A microfluidic platform for generation of sharp gradients in open-access culture

Abstract

Control of the 3D microenvironment for cultured cells is essential for understanding the complex relationships that biomolecular concentration gradients have on cellular growth, regeneration, and differentiation. This paper reports a microfluidic device for delivering gradients of soluble molecules to cells in an open reservoir without exposing the cells to flow. The cells are cultured on a polyester membrane that shields them from the flow that delivers the gradient. A novel "lid" design is implemented which prevents leakage from around the membrane without requiring sealing agents or adhesives. Once layers are molded, device fabrication can be performed within minutes while at room temperature. Surface gradients were characterized with epifluorescence microscopy; image analysis verified that sharp gradients (∼33 μm wide) can be reproducibly generated. We show that heterogeneous laminar flow patterns of Orange and Green Cell Tracker (CT) applied beneath the membrane can be localized to cells cultured on the other side; concentration profile scans show the extent of CT diffusion parallel to the membrane's surface to be 10-20 μm. Our device is ideal for conventional cell culture because the cell culture surface is readily accessible to physical manipulation (e.g., micropipette access), the cell culture medium is in direct contact with the incubator atmosphere (i.e., no special protocols for ensuring proper equilibration of gas concentrations are required), and the cells are not subjected to flow-induced shear forces, which are advantageous attributes not commonly found in closed-channel microfluidic designs.

An open-chamber flow-focusing device for focal stimulation of micropatterned cells

“…30 To address the numerous drawbacks of closed microfluidic systems, several groups have developed "open" devices that operate within an open architecture without sealed channels and chambers. 8,10,31,32 A central goal is for devices to deliver localized chemical stimulation to large cell culture surface areas and tissues in open architectures. Many designs seek to create a userfriendly experience for the researcher and a more benign microenvironment for the cells.…”

Biochemical perturbations of the mitotic spindle in Xenopus extracts using a diffusion-based microfluidic assay

“…In this regard, a microfluidic device offers powerful tools to modify the extracellular environment in a controlled manner through the use of concentration gradient generators. [1][2][3][4] These methods allow the manipulation of liquid solutions of varying compositions to rapidly and locally change the conditions in a small-scale system and monitor the resulting kinetic evolution. To control the extracellular environment of cells (bacteria or migrating cells), such strategies generally rely on the application of a hydrodynamic flow and require immobilized or adhering cells.…”

A modular cell culture device for generating arrays of gradients using stacked microfluidic flows

“…In order to avoid problems with enclosed channel designs, some designs utilize open microchambers to facilitate cell seeding and culture. 12,14,[18][19][20] Another effective solution has been to implement reversible sealing techniques such as clamping 21,22 or vacuum sealing, [23][24][25][26] which simplify integration of cell cultures with "add-on" microfluidic devices. More importantly, none of the devices presented to date has an architecture that is amenable to combinatorial testing of various gradients.…”

“…culture and shear flow confounding cell responses have led to the development of a second generation of microfluidic devices. 11 Several of these designs incorporate high-resistance microchannels [12][13][14] or porous barriers such as gels or membranes [15][16][17][18] to limit the exposure of cell cultures to shear forces. In order to avoid problems with enclosed channel designs, some designs utilize open microchambers to facilitate cell seeding and culture.…”