I'm Jane Clifford is econ. I'm professor and chair of biochemistry and and molecular biology at the Drexel University College of Medicine, And my name is Ola iv and I'm an MD PhD student in Dr.Clifford's lab. The video that Oli and I are prepared for you demonstrates the ex vivo culture of corneas obtained from rabbits and human don donors.
We have utilized these corneas for studying herpes virus infection and they are certainly applicable for other diseases of the corn for study of other diseases of the cornea, and it's a particularly relevant ex vivo model as compared with Cells and mono culture. Herpes keratitis is one of the most severe Pathologies associated with a herpes simplex virus type one. It is currently the leading cause of cornea derived and infection associated blindness in the developed world.
Herpes keratitis is traditionally studied in two types of experimental models. The in vitro model in which cultured mono layers of corneal epithelial cells are infected in a Petri dish offers simplicity, a high level of replicability, fast experiments, and relatively low costs. On the other hand, the in vivo model of herpes keratitis in which animals such as rabbits or mice are inoculated directly in the cornea, offers a highly sophisticated physiological system, but has higher costs, longer experiments, necessary animal care, and a greater degree of variability.
In this video article, we provide a detailed demonstration of a new ex vivo model of acute corneal epithelial HSV one infection, which combines the strengths of both the in vitro and the in vivo models. The ex vivo model utilizes intact corneas organ typically maintained in culture and infected with HSV. The use of the ex vivo model allows for highly physiologically based conclusions, yet it is rather inexpensive and requires STEM commitment comparable to that of the in vitro Model.
Prepare the following before Starting the procedure. Petri dish phosphate buffered saline, corneal culture and scaffold media spot plate strip of gauze, fresh eyeball, scalpel, Forceps, and sharp scissors. Wet a narrow Strip of gauze with PBS and wrap it around the eyeball to securely hold it.
During the procedure, use a sharp scalpel to make a tangential incision in the sclera about half a centimeter away from theus. Use scissors to completely excise the corneal scleral button. As the incision is extended, the pressure within the eyeball will decrease, making it difficult to hold and manipulate the tissue.
For this reason, we recommend using a sharpened reliable pair of scissors at this step. Once the corneal scleral button is isolated, tease away the iris while holding the scleral rim with forceps. Avoid damaging the endothelium or exerting excessive stress On the cornea.
Prepare A 1%agros in medium solution and keep it ready. At 55 degrees Celsius, thoroughly rinse the cornea in sterile PBS containing penicillin and streptomycin. Place the cornea epithelial side down into the well of a sterile Spotlight.
Add agros containing Medium to the endothelial concavity of the cornea. Just enough to fill it completely. Allow about one minute for the agros to solidify.
Place the cornea with the supporting agros scaffold epithelial side up and to a tissue culture dish and add supplemented MEM medium to cover the epithelial surface. The cornea may be cultured in this way for over a week with medium changes every 48 hours. Add one milliliter Of medium containing the desired amount of virus into a tissue culture dish.
Place the Cornea with the agro scaffold epithelial side down into the virus containing medium. Place the cornea in the tissue culture incubator to infect for one hour with intermittent rocking every 10 to 15 minutes after one hour. Aspirate the virus containing medium and rinse two to three times with PBS to remove any residual viral particles.
When rinsing with PBS, be careful not to dislodge the cornea from the agro scaffold. Add fresh medium to cover the epithelial surface. Experimental treatments may be started before or after infection depending on the nature of the experiment.
Culture medium Is changed every 48 hours. Mix the medium well and use it immediately for a plaque assay Alternatively stored at minus 80 degrees Celsius for later use. If collecting samples at different time points, Rinse the cornea well with PBS.
Remove the agro scaffold, Excites the ring of scleral tissue from the cornea to ensure that only corneal epithelial material is collected. Using a scalpel Scrape off the corneal epithelial cells into the appropriate buffer, depending on the type of material to be isolated. Removal of the scleral rim allows for the corneal str to rapidly swell by absorbing water.
Since the swelling may complicate the scraping, we recommend performing the step quickly rinse the Petri dish to ensure that the Entire sample has been collected. Remove the agro scaffold and rinse The cornea. Well with PBS.
Use the cornea to make a paraffin tissue block or a frozen tissue block as demonstrated here depending on your experimental goal. Embed the cornea in optimal cutting Temperature compound within a specimen mold Flash freeze the cornea in an ethanol dry ice bath. What we've shown here In this video is the preparation of corneas, the dissection of the cornea, placing it in exvivo culture and infecting corneas with herpes virus.
This has provided the technique for a model that has been very useful to us in studying herpes virus infection, ex vivo, and we hope it will be useful to others for their experiments with corneal cells.
