Dear Maayan,

Storage of Lentivirus: Virus can be stored at 4°C for a short time (less than a week) before using after reception. Since Lentiviruses are sensitive to freeze-thawing and the titer drops with repeated freeze-thawing, aliquot viral stock should be stored at -80°C freezer immediately upon arrival for long-term usage. While virus titer redetection is suggested before using if the lentiviruses have been stored for more than 12 months.

Dilution of Lentivirus: Dissolve virus in ice water if virus dilution is required. After dissolving, mix the virus with medium, sterile PBS or normal saline solution, keeping at 4°C (using within a week).

Precautions:

· Avoid lentivirus exposure to environmental extremes (pH, chelating agents like EDTA, temperature, organic solvents, protein denaturants, strong detergents, etc.)

· Avoid introducing air into the lentivirus samples during vortex, blowing bubbles or similar operations, which may result in protein denaturation.

· Avoid repeated freezing and thawing.

· Avoid exposing to “regular” plastics (especially polystyrene or hydrophobic plastics) for prolonged periods in liquid phase. Most lentivirus viruses are very sticky and loss can occur if exposed to regular plastics, including tubes, cell culture plates, pipette tips, if not frozen. It is best to store lentivirus in siliconized or low protein binding tubes. Pluronic F-68 used at 0.01%-0.1% in the formulation buffer will minimize sticking if regular plastics are used.

· Avoid diluting lentivirus into low salt solution. Some lentiviruses aggregate in low salt solution, which will be non-infectious.

Genemedi got a rich experience in lentivirus production and construction of stable cell lines, you could find more information on https://www.genemedi.net/i/lentivirus-packaging

Hope this may help you.

Hey,

Yes, I always freeze lentiviruses. Normally I don't concentrate, I just harvest the virus, throw it a screwcap tube and put it in the minus 80. No snap freeze, no additional additives. I always make virus in small scale (24 well), so this is quite an easy setup. The added bonus here is, that I don't have to filter the medium to remove 293T cells that may float around in the sup: these get killed by the freeze-thaw cycle (if you infect straight after virus production, it is best to filter to remove these cells).

Lentivirus is not that sensitive to freeze-thawing. In a previous lab they tested out how much activity was lost upon freeze-thawing. This was about 5-10% per cycle. I think it's closer to 5%.

cheers

Hi, I freeze them too, but I concentrate them first. I centrifuge the harvested medium from 293T cellsand resuspend the pellet in small amount of PBS1X (not more than 150 microlitres, starting from 6 wells of 10mm diameter), then do small aliquotes (5-10 uL) and save them at -80, in order not to freeze-thaw them more than one time, because some collegues of mine noticed that they were sensitive to that. You can use it also after long time, but consider that after one year for instance the titer may go down a little bit, so, in that case,  I recommend to re-titer it before using it.

hope it helped, cheers

Hi, I freeze them too.  You should feeze them at the time when you want to infect cells.  I filter my media do get rid of any cell debris and floated cells with a 0.45um syringe filter, put the media into a screw cap tube and just directly put into -80 freezer. 

The step of concentrating depends very much on the construct you are trying to insert into the targeted cell's genome.  From my understanding, constructs that have a size 7kbps, concentrate the virus.

Good luck

Our lab also freezes lenti at -80, and as another option for concentrating, we use centrifugal filters to reduce the volume without going all the way to a pellet, such as Amicon Ultra filters.

You can freeze lentiviral vectors, as straight supernatant from producer cells or after any method of concentration.  Freeze rapidly (some recommend snap freezing in liquid N2 or dry ice / ethanol baths) and store at -80C.

Freeze-thaw cycles definitely can affect your titre, especially of concentrated vector where we have seen the titre drop by up to 50% (so we always titrate vector after freezing first).  It is important to consider your envelope too; VSV-G is relatively stable and so you can concentrate by ultracentrifugation and you can freeze it.  Others are not so stable and will fall apart on spinning and a freeze thaw will have worse effects.

We usually concentrate vector as we need high MOIs for many of our experiments, and as freeze-thawing more than once ruins out titres, we mainly freeze at -80 in small aliquots and use each tube just once.  Just a word of caution; a colleague made lots and lots of 5ul aliquots and found his titres kept falling. In the end he realised that because his samples were at the front of the freezer, every time people opened the door of the freezer, his small aliquots would start to defrost, and this happened repeatedly, killing his vector.

For short term storage, we find less of a loss in titre by keeping the vector at 4degrees (fridge) for 3 or 4 days, rather than freezing and thawing, but for longer storage definitely store at -80.  

In  my experience, viruses were still infectious as long as four weeks at 4 degrees. Titer might have gone down but for making stable cells with antibiotic selection this was more than satisfactory. 

Dear Maayan,

Storage of Lentivirus: Virus can be stored at 4°C for a short time (less than a week) before using after reception. Since Lentiviruses are sensitive to freeze-thawing and the titer drops with repeated freeze-thawing, aliquot viral stock should be stored at -80°C freezer immediately upon arrival for long-term usage. While virus titer redetection is suggested before using if the lentiviruses have been stored for more than 12 months.

Dilution of Lentivirus: Dissolve virus in ice water if virus dilution is required. After dissolving, mix the virus with medium, sterile PBS or normal saline solution, keeping at 4°C (using within a week).

Precautions:

· Avoid lentivirus exposure to environmental extremes (pH, chelating agents like EDTA, temperature, organic solvents, protein denaturants, strong detergents, etc.)

· Avoid introducing air into the lentivirus samples during vortex, blowing bubbles or similar operations, which may result in protein denaturation.

· Avoid repeated freezing and thawing.

· Avoid exposing to “regular” plastics (especially polystyrene or hydrophobic plastics) for prolonged periods in liquid phase. Most lentivirus viruses are very sticky and loss can occur if exposed to regular plastics, including tubes, cell culture plates, pipette tips, if not frozen. It is best to store lentivirus in siliconized or low protein binding tubes. Pluronic F-68 used at 0.01%-0.1% in the formulation buffer will minimize sticking if regular plastics are used.

· Avoid diluting lentivirus into low salt solution. Some lentiviruses aggregate in low salt solution, which will be non-infectious.

Genemedi got a rich experience in lentivirus production and construction of stable cell lines, you could find more information on https://www.genemedi.net/i/lentivirus-packaging

Hope this may help you.