Anterior Eye Segment Culturing: A Method to Culture Cornea Bearing Anterior Segment of Porcine Eye
Transcription
After placing the anterior segment in the Petri dish, wet a cotton swab in the media, and gently dab in the center of the cornea to remove any pigment. Hold the eyes with forceps, and remove the extra pigment around the sclera by wiping. Place the inverted anterior segment over the elevated region of the bottom dish, ensuring that the cornea remains in the center of the elevated region.
Position the clamping ring on top of the newly placed anterior segment, and gently tighten the screws with the L-key. Attach both the fluidic connectors with O-rings to threaded ports on the bottom of the dish. To the first fluidic connector, attach 18-gauge 90-degree bent needle hub, a length of tubing, an 18-gauge needle hub, a nylon syringe filter, a three-way valve, and a 20-millimeter syringe filled with media.
To the second fluidic connector, attach 18-gauge 90-degree bent needle hub, a length of tubing, an 18-gauge needle hub, a three-way valve, as demonstrated for the first fluid connector, and then, attach the barrel portion of a sterile 10-milliliter syringe, which will act as a reservoir to catch the liquid and bubbles.
Keeping the three-way valves open appropriately to the syringes, gently push the media through the system using the first fluidic's connector port. To inflate the anterior segment, fill the tubing with media, and eventually fill the reservoir. Remove the bubbles by gently pushing the media into the dish, and invert the dish to push out the bubbles.
Place the anterior segment organ culture dish into the cell culture incubator. Direct the tubing lines out through the bottom of the incubator door to prevent interference with the door opening and closing. Position the tubing lines with the reservoir at the pressure transducer instrument. Connect the side three-way valve to the pressure transducer setup while PBS flows through the line to avoid the air bubbles from entering the tubing lines.
