Method: Maintenance of B95-8 Cell Line and Obtaining Virus for Lymphocyte Transformation

Rosalie Veile

Principle:

The B95-8 cell line was initiated by exposing marmoset blood leukocytes to Epstein-Barr virus (EBV) extracted from a human leukocyte line. B95-8 is a continuous line and releases high titres of transforming EBV. Thus it provides a source of EBV to establish continuous lymphocytic cell lines from human donors.

Safety considerations:

B95-8 must be handled with precautions since EBV can infect primates. A Biological Safety cabinet must be used when passaging the culture and harvesting the virus. Use bleach to kill unused virus. All material that comes in contact with virus must be autoclaved. In addition, the door of the room should remain closed to prevent outside contaminants from entering the room and to prevent any harmful viruses from leaving the area. Gloves should always be worn in dealing with any human or hybrid cell line because latent virus genomes can be present.

Special Reagents:

The B95-8 cell line is kept in culture in the lab; it was ordered from the American Type Culture Collection, Cat. No. ATCC CRL 1612.

Time required:

5 minutes twice a week to feed and split the culture to maintain the correct cell density of 1.0-2.0 X 106 cells/ml.

Procedure:

1. B95-8 should be grown in growth media (RPMI-1640 + 16% fetal bovine serum). The culture should be passaged twice a week: on Mondays and Thursdays, or on Tuesdays and Fridays. Passaging (subculturing) cells denotes the transplantation of cells from one culture vessel to another. Aspirate half of old media and replace it with an equal volume of new media.
2. To maintain a culture at a density of around 1 X 106 cells/ml it is necessary to split it1:4 once a week. For example: to a culture with a cell density greater than 1.5 X 106 cells/ml, one fourth is diluted with 3 parts growth media (10 ml cells+ 30 ml media). Save the old flask as a backup in case the new culture becomes contaminated. When the subculture is passaged the next time, dispose of the old flask.
3. Media containing fresh virus can be prepared at the same time the culture is passaged: Using a 10 ml or 25 ml disposable pipette, remove and transfer the media (above the cells) to a 50 ml centrifuge tube. Always be careful not to pull up any cells at bottom of the culture flask.Reserve 25 ml of media in the flask and add a equal amount of new growth media to maintain the culture.
4. Centrifuge the tube with the media-containing-virus for 10 minutes at 1200 rpm (no brake) at room temperature, using the TJ-6 centrifuge. Centrifuging the media will pellet any marmoset cells to the bottom of the centrifuge tube.
5. With a 10 ml pipette, transfer all but the bottom 10 ml of virus in the centrifuge tube to a 150 ml 0.22 µm cellulose acetate filter. Filter and store the virus at 4 degrees C for up to 7 days.

• Growth media:

  To 1 liter of sterile RPMI-1640, add:
  165.0 ml fetal bovine serum
  1.2 ml gentamicin reagent
  12.0 ml L-glutamine
   
  Filter sterilize and store at 4 degrees C, for up to 2
  weeks.

References:

CRI Methods Manual: RFLP Project 1989.