I am using a protocol similar to yours. Currently for bovine PBMC but also for human PBMC and dendritic cells (for clinical trials) . Cells like DC -  and I assume HPSCs - will always be more prone to damage. I found the following crucial to keep losses at a minimum :

- keep everthing cool,  i. e.  precool yourisoprop container,  cell suspension,  FBS,  FBS/DMSO and the cryovials to 4°C

- put 80:20 FBS:DMSO in the vials (precool again) then add same volume of cells in cold FBS to minimize time for cells in DMSO. 

- transfer to isoprop container and that to -80°C immediately. We don't even fill the entire container but only up yo 10 tubes to be faster. 

- I believe that a lot of damage is done during thawing - with faster thawing being better.  We prepare a tube with 37°C medium,  thaw the vials with frozen cells in a 37°C waterbath and as soon as the ice block inside moves chuck it all into the prepared medium. 

Good luck. If you try it,  let me know if it worked.

Cheers, 

Axel

I agree with Axel, especially the last point - faster thawing - are very important.

good luck

Jochem

Thanks Axel and Jochem for sharing your protocol and advice

we have long experience freezing PBMCs and had no problems at  all. But, with cord blood,bone marrow and also DCs as I also worked with them, the recovery as I mentioned earlier not high and a lot of clumbs due to dead cells.

I wil try your protocol Axel and I will let you know .

Thanks again and bests regards,

Ameera

Using 7.5% DMSO will give less dead cells and less clumbs.

90% FBS and 10% DMSO do not perform as well as Cryostor CS10 in our hands.  Also, be sure to heat inactivate your FBS (this is obvious, but just trying to help).  We do all the same things as mentioned above to reduce cell loss.  Also, try freezing in 0.5 ml rather than 1 ml.  When thawed, gently mix in the warm medium.  Spin cells for 10 minutes at 100 x g. 

https://www.researchgate.net/publication/34064797_An_evaluation_of_short_term_storage_conditions_and_cryopreservation_media_for_human_umbilical_cord_blood_

Article An evaluation of short term storage conditions and cryoprese...

Thanks Paula, James and Jean for your help and valuable comments and links. I will be back and let you know of my experience in due time.

Dear Jean, can you help me to download the refereed documents as I couldn't ! 

Hi, working with cord blood mononuclear cells, I was also freezing in cold 90% heat inactivated  and filtered FBS+10% DMSO, like you. For thawing, in our hand the best results were obtained with a drop by drop addition of RPMI+20%FBS (at 37C) for the first 5 ml, mixing by gentle circular moves between each addition of medium, then 3 drops at a time untill 10 ml, mixing similarly, then 1 ml and more until 40 ml. It had to be performed pretty rapidly, in less than 10-15 min. Then Centrifugation and 2nd wash before putting them in culture.

Thanks Natacha, I will try your protocol, and let you know if it works

Okay it seems your question is regarding the freezing protocol. Try not to transfer it straight into liquid nitrogen from the -80. Maybe if transferred into ln2 vapour storage instead, better still if you have a controlled rate freezer, use it to slowly bring the sample down about -140 degrees celsius to -180 degrees celsius then transfer to LN2 vapour phase storage. Think the rapid freezing rate from -80 to -196 is killing most of the cells.

And as suggested, rapid thawing of the samples works best in a 37 degree celsius water bath.

Agree with@James A Lederer

Hope you good luck

Greeting