Cultura de organotypic método para estudar o desenvolvimento de tecidos embrionários de galinha

The overall goal of this protocol is to provide access to embryonic chicken eyes in an in-vitro setting to enable in-depth study of complex developmental pathways in the embryo. In the embryonic chicken, studying the eye is often difficult, due to eyelid development, embryo movement, and the growth of extra embryonic tissues. Compounds such as drugs and inhibitors can also have lethal effects on the embryo, making it difficult to use these compounds to modulate gene pathways.

Advances in in-vitro tissue culturing have allowed scientists to isolate these tissues and eliminate many of these aforementioned barriers. Our protocol is advantageous in that it allows us to study the eye as a whole, including all of the tissues that have the potential to interact during development, such as the neural retina and the retinal pigmented epithelium. Begin by autoclaving the materials to ensure that all solutions and equipment are sterile prior to the procedure.

To do this, autoclave two liters of distilled water and one liter of chick saline, along with a box of tissues, semi-porous filter paper, wire mesh, water containers, and disposable glass pipettes. Thoroughly sterilize the incubator shelves and door using 70%ethanol, then fill the containers with autoclaved, distilled water, and preheat the incubator to 37 degrees Celsius. Prewarm the nutrient medium, along with a tube of the antibiotic penicillin streptomycin in the incubator.

Lay a paper towel across the workspace and sterilize with 70%ethanol. Sterilize all forceps and scissors. With the chicken head in autoclaved chick saline, bisect the chick head in the midline, leaving some tissue in tact around each eye.

The extra tissue will help the eye maintain some tension in the culture, as well as assisting with nutrient uptake throughout the eye and preventing the eye from sliding off the filter paper. These tissues also serve as landmarks when orienting the eye in later analysis. Leave the dissected tissue in room-temperature chick saline while preparing the culture.

Prepare the culture dish. Place two 35-millimeter Petri dishes inside one 100-millimeter Petri dish. Place one wire mesh stage in each small dish.

Place the half-head with the eye onto a piece of filter paper, tilting slightly to ensure the eye will not slide off the paper. Transfer the tissue and filter paper to the small Petri dish atop the wire mesh stage. Repeat with the other half-head of the same embryo.

Add culture medium to each of the two small Petri dishes. Bring the medium up to the level of the tissue paper. Do not submerge the tissue in the medium.

Add 50 microliters of the antibiotic penicillin streptomycin stock solution to the nutrient medium in each dish in order to prevent bacterial infection. Place a folded piece of thin tissue on the inner edge of the larger Petri dish and moisten it with distilled water. Incubate the culture in the dark incubator at 37 degrees Celsius for up to four days.

Following culturing, remove the tissue from the filter paper and fix in 4%paraformaldehyde. The first column shows an HH34 eye, representing the embryonic chick eye at the beginning of culturing. The second column shows an embryonic chick eye cultured for four days, beginning at HH34.

The third column shows an HH38 eye, which corresponds to four days of in-ovo development beyond HH34. Panels A to F are unstained, while panels G to I are stained for the alkaline phosphatase enzyme. The top row, panels A to C, shows that the eye continues to develop throughout culture, evidenced by the appearance of feather buds and the growth of the eyelids, indicated by arrows in each panel.

The middle row, panels D to F, shows that the conjunctival papillae begin to degenerate in culture. The bottom row, panels G to I, shows that the spatiotemporal pattern of scleral ossicle induction is maintained in culture, serving as an example of a developmental pathway that can be studied using this method. This protocol allows tissues to maintain key spacial and temporal aspects of development throughout the entirety of culture.

This protocol represents a key advancement in the culturing of whole sensory organs, providing alternative methodology for studying the intricate developmental pathways involved.