Successful transfection of Lymphoblastoid cell line (Preprint)
Authors: Zraikat Manar 
Published: July 2021

BACKGROUND

Immortalization is the stage that the cell goes through before full transformation [1]. Human resting B lymphocytes from peripheral blood are easily infected by EBV to be induced into actively proliferating B-lymphoblastoid cell lines (LCLs) [2]. Most LCLs from normal individuals are mortal because their telomeres shorten. Some LCLs are immortalized by developing strong telomerase activity but cannot form tumors in nude mice. Some post-immortal LCLs [3] additionally become tumorigenic and develop the ability to grow in nude mice, and considered as transformed cells [4]. EBV was the first virus to be completely sequenced in 1984 [5] [6]. The linear form of EBV genome is composed of linear double stranded DNA molecule of 172 kilobase (Kb) coding for around 100 viral proteins, and has 0.5 Kb terminal repeats (TR) which become joined in a circular form and 3 kb internal direct repeats [ITR] that divide the viral genome into long and short regions [7].
Retroviral transfection first involves introduction of the retroviral plasmid into the retroviral packaging cells then infecting the target cells with the retroviruses that is produced. The calcium phosphate–mediated transfection protocol result in many copies of the retroviral vector plasmid DNA to be taken up by packaging cell. This results in a high level of expression with consequent high retroviral titer. And more efficient infection [8] [9].

OBJECTIVE

The Lymphoblastoid cell line (LCL) is the immortal cell line that is produced after infecting the B lymphocytes with Epstein bar virus. This kind of cells known to be hard to be transfected practically. In this paper I am describing the protocols followed in my lab starting from infecting the B-lymphocytes with Epstein Barr virus (EBV) ending with successful transfection of LCL with retroviral vector contain lac Z insert.
The importance of this work is that it describes a away to prepare representative model for EBV associated lymphoma through preparing LCLs then retrovirally infecting these cells with retroviral vector.

METHODS

Materials and methods
EBV infection protocol
The method of EBV immortalization of B lymphocytes used in this study involved, first, isolation of mononuclear cells (lymphocytes) from buffy coat (catalog # MBS170216) , second, preparation of EBV stocks from B95-8 marmoset cell lines [6] through culturing the cells in RPMI1640 media supplemented with 10% fetal bovine serum [FBS]. Cultures were maintained in the incubator under 37° C with 5% CO2 for 12-15 days. After centrifugation the supernatant is filtered and stored at 4° C until use. Third, immortalization of lymphocytes was achieved by infecting the mononuclear cells with the EBV stock [11] by incubating the cells with an equal volume of virus supernatant at 37° C with 5% CO2 to produce the (LCL).
During centrifugation of the buffy coat (catalog # MBS170216) at 3200 round per minute (rpm) at room temperature for 15 minutes, erythrocytes and granulocytes were aggregated and rapidly settled at the bottom of the tube. Lymphocytes and other mononuclear cells remained at the plasma-erythrocyte interface which were then collected by Pasture pipette and transferred to new clean tube containing 5 ml washing media and centrifuged at 1000 rpm at 4 degrees for 10 minutes. The washing step was repeated twice and viable cell count was performed. The freezing medium was added to the cells and the cell suspension was transferred to cryogenic tubes Finally, the cells were stored in liquid nitrogen for future processing.
Maintenance of marmoset cells and EBV stock preparation
An ampoule of the B95-8 marmoset cell line was thawed and using routine sub-culturing methods the culture was maintained between 3 x 105 and 9 x 105 cells/ml by diluting the cells 1:2 - 1:4 every 3-4 days in RPM1-1640 (containing 5% FBS +1% v/v 200mM L- Glutamine) [12].
B95-8 cells exhibited both suspension and monolayer growth characteristics and therefore the culture flasks were incubated horizontally. Adherent marmoset cells were dislodged using trypsin/EDTA. When the cells reached sufficient numbers they where diluted to 2 x 105/ml in RPM1-1640 + 2% FBS. +1% v/v 200mM L.Glutamine) in 75cm2 culture flasks The flasks were then incubated horizontally over night in an atmosphere of 5%CO2, at 37ºC.
The vented caps of the flask were sealed with adhesive tape and transferred to a 33ºC incubator for 2-3 weeks. The medium was not changed and the culture flasks were not gassed during this time. After 2-3 weeks incubation, the culture medium turns bright yellow in color.
On the day of virus harvesting the flasks were up-ended to allow the cells to settle, then the supernatant was decanted carefully into 50ml tubes and centrifuged at (1,500 rpm) for 10 minutes at room temperature to remove all cell debris.
The supernatant was filtered through a 0.45 µm filter to remove any remaining cell debris and large particles. The supernatant was dispensed into 1ml aliquots in 2ml cryogenic ampoules and frozen rapidly in vapor phase nitrogen [13].
Transformation of B lymphocytes
Ampoules of frozen (LCL) cells were removed from liquid nitrogen and allowed to warm at room temperature in a Class II cabinet for 30-60 seconds then warmed in a 37°C water bath. The contents were transferred to a 15ml centrifuge tube containing RPM1 1640 and 1% v/v 200 mM L-Glutamine. After centrifugation, (1000 rpm) for 5 minutes, the cells were resuspended in transformation medium plus viral supernatant at a V/V ratio of 4:1 and a final cell concentration of 1-2 x 106 cells/ml and transferred into of a 24 well, flat bottomed, tissue culture plate.
Plates were incubated in a sealed, sterile plastic box, gassed with 5% C02, 95% air then the cultures were left undisturbed for two to three days.
The cultures were fed twice a week by removing 500µl of medium and adding 500µl of fresh maintenance medium without disturbing cells when changing the medium. After two to three weeks, the contents of each well were transferred into an individual 25cm2 culture flask containing 5 ml of maintenance medium (figure 1). The lymphoblastoid cells tend to form floating clumps after increase in the cell density (figure 2).

