TITLE: Lentivirus Production
AUTHORS: Anita Adami

[DESCRIPTION]
This protocol is about the production of lentivirus.

[STEPS]
SECTION: PEI stock solution preperation
1. Dissolve Polyethyleneimine [Polysciences PN 23966] by swirling using de-ionized H2O heated to 80 °C  and using  ~90% of volume required to reach a final concentration of 1 1501 [1 μg/μl].
SECTION: PEI stock solution preperation
2. Cool to Room temperature.

SECTION: PEI stock solution preperation
3. Set to pH 7.0 by adding HCl.

SECTION: PEI stock solution preperation
4. Add de-ionized H2O to reach final concentration of 1 µg/µL.

SECTION: PEI stock solution preperation
5. Filter using a 0.22 μm membrane and divide into aliquots. Store in -20 °C freezer.

SECTION: PEI stock solution preperation
6. Thaw in 37 °C water bath until precipitates have fully dissolved.

SECTION: PEI stock solution preperation
7. Store thawed aliquot in fridge [4 °C].

SECTION: PEI stock solution preperation
8. Re-dissolve precipitates by incubating at 37 °C before use if needed.

SECTION: Lentivirus production [Day -4 or -3]
9. Make media, warm in water bath.
SECTION: Lentivirus production [Day -4 or -3]
10. Seed cells on Thursday [0.9 x 106] or Friday [1.8 x 106] before [25 ml media]. This will yield approx. 20 million cells per T175. Seed 8-9 flasks for a 6 virus batch.
SECTION: Lentivirus production [Day -4 or -3]
11. Wash with 10 mL  [on the roof, then submerge carefully].

SECTION: Lentivirus production [Day -4 or -3]
12. 2 mL, 5 min in incubator, hit on the side.

SECTION: Lentivirus production [Day -4 or -3]
13. 10 mL, spin 400 x g, 5 min .

SECTION: Lentivirus production [Day -4 or -3]
14. Resuspend in 5 mL, count, [80 ul PBS + 10 ul cell susp, i.e. 1:10 dil].

SECTION: Lentivirus production [Day -4 or -3]
15. Add the right number of cells to each flask [see above], plus 10 mL - 20 mL .

SECTION: Lentivirus production [Day 0: Seeding of cells]
16. 
 

SECTION: Lentivirus production [Day 0: Seeding of cells]
17. Warm media in water bath.
SECTION: Lentivirus production [Day 0: Seeding of cells]
18. Wash with 10 mL [on the roof, then submerge carefully].

SECTION: Lentivirus production [Day 0: Seeding of cells]
19. 2 mL, 5 min in incubator, hit on the side.
SECTION: Lentivirus production [Day 0: Seeding of cells]
20. Add 5 mL - 7 mL to each flask to neutralize trypsin.

SECTION: Lentivirus production [Day 0: Seeding of cells]
21. Rinse flasks and transfer cells to a [or two] 50 ml tube.
SECTION: Lentivirus production [Day 0: Seeding of cells]
22. Spin down at 400 x g, 5 min, 20 °C .
 


SECTION: Lentivirus production [Day 0: Seeding of cells]
23. Resuspend cells in appropriate volumne [about 20 mL].

SECTION: Lentivirus production [Day 0: Seeding of cells]
24. Count.
SECTION: Lentivirus production [Day 0: Seeding of cells]
25. Add 20 mL to each flask [DMEM Glutamax + 10% FBS + Penicillin 100 U/ml + Streptomycin 100 U/ml].

SECTION: Lentivirus production [Day 0: Seeding of cells]
26. Plate 12.5 1478 per T175 flask. Need 12 for 6 virus batch.

SECTION: Lentivirus production [Day 0: Seeding of cells]
27. Add the calculated cell suspension to the flask with the media.
 

