髄膜炎菌 人工多能性幹細胞由来脳内皮細胞の感染

This protocol demonstrates the usage of iPSC-derived brain-like endothelial cells to examine host pathogen interactions at the blood-brain barriers, such as the meningeal blood-CSF barriers, in a model that can better mimic brain endothelial cells in vivo. In vitro examination of brain endothelial cell barrier properties and interactions with human-specific pathogens can be challenging due to limitations in modeling. iPSC-derived brain endothelial cells are particularly useful for these applications as they possess superior barrier phenotypes and are of human origin.

Maintain the induced pluripotent stem cells, or iPSCs, at 37 degrees Celsius and 5%carbon dioxide in six-well plates with two milliliters of stem cell maintenance medium. To passage the cells, aspirate the medium from a well that is not confluent and add one milliliter of non-enzymatic cell dissociation reagent. Then incubate the plate at 37 degrees Celsius for seven minutes.

Meanwhile, replace the matrix gel solution on a new six-well plate with two milliliters of fresh stem cell maintenance medium per well. Aspirate the cell dissociation reagent, taking care not to aspirate cells that are still attached to the plate. Rinse the bottom of the well with six milliliters of stem cell maintenance medium until all cells are completely detached.

Seed the new six-well plate with varying densities, typically one to six, or one to 12 for normal maintenance. Shake the plate back and forth and left to right, pausing in between alternating shaking motions until the medium has settled. Then put it in the incubator.

Add one milliliter of enzymatic cell dissociation reagent into each well and incubate the plate at 37 degrees Celsius for seven minutes. To deactivate the cell dissociation reagent, transfer one milliliter of the cell suspension into a 15-milliliter tube with at least two milliliters of fresh stem cell maintenance medium. Spin down the cell suspension at 1500 G for five minutes.

Then resuspend the cell pellet in one milliliter of stem cell maintenance medium and count the cells with a hemocytometer. Resuspend 7.5 times 10 to the fifth cells in 12 milliliters of stem cell maintenance medium and 10 micromolar rock inhibitor. Aspirate matrix from a T75 flask and transfer the cell suspension to the flask.

Shake the flask to distribute the cells and place it in the incubator. Refresh the media every day for the next two days to remove rock inhibitor and support the growth of stem cell colonies. On the third day, begin differentiation by changing the media to an unconditioned medium.

Change the medium daily for the next five days. Six days after starting differentiation, selectively expand the endothelial cell population by switching to endothelial cell or EC medium with 20 nanograms per milliliter bFGF and 10 micromolar retinoic acid. Incubate the cells for two days.

Prepare collagen IV and fibronectin solution according to manuscript directions, and use it to coat cell culture plates and membrane inserts. Aspirate the EC medium from the cells and add 12 milliliters of cell dissociation reagent. Incubate the flask at 37 degrees Celsius until 90%of the cells have detached.

During the incubation time, remove the coating solution from the previously prepared plates and inserts, and let them dry in a sterile hood. Once the cells have detached, use a 10-milliliter pipette to create a single cell suspension. Transfer the cells to a 50-milliliter tube and dilute them with an equal volume of human endothelial serum-free medium.

Then count the cells with a hemocytometer. Pellet the cells at 1500 G for 10 minutes. Then resuspend them in freshly prepared EC medium with bFGF and retinoic acid for a concentration of 2 million cells per milliliter.

Add 500 microliters of the cell suspension to the top of a 12-well insert and 1.5 milliliters of medium to the bottom. Distribute the cells evenly across the insert and incubate the plate at 37 degrees Celsius and 5%carbon dioxide. On the next day, change the media to EC without bFGF or retinoic acid.

Place the epithelial volt/ohm meter in the biosafety hood and connect the electrodes. Disinfect the electrodes by submerging them in 70%ethanol for at least five minutes and let them dry completely. Remove the cells from the incubator and immediately measure the transendothelial electrical resistance, or TEER, by placing the shorter electrode on top of the insert and the longer electrode into the medium surrounding the insert.

On the day before the infection experiment, start an overnight culture of the bacteria from frozen stock. Streak Neisseria meningitidis onto Columbia Agar with 5%sheep blood and incubate at 37 degrees Celsius and 5%carbon dioxide overnight. Pathogenic bacteria, such as Neisseria meningitidis, have the potential to cause disease in humans.

It's important to properly train individuals working with such microorganisms and always use best practices and wear proper PPE. On the next day, prepare fresh PPM plus media according to manuscript directions and inoculate 10 milliliters of the media with the bacteria and incubate the culture. While the bacteria are incubating, replace the medium in the iPSC-derived brain endothelial cells, then centrifuge the bacterial culture at 4, 000 G for 10 minutes.

Aspirate the media and resuspend the pellet in 250 microliters of PBS. Adjust to an OD600 of approximately 0.4, which is roughly one times 10 to the eighth colony-forming units per milliliter. Dilute the bacteria to the desired multiplicity of infection with EC medium and infect the cells with 100 microliters of the prepared bacterial suspension per well.

Then incubate the cells for the desired time of infection. When differentiated properly, the iPSC-derived brain endothelial cells exhibit tight barrier properties that are often greater than 2000 ohm centimeter squared and endothelial markers, such as VE-Cadherin and CD-31. Additionally, they express and localize the tight junction markers Claudin-5, Occludin, and ZO-1, as well as transporters, such as Glut-1.

Upon infection with Neisseria meningitidis, the cells upregulate neutrophilic, pro-inflammatory cytokines, such as CXCL8, CXCL1, CXCL2, CCL20, and IL6. When performing cell differentiation, daily maintenance and achieving the initial optimal seeding density when splitting the cells is critical. Following the steps outlined here, researchers can collect a variety of samples to investigate many aspects of post-pathogen interaction from inflammatory responses to brain endothelial cell dysfunction.

The advent of brain endothelial cells derived from iPSCs have opened the doors for the brain barriers field to investigate a wide range of diseases where endothelial cells are compromised. As certain CNS pathogens are human-specific, we demonstrate the use of a human-based system with the human-specific pathogen Neisseria meningitidis