Mucociliary epithelium lines internal organs of our body and provides the first line of defense by clearing foreign particles. This protocol makes it possible to generate embryonic cell derived mucociliary epithelial organoid in 24 hours. The key advantage of our method is that we can monitor the live progression of cell transitions on the surface of organoids.
This reproducible, scalable, and rapid protocol for the developing organoid will allow researchers to address fundamental questions for the biology of mucociliary epithelium. To begin, obtain X.Laevis embryos by manually collecting eggs from stimulated female frogs and performing in vitro fertilization. De-jelly the fertilized embryos with gentle agitation in 2%cysteine, in one-third X-modified Barth's saline for about five minutes.
Culture the embryos in one-third XMBS at the preferred temperature until the first signs of stage 10 are detected, such as the appearance of dark pigmented cells around the blastopore at the vegetal view. Select and gather embryos as they reach early stage 10 using hair tools under a stereoscope. Transfer the selected embryos into a DFA-filled Petri dish with a disposable transfer pipette.
Then remove the vitelline membrane of the embryos using sharp forceps from the vegetal side, without disrupting the animal side of the embryo. To isolate the animal cap, position the animal side of the embryo up. Visually estimate the extent of the animal cap to be excised and make the first incision along the edge with a hair knife, pulling the knife outward to make the cut.
Repeat this process to create a chain of small cuts to excise the animal cap. Trim the thick-layered edge of the animal cap using a hair knife to prevent the inclusion of mesoderm precursors. To separate deep ectoderm cells from the animal cap, transfer the excised animal caps to a Petri dish filled with calcium and magnesium-free DFA with a disposable transfer pipette.
Position the animal caps to face animal side up, maintaining a generous distance from other explants. Wait for five to ten minutes, and then monitor the explants under a stereoscope. Once the loosened deep cells have been released from the edge of the dark-pigmented superficial layer, begin lifting the superficial layer away from the light-colored deep ectoderm cells.
Carefully detach the superficial layer with a hair knife, starting at the edge. Then collect the deep ectoderm cells with as little calcium and magnesium-free DFA as possible. Transfer collected deep ectoderm cells to non-adhesive PCR tubes containing 200 microliters of DFA and gently pipette the media two to three times to disperse the transferred cells.
Close the tubes and keep them upright to induce spontaneous aggregation at the bottom. Monitor the aggregation process under a stereo microscope. Cells typically gather at the bottom of the PCR tube within an hour and assemble into spherical aggregates within two to three hours depending on the size.
To conduct live imaging or drug testing during the development of the mucociliary epithelial organoids, collect aggregates at two hours post-aggregation using a 200 microliter pipette fitted with enlarged tips. To allow aggregates to develop into mucociliary epithelial organoids in culture, collect them from the PCR tube at five hours post-aggregation and transfer them to a DFA-filled Petri dish. Position the aggregates far apart from each other to prevent fusing.
Within 24 hours of culture at room temperature, without any added factors, mature mucociliary epithelial organoids can be observed rotating due to the beating cilia that covers the surface of the differentiated epithelium. Prepare a glass-bottom imaging chamber by gluing a cover glass to a custom milled acrylic chamber with silicon grease, tightly sealing the chamber to prevent leakage of the culture media, then fill the imaging chamber with DFA. Pick up one hexagonal transmission electron microscopy or TEM grid using forceps and apply a tiny amount of grease to the edge of the grid.
Press down lightly to secure the TEM grid to the bottom of the imaging chamber. Transfer the aggregates to the imaging chamber and position them within the grid. Fill the chamber with DFA and seal it with a cover glass and grease.
To follow the progression of mucociliary epithelial organoid formation, collect time-lapsed z-stack images of aggregates using a confocal microscope. Mucociliary epithelial organoids were generated from multipotent progenitors of early gastrula stage X.Laevis embryos. The organoids have a mature epidermis that is indistinguishable from that of a tadpole including a fully differentiated epithelium, mucus secreting goblet cells, multiciliated cells and small secretory cells.
The dynamics of organoid development were followed by live imaging. To examine the epithelialization that emerges at the early stage of organoid formation, the embryos were labeled with fluorescently tagged tight junction proteins and membrane localizing proteins. With dual labeling the sequential steps of ZO-1 positive tight junction formation can be marked and quantitatively analyzed during epithelialization.
Some regions of cell-cell adhesion have scattered puncta of ZO-1 at different stages of epithelialization. In contrast, other areas have fully assembled contiguous ZO-1 expression. Over time the puncta coalesce and connect to form contiguous tight junctions, which maintain their morphology even during cell division.
As the tight junctions mature, cells dynamically move in and out of the surface along the apical planes of the organoids. Multi-scale analysis is possible by tracking cells spatio-temporally on the surface of differentiating organoids. When attempting this protocol, remember to put the dark-pigmented side of the isolate animal cap facing up and monitor it under a stereoscope.
It is critical to separate the superficial layer of the animal cap at the right timing in calcium/magnesium free DFA media. This simple protocol offers handy ways to study dynamic cell behaviors as well as the molecular and physical mechanisms regulating mucociliary epithelium.
