Agile manufacturing of PDMS microfluidic devices for cell culture without a clean room

Microfluidic devices control fluid flow and the cellular microenvironment, and their fabrication usually relies on photolithography in a clean room. For devices with features of hundreds of micrometers to millimeters, this route is slow and costly, especially during prototyping, when the design changes after each test. Here, we describe and benchmark a workflow to fabricate polydimethylsiloxane (PDMS) devices from laser-cut master molds, with equipment accessible to most biology laboratories. We cut three inexpensive materials, poly(methyl methacrylate) (PMMA), polystyrene and polyvinyl chloride (PVC) tape, over a grid of laser power and speed and measured each cut by scanning electron microscopy. Cut depth and width increased line-arly with the laser energy delivered per unit length, which gives a simple rule to choose the settings for each material. We then bonded the PDMS replicas to glass without a plasma cleaner, by stopping curing early, once the replica could be demolded, and completing it in contact with the glass. Without any surface treatment, these devices held up to about 400 mbar; activating the glass with a handheld corona device raised this to about 900 mbar, enough for perfusion. The master material set the surface of the device: PMMA gave smooth replicas, whereas PVC tape gave rough walls. A three-channel device molded on PMMA held two cell-laden gels in place with phase guides and supported the co-culture of cancer cells and fibroblasts under perfusion. Further analysis of flow laminarity and different mixer designs confirmed the robustness of this method. This workflow takes a digital design to a working de-vice within a day and lets biology laboratories adapt microfluidic devices to their experiments without the need of a clean room.