Hi Syed,

As far as I am concern I have found the same when I was developing the production of lentiviral particles. Normally people who concentrate the virusses it's because they collect the viral particles in a DMEM media form the 293t plates and their cells have another growth media that it's not compatible with DMEM. Also if you concentrate and you me a title of the nº of viral particles that you have per/ml or /ul you can recreate the assay as many times as you want. As I told you this is the information that I have regarding my short experience with this procedure. Have the best luck with it.

Dear Francisco, 

Thanks for your input, may be you are right but actually my plasmids (expression/packaging) are of more than 10.5 kb so I am interested to know since plasmids are big in size,so virus assembly may have not enough in number, thus in that way do concentration helps in assembly?

Thanks,  Syed

Hi Syed,

there are few reasons and a couple of methods to concentrate a lentiviral preparation, anyway concentration will only gather the viral particles decreasing the final volume of the preparation. Therefore the result is an increased tfu/ml diminishing the volume, nevertheless the lentivirus quality depends exclusively by the 293 trasfection, by concentration you could only increase tfu/ml and purify the virus if you need to work more clenly or if you'll utilize in vivo. And you can utilize this technique by ultracentrifugation or by commercial virus concentrator based in PEG (Clontech and other), I suggest you to visit Trono web site in regards.

I hope this will help you.

Syed, the plasmid that I am using are even 15Kb and my lentiviral transduction is working ok. You know that there is a limit between LTR that should not exceed 14kb as the maximum. The bigger your plasmid is, the lower your transduction efficiency will be.

Dear Cesaer & Francisco, 

Many thanks for your kind replies. Actually I am trying to transduce Neural Progenitor Cells, and unfortunately I am getting very less transduction (picture attached). Here the Red one is my mCherry lentivirus construct, my NPCs are already GFP labelled (green). You can see here, how less mCherry positive cells I have in this case.

My expression vectors are 11.5 kb (CRISPR) and 14 kb (mCherry-control). Although yet I have not titred the viral stock, which I am going to do tomorrow. Also I am planing to transduce the NPC with concentrated stock. May be concentration can help me in getting better result + a friend suggested to centrifuge the NPCs as soon as I transduce the cells for 90 mints at 2500 rpm, + incubate the cells at 32C for 24 hrs. in next experiment  I am planing to take these additional steps in consideration. Please if you guys know something regarding NPCs transduction kindly suggest. 

many thanks

Syed

Few more pictures: @ https://www.researchgate.net/post/Facing_difficulties_in_Neural_Progenitor_Stem_Cells_transduction_with_Lentivirus

Hi Syed, unfortunately NPC are not my field so I can not help within. Please try to contact the corresponding author (Macia A,) in this paper http://www.ncbi.nlm.nih.gov/pubmed/21041477 

I know she has been working with NPC for a long time and she might help, she is a kind person.

Also, it has been described that the use of Polybrene during the transduction time could increase the infection rate, you may wanna try this (for my cells it really works). I have used 2 different protocols:

1- Grow my cells to a 60-70% confluence and then change the growth media by the media containing the viral particle for at least 4-6 hours and then replace it again with growth media (adding polybrene 2ug/ml)

2- Detach the cells from the plate, spin them down 5' 12000 rpm and then resuspend it in the viral media with polybrene and plate it again.

The second protocol has worked better for me and my cell type.

Best luck

Hi Fransisco, many thanks for your quick reply. I am too using Polybrene 6ug/ml. But I do like this:

1. Day 1 seeding of cells

2. Day 2 transduction with lenti with Polybrene, for 24 hrs.

3. Post 24 hrs transduction replace media with fresh DMEM and wait for 3 days for genes to become mature. Then lurk selection with 0.5 ug/ml.

Hi Syed,

I normaly use 10mg/ml Polybrene, freshly defrost, normally I after adding virus I centrifuge 90min at 1.800rpm at 32°C and leave the ingection fro 48hr  at 37°C. For sure increasing tfu/ml concentrating the lentivirus would help youn to increase trasduction efficiency.

Good luck

   Cesare

Hi Cesarean, 

Many thanks for your reply, actually I have serious concern regarding NSCs, can I put those in 32C. Yesterday I read 2 articles not specific about the stem cells but they mentioned some cell loses viability/survival and also some time the change in property is observed, and since mines is stem cell I am thinking of to drop temperature change plan but to with centrifugation. 

Many thanks

syed

Hi Syed,

often I transduced cells without centrifugation, just adding 500ml of medium containing virus and than leaving the cells 48hr in incubator, this could you help collecting more healthy cells.

 Cesare

Hi Cesare, 

Just I wish to ask one important thing don't you add any fresh media in 24 well but just 500ml of Virus Containing collected Supernatant? Many thanks

Meanwhile, I did a control experiment to see the transduction efficacy of my mCherry Lentivirus (as a control) in 293 Cells, and can say you it is almost 90-95% transduce. In this case I concentrated the virus, but no 32C incubation (for stabilization of LV particles), and no centrifugation. Now will start the same mCherry control Experiment in NPCs today, but thinking off to do centrifugation step additionally (but the same virus, without 32C stabilization, and will use 6ug/ml polybrene. In the 293 study i used 2ug/ml polybrene). May it will enhance transduction in NPCs as they are hard to transduce. 

Please kind find the attachment of 293 mCherry lentivirus transduce cells.

Looking Forward,

Syed

Lentivirus is concentrated mainly for two reasons:

1. Where volume needs to be minimal, for example in animal studies. You can't go injecting mLs of virus into a mouse!

2. Non-concentrated virus is usually produced by HEK 293s, which require 10% serum in their media. So when you use crude LV preps, you're adding serum to the target cells. I'm not a neurobiologist, but I think in some cases serum can encourage stem cells to differentiate.

I've transduced NSCs before with lentivirus and they infected fine. Use polybrene with caution - it can cause some cell types to stop growing.

Dear Muhling Many thanks for your kind reply, With concentrated virus I have already got the result in  my NSCs. 

Thanks Again

Syed