Primary Cell Cultures to Study the Regeneration Potential of Murine Müller Glia after MicroRNA Treatment

This protocol allows to study the potential and the capability of Muller glia to convert into retinal progenitor cells after treatment with specific factors such as microRNAs. The advantage of this technique is that microRNA candidates can be tested for their efficiency and outcome before their usage in in vivo applications. Helping to demonstrate the procedure will be Seoyoung Kang, a PhD student from the laboratory of Stefanie Wohl.

First, dip the removed eyeball briefly into a tube with ethanol to avoid carryover of bacteria from the animal. Then, wash the eyeball briefly in the 10 centimeter Petri dish before placing it into the 24 well plate on ice. Once the eyeballs are removed and cleaned, place one eye in the dissection dish placed under a dissecting microscope with a light source.

Now fix one eyeball by grabbing the optic nerve and the surrounding connective tissue around the sclera with Dumont 5 fine forceps and press it carefully against the dissection dish. Next, make a hole in the cornea center using a 30 gauge needle to allow easier access for the venous scissors. Then, dissect the cornea around the ciliary body using venous scissors and carefully remove the cornea, lens, iris and vitreous body with Dumont 5 fine forceps.

Later dissect the sclera with venous scissors until the optic nerve is reached and carefully extract the retina using Dumont 5 fine forceps. Now use a second Dumont 5 fine forceps to push against the retina and completely remove the vitreous body. Transfer the retinas cut about 2.5 centimeters of the tip of a sterile transfer pipette to enlarge the diameter.

Now using this tip, pick up whole retinas without damaging the tissue and transfer the retinas into a new sterile Petri dish with cold HBSS and rock the dish. Next, using a new sterile transfer pipette, carefully push the retinas around to wash off retinal pigment epithelial cells. Immediately place the isolated retina in a new clean well of the 24 well plate filled with one milliliter of HBSS while keeping the 24 well plate on the ice during the dissection.

Prepare Papain DNase I dissociation mixture as described in the manuscript. Now with an enlarged tip transfer pipette, pick up the retinas. Wait until the retinas settle at the bottom of the tip and release the retinas without excessive HBSS into the tube containing Papain DNase I mixture.

Next, place the tube on a nutator in the incubator and incubate for 10 minutes at 37 degrees Celsius and with 5%carbon dioxide. Next, dissociate the cells by carefully pipetting up and down with a one milliliter pipette. After cells are dissociated, add 275 microliters of ovomucoid protease inhibitor from the Papain dissociation kit to neutralize the Papain and mix gently by pipetting up and down.

Place tubes into a centrifuge and spin at four degrees Celsius for eight minutes at a relative centrifugal force of 300. Add epidermal growth factor to the calculated volume of growth medium pre warmed at 37 degrees Celsius. Remove the tubes carefully from the centrifuge.

Without touching the pellet at the bottom of the tube, remove the supernatant carefully and entirely. Now resuspend the cell pellet with 500 microliters of epidermal growth factor supplemented growth medium. Then, transfer the cell suspension into one well of the labeled 12 well plate.

Rinse the tube with another 500 microliters of the epidermal growth factor supplemented growth medium and add it to the well. Rock the well plate carefully then place the plate into the incubator at 37 degrees Celsius with carbon dioxide. Begin by checking for 90%to 100%cell confluency.

Next, remove the medium and add one milliliter of cold HBSS to wash the well. Gently rock the plate and remove HBSS without any left traces. Later, add 500 microliters of a pre warmed trypsin containing solution to detach the cells from the well.

Rock gently and incubate for two minutes in 37 degrees Celsius incubator. After moving the plate from the incubator to the bio safety cabinet, aspirate the trypsin containing solution while tilting. Disperse it carefully and slowly over the well several times until the cells detach completely.

Next, transfer this cell suspension to a sterile 1.5 milliliter tube and put tubes into the centrifuge. Spin at 300 times g for eight minutes at four degrees Celsius and bring tubes back into the bio safety cabinet. Remove the supernatant without touching the pellet.

Now carefully resuspend the cell pellet by adding 600 microliters of pre warmed growth medium and pipetting up and down approximately 30 to 40 times. Finally, seed 100 microliters of the obtained cell suspension in the center of six coated cover slips of the 24 well plate. Place the plate on the incubator and let the cells settle for subsequent transfection using micro RNA mimics.

The figure shows control conditions five days after transfection with a few Ascl1-Tomato positive cells present. After a miR-25 treatment however, many more Ascl1-Tomato positive cells are found. The quantification revealed a fourfold increase in the number of Ascl1-Tomato positive cells in the miR-25 treated wells compared to controls.

Most importantly, the vast majority of the Ascl1-Tomato positive cells found in the riR-25 treated wells adopted a neuronal morphology. Features of this neuronal morphology included reduced cell soma size, the development of fine processes and the formation of tiny networks. The quantification revealed that about 70%of all Ascl1-Tomato positive cells had neuronal features.

Immunofluorescent labeling shows that Ascl1-Tomato positive cells with neuronal morphology express the neural markers OTX2 and MAP2, confirming neuronal identity. The quantification of these cells revealed that after miR-25 overexpression, about 40 Ascl1-Tomato positive neurons per field are present as compared to five neuronal cells per field in controls. The identified neuronal cells constitute about 70%of the total Ascl1-Tomato positive cell population of the miR-25 treated samples.

Furthermore, quantification of the absolute number of OTX2 and MAP2 coexpressing neurons showed that after miR-25 treatment, about 60 neurons per field were found as compared to 10 neurons per field in controls. It is imperative to work in a clean and sanitized environment with clean and sterile tools and to work carefully by avoiding any harsh treatment. Downstream applications can be for instance, protein quantification, DNA or RNA analysis, or electrophysiological studies.

This technique helped us to explore initial questions about nuclear reprogramming which belongs to the field of regenerative medicine. And there is a long range of factors and conditions that can be tested in order to explore new questions.