WARF: P08003US
A Three-Dimensional Culture Device for Controlling Soluble Factor Microenvironments

INVENTORS

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David Beebe, Vinay Abhyankar, Michael Toepke

Chemical gradients play a critical role in maintaining and regulating biological activity in vivo. A disruption in these chemical gradients can result in a variety of biological imperfections, such as cancer. Cells respond to the gradients by secreting soluble signaling factors that affect either the cell itself or other cells further away.

The response of cells to different chemical cues can be studied by generating gradients with the use of laminar flow based devices, which produce streams having no disruption between parallel layers. However, the continuous flow produced by these devices can wash away signaling factors, making them unsuitable when soluble factors are important in regulating cell behavior.

Additionally, current cell culture platforms use a two-dimensional approach to study how cells respond to gradients. A more physiologically-relevant three-dimensional approach would provide better insight into highly regulated processes, such as cellular differentiation and development, where soluble factors are of key importance.

Significant efforts have been made in developing three-dimensional scaffolds that create defined chemical environments as a means to study migration and reorganization of cell populations in the three-dimensional matrix. However, current three-dimensional scaffolds do not offer the ability to spatially and temporally control soluble factors and gradients. UW-Madison researchers have developed a device and method to dynamically control the soluble factor microenvironment within a three-dimensional gel construct. The gel material acts as a highly fluidic resistant membrane to minimize convective flow, resulting in the diffusive transport of soluble factors and chemicals. This approach utilizes diffusion and channel geometry to generate and maintain stable linear and non-linear concentrations within a three-dimensional gel.

The device’s design consists of two fluid reservoirs, containing the source and sink solutions, and a connecting fluid channel. Concentrations in the source and sink are held constant resulting in a steady state concentration profile within the connecting fluid channel. The source reservoir must be replenished in an appropriate manner periodically to maintain the gradient. The geometry of the channel that connects the source and sink determines the steady state profile; straight channels yield linear profiles, v-shaped channels produce logarithmic profiles, and channels with exponential wall profiles produce exponential concentration profiles.

• The total U.S. microfluidics and lab-on-a-chip Market in 2005 was $84.3M and forecast to reach $200M by 2012.
• The cell market segment includes cell manipulation/lysis/cytometry/electrophysiology and cell culture/microbiology.
• In 2005, this segment represented 12% of the total U.S. microfluidics and lab-on-a-chip market space with an expectation that in the future, more researchers will be utilizing microfluidics for protein and cellular analysis.
• In addition, many companies are targeting solutions in this market space because the current cell-based assays are extremely time consuming and require a high sample content.

• Complex three-dimensional cell culture assays that allow studies of highly regulated processes such as embryonic development, stem cell differentiation, immune response and cancer metastasis

• Lack of fluid flow greatly reduces the volume of reagents used, thereby reducing reagent costs.
• Three-dimensional design and diffusion-regulated flow provides in vivo-like conditions more suitable for studies than those offered by two-dimensional designs.
• Provides robust spatial and temporal control over the soluble factor in a three-dimensional microenvironment by coupling stable gradients and localized soluble factor pulses within the same platform
• Sustainable gradients over long periods (10 days or more) are important to create a more in vivo-like environment for cell differentiation, migration and invasion assays.
• Provides broad accessibility and easy scale-up and integration into existing systems
• A wide range of materials such as agarose, collagen, Matrigel^TM, or engineered polymers can be used as gel construct material.
   

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