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Abstract

Introduction

Protocol

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Acknowledgements

Materials

References

Bioengineering

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

Published: October 20th, 2018

DOI:

10.3791/58151

1Wyss Institute for Biologically Inspired Engineering, Harvard University, 2Apple, Inc, 3Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard University, 4Vascular Biology Program and Department of Surgery, Boston Children's Hospital and Harvard Medical School
* These authors contributed equally

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....

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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.
    .......

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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.......

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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.......

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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....

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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

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