The iuvo Microconduit Array (MCA)

As improvements in technologies are made, screening labs are empowered to run more complex cellular assays that incorporate better biology. The mainstay is immortalized cell lines, but technological advances have resulted in increased use of primary cells and more complex assays such as mixed culture and three dimensional cellular assays. Each has advantages as well as limitations. BellBrook Labs has developed iuvo™, a microfluidic device that is compatible with commonly used automated instrumentation and addresses many of the limitations screening labs encounter when trying to adapt cellular assays to the demands of high throughput compound screening.

Cell lines are commonly used in HTS. They are easy to work with, can be grown in vast quantities, are easily replaced from frozen stocks, and they display homogeneity. Further, there is a lot of accompanying documentation available such as cell history, growth behavior, and functional data. However, cell lines do have disadvantages. They are subject to phenotypic and genotypic drift, especially those that have been stored in cell banks for long periods of time, and they lack the physiological relevance that primary cells have to offer. Phenotypic drift is especially of concern when the cell line is used as a disease model, as the phenotype has diverged from the original cell line and may provide misleading results.

There is a strong desire to use primary cells in HTS due to its physiological relevance. Disadvantages to using primary cells are that the availability of fresh cells is limited and unpredictable, and primary cells can be difficult to culture. Limited supply makes these cells precious and expensive, driving screening labs to miniaturize assays to preserve cells.

Complex cellular assays, especially those that are heterogeneous, can be more challenging when miniaturized. Immunocytochemisty, like other heterogeneous assays, requires multiple steps including reagent additions and washes. In a highly miniaturized format this becomes very difficult without disrupting cellular monolayers. Adding 3D to mixed cultures provides a system that more closely simulates in vivo growth. 3D systems typically require a matrix, such as collagen or Matrigel® to provide a scaffold upon which cells grow.

Microfluidics, with its exquisite control of fluid flows, is well-suited to miniaturized heterogeneous assays. However, adoption in high throughput screening has been limited by the tubing and specialized instrumentation required for microfluidic platforms. What has been needed is a technology that marries the benefits of microfluidics with instrumentation that HTS labs have already invested in and routinely use when performing compound screens. The iuvo™ Microconduit Array (MCA) is such a technology. iuvo™ is based on surface tension effects, where the difference in pressure inside droplets of unequal volume drives flow in passive structures. This principle is called passive pumping.

Figure 1. Passive pumping and the micro-conduit array

Passive pumping allows placement of a droplet to drive fluid exchange in microchannels. The foundation of passive pumping is described by the Young-LaPlace equation, which predicts that for similar conditions, a smaller drop will have a greater internal pressure than a large droplet. (A) Schematic of a microchannel showing (B) placement of a large drop at the output port that fills the channel via capillary action. (C) Placement of a small droplet on the input port resulting in (D) fluid replacement in the channel, with excess liquid being forced out the other end, or output port. (E) The MCA conforms to ANSI/SBS standards for the tray footprint and access port positions.

The first MCA to be developed is the iuvo™ Single Array. The device is represented in Figure 1. The 1µL channel volumes conserve cells and reagents, and provide a microscale environment that facilitates autocrine and paracrine signaling. The iuvo™ Single Array has 192 channels per device, and is compatible with hand-held pipettes and with 1-, 8-, and 96-tip liquid handling robots. Gentle washing through passive pumping makes the single array well-suited for highly-miniaturized heterogeneous assays, such as an angiogenesis model using co-culture of two primary cell types followed by immunocytochemistry staining (Figure 2). Stromal cells involved in angiogenesis play an important role in wound healing as well as tumor progression. To model vascularization, a mixed coculture model of angiogenesis was adapted to microchannels.

Figure 2. Primary HUVEC cells cultured with primary dermal fibroblasts showing tubule formation in angiogenesis

The iuvo™ Single Array also simplifies 3D matrix assays and reduces matrix volume. Tube-based microfluidics are not well suited for 3D matrices because of clogging as the matrix gels. The tubeless nature of passive pumping though, especially with disposable tips, makes 3D feasible for higher throughput assays. Further, the entire assay volume is viewable by inverted microscope. Cells in multiwell plates are farther than 200 µm away from the cover glass, so most cells can’t be imaged without going down to a lower power objective, such as 4X. The iuvo™ Single Array channel height is less than 250 µm; therefore, cells may be imaged using a 20X objective.

Cellular assays with greater predictive capacity are greatly needed in the drug discovery industry and in biomedical research in general. More complex cell models involving chemotaxis or three dimensional extracellular matrix can deliver such results, but are currently difficult to perform because of the limitations of conventional assay formats. Throughput of these assays is hindered by mechanical manipulations required of the researcher. iuvo™ is an expanding platform designed to remove these limitations. Devices under development will enable screening labs to perform more complex cellular assays, such as chemotaxis, and reconstituted tissue assays. 

For additional information about the iuvo™ Microconduit Array platform, please view the iuvo™ video found at bellbrooklabs.com/iuvo.html.