Lymphocyte Transformation Using ATCC VR-1492

Epstein-Barr virus (EBV), also referred to as Human herpesvirus 4, is a gamma herpesvirus of the genus Lymphocryptovirus. It is the cause of infectious mononucleosis and has been associated with several human neoplastic diseases, including Burkitt’s and Hodgkin’s lymphomas. Most adults are seropositive for EBV, demonstrating its ubiquitous presence in the human population. Infection is usually latent. However, explanted lymphoma cells sometimes produce infectious virus. Several decades ago, supernatants from cell lines derived from lymphoma explants were used to confirm the neoplastic potential of EBV infection by virus-mediated immortalization of B lymphocytes from human blood in cell culture. Since then, EBV transformation of B lymphocytes in vitro has become a routine procedure for obtaining permanent human cell lines for use in diagnostics and research.^1,2

In 1972, G. Miller and colleagues found that EBV-transformed marmoset lymphocytes produce more virus than infected human lymphocytes, providing a ready source of the virus in the B95-8 cell line they established.^3,4 EBV-containing supernatants from the B95-8 cell line are distributed by ATCC as VR-1492. Prior to distribution, each preparation is tested to verify that the content of one vial (1 mL) is capable of inducing transformation in a sample of approximately 10⁶ normal human lymphocytes isolated from frozen normal human blood (approximately 0.5-1 mL).

This technical document describes the procedure recommended for transformation with this product as described by Caputo et al. in 1995, which is reported to have a success rate of 95%.⁵ This high success rate is attributed to the use of a feeder layer to support the culture during the transformation process. Other laboratories have reported other modifications that may improve transformation success rate.^6-9 These are generally aimed at ensuring reagent quality or preventing the development of cytotoxic T lymphocytes reactive with EBV antigens that might kill virally infected B cells before a transformed cell line can be established. Because blood samples vary in quality (and quantity), each laboratory must verify the efficacy of any EBV transformation methods it adopts and modify them as needed for specific applications.

Supplies available from ATCC

• Dimethylsulfoxide (DMSO) (ATCC 4-X or equivalent) 
• Iscove's Modified Dulbecco's Medium (IMDM) (ATCC 30-2005 or equivalent)
• RPMI-1640 (ATCC 30-2001 or equivalent)
• Fetal Bovine Serum (FBS) (ATCC 30-2020 or equivalent)
• Penicillin/streptomycin solution, 10,000 I.U./mL penicillin, 10,000 μg/mL streptomycin (ATCC 30-2300)
• PBS, calcium- and magnesium-free (ATCC 30-2200 or equivalent)
• Filtered supernatant from EBV-expressing cells (ATCC VR-1492)
• Irradiated MRC-5 feeder layer cells (ATCC 55-X)
  Note: Irradiated MRC-5 cells are packaged as frozen suspensions, 1-mL per vial. Each vial is sufficient for seeding up to 225 cm² of surface area (9 - T-25 flasks)

Additional supplies

• Cryovials to hold 0.5-1 mL
• Defibrinated or anticoagulant-treated whole human blood
• Histopaque-1077 Ficoll gradient solution (Sigma-Aldrich or equivalent)
• 25 cm² (T-25) flasks, cell culture treated
• 75 cm² (T-75) flasks, cell culture treated
• 15 mL sterile polystyrene centrifuge tubes, capped

Precautions

All work should be completed using aseptic technique under BSL-2 conditions using universal precautions for handling blood products.¹⁰

B95-8 cells may also carry and secrete a transmissible type D retrovirus, squirrel monkey retrovirus–B95-8 (SMRV-B95-8), which can infect human B and T cell lines. This retrovirus does not interfere with EBV’s transforming capabilities.¹¹

Procedure

Freeze blood sample for later use (optional)

1. Dispense 0.1 mL DMSO into labeled cryovials for blood storage.
2. Pool defibrinated or anticoagulant-treated blood from containers.
3. Dispense 0.9 mL of blood per cryovial, mixing periodically to ensure an even cell suspension.
4. Cap the vials and mix gently.
5. Freeze the vials in a cryopreservation container in ethyl alcohol at -80°C for 3-18 hours or in a controlled rate freezer at 1°C per minute.
6. Store vials of cryopreserved blood in liquid nitrogen.

Prepare feeder layers 1-5 days prior to use for transformation

1. Prepare complete culture medium by addition of FBS to either EMEM or IMDM to 10% v/v and penicillin/streptomycin solution to 1% v/v (1X).
2. Thaw a vial of Irradiated MRC-5 feeder layer cells (ATCC 55-X) and mix with 27 mL of complete culture medium in a sterile 50 mL conical tube.
3. Dispense 3 mL of cell suspension per T-25 flask (~4 × 10⁵ cells).
4. Examine flasks microscopically next day. Cultures should be about 30% confluent.
5. Use for transformation from 1-5 days after planting.

