Accessible Silicone Chip-to-Membrane Sealing Procedure for Flexible, Reliable Bonding
March 20th, 2026
A novel sealing protocol to produce water-tight silicone-based seals for membranes of Polydimethylsiloxane (PDMS) chip devices. Intended for labs setting-up chip models and small-scale chip platform development. We demonstrate the protocol using plastic and native tissue membranes. This procedure only requires PDMS, toluene, mold, membrane, and a vacuum chamber.
We aim to fill a gap in organ-on-chip design, providing a new sealing procedure for PDMS devices with a porous membrane. Currently, chip sealing is challenging, but our process forces silicone through the pores of the membrane, meaning that you only deal with silicone-silicone bonding. If the membrane is porous, such as for a lung or gut-on-chip, then only PDMS, toluene, and a vacuum is required.
To begin, thoroughly mix liquid PDMS base with curing agent in a 10-to-1 ratio. Using a vacuum chamber, degas the mixture until all air bubbles are removed. Using a wide-nozzle syringe, fill the previously prepared molds with the degassed PDMS mixture.
Place the filled molds in a vacuum chamber and degas for approximately 10 minutes until all bubbles are removed. Using a dry oven, cure the molds for two hours at 65 degrees Celsius. After curing, remove the solid chip-halves from the molds using a scalpel.
Trim the flashing from the edges of each chip-half. Then use a biopsy punch to add four inlets to the top half at each end of the chamber and at the two adjacent rings, indicating the bottom chamber inlet locations. Place a 3D-printed stencil over a PET or PC membrane.
Using a scalpel, cut the membrane into shape while firmly holding the stencil in place. Alternatively, prepare an extracted ECM membrane and dehydrate it. Place the stencil over the membrane and cut it into shape while firmly holding the stencil in place.
Wear protective goggles and nitrile gloves. Mix liquid PDMS with toluene in a 7-to-5 ratio to prepare the mortar. Using a wide-nozzle syringe, mix thoroughly by syringing up and down until consistent.
Place all laboratory materials used in handling toluene in a fume hood for 24 hours to permit evaporation. Dispose of the materials in approved and labeled containers through specialist waste services. Now use a pipette tip to swiftly apply the prepared PDMS mortar to the surface of the chip-half where the membrane will be placed.
Using clean forceps, hold the prepared membrane at one end. Position the membrane tip onto the wet mortar at the correct location on the chip and allow it to fall into place. Gently hold the membrane in place.
Reapply PDMS mortar over the top of the membrane, moving away from the chamber with each stroke to remove bubbles and secure the membrane without shifting its position. Immediately place the membrane-bound chip-halves into a vacuum chamber after mortar application and cure them at room temperature under 25-millibar vacuum for 72 hours. Turn on the vacuum pump, pressure modulator, and plasma oven.
Set the chamber pressure to 320 millibars. Then place both the membrane-bound chip-half and the second chip-half inside for 30 seconds of oxygen plasma treatment. After removing the chips from the vacuum chamber, swiftly press the two halves together under light hand pressure to complete chip assembly.
PDMS mortar dyed with Nile red was used to visualize the intrusion of the liquid mortar into the 0.4-micrometer pores of the PET and PC membranes during room temperature vacuum sealing. Imaging under a light microscope showed that the silicone had successfully invaded the pores and thinly coated the surface. Basement membrane-containing extracellular matrix scaffolds also successfully bound within the chip device, demonstrating the broad applicability of this procedure for achieving strong, flexible bonding across diverse membrane types.
The extracellular matrix membrane integrity test revealed that the membrane withstood one millimeter of cyclical horizontal stretch induced by vacuum overnight. To validate the performance of the chip as an airway-on-chip for air-liquid interface cell culture, human lung Calu-3 epithelial cells were grown to confluence and air exposed in the device. After 10 days of apical air exposure, the cells differentiated into mucus-producing cells, as demonstrated by Alcian blue staining.
When epithelial cells are co-cultured with fibroblasts on the extracellular matrix membrane, epithelial cells formed a confluent monolayer on the inner side, while fibroblasts were distributed less densely on the outer surface and retained a spindle-like morphology. This procedure facilitates the production of organ-on-chip devices in the majority of live environments. It's accessible for new developers and also for lower income labs.
Epithelial cells can be cultured in the device at the air-liquid interface, co-cultured with other cell types, and their barrier integrity readily investigated.
Overview
This article presents a novel, accessible workflow for sealing PDMS-based Organ-on-Chip devices with porous membranes, enabling robust, leak-free assembly suitable for air-liquid interface (ALI) cultures. The method is designed to facilitate chip development for new researchers and resource-limited laboratories, supporting applications such as Airway-on-Chip, Gut-on-Chip, and Skin-on-Chip models.
Key Study Components
Area of Science
- Biomedical engineering
- Organ-on-Chip technology
- Cell culture device fabrication
Background
- Organ-on-Chip platforms are increasingly used for modeling human tissues and disease.
- Reliable, leak-free sealing of chip devices remains a major technical challenge, especially with porous membranes.
- Existing methods often require specialized materials or equipment, limiting accessibility for new or low-resource labs.
- There is a need for broadly applicable, simple sealing techniques compatible with various membrane types.
Purpose of Study
- To develop and demonstrate a robust, reproducible sealing workflow for PDMS Organ-on-Chip devices using porous membranes.
- To enable the fabrication of ALI chips suitable for cell culture, including airway, gut, and skin models.
- To validate the method's compatibility with both synthetic (PET, PC) and biological (ECM) membranes.
Methods Used
- Preparation of PDMS chip halves using 3D-printed molds and standard curing protocols.
- Cutting and shaping of porous PET, PC, or ECM membranes using stencils.
- Preparation of a PDMS-toluene mortar, degassed and applied to chip surfaces and membranes.
- Vacuum-assisted infiltration of PDMS mortar into membrane pores, followed by room temperature curing under vacuum to avoid thermal stress.
- Oxygen plasma treatment and manual assembly of chip halves for final device construction.
- Validation using Nile red dye visualization, mechanical stretch testing, and cell culture assays with Calu-3 epithelial cells and fibroblasts.
Main Results
- The PDMS mortar successfully infiltrated and bonded to 0.4 μm pores in PET and PC membranes, as visualized by microscopy.
- ECM membranes were also effectively sealed and withstood cyclical mechanical stretch overnight.
- Assembled chips supported ALI culture of Calu-3 epithelial cells, which differentiated into mucus-producing cells after 10 days of air exposure.
- Co-culture with fibroblasts was achieved, with distinct localization of cell types on the ECM membrane.
- The workflow is accessible, cost-effective, and suitable for a wide range of laboratory environments.
Conclusions
- This sealing procedure enables reliable, flexible bonding of PDMS chips to diverse porous membranes.
- The method supports robust ALI cell culture and co-culture applications in Organ-on-Chip devices.
- It lowers technical barriers for new entrants and resource-limited labs in the Organ-on-Chip field.
