The overall goal of this method is to generate a choroid plexus epithelial cell-based model of the human blood-cerebrospinal fluid barrier. Enabling the study of bacterial infections from the basal-lateral side. This method can help to answer key questions about infectious diseases of the Central Nervous System, such as which processes are involved during bacterial interactions with the choroid plexus epithelium.
The main advantage of this technique is that it allows analysis of the interactions of pathogens with the basal lateral black side of epithelial cells. This method can also be applied to other studies such as, to the investigation of the transmigration of the immune cells and tumor cells across the choroid plexus epithelium. I first had the idea for using the human chorcoid plexus papilloma, or HIBCCP cells for this method when I read the 2005 manuscript by Shibata et.al.
describing the cell line. Begin by using sterile forceps to place 0.33 centimeters squared growth area cell culture filter inserts, with three micron size pores upside down into individual wells of a twelve well plate. Then, with a serological pipette, flood the well with about three milliliters of the pre-warmed medium, aspirating any extra solution, until the lower compartment is just filled.
Next, add 100 micrometers of 37 degree subculture medium supplemented with ten percent FCS on top of the filter. When adding the medium it is very important to avoid the generation of air bubbles, since the bubbles will prevent the cells from being fed sufficiently. When the medium has been added to all of the inserts, cover the plate and transfer the inserts to a 37 degree Celsius five percent CO2 incubator.
Now, wash a flask of HIBCPP cells with ten milliliters of PVS, two times with swirling. After the second wash, add three milliliters of 0.25 percent tripsyn EDTA to the cells and swirl the flask. Then, incubate the cells for about twenty minutes.
At the end of the incubation, confirm that the cells have lifted from the bottom of the flask and display a round shape. The cells do not detach completely from each other and are often found in agglomerates. Re-suspend the culture with 17 milliliters of medium to stop the reaction and transfer the suspension to a 50 milliliter tube.
Then, spin down the cells, and re-suspend the pellet in an appropriate volume of medium for counting. Next, dilute the cells to a one times ten to the sixth cells per milliliter concentration, and invert the tube to evenly distribute the cells. Then, add 80 micro liters of cells to the top of each filter insert.
When all of the inserts have been seeded, cover the plate, and return it to the incubator for overnight culture. The next day, add one milliliter of medium to each well of a 24 well plate. Then, using forceps, lift each insert and discard the medium inside, placing the inserts right side up in individual wells of the 24 well plate.
When the filter inserts are flipped from the 12 well plate into the 24 well plate, be careful not to touch the filter membrane with the cells growing on top. Fill the inserts with 0.5 milliliters of fresh medium, and return the plate to the incubator. Transfer the inserts into a new 24 well plate with fresh medium every two days, thereby replacing the medium in the upper compartment.
To measure the trans-epithelial electrical resistance, or TEER of the cells, first immerse the electrode tips of an epithelial tissue volt-ohm meter into 80 percent ethanol. After 15 minutes, remove the electrode from the ethanol to allow the electrode to dry. Then, place the tips into an aliquot of subculture medium for another 15 minutes.
When the electrode has equilibrated, position the longer arm so that it touches the bottom of the lower compartment of one well of the cell culture plate, and the shorter arm so that it reaches into the filter insert compartment. Measure the resistance values of the cell culture filter inserts in medium without cells to obtain the blank values. Then, measure the TEER of each of the seeded inserts.
After measuring, place the electrode back into 80 percent ethanol for 15 minutes, followed by storage in a dry tube. When the TEER values of the HIBCPP seeded inserts exceed 70 ohms per centimeter squared, renew the medium using fresh culture medium supplemented with five micro grams per milliliter of insulin and one percent FCS, and return the plate to the cell culturing incubator overnight. To determine the paracellular permeability of the insert cultures, add 50 micro grams per milliliter of freshly prepared FITC-inulin in fresh serum low culture medium to the upper filter compartment of each insert, and return the cells to the incubator.
When the cultures have reached a TEER of around 500 ohms per centimeter squared, infect the cells with a bacterial suspension of interest at a multiplicity of infection of ten, and return the cells to the incubator for the appropriate culture period. Then, wash the cells three times by transferring the filter into a new well with one milliliter of serum-free medium. Each time, place the medium in the filter compartment with 500 micro liters of the same medium, finally, determine the concentration of inulin that passed from the filter compartment through the cell layer after the bacterial infection by collecting medium samples from the lower well of each cell culture filter insert, and measuring all the samples in a micro plate reader against ten one and two FITC-inulin standard dilutions.
HIBCCP cells exhibit specific barrier functions that enable them to restrict their molecular exchanges to a modest degree. For example, immunofluorescent analyses of the type-junction associated with the protein, ZO-1, Occludin, and Claudin-1, reveal an uninterrupted signal at the sites of cell to cell contact. Illustrating the interconnectedness of the cells by continuous strands of tight junctions.
When cultured under the appropriate conditions, HIBCPP cells display a high membrane potential, that reaches up to about 500 ohms per centimeter squared without treatment, maintaining the typical barrier functions of the blood-cerebrospinal fluid barrier, Like the formation of a membrane potential, as well as a low-permeability for macro molecules. As increasing concentrations of DMSO are applied, however, the TEER values decrease in a dose-dependent manner, with a similar significant drop in the TEER value observed after Cytochalasin D treatment. More, the addition of two volume percent DMSO, or Cytochalasin D, results in a corresponding significant permeability increase for FITC-inulin flux.
Further, the bacterial infection of the HIBCCP cells results in a significant invasion of the cell layer by two pathogenic bacterial strains. MC58, and its capsule-deficient mutant. Whereas only a minor invasion is observed for the apathogenic alpha 14 strain.
While attempting this procedure, it's important to remember not to touch the filter membrane after seeding the cells as the intact HIBCCP layer will be disrupted by the contact. Following this procedure, other methods like vitro transmigration assays can be performed to answer additional questions about the mechanisms of immune and tumor cell migration across choroid plexus epithelial cells. After its development, this technique paved the way for researchers in the field pharmacology to explore the transfer of substrates across the choroid plexus epithelium in vitro.
After watching this video, you should have a good understanding of how to prepare a HIBCCP cell-based model of the blood-cerebrospinal fluid barrier for basal lateral infection with pathogens. Don't forget that working with human pathogens can be extremely hazardous and that precautions such as working under an appropriate safety hood should always be taken while performing this procedure.