Prepare lymphocytes for transformation

1. Mix 3 mL of whole blood (fresh or thawed) with an equal volume of PBS or RPMI-1640.
2. Separate lymphocytes from red blood cells and granulocytes using Ficoll.
   1. Carefully layer the diluted whole blood over 4.5 mL of room temperature Histopaque-1077 in a 15 mL polystyrene centrifuge tube.
   2. Centrifuge at 400 × g for 40 minutes at room temperature.
   3. Remove and discard the upper, clear plasma layer to within 0.3 cm² of intermediate layer.
   4. Transfer the opaque lymphocyte band at the intermediate layer and the Histopaque-1077 layer down to within 0.3 cm² of the pellet at the bottom of the tube to a new 15 mL centrifuge tube.
   5. Fill the tube with IMDM with 20% FBS and penicillin/streptomycin; mix the tube well and centrifuge at 260 × g for 15 minutes. Remove and discard the supernatant.
   6. Resuspend the cell pellet in 10 mL IMDM with 10% FBS and penicillin/streptomycin; mix and collect cells again by centrifugation at 260 × g for 15 minutes. Remove and discard the supernatant.
   7. Resuspend the cell pellet in 3 mL IMDM with 10% FBS and 1X penicillin/streptomycin.
3. Perform a viable count on the resulting lymphocyte preparation using a dye exclusion method.
4. Use cells immediately for transformation or freeze them for future use as for whole blood.

Set up transformation cultures (2 flasks per sample are recommended)

1. Thaw 1 vial of Human gammaherpesvirus 4 (HHV-4) (ATCC VR-1492) (1 mL) per transformation flask.
2. Remove medium from feeder layer(s).
3. Per flask add:
   1. 2 mL IMDM with 20% FBS
   2. 1 mL lymphocyte suspension containing 1-6 × 10⁶ lymphocytes.
   3. 1 mL (1 vial) Human gammaherpesvirus 4 (HHV-4) (ATCC VR-1492)
4. Mix gently and incubate flasks at 37°C under a 5% CO₂ atmosphere.

Maintain and observe cultures for transformed cells

1. Seven days after initiation, observe each flask for signs of transformation and add 4 mL of fresh medium (IMDM with 20% FBS and 1X penicillin/streptomycin).
   1. Early signs of lymphocyte transformation include cone-shaped cells, cells with spike-like projections and clusters of refractile, healthy-looking cells.
   2. Later signs of transformation include increased turbidity of the culture and acidification of the culture medium, indicated by yellowing.
2. Every 3-4 days thereafter, examine cultures for signs of transformation and add fresh medium.
   1. Carefully withdraw 3-4 mL of medium without removing cells.
   2. Add back the same volume of complete culture medium (IMDM with 20% FBS and 1X penicillin/streptomycin).

Expand transformed cultures to establish lymphoblast cell lines

1. Transfer cells from a densely populated T-25 flask to a T-75 flask and add sufficient IMDM with 10% FBS and penicillin/streptomycin to obtain a cell density of 1-3 × 10⁵ cells per mL.
2. Maintain cell density in the T-75 flask at 1-3 × 10⁵ cells per mL by addition of culture medium. A maximum density of 1-2 × 10⁶ is achievable and maintainable.
3. Expand to additional flasks to maintain cell density at 1-3 × 10⁵ cells per mL.

Freeze transformed cells at 3-5 × 10⁶ cells per mL for preservation and later use

1. Pool the cultures that are to be frozen. Complete a viable cell count to determine the density and viability of the cells in the suspension and the total number of cells reserved to be frozen.
2. Collect cells from culture fluids by centrifugation at 260 × g for 15 minutes.
3. Discard the supernatant and resuspend the cell pellet in a volume of chilled freezing medium (IMDM with 10% FBS, penicillin/ streptomycin, and 10% DMSO) that will give a cell density of 3-5 × 10⁶ cells per mL.
4. Dispense the resulting cell suspension into labeled cryovials.
5. Freeze vials in a cryopreservation container in ethyl alcohol at -80°C for 3-18 hours or in a controlled rate freezer at 1°C per minute and then store them in liquid nitrogen.

References

1. Kieff ED, Rickinson AB. Epstein-Barr Virus and its Replication. In Knipe DM and Howley PM ed. Fields Virology, Fifth Edition, Philadelphia, PA: Lippincott, Williams & Wilkins; 2007:2603- 2654.
2. Rickinson AB, Kieff E. Epstein-Barr Virus. In Knipe DM and Howley PM ed. Fields Virology, Fifth Edition, Philadelphia, PA: Lippincott, Williams & Wilkins; 2007:2655-2700.
3. Miller G, et al. Epstein-Barr virus: Transformation, cytopathic changes, and viral antigens in squirrel monkey and marmoset leukocytes. Proc. Natl. Acad. Sci. USA 69:383-387, 1972.
4. Miller G, Lipman M. Release of infectious Epstein-Barr virus by transformed marmoset leukocytes. Proc. Nat. Acad. Sci. USA 70:190-194, 1973.
5. Caputo JL, et al. An effective method for establishing human B lymphoblastic cell lines using Epstein-Barr virus. J. Tiss. Cult. Meth. 13:39-44, 1991.
6. Bird AG, et al. Cyclosporin A promotes spontaneous outgrowth in vitro of Epstein-Barr virus-induced B-cell lines. Nature 289:300-301, 1981.
7. Penno MB, et al. Cryopreservation of whole blood and isolated lymphocytes for B-cell immortalization. J. Tiss. Cult. Meth. 15:43-48, 1993.
8. Ling PD, and Huls HM. Isolation and Immortalization of Lymphocytes. Current Protocols in Molecular Biology. Supplement 7 28.2.1-28.2.5, 2005.
9. Amoli, et al. EBV Immortalization of human B lymphocytes separated from small volumes of cryo-preserved whole blood. Int. J. of Epidemiology 37:i41–i45, 2008.
10. Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. U.S. Government Printing Office, Washington, D. C., 2007.
11. Sun R, et al. Transmissible retrovirus in Epstein-Barr virus-producer B95-8 cells. Virology 209(2):374-83, 1995.