Next-generation microwell and nanowell arrays for single-cell applications

Microwell arrays allow simultaneous, parallel study of a large number of single cells in replicate, making it possible to analyze each cell independently, while minimizing use of costly reagents. Isolation with this method is achieved by sweeping suspended cells at slow speeds across capture wells. When sized correctly, arrays tend to capture and retain exactly one cell per well¹, enabling a range of downstream analyses, such as monoclonal cell culture or single-cell multi-omics.

Beyond their versatility, the most obvious advantage of cell isolation with a microwell array is scale. While other techniques deposit individual cells into wells with a maximum density of 1536 wells per 13cm x 9cm microplate², microwell arrays can be fabricated at densities as high as hundreds of wells per square millimeter. Such a high data density opens a new world of analysis, allowing genomics, transcriptomics, and proteomics data from large populations of cells to be collected and analyzed at single-cell resolution.

Materials and Fabrication Methods

For custom research applications, microwell array capture devices are typically built in polydimethylsiloxane (PDMS), with photoetched silicon wafers used as molds. However, because PDMS lacks the durability and shelf-life for most commercial applications, device manufacturers may wish to build capture devices directly in photoetched silicon, laser-etched glass, nanoimprint-lithographed thermoplastic, or compression-molded thermoplastic.

Among these methods, compression molded thermoplastics stand out as an excellent option in terms of both price and product performance.

How Compression-Molding Allows Precision at the Micron Scale

If their only plastics experience is with injection-molded parts, device designers may be surprised to learn that micrometer-scale features can be molded in thermoplastic materials. The flow characteristics of molten plastic mean that injection molding struggles to fill the small, high aspect-ratio structures required for a dense array of micrometer-scale wells. This and related issues lead to incomplete feature formation in microscopic structures. Preventing these issues requires a very low feature aspect ratio and high draft angle, which limits both the capture efficiency and well density of an injection-molded microwell array.

With careful process control, however, compression molding sidesteps these issues so that the micron-scale, zero-draft-angle geometry produced with other microfabrication methods is transferred to plastic parts at high production volumes. For example, arrays of 50µm-diameter wells can be spaced only 2µm apart—a density of 427 wells per square millimeter.

Advantages of Microwell Arrays with Precision Geometry

Tight well spacing dramatically increases the number of single-cell isolation sites available per device. High surface utilization allows higher data density, enhancing sample throughput and data collection efficiency.

The geometry of wells can also be fine-tuned to maximize single-cell capture and retention¹. Wells with high aspect ratio and zero draft are ideal for capturing and retaining either cells or functionalized nanoparticles, and well diameter can be optimized to capture specific-sized cells, beads, or nanoparticles. Efficient capture leads to a further increase in data density, because the majority of wells are utilized.

The high aspect ratio and dense well spacing made possible by compression molding also allows superior fluorescence imaging performance compared to microwell arrays with high draft angle and limited well density, such as those produced by injection molding. This is because tightly packed wells have less refractive material around them, and because internal reflections off of a zero-draft, vertical well surface are less likely to interfere with imaging. While extremely small, tightly packed wells may experience some fluorescent cross-talk due simply to proximity, precision geometry facilitates easier software deconvolution. The practical result is better signal quality and lower limits of detection.

Micron-scale labelled fiducial markings can also be incorporated into arrays, enabling one-to-one tracking of well positions across multiple scans of a single array or when comparing data across multiple arrays. Additionally, high-precision registration features permit precise alignment for transfer of contents between arrays, such as when moving cells from capture to culture wells.

For clinical use and kit-based research applications, fitted two-part devices can be used to minimize process complexity, because a technician or automated system can easily transfer captured cells into culture wells by flipping the device over¹. Similarly, concentric wells can simplify multi-step loading procedures by employing a smaller well for capture of functionalized beads and a larger, concentric well above it for cell capture and analysis.

Microwell Array Cell Capture Applications

Microwell arrays can be incorporated into a wide range of device formats, including:

•   Open slides
•   Thin films
•   Flow cells
•   Multi-well plates, including 6-well, 24-well, 96-well, 384-well, and 1536-well
•   Transwell plates
•   Lab-on-a-chip devices
•   More complex devices

These formats, in turn, can be employed in a variety of applications. In addition to the single-cell monoclonal culture methods discussed above, single-cell capture with microwell arrays can feed into downstream applications such as dPCR, sequencing, cytokine-response drug discovery, pluripotent stem cell culture, FRET detection of protein interactions, biopanning, and other antibody discovery methods.

Considerations for Microwell Array Device Design & Manufacture

When considering the potential advantages of microwell arrays for single-cell capture, device manufacturers should keep their application requirements in mind, including:

•   Optimal well size and spacing for efficient capture of target cells
•   Array density required for desired throughput
•   Compatibility with optical imaging systems
•   Special features, such as fitted dual-well or concentric-well design
•   Fluid handling pumps and connections
•   Alignment, fiduciary, and registration requirements
•   Choice of thermoplastic material

By thinking about these factors at the design stage, product developers can fully utilize the favorable properties of compression-molded microwell arrays. The right design and manufacturing process will lead to more effective, efficient, and scalable solutions for single-cell isolation that are cost-effective and can feed into a range of downstream applications.

References

1. Lin, C.-H. et al. A Microfluidic Dual-well Device for High-throughput Single-cell Capture and Culture. Lab Chip (2015).
2.  Sinkala, E., Scherzinger, J., Lautscham, L. & Riba, J. Combined Single-cell Dispensing and 3D Full Well Imaging for Cell Lines with >99.99% Probability of Monoclonality. Preprint at https://www.bpes.co.uk/wp-content/uploads/2021/01/UP.SIGHT-app-note_final_01252021_english.pdf.