The overall goal of this procedure is to excise the embryonic heart for ex vivo culture. This is accomplished by first retrieving the uterine horn from a pregnant female mouse. Next, an embryo is retrieved and prepared for dissection.
Then the embryonic chest wall is opened to visualize the heart. Finally, the heart is excised and placed in a culture well containing dilute matrigel. Ultimately, results can be obtained that show a healthy live heart through viewing under a bright field microscope.
The main advantage of this technique over other methods like rocking cultures, is that the heart can be treated with either local agents like a protein conjugated to bead, or with global agents, such as adding a small molecule to the matrigel. The implications of this technique, it extends towards therapy for cardiovascular diseases because it allows the events within specialized segments of the heart to be examined in the context of the whole intact organ. After euthanizing a timed pregnant mouse at the desired embryonic day, using a technique approved by your institute liberally spray the female with 70%ethanol before opening the abdominal cavity and retrieving and excising the uterine horn.
Place the uterine horn in a Petri dish containing cold one XPBS and rinse it. Place the dish on ice until ready to dissect. Then cut open the uterine horn via the midline to expose the attached embryonic sacks.
Cut open an embryonic sack and place an embryo in a second Petri dish with cold PBS. Place the dish with the uterine horn on ice. After decapitating the embryo, place the embryo on its back and cut.
Open the chest wall to visualize the heart and lungs. Depending on the stage, the chest wall may be transparent. If genotyping is to be done, save the embryo's tail.
Using forceps carefully lift the heart and cut the vessels below. Then cut above the great vessels to free the heart. If necessary, remove extraneous tissues.
Place the heart in one well of a 24 well dish filled with cold PBS and sitting on ice. Repeat the dissection to isolate all the embryonic hearts. In a laminar flow hood, combine cold matri gel and the desired culture medium at a one-to-one ratio while keeping the dilute matri gel on ice.
Pipette 500 to 1000 microliters of the solution into the wells of a 24 well culture. Dish that on ice. Carefully pick and place the excised hearts into the individual wells of the matrigel containing culture dish.
If orientation of an embedded heart is crucial, use 70%ethanol to liberally spray an inverted microscope and the surrounding space. Then after working in the hood to place the heart into the well of the mat gel on ice. Place the plate on a microscope stage and quickly orient the heart as the mat gel begins to solidify.
Place the culture dish in a humidified 37 degrees Celsius incubator and allow the matri gel to solidify for approximately 30 minutes. Add one milliliter of pre warmed culture, medium to each well containing a heart. The hearts can remain in culture for at least four days.
If desired, add a fluorescent live cell die, such as cyto 16. To visualize the live heart photography will be limited to the side of the heart that is visible based on the position in which it was embedded. If whole mount imaging or sectioning will be performed, remove the culture medium.
Replace it with cold 4%formaldehyde, and place the dish on ice for 30 minutes. After the matrigel dissolves, replace the fixative and matrigel with fresh formaldehyde and fix the hearts overnight at four degrees Celsius. Wash the hearts with PBS or Triss and store at four degrees Celsius until further analyses.
When cultured ex vivo for 24 hours, the coronary vasculature appears similar to a heart excised from a comparably aged embryo that was maintained in utero as shown here. Hemat, toin and eoin analysis of hearts excised at E 16.5 and cultured ex vivo for four days demonstrates that the overall morphology, including the walls of the great arteries and the ventricular and interventricular septal myocardium of the heart remains intact despite the lack of circulation. The coronary osteo are apparent as indicated by the arrowhead.
However, DNA fragmentation indicated with arrows and observed with both h and d and DAPI is apparent in the compact myocardium and around the great vessels. Furthermore, the myocardium is less dense as compared with a heart from an E 18.5 embryo that developed in utero even though the ex vivo cultured heart was still beating. Thus, there are limitations to the length of time for or the stage at which these hearts can be maintained.
This culture technique can also be used to fluorescently label hearts while in culture following incubation with an endothelial specific fluorescent dye hearts are cultured for 48 hours. In this instance, visualization is limited based on the orientation of the heart and the opacity of the tissue. Subsequent histological sections through the ventricular myocardium confirm that cyto 16 can penetrate the outer cell layers to label sub epicardial vessels within the ventricles and co localizes with is selectin as demonstrated here After ex vivo culturing for 30 hours, a heart excised from an E 14.5 embryo shows consistent contractility and pacing between the atria and ventricles.
When performing this technique, it's important to be gentle and careful when excising the heart. After watching this video, you should have a good understanding of how to excise and culture an embryonic mouse heart.
