12%, 40%, 60%, and 100%) were included; control channel height was fixed per chip version. Flow channels were filled with blue dye; valve actuation was tested by applying control pressures of 1.00, 1.25, 1.50, and 1.75 bar and observing valve closure under a microscope (presence/absence of dye). Leak testing of closed valves indicated leak-free performance to a detection limit of 10 nL/min. Beyond characterization, two macrovalve designs were used to demonstrate functions: (1) a peristaltic pump and a mixing/metering device using a valve design that closes a 400 µm high, 1000 µm wide channel, and (2) a recirculation chip for a proof-of-concept endothelial cell culture using a valve design closing a 200 µm high, 1000 µm wide channel; molds for the cell culture device received chloroform smoothing. Performance metrics included maximum pump rate, mixing time for a known volume, and multi-day cell culture under continuous perfusion with automated medium refreshment.

- Macrovalves fabricated via micromilled direct positive molds can fully close rounded PDMS flow channels up to 700 µm high and 1000 µm wide. - Valves are leak-free when closed to a detection limit of 10 nL/min and suitable for pumping operations regardless of the optional mold-smoothing step. - Systematic characterization across flow widths of 250, 500, 750, and 1000 µm and heights up to 70% of width showed: bridging is reliable even with narrow control channels (10–12% of flow width) when flow height exceeds 20% of width; the most robust closure occurs when the control channel width equals the flow channel width (at the cost of footprint); a control channel width of ~60% of flow width suffices at higher actuation pressures (1.5–1.75 bar). - Demonstrations: • Peristaltic pump achieved up to 48 µL/min using a valve design that closes a 400 µm high, 1000 µm wide channel. • Mixing and metering device achieved complete mixing of 6.4 µL within 17 s. • Proof-of-concept endothelial cell culture maintained over multiple days under continuous perfusion with automated medium refreshment, indicating biocompatibility of integrated macrovalves. - Practical fabrication insights: micromilling yields staircase roughness (~10 µm step height) on rounded features, which can be smoothed via a 5-minute chloroform treatment on PMMA molds; smoothing was applied for the cell culture recirculation chip.

The work demonstrates that cleanroom-free, micromilled direct positive molds enable Quake-style PDMS macrovalves compatible with organ-on-chip-relevant dimensions, overcoming height and stroke-volume limitations of reflow-photoresist-based microvalves. By achieving closure of channels up to 700 µm high and 1000 µm wide and delivering pump rates in the tens of microliters per minute, the macrovalves meet and exceed typical OoC flow requirements, enabling automated pumping and rapid mixing in larger channels. The systematic design guide relating flow channel dimensions, control channel widths, and actuation pressures provides practical rules for engineers to balance valve robustness against footprint (e.g., ~60% control width at elevated pressures for compact designs). The ability to fabricate devices without a cleanroom, using rapid micromilling, reduces cost and complexity and supports scalability and commercialization of PDMS-based OoC platforms. Biocompatibility demonstrated through multi-day endothelial culture under perfusion suggests direct applicability to automated, multiplexed OoC experiments that reduce manual handling and increase throughput.

This study introduces a cleanroom-free method to fabricate PDMS macrovalves via micromilled direct positive molds and provides a systematic characterization linking geometry and actuation pressure to valve performance. The approach enables sealing of large, rounded channels (up to 700 µm high and 1000 µm wide), supports high-flow peristaltic pumping (up to 48 µL/min), rapid mixing (6.4 µL in 17 s), and is compatible with cell culture under continuous perfusion, demonstrating biocompatibility. These results bridge microfluidic large-scale integration with organ-on-chip needs, offering a practical and scalable route to automated fluid handling in PDMS OoCs. Future work could explore long-term durability and cycling of macrovalves, broader ranges of pressures and geometries, integration into highly multiplexed OoC arrays, further surface optimization for sensitive cell types, and evaluation across varied biological assays and media to fully standardize and commercialize the platform.

- Characterization was performed for specific pressure ranges (1.0–1.75 bar) and a defined set of geometric ratios (flow channel heights up to 70% of width); performance outside these ranges was not reported. - Although leak-free operation to 10 nL/min was shown, long-term durability, cycling endurance, and failure modes were not detailed. - PDMS material considerations (e.g., absorption of small molecules) remain a general concern for some applications; the study focuses on PDMS due to its fabrication advantages. - Mold surface roughness from micromilling may require solvent smoothing for certain applications; smoothing was applied for the cell culture recirculation device but not for characterization, pump, or mixer devices. - Biological validation was limited to an initial endothelial cell culture demonstration; broader applicability across cell types and longer-term cultures was not assessed in this report.