Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

Summary

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.

Abstract

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.

Introduction

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 vitro^1,2. Critical to engineering this functionality are two perfused microfluidic channels separated by a semi-permeable membrane (Figure 1). This design enables recreation of tissu....

Protocol

1. General Preparation

1. To avoid debris, clean work area using packing tape and wipe down area with a cleanroom wipe and isopropyl alcohol.
2. For all steps requiring PDMS, mix PDMS at a 10:1 ratio (10 g of cross linking agent, 100 g of elastomer base). Mix by hand or with a commercially available mixer. Use a planetary centrifugal mixer here: mixing for 2 minutes at 2000 rpm, then degassing the PDMS for 2 minutes at 2200 rpm.
3. Clean all molds with air gun to blow out debris prior to use.
   CAUTION: Do not use metal forceps to remove debris as it will damage the surface of the molds.

2. ....

Results

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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Personal Protective Equipment
Hairnet	VWR	89107-770
Tyvek lab coat	VWR	13450-506
Extended cuff gloves	VWR	89521-898
Equipment
Cutting mat	VWR	102096-430
Tile cutter	McMaster-Carr	26765A31
Mold-in-place (MIP) top molds	Protolabs, Inc.	custom	printed in Prototherm 12120
Mold-in-place (MIP) bottom molds	Protolabs, Inc.	custom	printed in Prototherm 12121
Duckbill curved forceps	VWR	63041-864
Sharp tipped forceps	Electron Microscopy Sciences	72700-D
Metal spatula	VWR	82027-528
Deep reactive ion etch (DRIE)  pillar array wafers	Sensera, Inc.	custom	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	McMaster-Carr	85585K33	cut to 45 mm square
PDMS blocks (40 x 40 x 5 mm)	n/a	custom
Laminar flow hood	Germfree	BVBI	cast in-house
Air gun
60°C level oven
Vacuum desiccator
Mass balance			accuracy to 0.1 g
Plasma machine	Diener	Nano	oxygen plasma capability is critical
Supplies
Sylgard 184 poly (dimethylsiloxane) (PDMS) base/curing agent kit	Ellsworth Adhesives	4019862
Mixing cup			Ensure adequate ventilation when handling prepolymer due to low levels of ethylbenzene
1 mL syringe	VWR	10099-395
Cleanroom wipes	VWR	TWTX1080
25 x 75 mm glass microscope slides	VWR	48311-703
Packing tape	VWR	500043-724
Scotch tape	VWR	500026-873
Die-cut Polyurethane (PU) strips	Atlantic Gasket, Inc.	custom: AGWI2X3	1/8” thick; 60 Durometer Black Polyurethane; 2” x 3”
Polycarbonate film .005” thick	McMaster-Carr	85585K102
100 x 100 x 15 mm square gridded petri dishes	VWR	60872-480
Aluminum foil
Optional Equipment
Thinky PDMS Mixer	Thinky	ARE-310
Mold-in place (MIP) jig	in-house		screw clamp compression jig
Automated membrane fabricator (AMF)	in-house		pneumatic compression piston array with programmable heater