The overall goal of this procedure is to culture organotypic hippocampal slices. This is accomplished by first dissecting the hippocampus. The second step of the procedure is to slice the hippocampus.
The third step of the procedure is to separate and sort the hippocampal slices. The final step of the procedure is to culture the hippocampal slices. Ultimately, results can be obtained that show healthy organic hippo hippocampal slices through visualization with a dissection scope, or a microscope.
Hello, my name is Jimena Opitz and I am a research scientist in Dr.Andres bar's lab at the University of Washington. In Seattle, I will be demonstrating the procedure for organotypic hippocampus, LICs culture. Generally, individuals new to this method will struggle because of the need for quickly dissect and slice the hypo campi and to prevent contamination, Sterile dissection solution, and slice culture.
Media can be prepared up to three weeks in advance and stored at four degrees Celsius as described in the written protocol. Before hippocampal slice, cultures can be prepared. It is necessary to properly sterilize both the tissue culture hood and the equipment that will be used in the dissection.
First, wipe the inner surface of the tissue culture hood with 70%ethanol. Then place the dissecting microscope and all dissection instruments inside the hood. Prepare the tissue slicer by placing a clean piece of Teflon sheet onto the stage and mounting a new blade.
Set the tissue slicer inside the tissue culture hood using a UV light, sterilize all surfaces and equipment for 15 minutes. Turn off the light after the UV sterilization process is complete. Working in the tissue culture hood.
First, prepare the six well tissue culture plates to be used for culturing the hippocampal slices. Pipette 750 microliters of SEM into each well of the six well plate. Then using sterile forceps, place a cell culture insert into each well of the tissue culture plate.
Taking care not to trap air bubbles underneath the insert. It is important that the membrane of each cell culture insert is wet with slice culture medium. If the membrane is dry, check that no air bubbles are present.
If the membrane is sitting above the meniscus of the SCM, more media can be added to the well. Place the prepared culture plates into a tissue culture incubator. Set at 35 degrees Celsius with 5%carbon dioxide until needed.
Finally, fill an ice bucket with ice salt mix and place it next to the decapitation area working inside the hood. Fill a 100 milliliter beaker with dissecting solution. Push the beaker into the ice salt mix and to bubble the solution with 5%carbon dioxide and 95%oxygen for 10 to 20 minutes until the color changes from pink to orange.
And the dissecting solution forms a slurry mix of frozen and liquid solution. Now the dissection environment and tissue culture plates are ready for dissection. P three to P seven wrap.
Pups are used for this procedure.Slice. Cultures can be prepared from up to three wrap pups at a time. This procedure will be illustrated using a single pup.
Quickly harvest the brain from the decapitated wrap pup and place it into the 100 milliliter beaker containing the dissecting solution Slurry Chill for one minute. During this time, prepare a dissection dish by adding around 10 milliliters of ice cold dissecting solution to the bottom of a 60 millimeter tissue culture dish Using a rounded spoon, micros spatula, transfer the brain from the 100 milliliter beaker to the 60 millimeter dish filled with cold dissecting medium. Ensure that the brain is fully covered by the dissecting solution.
Place the dish containing the brain under the dissecting microscope. Look through the dissecting microscope and locate the brain. Use sterile dissecting forceps to move the brain into the center of the field of view.
Hold the brain at the midline with the dissecting forceps pressed to the bottom of the dish. Using the hippocampus dissecting tool, gently peel the cortical hemispheres away from the midbrain while leaving the midbrain in place, and ideally without detaching the cortical hemispheres from the rest of the brain, the hippocampus is now exposed. Take the hippocampus dissecting tool and scoop out the hippocampus from one hemisphere.
Then use the dissecting needle to clean away as much non hippocampal tissue as possible. Once the first hippocampus is isolated and cleaned, repeat this process for the second hippocampus from the other hemisphere. Next, use sterile scissors to snip off the tip of a P 1000 filter pipette tip.
Place the pipette tip onto a sterile P 1000 pipette. Gently aspirate one hippocampus and pipette it onto the Teflon sheet on the tissue slicer. Then similarly, place the second hippocampus onto the slicer.
Position the hippocampal halves on their concave sides and align them perpendicular to the blade aspirate excess liquid from the Teflon sheet. Set the ver or micrometer at the zero position. Slice the hippocampi into 400 micrometer sections.
Prepare another snipped P 1000 filter tip and mount it on a sterile P 1000 pipette. Pour approximately 10 milliliters of cold SCM into a 60 millimeter sterile tissue culture dish. Using the pipette, add some media and transfer the hippocampal slices from the slicer to the dish.
Avoid making bubbles after repeating the transfer process for all the slices, place the dish under the dissecting microscope. Finally, using an iris spatula and a straight spatula. Gently separate the slices from each other.
Separate the well-defined and undamaged slices from any damaged slices. The well-defined hippocampal slices are now ready to be cultured. Remove the previously prepared six well tissue culture plates containing SCM and cell culture inserts from the incubator using a P 1000 with a snipped wide boar tip transfer four to five individual slices per membrane.
When necessary, use the iris spatula to separate slices. Be careful not to place the slices too close to the insert wall or in close proximity to each other. Taking care not to touch the slices, aspirate any excess medium from the upper surface of the cell culture.
Insert membrane. Finally, return the plate, which now contains the hippocampal slice cultures to the tissue culture incubator. Set at 35 degrees Celsius and 5%carbon dioxide.
After 48 hours, remove the plate from the incubator working inside the tissue culture hood. Aspirate the media with a posterior pipette. Add 750 microliters of fresh prewarm SCM per.
Well again, ensure that no bubbles are formed under the cell culture insert membrane. This is an example of a healthy hippocampal slice ready to be cultured. The CA one, CA three and dentate gyrus, regions of the hippocampus are clearly visible and undamaged.
Shown here is a damaged hippocampal slice not used for culture. Finally, this is an example of a healthy hippocampal slice after four days in culture. Note that the slice looks white under the dissecting microscope and the surface of the slice is clean.
The CA one, CA three and dentate gyrus, regions of the hippocampus are clearly visible and undamaged. Once master it. This technique can be done in about one hour if it's performed properly.
While attempting this procedure, it is very important to remember to use appropriate steroid techniques and to be gentle with the tissue. After watching this video, you should have a good understanding of how to culture organotypic. Hippocampal lysis.
Thanks and good luck with your experiments.
