Published: October 20th, 2018
Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.
A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.
Here, we describe the fabrication of dual channel, vascularized Organ-on-a-Chip (Organ Chip) microfluidic culture devices using a scalable protocol amenable for use by research groups lacking access to cleanrooms and traditional soft lithography tools. These devices have been developed to recapitulate human organ-level functions for understanding normal and disease physiology, as well as drug responses in vitro1,2. Critical to engineering this functionality are two perfused microfluidic channels separated by a semi-permeable membrane (Figure 1). This design enables recreation of tissu....
1. General Preparation
The protocol presented here describes the scalable fabrication of PDMS Organ Chips. These devices enable culture of two distinct perfused tissue types on an elastic porous membrane (Figure 1). The PDMS channels are cast using 3D printed molds, which accelerates prototyping of new designs (Figure 2A and 2B). Top channels are cast in molds under compression against a compliant polyurethane gasket to produce co.......
The fabrication process relies on high resolution 3D printed molds to pattern the PDMS top and bottom Organ Chip body components coupled with micromolded porous PDMS membranes. This critical approach was selected due to ease of prototyping combined with rapid transition into scaled up fabrication and replacement of tooling. The top component molds are designed to pattern ports in precise locations with defined vertical profiles during the casting step. This not only avoids the labor involved in manually punching access p.......
We thank M. Rousseau and S. Kroll for help with photography and videography and M. Ingram, J. Nguyen, D. Shea, and N. Wen for contributions to initial fabrication protocol development. This research was sponsored by the Wyss Institute for Biologically Inspired Engineering at Harvard University and the Defense Advanced Research Projects Agency under Cooperative Agreements #W911NF-12-2-0036 and #W911NF-16-C-0050, and FDA grant #HHSF223201310079C, NIH grants #R01-EB020004 and #UG3-HL141797-01, and Bill and Melinda Gates Foundation grants #OPP1163237 and #OPP1173198 to DEI. The views and conclusions contained in this document are those of the authors and should not be int....
|Personal Protective Equipment
|Tyvek lab coat
|Extended cuff gloves
|Mold-in-place (MIP) top molds
|printed in Prototherm 12120
|Mold-in-place (MIP) bottom molds
|printed in Prototherm 12121
|Duckbill curved forceps
|Sharp tipped forceps
|Electron Microscopy Sciences
|Deep reactive ion etch (DRIE) pillar array wafers
|Four 50 x 50 mm pillar arrays per wafer; pillars 7 um wide, 50 um tall, spaced hexagonally 40 um apart
|Textured polycarbonate .01” thick
|cut to 45 mm square
|PDMS blocks (40 x 40 x 5 mm)
|Laminar flow hood
|60°C level oven
|accuracy to 0.1 g
|oxygen plasma capability is critical
|Sylgard 184 poly (dimethylsiloxane) (PDMS) base/curing agent kit
|Ensure adequate ventilation when handling prepolymer due to low levels of ethylbenzene
|1 mL syringe
|25 x 75 mm glass microscope slides
|Die-cut Polyurethane (PU) strips
|Atlantic Gasket, Inc.
|1/8” thick; 60 Durometer Black Polyurethane; 2” x 3”
|Polycarbonate film .005” thick
|100 x 100 x 15 mm square gridded petri dishes
|Thinky PDMS Mixer
|Mold-in place (MIP) jig
|screw clamp compression jig
|Automated membrane fabricator (AMF)
|pneumatic compression piston array with programmable heater
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