An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy is the most common microvascular complication of diabetes.This method can help answer key questions on the pathophysiology of the end stage disease proliferative diabetic retinopathy.This technique allows the deconstruction of the native three dimensional tissue landscape and the investigation of tissue pathophysiology in live patient material with direct clinical relevance.Refer to the text protocol for details on the surgery and the instrumentation utilized in the dissection.To begin, place fibrovascular tissue that has been excised from human subjects undergoing transconjunctival microincision vitreoretinal surgery in a cell culture dish containing sterile PBS.Using an upright dissection stereo microscope, cut the fibrovascular tissue into approximately one square millimeter pieces.Submerge the tissue in PBS and hold the tissue in place with micro dissection tweezers.Make clear cuts in the tissue with a sterile scalpel taking care to avoid tearing the fibrovascular tissue.Place each individual piece of tissue into a well of a 12 well cell culture plate containing one milliliter of sterile PBS.First, prepare 25 microliter aliquots of fibrinogen solution at room temperature.Place one piece of fibrovascular tissue at the center of the well of a 24 well plate and remove any excess PBS.Next, add 25 microliters of TA solution to one of the prepared fibrinogen aliquots.And using another pipette set to 50 microliters mix by pipetting.Dispense the mixture onto the piece of fibrovascular tissue and pipette up and down to insure the tissue piece is contained within the droplet.The time of fibrinogen formation critical for the three dimensionality of the ex vivo culture, is tested in an empty droplet prior tissue embedding as described in the protocol.Incubate the plate in a cell culture incubator.After 30 to 60 minutes tilt the plate to check that the fibrin gel is completely formed.Next, overlay the fibrovascular tissue fibrin gels with ex vivo culture medium supplemented with aprotinin.After this, return the culture plates to the cell culture incubator for the desired time period.First, replace the ex vivo culture medium with one milliliter of paraformaldehyde solution per well to fix the cultures.After one hour, rinse the gels with three five-minute PBS washes.Next, replace the PBS wash with two milliliters of sodium azide solution per well.Store the fixed gels at four degrees Celsius.Use a stainless steel squared edge spatula to lift the droplets from the plate carefully, starting at the edges before lifting the center of the droplet.Using a round edged spatula transfer the droplets to individual wells of a 12 well plate containing one milliliter of PBS.After this, tilt the plate on a support and postfix the droplets with one milliliter of ice-cold acetone methanol solution for one minute.Then, rinse with three to five two milliliter washes of PBS.Centrifuge the blocking solution for 15 minutes to remove any debris.Then, tilt the plate on a support and incubate the droplets in 500 microliters of blocking solution for two hours at room temperature.Prepare the primary antibody mixture according to the text protocol.Then use a round edged spatula to transfer the droplets to a round bottom 96 well plate.Incubate the droplets in 30 microliters of primary antibody mixture per droplet overnight at four degrees celsius.The next day, transfer the droplets to a 12 well plate containing two milliliters of washing solution per well.Rinse the droplets with three five-minute washes.Then, wash the droplets with nine 30-minute washes.The following day, rinse the droplets three times with PBS.Prepare the appropriate fluorophore-conjugated secondary antibody mixture according to the text protocol.Transfer the droplets to a round bottom 96 well plate, as previously demonstrated, and incubate the plate with 30 microliters of secondary antibody mixture for four hours at room temperature, protected from light.After four hours, transfer the droplets to a 12 well plate containing two milliliters of washing solution per well.First, rinse the droplets with three five-minute washes.Then, wash the droplets with four 30-minute washes.The next day, rinse the droplets five times with washing solution for 30 minutes, and three times with PBS for 30 minutes.The next day, transfer the droplets to a round bottom 96 well plate, as previously demonstrated.Counterstain the droplets with Hoechst nuclear stain for 30 minutes at room temperature.Transfer the droplets to a 12 well plate containing two milliliters of PBS per well and rinse three times with PBS.Next, apply a narrow layer of quick hardening mounting medium along the edges of a square cover glass and allow it to dry for one minute.Then, rinse the droplet by dipping it into a well of a 12 well plate containing deionized water.And transfer the droplet to a microscope slide.Next, dispense 15 microliters of non-hardening anti-fade mounting medium onto the droplet.Gently position the cover glass over the droplet with the mounting medium facing the slide and let it settle.Let the slides dry for two hours at room temperature and store them at four degrees Celsius overnight.Finally, image the slides with an upright epifluorescence microscope equipped with an optical sectioning function at 20x or 40x objective.In this protocol fibrovascular tissue was prepared and imaged.The representative data showed that PDR ex vivo cultures induce the sprouting of CD-31 positive endothelium and vasculature preservation in response to VEGFA.Conversely, TGF