Hi, my name is Nicole Dunca. I'm head of the Department of Neuro at the Institute for Anatomy of the University of D book ASIN in Germany. Hi, I'm I'm a PhD student at Professor Dunca Lab at the Department of Neuroanatomy.
Today we'll show you a handy system for culturing neuro retinas close to the NI situation as organotypic retinal hormones. So let's get Started. Targeted ablation Of genes and analysis of animal models is a classical strategy for enrolling specific retinal gene function.
However, transgenic mouse models often display early lithology or suffer from severe malformations preventing an analysis beyond embryonic or early postnatal stages. Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects.
However important cell cell interactions are destroyed in dissociated cultures. By contrast, organotypic retinal hormone cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved. In our video article, we provide a step-by-step demonstration of the micro dissection of morine eyes, including removal of lens and vitreous.
After the 15 day old mouse Emyo has Been killed by decapitation. The eyes are nucleated by the use of fine curved Forceps, Peeling The ice from the eye socket. Two days after birth, Young mouse pups are killed by decapitation up to postnatal.
Day 15, the time point when mice open their eyes, the isolates have to be mechanically opened by the use of forceps. The tip of fine curve forceps are inserted into the orbit and the connective tissue surrounding the eye is removed. As at postnatal day two, the orbital bones still cartilaginous and soft.
It is important not to apply too much pressure with the forceps while trying To remove the eyes. 15 day old mouse pups are killed by cervical dislocation. Afterwards, the isolates Are enlarged by two transfers, cuts with spring scissors.
Then the eyes are nucleated by the help of curved forceps, applying pressure to the orbit and transferred to a Petri dish. With PBS, adult mice are Killed by cervical dislocation And the eyes can be directly nucleated by the Help of curved forceps applying Pressure to the orbit. We are going to demonstrate The peculiarities in this section of morine eyes of different developmental stages from left to right morine eye at embryonic day 15, postnatal day two, postnatal day 15, and an adult eye in a small Petri dish.
With sterile PBS, the surrounding eye layers have to be removed during dissection, both forearms should rest on the bench top. To stabilize the hands, the eye is turned with the lens site facing the bottom of the Petri dish. When dissecting embryonic eyes, the site where the developing optic nerve relieve the eye is often covered by connective tissue, which needs to be removed to create space for entering the subretinal space between retina and pigment epithelium by the tips of very fine forceps, the sclera and the pigment epithelium Are still very soft and easy to remove.
Make however sure to completely remove The triangular Trent like capillary plexus underneath the vitreous body, together with the vitreous. This sticky plexus is difficult to remove separately from the remaining retinal cup without damaging the retina, but it'll hinder the excess of exogenously applied substances to the retinal layers. During pharmacological Manipulations at postnatal day two, The optic nerve at the back of the eye needs to be pinched off by forceps.
The surrounding eye layers, including the sclera and the pigment epithelium are still soft and easy to remove. Take however care not to damage the retina. When entering the subretinal space via the optic Nerve hole, The pigment epithelium is removed together with the choroid membrane and the sclera by carefully tearing to either side with both forceps.
The eye cup is then turned to the lens side, the lens and vitreous grasped with forceps. And while tearing, the eye cup is holding Place with the other forceps. At postnatal day 15, The optic nerve frequently keeps attached to the eye during a nucleation and has to be pinched off by forceps as close to the basis as possible.
The sclera is already tighter and thus entering of the subretinal space. At the hole where the optic nerve has been removed and peeling of sclera choroid membrane and pigment epithelium becomes more Difficult than at P two. Peel off the layers up to the level of cornea, then turn the eye cup to the lens site and remove the cornea together with pigment, epithelium, choroid membrane, and sclera.
While holding up the remaining retinal cup by the other forceps, grasp the vitreous together with the small lens and while tearing with forceps, keep the retinal hormone mount in place with the Other forceps in the adult eye. The optic nerve, which needs to be pinched off to get access to the subretinal space, is much thicker compared to postnatal stages. The clearer is extremely firm and penetration with forceps to enter the subretinal space will turn out to be one difficult part in dissection, tearing off the pigment epithelium together with the current membrane and the sclera becomes harder and needs more Controlled force.
