The overall goal of this simple method is to establish B lymphoblastoid cell lines, or B-LCLs with high immortalization efficiency using only a small amount of blood and saving time between initiation and cryopreservation. This is a novel method for immortalizing human B cells from whole blood by lysing red blood cells. B-LCLs provides a valuable source of bio-materials to carry out various studies.
The main advantage of this method is that only 0.5 milliliters of blood is sufficient to obtain B-LCLs with immortalization efficiency and high cell viability. To establish B-LCLs by Epstein-Barr virus, or EBV, transforming B lymphocytes, the widely used method into separate peripheral blood mononuclear cells or PBMC, by Ficoll density gradient centrifugation. However, when the sample is less than one milliliter of the red blood cell lyse, the addition will fail.
We first had the idea for this method when we that to isolate the PBMC from small red blood cells is difficult using a density gradient separation method. To prepare EBV, on day one begin by thawing B95-8 cells and culture them in a T25 culture flask in six milliliters of complete RPMI 1640 Medium supplemented with 10%FBS, two millimolar of L-glutamine, and antibiotics. Incubate the culture at 37 degrees Celsius and 5%carbon dioxide.
On day two, under a microscope, observe the cell morphology. Cells are in good condition when they are bright and round. Add 10 milliliters of complete RPMI 1640 Medium to the culture.
On day four, add 10 milliliters of fresh medium and transfer the cells to a T75 culture flask. Then, on day eight, add 20 milliliters more of medium and maintain the cells for eight days without adding fresh medium. On day 16, check the cells under the microscope.
Then collection 12 milliliters of cells into 15 milliliter centrifuge tubes. Next, cryopreserve the cells at minus 80 degrees Celsius for one hour and rapidly thaw the cultures at 37 degrees Celsius. Repeat the freeze thaw, three more times until the virus particles are released from the cells.
Centrifuge the cultures at 240 times g at room temperature for 10 minutes. Then, filter the supernatant through 0.22 micron filter. Aliquot 12 milliliters of the filtrate per tube and store the tubes at minus 80 degrees Celsius.
To a 50 milliliter tube, add 0.5 milliliters of whole blood that has been anticoagulated by EDTA K2.Add one milliliter of red blood cell lysis buffer to the whole blood and mix well. Incubate the mixture at room temperature for four minutes. Then quickly add five to seven milliliters of 1x PBS to stop the lysis reaction.
After adding red blood cell lysis buffer to blood, be sure to mix well. Invertation ensures that the reaction is terminated in a timely manner. Next, centrifuge the tube at 240 times g at room temperature for five minute.
The white blood cells will precipitate at the bottom of the tube. Discard the supernatant and wash the precipitate by adding seven milliliters of 1640 Basic Medium. Then centrifuge the solution before repeating the wash.
With 300 microliters of transformation medium, resuspend the cell pellet. Then to carry out EBV infection, add 60 microliters of 20 micrograms per milliliter of Cyclosporin A to the cell suspension. Rapid thaw an aliquot of EBV at 37 degrees Celsius and add 240 microliters of the virus to the cells.
Thoroughly mix the suspension and transfer 200 microliters per well to a 96 well plate. Then incubate the plate at 37 degrees Celsius and 5%carbon dioxide. For the first month after EBV infection, change half of the medium in the cell culture once per week.
On day seven, only a thick layer of cell fragments is visible under the microscope. After one month of infection, lymphoblastoid cell clusters are visible under a thick layer of cell fragments. From this point forward, change half of the medium every three days until the cell clusters are clearly visible under the microscope.
After resuspending the cells, transfer the suspension to the wells of a 24 well plate. Then depending on the rate of cell growth, when the culture medium turns yellow, double the volume of complete RPMI 1640 Medium. When lymphoblastoid cell clusters are visible to the naked eye, transfer the culture to a T25 culture flask.
As the culture medium turns yellow, double the volume of the medium until the volume expands to 10 to 15 milliliters. Before freezing, change the medium 24 hours ahead a time. Then to freeze the cells, centrifuge the cell suspension and use a cell counter to count the cells.
With the frozen stock solution containing 90%FBS and 10%DMSO, adjust the suspension to a density of five to 10 times 10 to the sixth cells per milliliter. Then freeze the suspension at a rate of one degree Celsius per minute to minus 80 degrees Celsius before transferring the frozen cells directly into liquid nitrogen. During the process of transformation, the morphological changes of cells are visible by light microscopy.
After seven days of infection, small clusters of cells are clearly visible using the density gradient separation method, while only a thick layer of cell fragments are visible from the red blood cell lysis method. 30 days post infection, lymphoblastoid cell clusters are visible under the thick layer of cell fragments. With more frequent medium changes, the cell debris is reduced and the cell clusters are clearly visible and look similar to the culture established by density gradient separation.
Finally the MTT assay was used to evaluate the viability of B-LCLs. The results shown here indicate that the B-LCLs cell cultured using the red blood lysis method were significantly more viable than the culture established using the density gradient separation method. Once mastered, the EBV viral transformation process can be done in 30 minutes when it is performed properly.
After watching this video you should have a good understanding of how to establish B-LCLs with a simple and time saving red blood cell lysis method without previous purification of lymphocytes. After its development, this technique accelerated the progress of the genomics, transcriptomics, and proteomics study of EBV associated diseases and this application in the production of mononuclear antibodies. Don't forget that working with human pathogens, such as EBV can be extremely hazardous and the precautions should always be taken while performing this procedure.
