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 tissue-tissue interfaces between at least two types of tissues, typically organ parenchymal cells on one side of the porous membrane and vascular endothelium on the other, as well as their exposure to fluid flow. In addition, because the elastomeric polymer, poly (dimethyl siloxane) (PDMS), is used to fabricate the Organ Chip body and membrane components, cyclic mechanical strain can be applied to the entire engineered tissue-tissue interface via the elastic membrane to mimic the natural physical microenvironment of living organs, such as breathing motions in the lung and peristalsis in the intestine.

Figure 1: Organ Chip cross section. Organ Chips consist of two channels separated by a porous, elastic membrane that can be seeded with cells on both sides. Top channel cross sections are 1 mm wide x 1 mm high, bottom channel cross sections are 1 mm wide x 0.2 mm high, and vacuum channels in both and bottom parts are 0.3 mm wide, 0.5 mm high, and spaced 0.3 mm from the fluidic channels. Please click here to view a larger version of this figure.

These stretchable, dual channel, Organ Chips have been used for demonstrating the impact of breathing motion on nanoparticle absorption in the lung and drug-induced pulmonary edema^3,4; effects of peristaltic motion on differentiation⁵ and bacterial overgrowth in the intestine^5,6,7; and influence of cyclic deformations due to the pulsation of the heart on differentiation and maturation of glomerular podocytes in the kidney⁸. Additionally, these two-lumen devices that contain an endothelium-lined vascular channel separated by an extracellular matrix (ECM)-coated membrane from parenchymal cells within a separately accessible channel are well suited for characterization of drug PK parameters and new target discovery, which has been limited in single perfusion channel systems. Moreover, multiple Organ Chips may be linked together via their vascular channels to effectively create a human body-on-chips, which could offer an attractive human in vitro platform for therapeutics development^9,10. Unlike most micro-physiological systems (MPS)^11,12,13, the Organ Chips contain two microfluidic channels separated by a porous membrane that facilitates vascular-parenchymal interactions to recapitulate in vivo organ function. This not only simplifies linking of different organs together by perfusing a common medium through the vascular channels, but the compartmentalization of tissues and fluids mimics in vivo functions and supports pharmacokinetic experimentation and modeling as well as in vitro–in vivo extrapolation^9,10 that is difficult or impossible in single channel MPS^14,15,16. The popularity of PDMS in microfluidic devices has led to the development of tools to overcome the material's inherent ability to absorb small molecules^10,17. However, the large numbers of chips required to support biological studies where the use of microbial agents and PDMS-absorbing compounds make reuse of Organ Chips difficult necessitates a scalable manufacturing process even for small research groups. The protocol described here presents a method for the device fabrication suitable for the use in academic laboratories, including those lacking access to cleanrooms and soft lithography. This protocol aims to broaden access to Organ Chips by a broad range of researchers seeking to use the stretchable, dual-channel devices for exploring basic biological processes as well as translational therapeutic development.

Leveraging best practices from micromanufacturing fields coupled with design for manufacturing, a robust approach was developed for fabricating Organ Chip devices in large quantities with high reproducibility and yield. The fabrication protocol described here provides a scalable method for Organ Chip production. We describe the use of an optional Mold-in-Place Jig (MiP; design details in Supplemental Materials) coupled with polyurethane gasket strips to enable scaling up of casting PDMS components. The glossy side of polyurethane strips produce optically smooth PDMS parts while the textured side facilitates demolding. We also describe the use of an optional Automated Membrane Fabricator (AMF) that provides uniform compression of membrane wafer molds during curing for fabricating up to 24 membranes per batch. The design is broadly applicable for studies of organs that are composed of tissues that experience mechanical strain and perfusion, and these chips can be produced with low chip-to-chip variability in quantities required to meet the needs of small and large research groups alike. The workflow is amenable to a batch or assembly line format, and readily compatible with quality assessment protocols for control of production processes, personnel training, and responsive troubleshooting. We hope that this protocol will expand access to the capabilities of dual channel, stretchable, Organ Chips for basic and translational research.

Protocol

1. General Preparation To avoid debris, clean work area using packing tape and wipe down area with a cleanroom wipe and isopropyl alcohol. 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. Clean all molds with air gun to blow out debris prior to use….

Representative 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

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