SECTION: Lentivirus production [Day 1: transfection of cells]
28. Check to see how confluent cells are [should preferably be 75% - 90%].
SECTION: Lentivirus production [Day 1: transfection of cells]
29. Make sure you have all calculations done prior to starting, including the volumne of PEI to be used [102 μm - 132 μm].
SECTION: Lentivirus production [Day 1: transfection of cells]
30. Mix packaging vectors [pMDL, psRev and pMD2G] and transfer vector in a 50 ml tube with serum free media [DMEM+P/S] or PBS, so that the total volumne [including PEI] becomes 3.6 ml.
SECTION: Lentivirus production [Day 1: transfection of cells]
31. Add PEI and mix by vortexing for a few seconds.
SECTION: Lentivirus production [Day 1: transfection of cells]
32. Incubate the DNA/PEI mix at Room temperature for 15 min .

SECTION: Lentivirus production [Day 1: transfection of cells]
33. Change media in flasks and add 16.2 mL .

SECTION: Lentivirus production [Day 1: transfection of cells]
34. Add 1.8 mL to each T175 flask, mix gently over cells by tilting the flask.

SECTION: Lentivirus production [Day 3: Harvest]
35. 
 

 

SECTION: Lentivirus production [Day 3: Harvest]
36. Check that they are alive and if they have the right flurophore [if applicable].
SECTION: Lentivirus production [Day 3: Harvest]
37. Collect media from the two flasks for each virus into one 50 ml tubes.
SECTION: Lentivirus production [Day 3: Harvest]
38. Spin the supernatant at 800 x g, 10 min, 4 °C .

SECTION: Lentivirus production [Day 3: Harvest]
39. While tubes are in the centrifuge, prepare in hood the requirements for the next step. Also, rinse the now empty culture flasks with Virkon and discard.
 

SECTION: Lentivirus production [Day 3: Harvest]
40. Remove the 50 ml tubes from centrifuge, and in the hood, pour the supernatant into open syringe with attached filter over the labelled Beckman UC tube. After each virus/50 ml tube, change the syringe and filter. Ensuring that the discarded syringe and filter are placed and rinsed in Virkon.
SECTION: Lentivirus production [Day 3: Harvest]
41. Put Beckman UC tubes into their chambers using forceps.
SECTION: Lentivirus production [Day 3: Harvest]
42. To avoid collapsing the tubes, fill up sample tubes with media. Fill up counterweight tubes with DPBS.
SECTION: Lentivirus production [Day 3: Harvest]
43. Balance the weight of the tubes carefully, down to 5 ug difference.
SECTION: Lentivirus production [Day 3: Harvest]
44. Clean everything in the hood thoroughly. Any plastics that have been used must be rinsed with Virkon before discarding. Spray pipettes, racks, pipette gun etc. with ethanol. Clean hood surfaces with Virkon first, then with ethanol.
SECTION: Lentivirus production [Day 3: Harvest]
45. Turn off the hood fully and then put the UV on.
SECTION: Ultracentrifuge
46. Put down rotor, spin it.
 

SECTION: Ultracentrifuge
47. Put down all 6 chambers into centrifuge [you do not have to weigh the empty ones].
SECTION: Ultracentrifuge
48. Check settings of centrifuge:

- 19500 x g, 120 min, 4 °C 
- Enter to save
- Ensure Acceleration is set to max
- Ensure Deceleration is set to max

SECTION: Ultracentrifuge
49. Wait until centrifuge reaches full speed before leaving.
SECTION: Ultracentrifuge
50. Set alarm for when centrifugation is done, don´t let the tubes stand longer.
SECTION: Ultracentrifuge
51. Deceleration of centrifuge takes approx. 5 min. After it reaches 3000G, you can release the vacuum to increase the speed of deceleration.

SECTION: Ultracentrifuge
52. Take out Beckman UC tubes using forceps, and pour out liquid into Virkon.
SECTION: Ultracentrifuge
53. Place tuned upside down on paper to dry, be careful to remove all liquid, also from edges and walls.
SECTION: Ultracentrifuge
54. Take a bottle of cold PBS from freezer and resuspend pellet in 80 µL [75 ul - 100 ul is ok]
SECTION: Ultracentrifuge
55. Seal tubes with Parafilm, vortex and put into fridge, leave them there for at least 120 min - but preferably  .

SECTION: Ultracentrifuge
56. Aliquot virus [10 ul/tube] and store -80 °C.

SECTION: Ultracentrifuge
57. Viruses can now be titrated and/or used.