Controlled 3D culture in Matrigel microbeads to analyze clonal acinar development.

3D culture systems are a valuable tool for modeling morphogenesis and carcinogenesis of epithelial tissue in a structurally appropriate context. We present a novel approach for 3D cell culture based on a flow-focusing microfluidic system that encapsulates epithelial cells in Matrigel beads. As a model we use prostatic and breast cells and assay for development of acini, polarized cellular spheres enclosing lumen. Each individual bead on average acts as a single 3D cell culture compartment generating one acinus per bead. Compared to standard protocols microfluidics provides increased control over the environment leading to more a uniform acini population. The increased facility of bead manipulation allowed us to isolate single cells which are self-sufficient to fully develop into acini in presence of Matrigel. Furthermore, combination of our microfluidic approach with large particle FACS opens new avenues in high throughput screening on single acini or spheroids.

Mapping the complex morphology of cell interactions with nanowire substrates using FIB-SEM.

Using high resolution focused ion beam scanning electron microscopy (FIB-SEM) we study the details of cell-nanostructure interactions using serial block face imaging. 3T3 Fibroblast cellular monolayers are cultured on flat glass as a control surface and on two types of nanostructured scaffold substrates made from silicon black (Nanograss) with low- and high nanowire density. After culturing for 72 hours the cells were fixed, heavy metal stained, embedded in resin, and processed with FIB-SEM block face imaging without removing the substrate. The sample preparation procedure, image acquisition and image post-processing were specifically optimised for cellular monolayers cultured on nanostructured substrates. Cells display a wide range of interactions with the nanostructures depending on the surface morphology, but also greatly varying from one cell to another on the same substrate, illustrating a wide phenotypic variability. Depending on the substrate and cell, we observe that cells could for instance: break the nanowires and engulf them, flatten the nanowires or simply reside on top of them. Given the complexity of interactions, we have categorised our observations and created an overview map. The results demonstrate that detailed nanoscale resolution images are required to begin understanding the wide variety of individual cells' interactions with a structured substrate. The map will provide a framework for light microscopy studies of such interactions indicating what modes of interactions must be considered.