Amplifying Recombinant adenovirus: Preparation of high titer stocks

 

(The probability of producing replication competent adenovirus (RCA) increases with each successive amplification. For this reason, use early amplification stocks whenever you need to make additional quantities of adenovirus)

  1. Plate HEK293 cells in eight T175 flasks. The cell monolayer should be 70-80% confluent when you infect them. (Approximately, a 70-80% confluent T175 flask contains 17-18 million cells).
  2. Incubate cells for 24-48 hrs in a 37 0C incubator until they become 70-80% confluent.
  3. To infect the cells, replace the medium in each flask with 10 ml of fresh growth medium that contains adenovirus. Infect cells at a multiplicity of infection (MOI) 5-10 (i.e., at 5-10 pfu/cell). For example, if the T175 flask contains ~ 18 x 10 6 cells, add 1.8 x 10 8 pfu adenovirus to get an MOI of 10 pfu/cell. Dilute the virus with media if necessary.
  4. After 5 hrs of incubation at 37 0C, add 5 ml more of the growth medium, to make the final volume 15ml.
  5. Incubate for 3-4 days at 37 0C incubator and check for cytopathic effect.
 

[Infected cells typically remain intact, but round up and may detach from the plate. These changes are collectively called cytopathic effect (CPE). Ideally the CPE should be complete within 72-96 hrs from the time of infection].

  1. When ~80% of the cells have detached, transfer the suspension to four -50 ml
 

tubes. Do not use trypsin. Infected cells still attached to the bottom can be dislodged into the medium by pipeting up and down.

  1. Centrifuge the suspension at 1000 rpm for 5 min at room temp.
  2. Collect the supernatant and store at –20 0C. (This supernatant contains viral particles, but titer may be low). Resuspend the pellet (from all 4 tubes) in a final volume of 10ml.
  3. The cell pellet (in 10 ml of media) is lysed by three consecutive freeze-thaw cycles. Freeze the cells in a dry ice / ethanol bath or liquid nitrogen; thaw cells by placing the tube in a 37 0C water bath. Do not allow the suspension to reach 37 0C. Vortex cells after each thaw.
  4. After the third cycle, centrifuge the cells at 1000 rpm for 5 min to pellet the debris. Discard the pellet and collect the supernatant. Aliquot and keep at –20 0C.
  5. This crude lysate can be used for infecting the target cells. Also this can be further purified using the BD Ad-purification kit according to the manufacturer’s instructions.

Determination of viral titer

  1. Approximately 24 hrs before the beginning of the titration protocol, plate 500,000 HEK cells per well in 6-well plates in 4-ml of regular growth medium. To control for random errors, plate 3-4 wells for each dilution of viral stock to be tested.
  2. Prepare serial dilutions of the virus as follows:
    1. make a 1: 100 dilution by adding 10 ml of viral stock to 990 ml of sterile DPBS (Dulbecco’s PBS with Ca 2+ and Mg 2+).
    2. Starting with a 1: 100 dilution, prepare serial 1:10 dilutions by transferring 100 ml of diluted virus to 900 ml of sterile DPBS. In general, an appropriate range of dilutions for testing is 10 -5 to 10 -10
  3. Remove the culture plates from the incubator and check wells to ensure that cells have attached to form an even monolayer, 70-80 % confluent. Remove the growth medium and add 0.2ml of adenovirus to the center of each well taking care not to dislodge any cells. Spread the virus evenly over the monolayer.
  4. Cover the plates and incubate the cells in a humidified CO 2 incubator for 1 hr to allow the virus to infect the cells.
  5. During the incubation, prepare the overlay medium as follows:
    1. Prepare a 5% agarose solution using Agarplaque-Plus TM Agarose (low melting point agarose, BD PharMingen) in DPBS. Heat the solution in a microwave until the agarose is melted; allowing the solution to reach boiling will help to ensure its sterility. Take care that all the agarose is melted, but do not overheat. Cool to 44 0C in a water bath.
    2. Warm 50-100 ml of HEK 293 growth medium to 44 0C.
    3. Add 45 ml of growth medium to 5 ml 5% agarose and mix well and allow to cool to 37 0C. This makes a 0.5% solution, which is used to overlay the infected cell monolayer to prevent virus progeny from spreading.
  1. Gently add 2-4 ml of 0.5% agarose solution to each well, taking care not to dislodge any cells. Allow plates to sit at room temperature for 20 min.
  2. When the agarose has set, incubate at 37 0C. Plaques should be visible within 7-10 days.
  3. To count the plaques: Prepare a 0.03% solution of neutral red in DPBS. Add 1 ml of the solution to each of the wells and incubate at 37 0C for 2-3 hrs
  4. Remove the stain by aspiration and invert the dishes to allow the plaques to clear. (Neutral red is taken up by healthy cells but not by dead cells. Therefore plaques appear as clear circles against a red or pink background).

Calculation of viral titer

 

Count the number of isolated plaques.

Then use the formula: #plaques = pfu/ml

d x v

d= dilution factor

v=volume of diluted virus added to the well

Sample:

An average of 50 plaques formed in the 1: 10,000 dilution wells

Volume of diluted virus added: 0.2 ml

50 = 2.5x 10 6 pfu/ml

0.0001x 0.2

Storage:

Crude virus: short term storage is at 4 0C ( little breakdown for 2 years)

Long term is at –20 0C. Do not store at –80 0C.

Purified virus: Very short term at 4 0C ( good for < 2 months)

Better aliquot and store at –80 0C. This is good for 10 freeze-thaws and

after that it starts losing particles.

Handling:

Work in TC hood to avoid contamination, take normal TC care when using adenovirus.