This article describes a method for culturing porcine anterior eye segments to create a three-dimensional model that simulates the eye's architecture under physiological conditions. The process involves careful preparation and perfusion of the eye segment to maintain its viability for experimental use.
For anterior eye segment culturing, begin with a suitable culture dish. The culture dish has an elevated stage at the center, connected to two fluid ports for inflow and outflow.
First, place a fresh porcine anterior eye segment containing cornea over the stage. Subsequently, place a clamp ring over the eye segment and secure it. Next, prepare a perfusion set-up comprising a syringe filled with a suitable culture medium. Connect the syringe to a valve, filter, and tubing with a bent needle hub at the other end.
Prepare a similarly assembled reservoir set-up using an empty syringe. Now, connect the needle hubs of the perfusion and the reservoir set-up to the inflow and outflow ports, respectively. Gently perfuse the media and allow the anterior segment to inflate.
Continue the perfusion and allow the bubbles and excess medium to flow into the reservoir. Thereafter, incubate the culture dish at the desired height and standard physiological conditions. This results in a three-dimensional model that simulates the complex architecture of the eye under physiological conditions.
Connect a pressure transducer to the reservoir valve to constantly monitor the eye pressure. The anterior eye segment is now ready to use for further experiments.
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.
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