运用

The overall goal of this procedure is to examine the effects of small molecule inhibitors on fetal organ development using an ex vivo droplet culture technique. This is accomplished by first opening the peritoneum of a pregnant mouse and removing the embryo containing uterus. Next, the embryos are removed from the uterus and are freed from the yolk and amniotic sacs.

Then the goad me neph complex is dissected and isolated from the embryo. Finally, the goad Meris complex is cultured in a droplet with or without a small molecule inhibitor. Ultimately, immunofluorescence microscopy is used to show changes in organ architecture using cell type specific antibodies.

So the advantage of our whole organ ex vivo upright droplet culture compared to say cell culture lines, is that it has the advantage of having three dimensional structure giving rise to cellular interactions due to locality, as well as extracellular matric signaling that are may be important for organ formation and patterning. Furthermore, our upright droplet technique allows for less reagents to be utilized, therefore reducing cost and potential toxicity due to side effects. This is an advantage over other whole organ techniques, and it still allows for the diffusion of pharmacological agents to the target organ.

Although this technique can provide insight into the vascular development of fetal testes, it can also be applied to other fetal organs like the lung, and we can also use it to investigate diverse biological processes. There are other cell signaling pathways during fetal development At E 11.5. After euthanizing the pregnant mouse, according to the text, use 70%ethanol to spray down the abdomen with fine scissors.

Make a V-shaped incision to open the belly skin and peritoneum and pull back the flap of tissue to expose the anterior organs. Locate the ovaries at both ends of the uterine horn, then with scissors cut at the ovary and the connective tissue to separate the uterus containing the embryos from the mother's body. Next, open the uterine wall by gently cutting the side opposite the placenta, exposing the yolk sacks.

Be careful not to puncture the yolk sack to avoid damaging or losing embryos. Then cut near the placenta to remove the embryo containing yolk sacks and place them into a dish containing PBS with calcium and magnesium, which will help support cell adhesion molecules to maintain tissue architecture and prevent the tissue from sticking to the tools With forceps, remove the yolk sac and amnion from the embryo by carefully puncturing the yolk sac to create a hole in which the embryo can slide out. Once an embryo is outside of the yolk sac, cut the umbilical cord.

From this point forward, use a microscope located in a sterile tissue culture hood. Remove the head of the embryo by using forceps to pinch on either side of the neck, discard the head, remove the tail, and place it into a new micro centrifuge tube for X-Y-P-C-R analysis to determine the sex of the embryo. Now secure the embryo in a supine position against the bottom of the culture dish by using a pair of forceps to pin the armpits of the embryo with another pair of forceps.

Remove the skin covering the abdomen and gently remove the liver intestines and other organs to expose the back body wall where the urogenital ridge is located. Use the forceps to scoop underneath the urogenital ridge by carefully opening the forceps and lifting up. Remove the urogenital ridge.

Take care not to cause damage to the gonads. Transfer the urogenital ridge into a dish of C-D-M-E-M to acclimate the tissue to the medium using 27 gauge needles to separate the gonad Sphs complex from the rest of the urogenital bridge. Use one needle to cut by pressing down and the other to guide the tissue correctly to allow optimal separation to culture.

The gonad with a small molecule inhibitor set two 20 microliter pipettes to 15 microliters each using a clean sterilized razor blade. Cut about one to two millimeters off one of the barrier pipette tips for transfer of the gonads and addition of control C-D-M-E-M. Line up the gonads with their long axis parallel to the pipette tip so they can easily be pipetted with a cutoff tip Label one side of a 35 millimeter dish lid as the control droplet and the other as the drug containing droplet.

Make sure the labels on the lids match the labels on tail tissue containing micro centrifuge tubes. Pipette 15 microliters of C-D-M-E-M containing a single gonad into a droplet in the lid. Taking care that gonads don't stick to the inside of the pipette tip and repeat with the second droplet on the other side.

Check under the microscope that the gonads have been transferred to the droplet designated as the control. Use the control pipette to add an additional 15 microliters of C-D-M-E-M containing DMSO to arrive at a 30 microliter droplet to the other droplet. Use the drug designated pipette to add 15 microliters of TKI two in C-D-M-E-M using the pipette.

Spread out the droplets in an expanding circular pattern until they are about 15 to 18 millimeters in diameter and the gonad is located roughly in the middle. Orient the gonad so that it lies on its side and the goad and the messines are easily distinguishable. Orient the lungs so that the two lobes lay flat and are held in place by surface tension while not touching one another.

Carefully place the culture dish lid with the droplets upright and laying flat on the surface of the water in a large humidifier dish, ensuring that there are no bubbles trapped underneath and that no water gets into the lid. To create a small humidified chamber immediately cover the large dish with its lid, ensuring that the small dish lids are able to move freely in the larger dish and that air exchange can occur. Once the two small lids are placed into the chamber, immediately placed the chamber into a 37 degree Celsius Carbon dioxide incubator for 48 hours.

Carry out PCR and immunofluorescence according to the text protocol. When cultured using the Exvivo droplet culture system, fetal gona sphs complexes have a high survival rate as shown here. Comparisons of E 11.5 gonads at initial incubation versus 24 or 48 hours in culture reveal a dramatic change in gonad shape and the appearance of stripe like cord structures in the control XY gonad as seen here.

The droplet culture system can recreate testis differentiation events ex vivo as it is possible to visualize SOX nine positive sertoli cells in XY gonads forming into tubo like cords and vasculature forming throughout the organ. In lungs, 48 hour culture results in increased branching of SOX nine eco herrin double positive epithelial branches. While there is some increase in apoptotic cells in lungs, in the same culture conditions, similar levels of apoptosis are seen in control cultured and in utero.

Gonads suggesting that the gonad is particularly amenable to the culture conditions. To examine the effects of vascularization and vascular remodeling in testis morphogenesis, the small molecule inhibitor TKI two, which blocks the activity of VEGF receptors was used. The results demonstrate that disruption of vascular remodeling in the fetal testis is effective in the droplet culture system, and subsequent defects in testis cord morphogenesis can be visualized Following this procedure.

Other methods like real-time quantitative PCR, next generation RNA sequencing or flow cytometry can be performed to answer additional questions like how different pharmacological reagents affect the expression of protein or genes of interest. Additionally, we can use time-lapse live imaging to visualize cellular dynamics in real time if transgenic fluorescent embryos are used. Additionally, a wide array of reagents is available to target candidate signaling pathways that may be involved in fetal organogenesis.

These techniques will help us to understand the underlying mechanisms that drive fetal development. After watching this video, you should be able to perform whole organ ex vivo upright droplet cultures in order to elucidate signaling pathways during fetal organogenesis. This technique involves dissection of mid gestational embryos, the isolation of target organs, and the preparation of upright droplet cultures for incubation of these organs with pharmaceutical reagents.