Search

Egg (oocyte) vitrification is the dominant method for preserving fertility for women of reproductive age. However, the method is typically performed by hand, requiring precise (∼0.1 to 10 μL) and time-sensitive (∼1 s) liquid exchange of cryoprotectants (CPA) around eggs as well as fine handling of eggs (∼100 μm) for immersion into liquid nitrogen (LN2). Here, we developed a microfluidic platform for programmable vitrification. Our platform is based on a millimeter-sized hanging droplet inside which a given egg is suspended and subjected to liquid exchanges within seconds. After programmable exposures to CPA, the egg is extracted from the liquid–air interface of the droplet using a motorized fine-tip instrument and immersed into LN2 for vitrification. To benchmark our platform with the manual method, we vitrified over a hundred mouse eggs and found comparable percentages (∼95%) for post-vitrification survivability. In addition, our platform performs real-time microscopy of the egg thereby enabling future studies where its morphology may be linked to functional outcomes. Our study contributes to the ongoing efforts to enhance the automation of embryology techniques towards broader applications in reproductive medicine both for clinical and research purposes. [ABSTRACT FROM AUTHOR]

The intestinal mucosal barrier forms a critical interface between lumen contents such as bacteria, drugs, and drug carriers and the underlying tissue. Current in vitro intestinal models, while recapitulating certain aspects of this barrier, generally present challenges with respect to imaging transport across mucus and uptake into enterocytes. A human mesofluidic small intestinal chip was designed to enable facile visualization of a mucosal interface created by growing primary human intestinal cells on a vertical hydrogel wall separating channels representing the intestinal lumen and circulatory flow. Type I collagen, fortified via cross-linking to prevent deformation and leaking during culture, was identified as a suitable gel wall material for supporting primary organoid-derived human duodenal epithelial cell attachment and monolayer formation. Addition of DAPT and PGE2 to culture medium paired with air-liquid interface culture increased the thickness of the mucus layer on epithelium grown within the device for 5 days from approximately 5 mm to 50 μm, making the model suitable for revealing intriguing features of interactions between luminal contents and the mucus barrier using live cell imaging. Time-lapse imaging of nanoparticle diffusion within mucus revealed a zone adjacent to the epithelium largely devoid of nanoparticles up to 4.5 hr after introduction to the lumen channel, as well as pockets of dimly lectin-stained mucus within which particles freely diffused, and apparent clumping of particles by mucus components. Multiple particle tracking conducted on the intact mucus layer in the chip revealed significant size-dependent differences in measured diffusion coefficients. E. coli introduced to the lumen channel were freely mobile within the mucus layer and appeared to intermittently contact the epithelial surface over 30 minute periods of culture. Mucus shedding into the lumen and turnover of mucus components within cells were visualized. Taken together, this system represents a powerful tool for visualization of interactions between luminal contents and an intact live mucosal barrier., Competing Interests: Conflicts of interest There are no conflicts to declare.