The overall goal of this protocol is to purify and isolate primary porcine brain endothelial cells in order to set up a tight in vitro blood-brain barrier model. This method can help answer key questions in the blood-brain barrier field, such as drug delivery, permeability, receptor trafficking, as well as the tightness and polarity of the BB.The main advantage of this model is the high yield of primary brain endothelial cells that can be used to create an in vitro blood-brain barrier model which has high TEER and low permeability. To begin this procedure, place the brains in a sterile flow bench and gently wash them with one liter of PBS in a beaker placed on ice.
Next, carefully remove the meninges from each brain using fine-tip forceps. Using a scalpel, scrape off gray matter from the brains one at at time and transfer the isolated material to Petri dishes containing 20 milliliters of DMEM/F-12 placed on ice. For initial fragmentation, run the gray matter material through a 50-milliliter syringe without the needle.
Repeat the procedure for all brains and pool the collected gray matter material together. Transfer the isolated gray matter material with DMEM/F-12 medium to the grinder tube of a handheld tissue homogenizer. Homogenize the material by making eight up-and-down strokes with a loose pestle, followed by eight up-and-down strokes with a tight pestle.
Next, isolate the capillaries by filtering the homogenate using a 500-milliliter blue cap bottle with filter holder and 140-micron filter mesh. Place the capillary-containing filters in Petri dishes with a digestion solution. Subsequently, incubate the Petri dishes at 37 degrees Celsius for one hour on an orbital shaker at 180 RPM, or stir them gently every 10 minutes.
After one hour, wash off the capillaries from the filters with suspension from the Petri dish. Then, split the suspension from the three dishes into two 50-milliliter tubes and stop the digestion by adding 10 milliliters of DMEM/F-12 to each 50-milliliter tube. Centrifuge the cell suspensions at 250 times g, four degrees Celsius for five minutes.
Afterward, aspirate the supernatants and resuspend each pellet in 10 milliliters of DMEM/F-12. Then, add a further 20 milliliters of DMEM/F-12 to each tube. To optimize the conditions for the attachment of PBECs, add coating solution to the T-75 flask, resulting in the final concentrations of collagen four at 150 micrograms per milliliter and fibronectin at 50 micrograms per milliliter and by distilled water, making sure that the solution covers the whole surface.
Next, thaw the capillaries in a 37-degree Celsius water bath. To remove freezing medium, spin the capillaries down at 250 times g, four degrees Celsius, for seven minutes. After that, carefully aspirate the supernatant and resuspend the pellet in one to two milliliters of the 10-milliliter aliquot.
Following that, transfer the capillary suspension to the coated T-75 flask, and gently tilt the flask to ensure equal distribution over the surface. Place the T-75 flask at 37 degrees Celsius and 5%carbon dioxide. After overnight incubation, prepare 10 milliliters of PBEC growth medium, supplemented with puromycin at four micrograms per milliliter for one T-75 flask and perform a medium change.
Then, prepare the permeable membrane inserts for PBEC seeding by coating them with collagen IV at 500 micrograms per milliliter, and fibronectin at 100 micrograms per milliliter, and by distilled water. Trypsin-ize the cells by adding two milliliters of trypsin-EDTA solution to the T-75 flask, and place it at 37 degrees Celsius, 5%carbon dioxide, for five to seven minutes. To detach the brain endothelial cells, gently tap the side of the T-75 flask with fingertips.
Subsequently, transfer the cell solution to a 15-milliliter tube, and spin down the brain endothelial cells by centrifugation at 250 times g, four degrees Celsius, for seven minutes. Afterward, carefully remove the supernatant, and resuspend the pellet in one milliliter of medium. Then, add a further two milliliters of medium to bring the total volume to three milliliters.
Count the number of cells manually using a cell-counting chamber or an automatic cell-counting system. For non-contact coculture, transfer inserts to the wells that have astrocytes, which were refreshed with astrocyte growth medium the day before. The next day, replace the medium on the permeable membrane inserts containing PBECs.
Then, carefully aspirate the medium from the wells and add 750 microliters of differentiation medium to each well. Following that, carefully aspirate medium from the inserts and add 500 microliters of differentiation medium per insert. Then, add the remaining 750 microliters of differentiation medium to each astrocyte well.
After that, incubate the cells with differentiation medium at 37 degrees Celsius, 5%carbon dioxide, until the next day. On the day after stimulating the cells with differentiation medium, prepare the tissue-resistance measurement chamber system for TEER measurements by rinsing the chamber two times with bi-distilled water, one time with 70%ethanol for five minutes and two more times with bi-distilled water. Next, connect the chamber to the system, add four milliliters of DMEM/F-12 to the tissue-resistance measurement chamber, and let it stay at room temperature for approximately 30 minutes.
After that, perform TEER measurements by carefully placing the inserts in the tissue-resistance measurement chamber. Here are the phase phase-contrast microscopy images of PBECs, cultures, and astrocytes viewed over five days. Purified capillary fragments on the first day of the initial culture showed presence of both capillaries and contaminating cells, which after medium change on day two and an additional day of growth, showed selection for PBECs, starting their growth from the capillary fragments.
Confluent monolayers of PBECs were usually achieved on day four through six, at which time PBECs showed spindle-shaped morphology and were longitudinally-aligned. Rat astrocytes seeded in the bottom wells normally proliferated to confluent layers within five days, and were used for non-contact coculture models after two weeks of growth. Once mastered, this technique can be done in seven hours.
After its development, this technique paved the way for researchers to explore drug delivery and receptor trafficking in tight and polarized brain endothelial cells. After watching this video, you should have a good understanding how to create an in vitro blood-brain barrier model that can be used for new studies that cannot carry out in vivo.
