Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Cells in traditional 2D environments show inconsistent shapes and organelle organization. These variations limit the accuracy of cell-based assays. In contrast, in vivo cells exist in 3D environments influenced by extracellular matrix interactions. This discrepancy leads to unreliable results in phenotypic studies. Researchers have sought better methods to mimic natural cell environments. Prior work has demonstrated that 3D conditions can stabilize cell morphology and behavior. However, standardizing single-cell placement in 3D remains a challenge. This gap motivated the development of a device to control cell positioning in 3D.

The aim of this study was to create a device for in vitro 3D cell culture that standardizes single-cell placement. The researchers wanted to improve cell-based assays by mimicking physiological conditions. They focused on normalizing cell shape, polarity, and internal organization. The study also aimed to facilitate high-content screening of cellular processes. By controlling cell positioning, they hoped to reduce variability in experiments. The device was intended to support visualization of organelles and dynamic processes. Applications in drug screening and personalized medicine were also considered. The goal was to provide a reproducible platform for diverse biological systems.

The device was fabricated using replica molding to create an array of microcavities. These microcavities, called 'eggcups,' were patterned onto a PDMS layer. The PDMS layer was adhered to a coverslip for stability. Fibronectin was applied to the cavities to promote cell adhesion. Cells were introduced into the cavities through centrifugation. The filling efficiency was optimized for each cell type. Mammalian cells, fission yeast, budding yeast, and C. elegans were tested. The device enabled observation of organelles like the nucleus and Golgi apparatus.

The device successfully standardized cell placement in a 3D environment. Cell morphology and internal organization became more uniform. Up to 80% of cavities were filled with single cells or embryos. Visualization of the nucleus and Golgi apparatus improved significantly. The cytokinetic ring closure during mitosis was observed in detail. Periodic myosin and actin accumulations were identified during ring closure. Compact phenotypes were observed for Golgi and nucleus alignment. The device was validated for multiple species with specific adaptations.

The device provides a reproducible platform for 3D cell culture and high-content screening. It allows consistent cell positioning and improved visualization of organelles. The method was successfully applied to mammalian cells, yeast, and C. elegans. The device supports the study of dynamic processes like cytokinesis. It may enhance drug screening and personalized medicine applications. The researchers propose that this approach improves the accuracy of cell-based assays. The findings suggest that 3D environments better reflect physiological conditions. The device's versatility supports diverse biological investigations.