Jul 08, 2025

Public workspaceLentiviral vector production V.1

 Forked from Nanoblade production
This protocol is a draft, published without a DOI.
  • maxime.smits 1,
  • Joris Van Asselberghs1
  • 1KU Leuven
  • center for molecular medicin KU Leuven
Joris Van Asselberghs
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Protocol Citation: maxime.smits , Joris Van Asselberghs 2025. Lentiviral vector production. protocols.io https://protocols.io/view/lentiviral-vector-production-g4zdbyx27
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: In development
We are still developing and optimizing this protocol
Created: July 08, 2025
Last Modified: July 08, 2025
Protocol Integer ID: 221957
Keywords: Nanoblades, Virus-like particles, lentiviral vector production lentiviral vector, lentivirus, type of retrovirus, retrovirus, gene delivery into cell, genetic material into the host cell, gene delivery, term transgene expression, host cell, genetic material, gene,
Abstract
Lentiviral vectors are derived from lentiviruses, a type of retrovirus, and are widely used for gene delivery into cells. They are particularly valuable because of their ability to transduce both dividing and non-dividing cells, and their capacity to integrate their genetic material into the host cell's genome, leading to stable and long-term transgene expression. 
Guidelines
Production of lentiviral vectors are being performed in a Bio Safety Lab 2 (BSL-2 lab)

BSL-2: Here, it is permitted to work with organisms that cause diseases. However, the lab is only accessible to people who work there and know the procedures. Doors are always closed during work and windows cannot be opened. The entire area is set up for efficient cleaning and disinfection. All waste is disinfected.
Materials
Materials

  • Sterile 1,5 mL Eppendorf tubes
  • Sterile 50 mL Falcon tubes
  • Petri-dishes (PD)
  • Pipetboy
  • Sterile pipets (5 mL, 25 mL)
  • Micro-pipets
  • Sterile Pipet tips (100 µL, 1000 µL)
  • 0,45 µM filter
  • Sterile syringes
  • Sterile vivaspin membranes
  • spectrophotometer/Nanodrop
  • Incubator
  • Microscope

Reagents

  • DMEM medium 2% FCS
  • OptiMEM
  • Gentamycin
  • Linear PEI
  • PBS
  • Plasmids (transfer, envelop, packaging)
  • HEK293
Troubleshooting
Safety warnings
  • Be careful not to mix bleach with alcohol-derived reagents. The key ingredient in household bleach is sodium hypochlorite. Sodium hypochlorite reacts with ethanol, isopropyl alcohol, and other types of alcohol to make chloroform (CHCl3), hydrochloric acid (HCl), and other compounds, such as dichloroacetate or chloroacetone.

Before start
  • Desinfect the flow with 70 % Ethanol before starting any work there.
  • Always wear two sets of gloves and a sterile lab coat.
  • Desinfect bottles and tip boxes with 70 % Ethanol, before entering the flow and after the work is finished.
  • Desinfect gloves regularly with 70 % Ethanol.
  • Waste is collected in a beaker filled with bleach.
DAY 0
SEEDING Petri-dishes (PD)

Seed HEK293T cells in a 10 cm PD in DMEM 2% FCS + 500 µL Gentamycin
Incubate on 37 °C at 5% CO2
DAY 1
TRANSFECTION of transfer, packaging, and envelop plasmids
Before starting, measure the DNA concentration and the purity of the plasmids in the molecular lab.
Make a DNA mixture in an 1,5 mL tube of the following plasmids:




ABC
1 PD5 PD
transferplasmid12,74 µG63,7 µG
packaging plasmid6,36 µG31,8 µG
evenveloppe plasmid3,18 µG15,9 µG




Write down the total volume of the DNA mixture
Transfer the DNA mixture in a 50 mL Falcon tube in the vector lab, under the flow
Add autoclaved PBS to the mixture with a final volume of 559,6 µL (1 PD) or 2798 µL (5 PD)
Dilute the linear PEI (323 mg/L) with autoclaved PBS:


ABC
1 PD5 PD
Linear PEI245,6 µL1228 µL
PBS313,6 µL1568 µL

Add the diluted linear PEI to the DNA mixtures GENTLY, and incubate for 5 to 10 minutes on RT
Add 5 mL (1 PD) or 25 mL (5 PD) of cold medium (DMEM 2% FCS + 500 µL Gentamycin or OptiMem + 500 µL Gentamycin) to the DNA-PEI mixtures GENTLY and mix
Check the seeded HEK293T cells under the microscope for confluency
Remove the medium on the HEK293T cells and add the transfection medium. Normally there is enough for 6 mL of medium per PD
Incubate on 37 °C at 5% CO2
Leave the medium flask at RT
DAY 2
REMOVING TRANSFECTION MEDIUM
Remove the Transfection medium and replace it with RT medium. 5 mL per PD should be sufficient
Incubate on 37 °C at 5% CO2
Also store the medium flask at 37 °C
DAY 3
1st HARVEST
Collect the supernatant from the PD using a 0,45 µM syringe and store at 4 °C
Add Pre-warmed medium to the cells.
Incubate on 37 °C at 5% CO2
DAY 4
2nd HARVEST
Collect the supernatant from the PD using a 0,45 µM syringe and store at 4 °C
Desinfect the PD by spraying 70% Ethanol or bleach on the cells, before throwing them in the yellow bin
The supernatant of both harvest days will be concentrated via vivaspin

1. First wash the vivaspin membrane by adding 10 mL of PBS and spinning down at 3000g for 3 minutes
2. Add the supernatant to the vivaspin membrane and spin down at 3000g, until there is about 0,5 - 1,0 mL remaining
Make aliquots of the desired size and freeze them in the -80°C ultrafreezer.
IMPORTANT REMARK: do no re-freeze aliquots! With every freeze-thaw cycle you have a  loss of lentiviral vector functionality