The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.
I'm Birgitt Schule, the Director of the Brain Donation Program at the Parkinson's Institute and Clinical Center. In this video, we describe a technique for an explant cell culture and duration of primary fibroblast from leptomeninges of postmortem human brain from patients with Parkinson's disease. These fibroblasts can be used for variety of applications, including cell-based assays.
This is important, because we can study cell lines from autopsy-proven cases of sporadic Parkinson's disease with Lewy body pathology. Begin by placing all required materials into the tissue culture hood. All objects entering the hood must be sprayed with 70%ethanol and wiped down.
The first step is to transfer the meninges from the tube to the cutting dish. Start by pouring approximately 10 milliliters of phosphate-buffered saline into a sterile 10-centimeter cutting dish. Here you can see the meninges.
Using sterile forceps, carefully transfer the meninges into the cutting dish. Perform this step slowly to prevent ripping. The next step is to wash the meninges in PBS to remove as much blood as possible.
If your sample is too bloody, you may consider transferring it to another cutting dish. Add another 10 milliliters of PBS into a fresh 10-centimeter dish. Repeat this wash to continue removing blood from the meninges.
Finally, transfer the meninges into a fresh 10-centimeter dish containing PBS. When finished, cover the dish and transfer it to the dissecting microscope in the laminar flow Hood. This next step is to continue sectioning the meninges into smaller pieces.
Using two sterile scalpels, gently separate the blood vessels from the meninges with a shallow scraping motion. Prepare a region large enough to cut roughly 18 3-millimeter by 3-millimeter pieces, as shown in this image. Then, take the scalpel and, using a rocking motion, section the prepared region into 3-millimeter by 3-millimeter pieces.
It is easier to do this in a stepwise process by first cutting the meninges into larger pieces and then cutting those pieces into smaller sections. Cover the cutting dish, and transfer it back into the tissue culture hood. Next, prepare to transfer the meninges into a six-well tissue culture plate that has been pretty coated with 1 milliliter of 0.1 percent gelatin for 30 to 60 minutes.
Add one milliliter of meninges media into each well. Then, with sterile forceps, transfer three to four meninges pieces into the center of each well. The cells grown on this plate will be a passage zero.
The next step uses coverslips to ensure that the meninges pieces are stationary and remain in contact with the culture dish. Place a sterile 15-millimeter circular coverslip over the meninges pieces. If necessary, reposition the meninges pieces with a coverslip.
Then, using the forceps, firmly press down on the coverslip to ensure that the pieces are touching the plate. This image shows the meninges pieces held in place with a coverslip. Place the cover over the six-well plate and carefully transfer the plate into an incubator set to 37 degrees Celsius and 5%carbon dioxide.
After two days in culture, add an additional one milliliter of fresh meninges media to each well. At seven days, aspirate the media. Then, refill each well with 2 milliliters of fresh media.
After day seven, continue to replenish the wells with 2 milliliters of fresh media every other day. This image shows the initial day of the meninges pieces and culture under an inverted microscope. After roughly six to nine days, cellular attachment in the first outgrowth can be observed.
Between 15 to 19 days, the cell number increases and growth becomes denser. Meningeal fibroblasts are characterized by the expression of fibronectin. These images show cultured meningeal cells that have been stained and are positive for fibronectin.
After watching this video, you should have a good understanding of how to derive leptomeninges explant cultures from postmortem human brain donors. This technique provides a simple and robust way to derive meningeal fibroblast within three to six weeks, and allows to bank 15 to 20 million cells at a low passage number.
