The overall goal of this procedure is to generate eps. Epstein-Barr virus transformed Lymphoblasts cell lines or LCL from human B cells. This is accomplished by first isolating peripheral blood mononuclear cells from human blood.
Next, the cells are infected with eps Epstein-Barr virus in the presence of FK 5 0 6 and immunosuppressive agents three to four days after infection with EP Epstein-Barr virus, a subpopulation of cells emerges that is marked as CD 23, high CD 58 positive, and is predicted to undergo proliferation CD 23 high. CD 58 positive cells can be enumerated by flow cytometry as a predictor of successful outcome. Proliferating cells can be further identified, micro and macroscopically by the clusters of lymphoblasts cell lines that form in successfully transformed cell cultures.
The final step is to expand lymphoblasts cell lines for cryo-preservation. In my laboratory, we generate lymphoblasts cell lines or LCLs to understand interactions between the host and Epstein-Barr virus. Lymphoblasts cell lines are also of broad general interest to the scientific community, particularly to virologists immunologists and cancer biologists.
As a postdoctoral O fellow in a du Macintosh lab, I'll be demonstrating the procedure for generating lymphoblasts cell lines. After drawing 10 milliliters of blood from a donor, add the heparinized blood to a 50 milliliter conical tube and then dilute it one to two. By adding 20 milliliters of room temperature PBS to the tube now underlay the diluted blood with 15 milliliters of FICO HIA lymphocytes separation medium, and then spin down the blood at 225 times G without the break at room temperature for 30 minutes to establish a gradient.
Next, remove the buffy coat and transfer it to a new 50 milliliter conical tube. Bring the volume up to 50 milliliters with PBS and then pellet the cells at 600 times G for 10 minutes. Pour off the supinate, re suspend the pellet in 50 milliliters of PBS, and then wash the cells two more times under the same conditions.
Now resuspend the cells in one milliliter of complete RPM. I use five microliters of cells to repair a one to 10 dilution in triam blue, and to count the live cells using a hemo cytometer, then use complete RPMI to adjust the volume to obtain a cell concentration of two times. Center the six cells per milliliter and transfer the cells to a 25 centimeter squared tissue culture flask.
To prepare the cells for infection with Epstein bar virus at FK 5 0 6 to the tissue culture flask to a final concentration of 20 M.Then place the flask in a 5%carbon dioxide incubator at 37 degrees Celsius for one hour. When the cells are almost finished incubating rapidly thaw an aliquot of Epstein bar virus, harvested from B 95 8 cells. Then remove the flask from the incubator and add the EP Epstein bar virus to the cells at one to 10 dilution now swell the flask to mix the epton bar virus into solution, and then place the flask upright in the incubator with the cap on, but slightly loose if using a non vented flask for two to three weeks prior to expansion of the culture for cryo-preservation.
A week after eps, Epstein Barr virus infection, clusters of cells are visible by light microscopy as seen in this representative figure of early microscopic clusters. As time progresses, the microscopic clusters become larger, such that clumps are visible macroscopically in the flask as seen in this figure where larger clusters of LCL can be observed to feed the cells, double the volume of the supportive medium in the culture flask on day 12. When the culture medium turns yellow, typically about once a week, it is generally time to feed the cells to expand the cells, increase the volume of the culture two to three fold.
Using complete RPMI expand the culture to 70 to 100 milliliters in a 75 centimeter square flask over the next few weeks to generate cells for cryopreservation, a successful outcome is predicted by the presence of CD 23, high CD 58 positive cells as seen in this scatterplot, approximately two to 3%of live cells on day three to four after exposure to eps Epstein-Barr virus have this profile other subpopulations of CD 23 positive, CD 23 negative, CD 58 positive and CD 58 negative cells observed after exposure to eps. Epstein-Barr virus show minimal proliferation and uninfected cells or cells infected with a non immortalizing strain of ep. Epstein-Barr virus do not demonstrate CD 23 high.
CD 58 positive cells. The cells may also be visualized by light microscopy. As mentioned, within a week of Epstein-Barr virus infection, small glasses of cells are visible in the flask by light microscopy.
As time progresses and the cells develop into LCL, the microscopic clusters become larger, such that clumps are visible macroscopically in the flask. All right. After watching this video, you should have a good understanding of how to isolate peripheral blood mononuclear cells from human blood and to generate Epstein-Barr virus transformed lymphoblasts cell lines from human B cells, While generation of lymphoblasts cell lines has been described by other laboratories.
The advantage of our protocol is that it provides the researchers with a method to predict successful outcome as early as three to four days after infection of B cells with Epstein-Barr virus.
