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Magnetically Attachable Polydimethylsiloxane Stencils (MAtS)
Vanderbilt researchers have developed a unique system for patterning cells or proteins in cell culture environments using magnetically attachable stencils (MAtS™) secured onto a culture surface by applying transbase magnets. The desired pattern is obtained by partially covering the culture surface using a MAtS™ stencil and subsequently applying cells or proteins in solution around the stencil. The efficacy of a stencil depends on the force sealing the stencil against the culture surface. Competitive stencil technologies achieve proper sealing by either 1) self adhesion of the stencil material, such as polymethylsiloxane or parylene-C to polystyrene and glass, or 2) mechanical compressive force. These techniques are insuffuicient because selfadhesion is not applicable to surfaces coated with water-based hydrophyllic biological substrates, and mechanical compression disrupts protein coatings on the cell culture surface. Additionally, mechanical compression methods impede access, and manipulation of the cells or proteins being patterned and are designed for use with specific multi-well plates. In the present technology, these limitations are obviated by use of attractive magnetic force on ferromagnetic elastomers.
In biological applications of cell culture, the physical positioning of cells, proteins and three-dimensional matrices at micrometer resolution is highly desirable. Currently this is achieved using two methods: 1) printing protein or cells by direct application of liquid materials in a defi ned pattern and 2) applying a stencil that excludes cells or proteins when they are subsequently applied to the culture surface. Both methods yield patterned applications. The first method requires high resolution technologies not routinely available to the cell biologist. The second method requires the application of mechanical compression or self-adhesion in order to seal the stencil against the culture surface. Techniques relying on self-adhesion are less obtrusive but not suitable for patterning on top of physiological matrices (such as fi bronectin, collagen and laminin) under the wet conditions required for cell culture. Conversely, techniques relying on compression generally obstruct access to the cell culture, require use of specific cell culture dishes and are destructive to the underlying matrix. To overcome these limitations, Vanderbilt researchers have developed a stencil that utilizes magnetic force to attach itself to any cell culture surface, such as a glass cover slip or multi-well plate. The magnetically attachable stencil overcomes the current limitations of stencil technology and provides all the features essential to patterning cells and proteins for cell culture, including:
1. Sterile and disposable application. Low material and production costs of MAtS™ make them suitable for sterile, disposable application.
2. Reproducible micro-scale resolution of the migration distance. MAtS™ create reproducible 400 and 800 micron gaps within a cell monolayer for reliable wound healing studies.
3. Unobtrusive use during cell culture procedures. MAtS™ are attached to cell culture surfaces by underlying magnets. The low profile of the stencil and magnet create a system that is unobtrusive to cell culture procedures.
4. Prevents cell damage and debris from the monolayer. By preventing cells from occupying precise areas, stencils enable collective migration or wound healing assays to be conducted without scratching away the cells.
5. Applicable to wet, proteincoated surfaces without disrupting protein coatings. MAtS™ are used to study cell migration on cell culture surfaces coated with pre-determined proteins such as an extracellular matrix protein (i.e., collagen or fibronectin).
The traditional wound healing assay (also known as the scratch assay) starts by scratching a narrow (500-900 micron) wound into a confluent monolayer of cells. The scratch divides the monolayer of cells into two populations that migrate toward each other until the cells have refilled the scratched area. This assay has two problems: 1) the cells and the scratch alter the composition of or totally remove any matrix proteins applied to the culture surface and 2) the scratch physically damages the cells and creates debris. In order to avoid the physical damage and debris caused by scratches, stencils have previously been developed to prevent cell adhesion at definable areas of the culture surface. Two basic designs have evolved. The first relies on self-adhesion of the stencil material to the cell culture surface. The limitation of this approach is that the stencils cannot adhere to wet protein coated surfaces. The second design that has evolved are multi-well plate inserts. The inserts press the stencil pattern against the culture surface by wedging tightly against the walls of the multi-well plate. This stencil design overcomes the need for self-adhesion but can only be used with its corresponding multi-well plate.
Of the two stencil designs, only the second is currently commercially available. Cell Biolabs sells their "CytoSelect™ 24-Well Wound Healing Assay" for $350 each. Their stencil creates rectangular open areas (0.9 mm by 4-5 mm) in a 24-well plate. Platypus Technologies markets "Oris™ Cell Migration Assay," which is a circular stencil (2 mm diameter) designed for use with a 96-well plate. Their kit sells for $198 each.
To the best of our knowledge, the only other upcoming contenders in this market are a recently-patented stencil made of Parylene-C that, like our product, is thin, unobtrusive and is a thin PDMS stencil that will auto-adhere to clean polystyrene or glass surfaces but not to wet, proteincoated surfaces.
In comparison with the abovementioned stencil technologies, the application of MAtS™ has the following advantages:
1. The pattern (gap) can be varied by design from small (200μm) to large (1000μm) and thereby accommodate the accurate analysis of slow as well as fast moving cells.
2. The coating or layer of underlying matrix protein is not disrupted by the stencil enabling studies of collective cell migration on physiologically relevant matrix.
3. The stencil does not impede access to the culture dish, the cells or the culture medium.
4. The stencil can be applied to any type of substrate, including culture dish, multi-well plate or glass cover slip or slide.
The simplicity of MAtS™ makes it suitable for several biological and biomedical markets. The initial market is expected to be the arena cof biology involved with cell migration--specifically, collective cell migration as measured by the traditional wound healing assay. This is a large market in which the application of MAtS™ is anticipated to grow as the advantages of the stencil open the door for new, better-controlled experiments. The second market is likely to be the smaller one of protein patterning for chemotactic and haptotactic assays. This market is smaller only because it is less developed. The simplicity and ease of use of MAtS™ will enable simple, cost-efficient research involving protein patterns. The disposable nature of MAtS™, along with the sterility, pre-ordered design at low cost and ready-made application, will facilitate wide-spread use.
According to Frost & Sullivan, the global market for cellular assay products was estimated to be $1.6 billion globally and $112.5 million in the U.S. in 2007. The functional assay segment accounted for 46.8% of revenues, and this was expected to grow at an annual rate of 9.6% through 2014.
Vanderbilt solicits interest from laboratory disposable materials manufacturers for further development and commercialization of this device based on licensing of the technology.
Intellectual Property Status
Currently an invention disclosure has been submitted to Vanderbilt; can be disclosed under NDA.
Inventors:Andries ZijlstraWilliam AshbyJohn Wikswo, Jr.Philip Samson