Microfabricated Platforms for Mechanically Dynamic Cell Culture

Summary

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

Abstract

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

Protocol

A. Device description and operation

Devices are fabricated using multilayer soft lithography¹ in polydimethylsiloxane (PDMS), and are capable of simultaneously generating a range of mechanical conditions in individual cell culture locations across the microfabricated array. In this protocol, the steps to fabricate an array of pneumatically-actuated microposts are first described, followed by steps to modify the device to enable mechanically dynamic culture in both two-dimensional (2D) and three-dimensional (3D) culture paradigms. The outlined microfabricated approach increases throughput over existing macroscale systems, and is....

Discussion

Though conceptually simple, device fabrication does take some experimental skill and practice. Particularly in the case of 2D cell culture, alignment of the multiple layers in the device can be challenging, especially over a large-area array. Practically speaking, we can reliably achieve a 100% alignment success rate using devices with 50 μm of tolerance in spacing between adjacent features in multiple layers. We have also successfully demonstrated alignment with tolerances as low as 15 μm, but the alignment tim.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sylgard 184 PDMS Monomer and Crosslinker Kit	Dow Corning
SU-8 masters
Silanization agent: (tridecafluoro-1, 1, 2, 2-tetrahydrooctyl)-1-trichlorosilane	United Chemical Technologies
Foam pads			Craft supply stores, 1-2 mm thick
Overhead inkjet transparencies	Grand Toy
Plexiglass plates
C-clamp			hardware store
Micromanipulator system	Siskiyou, Inc.
Custom-made vaccum mount
Vision system, Navitar 12x zoom	Navitar
Connecting tubes	VWR international	Clay Adam Intramedic PE190
Blunt 18G needles	Small Parts (www.smallparts.com)
Eccentric diaphragm micropump	Schwarzer Precision	SP 500 EC-LC4.5V DC
Solenoid valves	Pneumadyne	S10MM-30-12-3
Solenoid manifold	Pneumadyne	MSV10-1
Function generator
3-(trimethoxysilyl) propyl methacrylate	Sigma-Aldrich
Polyethylene glycol diacrylate 3.4 kDa	Laysan Bio Inc.
Polyethylene glycol 8 kDa	Sigma-Aldrich
Irgacure 2959	Ciba Specialty Chemicals
1-vinyl-2-pyrolidinone	Sigma-Aldrich
Standard cell culture reagents