Retroviral transfection pFB-Neo-LacZ into 293 packaging cells.
A modification of the calcium phosphate–mediated transfection protocol was used to transiently transfect pFB-Neo-LacZ into 293 packaging cells.
The 293 cells were seeded (6.25 × 105 cells/ml) in a 60-mm tissue culture dish the day before transfection and fed with fresh complete medium to ensure that on the day of the transfection, the cells are thoroughly separated on the dish to increase the ability to take up DNA. The retroviral vector plasmid DNA was Ethanol precipitated and air dried then resuspend in 450 µl sterile water before 50 µl of 2.5 M CaCl was added.
500 µl of 2× HeBS was placed in a sterile 15-ml conical tube. A mechanical pipettor attached to 2-ml pipet was used to bubble the 2× HeBS while adding the DNA/CaCl2 solution dropwise with a Pasteur pipet then immediately the solution was vortexed for 5 sec and allowed to precipitate for 20 min at room temperature. Then the precipitate was distributed evenly over a 6-cm plate. the cells were incubated overnight under standard growth conditions. Next day the medium was removed. and 4 ml complete medium was added. The retroviral supernatant was harvested ∼48 hr following transfection by gently removing the supernatant from the cells. The supernatant was filtered through a 0.45-µm filter and transferred to ice. The transfected cells were assayed for transfection efficiency after retroviral supernatant was harvested, by lacZ histochemical staining (figure 3).
Retroviral infection of LCL cells by cocultivation
LCL cells were infected by cocultivating them with irradiated retroviral producer cells for 48 hr. Briefly, 3-ml infection cocktail was prepared twenty-four hours after 293 cells have been transfected. The infection cocktail contained, Retroviral supernatant ≤1.5 ml, 2 ug/ml polybrene in PBS, 105 to 106 cells/ml LCL, LCL growth medium. The 293 packaging cells were irradiated prior to addition of infection cocktail. The medium was aspirated from the transfected LCL cells and the infection cocktail was gently added to the cells and incubated for 24 hr. 2 ml medium containing many LCL cells was removed twenty-four hours after the infection cocktail was added and transferred to a conical tube and centrifuged for 5 min at 500 × g, 4°C. The supernatant was removed, and the cell pellet was resuspended in freshly prepared infection cocktail. The fresh infection cocktail containing the LCLs was added to the wall of the plate of 293 cells and incubated for 24 hr. At 72 hr post transfection, the LCLs were collected from the dish by gentle pipetting. The suspension of nonadherent cells was Centrifuged for 5 min at 500 × g, 4°C. The cells were Resuspend in LCL cell growth medium. The LCLs were plated in 60-mm tissue culture dishes and incubated for 48 hr. LacZ expression assay was done with promising results (figure 4).

RESULTS

The first part of this work describes the immortalization of B lymphocytes by EBV to form LCLs. Immortalization of B lymphocytes is an acquired ability of cells to proliferate indefinitely in culture [3] [4]. The importance of LCLs is that these cells could be a suitable model to represent different kinds of lymphoma. Epstein Barr virus-associated B-cell lymphoproliferative disorders include many kinds of lymphoma such as, Hodgkin's lymphoma, Burkitt's lymphoma and post-transplant lymphoprolifrative diseases. [14].
The second part of paper describes the retroviral transfection of the LCLs using CaCl precipitation and coculturing LCLs with the packaging 293 cells. The importance of this part of the described protocol is that the LCLs alone are not considered alone enough to represent most kinds of lymphoma, there is always necessity to make further modifications on the cell to make the model mimic the lymphoma tumour better and consequently make the model more representative to the real tumour in vivo. This paper suggests a protocol to transfect LCLs cells based on my experience in the lab and shows some successful resells.

CONCLUSIONS

Conclusion
This work gives a detailed reproducible protocol describes the EBV infection of B-lymphocytes to produce continues cell line of lymphoblastoid cell line. Furthermore this paper describes the process of retroviral transfection of LCLs using retroviral vector carries LacZ and shows successful transfection process.