The overall goal of this procedure is to demonstrate the use of stretch injury as a model of traumatic brain injury. This is accomplished by first cultivating, mirroring microvascular endothelial cells and flexible bottoms wells. The second step is to differentiate the cells by reduction of fetal calf serum in the culture medium.
Next, the cells are injured using the cell stretcher device. The final step is to assess the degree of injury in the cells. Ultimately, stretch induced injury in brain endothelial cells is used to show injury in the blood-brain barrier in vitro.
The implications of this technique extend towards therapy of traumatic brain injury because it models the actual impact that occurs during TBI in the blood-brain barrier using stretch injury of marine brain endothelial cells. Demonstrating the procedure will be Dr.Elaine Salvador, a postdoc from my laboratory. Begin this procedure by cultivating the marine brain microvascular endothelial cells in a T 75 culture flask.
Change the medium twice a week until confluence is reached. Next, wash the cells with PBS. Then remove the PBS and tryps.
Anize the cells with two milliliters of warm tryin EDTA solution. Incubate the cells at 37 degrees Celsius for five minutes or until the cell layer is dispersed. After that, at eight milliliters of culture medium into the cells, tap the flask several times to detach the cells.
View the cells under the microscope to ensure complete detachment from the flask. Then pipette the medium with detached cells up and down. Swirl the flask to mix the cell suspension afterward.
Add 20 microliters of the cell suspension into a hemo cytometer, and count the number of cells. Determine the cell density and see 20, 000 cells per square centimeter into the well. Next, transfer the cell suspension into collagen.
One pre-coded six. Well flexible bottoms culture plates in a total volume of three milliliters per well. Change the culture medium twice per week and grow the cells at 37 degrees Celsius for one week until confluent.
For cell differentiation, change the culture medium of the cells with differentiation medium, and incubate the cells at 37 degrees Celsius for 24 hours. In this procedure, turn on the cell injury controller device, set the delay to 50 milliseconds. Next, set the regulator pressure to 15 PSI and press the trigger a couple of times until the registered peak pressure becomes stable.
Then set the regulator pressure to the desired value. After that, place the six well flexible bottoms culture plate into the tray holder. Make sure that the well selector is set to the correct well size.
Then place the adapter plug firmly over the, well hold the plug firmly in place with one hand with the other hand pushing the trigger record the peak pressure generated afterward. Immediately put the plate back into the 37 degree Celsius incubator for the desired length of time, or use immediately for subsequent experiments or evaluation to assess the stretch injury by DI uptake assay. Immediately after the cells are stretched at 30 microliters of one milligram per milliliter of the viability stain that acts as a cytotoxicity marker to the cell culture medium.
To assess the stretch injury by lactate dehydrogenase release. After stretching, the cells immediately remove 200 microliters of cell culture medium from the, well do the same for every time point post-injury you would like to include in your investigation of LDH release. Next, centrifuge the cell culture medium at the highest setting of a micro centrifuge for five minutes.
To remove any cell debris, remove the supernatant and use it for the subsequent steps. Once the necessary samples from various time points have been taken, lys the cells using the lysis solution included in the LDH assay kit. After that, add 100 microliters of the assay medium included in the assay kit to every well of a 96 well plate provided in the kit.
Then add 100 microliters of the cell-free cell culture medium into two parallel wells of the 96. Well plate incubate the plate at 37 degrees Celsius for 30 minutes and read the absorbance at 492 nanometers. Shown here is the light microscopy examination of normal against injured cells.
The normal unstretched confluence cell monolayer is tightly packed and elongated when cells are stretched by applying a peak pressure pulse of 1.8 to 2.0 PSI, they appear less compact. When the cells are moderately injured with a 2.5 to 3.0 peak pressure pulse, some of them appear swollen and deformed. The cells become retracted with a severe stretch of 3.5 to 4.5 PSI.
Here is the fluorescence microscopic examination of normal against injured cells. The cells were treated with viability stain two hours after injury. These are the control unstretched cells, the moderately stretched cells, and the severely stretched cells.
This figure shows the LDH enzyme release into the snat after stretch injury LDH released into the culture. Medium was measured at various time intervals after stretch induced injury and was expressed as a percentage of the total releasable L-D-H-L-D-H released from cells that were subjected to low and moderate stretch. Did not differ significantly from that of the unstretched controls and from each other.
Cells that were severely stretched released a significantly greater amount of LDH as compared to all other samples except for the moderately stretched sample at one hour After its development. This technique paved the way for researchers in the field of neurobiology to explore traumatic brain injury in marine brain endothelial cells in vitro.