The removal of the vitreous is much easier compared to embryonic eyes, but the vitreous needs to be grasped at the sides. Do not pierce the vitreous with the tips of the forceps as its content will stick to the forceps hindering its Removal. Before starting The culture, the hyaluronidase containing inner and auto limiting membrane of glial cells need to be partially digested to facilitate the penetration of exogenously applied substances for this purpose, pipe H 200 microliter pH balance, ous modified eagles medium containing 0.5 milligram per milliliter hyaluronidase in each well of a 96 well plate to transfer the retinal home mound without damage.
The tip of a one milliliter pipette is cut a few millimeters to widen the opening and the cutting edges are smoothened by insertion and twisting of a second pipette Tip. The retina hormones are transferred From the 96 well collection plate to the 96 well plate containing medium with high days and pre incubated for 15 minutes at 37 degrees centigrade. Then the hormones are Transferred to a 24 well plate Containing two milliliters chemically defined to BECUs modified Eagles.
Medium Per well. The retinal hormone Cultures Are returned to an incubator and maintained for 24 to 48 hours at 37 degrees centigrade in a 5%CO2 Atmosphere, 850 microliters PBS and 50 microliters bovine zero album. Mine are pipetted to two milliliters eph tubes with round bottom.
The use of eph tubes was round bottom instead of classical chronic tubes improved the precipitation of cells during centrifugation, resulting in a more stable cell palette. After the desired culture time, the retinal home mos are transferred from the 24 well plate to the two milliliter eend off tubes containing PBS and bovine X album Mine. The eph tubes containing The retinal home mos are placed into a heating block heated up at 37 degrees centigrade, 25 microliter collagenase, and 25 microliters.
Hyaluronic days are added to each eph cube and the dissociation of the retinal hormones into single cell suspension is started by three pars through siliconized Poster pipettes. 10 microliter Trypsin are added and the cell suspension incubated for three to five minutes. The incubation time for zyme dissociation varies and depends on the size of the eyes and the developmental stage respectively.
Check the stage of enzymatic digestion of the tissue by gently pipetting up and Down After a couple of minutes, 10 microliters DNAs are edit after another incubation period of about three to five minutes. The cell suspension is further homogenized by slowly pipetting up and Down. When the cell Suspension appears homogeneous, the digestion of the tissue is stopped by addition of 10 microliters EDTA and the eol tubes are removed From the heater.
One milliliter fresh Ice cold 8%Perfomal Hyde are added to each EOL tube and the cell suspensions are fixed at room temperature on a Rotation shaker. After one hour Fixation, the cell suspensions are removed from the rotation shaker and placed into a cooling centrifuge. The cell suspensions are centrifuge for five minutes at 40 degrees centigrade and at 0.2 relative and ugal force, the palliated cell suspension is removed from the centrifuge.
The SUP natant is discarded and the pate is resuspended in one milliliter. PBS containing three milligram per milliliter BSA. After repeating these washing Steps twice, the pellet is finally resuspended in 500 microliters.
PBS containing three milligram per milliliter. BSA five millimolar EDTA and 0.1%sodium acid if an immunochemical staining will follow, do not add sodium acid to the Resus suspension buffer. And this results in loss Of Staining quality.
A frosted end Microscope slide, a cytosine filter with one or two holes and a cytosine funnel row inserted into a cytosine clip And the slight clip is closed. Make sure that the holes of the funnel and the Cytosine filter are aligned. All slide clips are placed into the cytosine center.
Future rotor homogenized is the associated cell suspension by gently pipetting up and Down. When pipetting a 100 microliter Equa of the cell suspension to the funnel, the tip of the pipet should reach all the way down to the bottom of the funnel. It is important not to push through the second pressure point of the pipet.
If this creates air bubbles in the cell spot, which Will Hamper cell counts, Then the lid is placed onto the rotor and the rotor lifted into the centrifuge. The single cell suspensions Are centrifuge at 700 RPM for seven minutes. The rotor is removed from the cytosine and the lid is opened.
The side clip is opened and the funnel and the slide Clip are removed. Then the Sail spot is marked by adding on the back of the slide. While the filter is still in place, the filter is removed and the slide with a sail spot is cover slip with darie in mounting medium.
Alternatively, the cell spot can be immuno chemically stained with BREU or neuro specific Antibodies. We Just demonstrated the advantages of organ organotypic retinal hormones providing a culture system close to the physiological in vivo situation with neuronal interactions and connections still preserved. This culture system significantly facilitates the accessibility for manipulations of morine eyes.
So that's it. Thanks for watching and good luck with your Experiments.
