Anterior Eye Segment Culturing: A Method to Culture Cornea Bearing Anterior Segment of Porcine Eye

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Setting up anterior segments in organ culture dishes

1. Place a single anterior segment (AS) in a Petri dish with AS inverted (cup up). Using a cotton swab, wet in the media and gently dab in the center of the cornea to remove any pigment. Using forceps to hold the eye and the same swab, wipe the cotton swab around the sclera to remove extra pigment.
2. Invert the AS and place on top of bottom part of the dish over the elevated region, centering the cornea over the elevated region in the dish. Place the clamping ring on top of the newly placed AS.
3. Place four screws into the corresponding holes to hold the ring in place with the AS under the ring. Gently hand-tighten the screws with the L-key.
   NOTE: The tightening step will happen daily throughout the experiment, so the goal of the initial tightening here is to ensure the media does not leak while avoiding breaking the clamping ring.
4. With a sterile Petri dish set, place the top over the dish and invert the setup. Attach the dish stand. Attach the fluidic connectors with O-rings to threaded ports on the bottom of the dish.
5. To one fluidic connector, attach an 18 G 90° bent needle hub, a length of tubing, an 18 G needle hub, a nylon syringe filter, a three-way valve, and a 20 mL syringe filled with media.
6. To the second fluidic connector, attach an 18 G 90° degree bent needle hub, length of tubing, an 18 G needle hub, a three-way valve, and the barrel portion of a sterile 10 mL syringe (this will act as a reservoir to catch liquid and bubbles from the ASOC).
7. With three-way valves open appropriately to the syringes, gently push media through the system using the fluidics connector port identified in step 1.5 to inflate the AS, fill media in the tubing, and eventually the reservoir.
   NOTE: If media leaks into the ASOC dish, the AS is not secured tightly enough with the clamping ring.
8. Remove bubbles by gently pushing media into the dish and inverting the dish to push out the bubbles into the reservoir.
9. Place the dish and stand upright. Place the bottom portion of a Petri dish underneath the feet of the stand, careful not to ensnarl the tubing.

2. Starting anterior segment organ culture

1. ASOC is now ready for incubation. Place the ASOC dish into the cell culture incubator (37 °C, 5% CO₂). Ensure the height of the ASOC dish in the incubator above the pressure transducers is known and accounted for to calculate intraocular pressure (IOP) accurately.
2. Direct the tubing lines out through the bottom of the 37 °C, 5% CO₂ incubator door so that they do not interfere with the opening and closing of the door. Attach the 20 mL syringe to the syringe pump set at 2.5 µL/min.
3. Position the tubing line with the reservoir at the pressure transducer instrument. Connect the side 3-way valve to the pressure transducer setup while flowing PBS through the line to avoid air bubbles entering the tubing lines.
   NOTE: Empty the PBS from the reservoirs after the system is set up to reduce the likelihood of reservoir contamination with microbial growth for the duration of the organ culture.
4. Initiate IOP data collection by first ensuring a microSD card is present for saving data files. Then, turn on the pressure transducer setup to begin data collection.

3. Daily maintenance of ASOC

1. After the ASOC has had 24 h to equilibrate, remove the dishes from the 37 °C, 5% CO₂ incubator and place them into the Biosafety cabinet (BSC) II cabinet.
   NOTE: On pressure data acquisition, these time periods look like spikes as the ASOCs are removed from the incubator (height change) and adjusted in the cabinet.
2. Check for leaks under each dish on the Petri plate. If present, check for tight fluidic connections under the dish and re-tighten if necessary. Check for leaks in the dish top using a sterile L-key to tighten the screws in the clamping ring.
   NOTE: The AS sclera tissue will compress and reduce thickness by 24 h, and the clamping ring will need to be tightened.
3. Aspirate the media from the dish well.
   NOTE: The trabecular meshwork is filtering fluid from the media being pumped into the ASOC. Therefore, media will be present in the ASOC dish along the edges.
4. Repeat steps 1.7 and 1.8 to remove any trapped air bubbles.
5. Refill syringes on the syringe pumps, ensure the syringe pumps are operating, and confirm the alignment of valves for perfusion into the ASOC. Return the ASOC dish to the 37 °C, 5% CO₂ incubator.
   NOTE: Optimally, these steps should be performed daily. However, the use of a 20 mL starting volume of media, the ASOC dish well volume, and a pump rate of 2.5 µL/min should be sufficient to let the system run for several days undisturbed.