Jul 24, 2020

Public workspaceLentiviral_vector_production_with_PEI

This protocol is a draft, published without a DOI.
  • 1Case Western Reserve University School of Medicine
  • MatreyekLab
Kenneth Matreyek
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Protocol Citation: Kenneth Matreyek, Anna Bruchez 2020. Lentiviral_vector_production_with_PEI. protocols.io https://protocols.io/view/lentiviral-vector-production-with-pei-bhmyj47w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 18, 2020
Last Modified: July 24, 2020
Protocol Integer ID: 38296
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Materials
MATERIALS
ReagentPEI MAX - Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40000)Fisher ScientificCatalog #NC1038561

Preparation
Preparation
1d
1d
This protocol will require transfecting HEK 293T cells. Make sure you have some 293T cells thawed and growing for at least a couple of days before doing this protocol. Furthermore, prepare stocks of PEI and Diluent if you have not done so already.
PEI solution: Concentration1 µg/µL PEI in H2O. I have used "Polysciences, Inc.Supplier Diversity Partner PEI MAX - Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000)"

  1. Dissolve PEI in endotoxin-free dH2O that has been heated to approximately Temperature80 °C .
  2. Let cool to room temperature.
  3. Adjust pH until it reaches Ph7 , filter sterilize (0.22 uM filter), aliquot, and store at Temperature-20 °C . A working stock can be kept at Temperature4 °C .
Diluent solution: Concentration10 millimolar (mM) HEPES , Concentration150 millimolar (mM) NaCl Ph7.05

For Amount500 mL :
  1. Get Amount445 mL distilled water
  2. Add Amount5 mL HEPES
  3. Add Amount15 mL Concentration5 Molarity (M) NaCl
  4. Filter sterilize (0.22 uM) filter. Surround in foil to protect from light. Store at room temperature.
Transfection
Transfection
20m
20m
Prepare the mixture in a sterile microcentrifuge tube.

If transfecting a 10cm plate (eg. when trying to make larger stocks of lentiviral vectors):

  1. Aim for a total volume of Diluent + DNA of Amount500 µL
  2. Add Amount10.5 µg total DNA . When using VSV-G, I do a 5:5:1 ratio packaging plasmid, transfer vector, and pMD-VSVG.
  3. Add Amount42 µL PEI to the diluted DNA mixture. Immediately pipet up and down and / or vortex.
  4. Incubate between Duration00:10:00 (minutes) and Duration00:15:00 (minutes). Don't go over Duration00:20:00 (minutes) .
  5. Trypsinize, count, and plate 10 million cells per 10cm plate, in a total volume of Amount10 mL .
  6. After the incubation is over, add the DNA + PEI mixture dropwise to the cells, and return to the incubator.


If transfecting an individual 6-well (eg. when trying to test out some lentiviral vectors):

  1. Aim for a total volume of Diluent + DNA of Amount80 µL
  2. Add Amount1.8 µg total DNA . When using VSV-G, I do a 5:5:1 ratio packaging plasmid, transfer vector, and pMD-VSVG.
  3. Add Amount7 µL PEI to the diluted DNA mixture. Immediately pipet up and down and / or vortex.
  4. Incubate between Duration00:10:00 (minutes) and Duration00:15:00 (minutes). Don't go over Duration00:20:00 (minutes) .
  5. Trypsinize, count, and plate 1.5 million cells per 6-well, in a total volume of Amount2 mL .
  6. After the incubation is over, add the DNA + PEI mixture dropwise to the cells, and return to the incubator.
Change media
Change media
1d
1d
The next day (between 12 and 24 hours), replace with fresh media. To increase lentivector concentration of the supernatants, use Amount6 mL for 10cm plates and Amount1.5 mL for 6-wells.

Collect the supernatants
Collect the supernatants
3d
3d
Collect media over the next four days and store pooled supernatant at Temperature4 °C . If using a viral envelope protein that is able to enter HEK 293T cells (eg. VSV-G or Ebola virus GP), it may be wise to collect multiple times in one day (Once when entering the lab, and one when going home), as the viral particles will be able to re-enter the producer cells essentially becoming a dead-end product. This may not be as important for viral envelopes with receptors that are not normally expressed in 293T cells (eg. SARS CoV), although its possible that virus stored at Temperature4 °C will still be more stable than virus left at Temperature37 °C .

Prepare the supernatants for use
Prepare the supernatants for use
30m
30m
The supernatants may still have detached 293T cells floating which may "contaminate" downstream cultures.
  1. Centrifuge the supernatant at greater than Centrifigation300 x g, 4°C, 00:03:00 .
  2. Filter the supernatant through a 0.45um filter.
  3. Aliquot and store the lentivector supernatant at Temperature-80 °C .