General Protocol for the Immortalization Of Human B-Lymphocytes Using EBV

Preparation of Epstein-Barr virus (EBV)

  1. The marmoset cell line, B95-8, is stored in a liquid nitrogen freezer at –180° C, preferably vapor-phase liquid nitrogen.
  2. Cells are thawed rapidly by submersing the cryovial containing the cells in pre-warmed sterile double distilled, deionized water (37° C, 1-2 min.) and the cell suspension then plated into a 25 cm2 tissue culture flask in 10 ml RPMI 1640 + 15% fetal bovine serum. Maintain the cells at 37° C, 5% CO2.
  3. After 24 hours, change 50% of the medium to further dilute the original cryopreservative, DMSO. At day 2-3 of culture or when the appropriate cell density has been achieved, expand the B95-8 cells to a 75 cm2 flask in 20 ml culture medium.

  4. At day 5-7 of culture or when appropriate cell density has been achieved, expand the B95-8 cells to a 150 cm2 flask in 200 ml culture medium.
  5. At day 12-15 of culture, harvest the supernatant by centrifuging out the cells. Important: Do not add or take off any medium between expansion to a150 cm2 flask and the harvest. The EBV will be in the supernatant medium.
  6. Prescreen the supernatant for mycoplasma using the Gen-Probe mycoplasma detection system or an appropriate mycoplasma screening method.
  7. Dilute the supernatant 1:1 with RPMI 1640 + 20% FBS + 400ng/ml cyclosporin. Note: The final concentration of serum should be 15-20% and cyclosporin should be 200ng/ml.
  8. Filter the diluted supernatant through a 0.22um filter to sterilize.
  9. Aliquot 3 ml EBV supernatant into cryovials and snap freeze at –180° C, ie. liquid nitrogen.

Blood Collection

  1. A volume of 10-20 ml of blood is recommended for whole blood samples to separate and recover at least 3 x 106 fresh lymphocytes. Extra separated lymphocytes can and will be cryopreserved in case the first transformation attempt fails in cases where the number of lymphocytes is adequate. When attempting transformations using cryopreserved lymphocytes, begin with 5-10 x106 cells.
  2. It is preferable to use a 21 1/2-gauge needle when drawing the blood to decrease cell lysis.
  3. ACD, yellow top, vacutainer tubes should be used to prevent clotting and are best for longer than usual storage prior to processing or for compromised shipping situations. Sodium Heparin, green top, (not Lithium Heparin (a lighter green top), vacutainer tubes may also be used with a high success rate.
  4. Blood samples should be kept at room temperature until the time of separation. Do not cool or transport the blood on ice.
  5. Separation should be initiated within 24 hours of the blood draw, however, successful isolation and subsequent transformation can often be accomplished up to about 72 hours post collection for research projects requiring genomic DNA with inherited polymorphisms only. The TCF recommends setting 48 hours as the maximum period post-collection time before processing to maintain a high successful transformation rate. For studies interested or focused on non-inherited polymorphisms or other types of DNA damage repair mechanisms, point break DNA damage mechanisms, or short term DNA damage and/or repair mechanisms, processing must begin as soon after collection as possible and in no situations should the samples be held overnight (the actual time period affecting this particular issue has not been determined yet but evidence has been obtained related to this issue and studies are on-going to address this point.

Lymphocyte Isolation and Transformation

Sample fractions to be saved will vary by project. Determination of sample fraction collection and treatment requirements should be finalized with all investigators prior to setting the final protocol(s). The following assumes plasma will NOT be required to be saved. If plasma is to be collected and retained, determine the volume required from the investigator. Contact the TCF because an additional step will need to be included. Store plasma samples in 3 ml cryo vials at –80°C.

  1. Aliquot approximately 10 ml of blood per 50 ml conical tube or 2-3 ml of blood if using 15 ml tubes.
  2. Dilute each tube of blood 1:2 by mixing with sterile phosphate buffered saline (PBS) and mix thoroughly by pipetting up and down in the tube.
  3. Carefully and slowly underlay each 50 ml tube with 10 ml of Ficoll-PaquePLUS from Pharmacia Biotech (or similar separation medium). Use 3 ml Ficoll-Paque for 15 ml tube. Important: When layering the sample do not mix Ficoll-Paque and the diluted blood sample.
  4. Centrifuge at 400 x g for 30-40 minutes at room temperature.
  5. Collect the interphase of Peripheral Blood Mononuclear Cells (PBNCs) or also referred to as Peripheral Blood Lymphocytes (PBLs). Avoid collecting Ficoll-Paque and supernatant. Collecting excess Ficoll-Paque causes granulocyte and possibly macrophage contamination and the supernatant contains unwanted platelets and plasma proteins.
  6. Wash the lymphocytes two times with 40 ml PBS. Centrifuge cells at 100 x g for 10 minutes each time.
  7. After cells are washed, resuspend the cells in an appropriate volume of PBS, count the cells using a hemacytometer or appropriate cell counter, and retain 3-5 x 106 cells for infection with the EBV supernatant. Use the higher side of this recommendation to increase transformation success rates.
  8. Centrifuge cells at 100 x g and resuspend in 3 ml of EBV supernatant.
  9. Incubate over night at 37° C, 5% CO2. The cells should be incubated in vented filter cap tissue culture flasks oriented in the upright position (flasks standing upright on the bottoms not laying down in the typical orientation when used for anchorage type cells). This is important throughout propagation.
  10. After 24 hours of incubation in the EBV supernatant, add 7 mls of transformation medium (RPMI 1640 + 20% FBS + 200ng/ml cyclosporin).
  11. After 5-7 days, take off 5 mls medium and add 5 mls fresh growth medium by letting the cells settle in the flask and carefully pipetting off the upper volume of medium. This step can be postponed for up to two (2)) + weeks. The medium should be allowed to become acidic, pH 6.5 to 6.8) and the cells allowed to clump and not be disrupted. Adequate judgement should be used in the propagation handling steps of these cultures. Continue to feed the cells in this way for the subsequent 2-3 weeks or until transformation is clearly achieved.
  12. Subsequently, the transformed lymphocyte cell line (LCL), also called lymphoblasts, can be propagated in RPMI 1640 + 10-15% FBS.
  13. Maintain the cells at a concentration between 4 x 105 - 1 x 106 cells/ml and expand as needed. Cryopreserve LCLs for future use and maintain in vapor –phase liquid nitrogen at –150° C or lower.
  14. Important: In order to keep the LCLs “young”, i.e., a low number of passages to avoid moving toward oligoclonal and monoclonal cultures, expand as quickly as possible and cryopreserve six (6) vials of cells. One week later, pull one of the vials for a recovery and performance test.



Notice of disclaimer.
This protocol has been used at the TCF for over seven years with an average transformation success exceeding 99% for all samples. For samples collected and submitted according to the recommended procedures and time period, the transformation success rate is 100% with over 8000 samples processed. This protocol is not copyrighted nor is it proprietary. It may be used and distributed without prior consent of the TCF. However, the TCF makes no guarantee, expressed or implied, that transformations will be achieved at all or with any guaranteed rate of success by using this protocol. For further information regarding any aspect of this protocol or services provided by the TCF, please contact Steve Oglesbee, Director, at 919-966-4324.

TCF General Protocol EBV.v10 2/7/2007