Standard Operating Procedure

 

Transformation of human B cells with Epstein Barr Virus

and use of resulting lymphoblastoid cell lines (LCL)

 

Hazards: Epstein Barr Virus (EBV) is a member of the herpesvirus family and is a ubiquitous human pathogen. It is typically transmitted orally, it infects and transforms B cells, causing life long latent infection.  Most adults have been infected with EBV and are immune to it.  However, EBV can cause infectious mononucleosis in adults who have never been exposed to the virus before. Although there is little evidence that infectious aerosols are a significant source of laboratory-acquired infections, it is prudent to avoid the generation of aerosols.

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Protection:

  1. Initial supervision and training is mandatory. Persons working with human samples must take the online training (http://www.dehs.umn.edu/bio_pracprin_blood_bpt.htm) and receive one-on-one training in the lab. 
  2. Immunization against HBV is recommended.  There is currently no immunization for EBV.  
  3. General Precautions:
    1. The EBV cell line and human blood samples should be handled in a laminar flow hood to the greatest extent possible.  Cells must be fixed with paraformaldehyde prior to running on the flow cytometer. 
    2. All hood surfaces and equipment are to be treated with a virocidal agent (Virex in a 1:10 mixture with water).
    3. Expose hood to UV light after use.
  4. Personal Precautions:
    1. Wear gloves all times when in contact with a specimen. Any cuts, sores or other breaks in the skin should be additionally covered with a bandage.
    2. Wear a lab coat to protect clothing and arms. 

 

 

Waste:

  1. Solid Waste (including plastics): All waste will be put in a clear plastic bag (keep next to hood) and autoclaved at the end of the experiment.  In hood, use a small plastic bag for pipette tips.  Autoclave at 121¡ C for 60 minutes.  Then it can be treated as normal waste.  We discourage putting human blood waste into the other autoclavable trash bags in use other places in the lab.
  2. Liquid Waste: Fluids generated from samples should be aspirated into a flask with bleach. (set up in the hood). 
  3. Sharps (glass pipets, slides, etc.) are placed into Biohazard sharps containers located under the hoods.  When full they are sealed and the exposed surface of the container is decontaminated with a virocidal agent before placing the container in a designated area to be picked up for incineration.

 

Spill Clean-Up:

1.     Spills are handled by covering the spill with absorbent material and soaking the area with a virocidal agent, such as Virexª or bleach.  Allow the area to soak for a minimum of 20 minutes, and dispose of the waste material in either an autoclave bag or incineration bag.  Make sure that any sharps in the spill are collected in a pan or container that will prevent injury and can be decontaminated.

2.     Large spills can be absorbed using an absorbent powder provided for such emergencies.  This material is then treated as above.

3.     If your skin is cut or punctured while handling potentially infectious materials:

                        -encourage the wound to bleed

                        -flush with water

                        -apply antiseptic

Then notify your supervisor, and immediately seek medical attention at Boynton Health Service (8:00am-5:00pm M-F, 9am-1pm Sat).  Report the incident to your supervisor as soon as it is possible, and fill out the appropriate paperwork. A State of Minnesota First Report of Incident form (UM1536), a U of MN Employee Incident Report Form (UM1534) and a U of MN Supervisor Incident Investigation Report form (UM1533) should be filled out and faxed to the proper locations within 24 hours.  These forms can be found online at http://www1.umn.edu/ohr/forms/index.html. Electronic copies are on the server in the EBV Folder on Hoquistlab server.

 

 

Procedure:

 

Grow-up B95-8 cell line to produce virus-containing supernatant.

  1. Thaw B95-8 (marmoset cell line infected with human EBV) into RP-10.
  2. Start a 20ml culture with 1x10e6 healthy growing B95-8.  Incubate 3 days.  Check viability (should be >95%).
  3.  Centrifuge 10 minutes at 1200 rpm to spin cells.  Filter supn. Through a 0.45 um filter.  Store in liquid nitrogen. (use up to one year)

Transform human PBMC

  1. Thaw human PBMC, wash, dilute to 5x10e6/ml (about 2 ml) in a 50 ml conical tube.  Add 2.5 culture supernatant from step 1.  Incubate 2 hr in a 37o water bath.
  2. Add 5 ml of RP10 containing 10ug/ml cyclosporin A (if individual was seropositive) or 10ug/ml Phytohemagglutinin (PHA) (if individual was seronegative).  Transfer to a T25 flask.  Stand flask upright in incubator.
  3. After 3 weeks mix cells and transfer 5ml to two new T25 flasks.  Add 5ml RP10 and incubate 1 more week.  Cells can be counted and frozen at this time.
  4. To maintain lymphoblastoid cell line (LCL), split cells 1:3 once a week.

LCL-interferon assay

  1. Thaw LCL and PBMC from the same donor.  Wash 2 times.  Mix PBMC and LCL at 10:1 or 5:1 ratio (2x10e6 PBMC/ml) in RP10.  Incubate for 16 hours.  Add Brefeldin A (5ug/ml) to the cultures for the last 4 hours.
  2. Harvest count and stain for IFNg using fixation/permabilization reagents.
  3. Analyze samples on flow cytometer.  Do not run unfixed samples through flow cytometer!