If you buy 1 vial of 10 million cells from a supplier, you can recover up to 8 million cells depending on your thawing protocol. Although not recommended, you could try to refreeze them but you need to put at least 4 million cells/vial to have a decent recovery the next time around. Trying to culture PBMCs so you have a substantial yield is also not a good idea as it might affect your experimental objectives/setup (like Andrei S. Babenka mentioned).

Try obtaining a leukopak/whole blood and then isolate PBMCs yourself. Leukopaks can give you >1000 million PBMCs. You could freeze 100s of vials (@10 million/vial) and use them at your pace. The only drawback is that all these cells would be from a single donor. If you would rather have multiple donors, get whole blood samples and a 50-100 mL draw should last you a while (average 1 million cells from 1 mL of whole blood). Many life science suppliers sell these and these options are more cost effective than buying pre-processed PBMCs (unless you are looking for specific PBMC types). PBMC isolation is pretty easy and you can find protocols online.

If you only need a few cells here and there and want to save a few hundred dollars, there is an alternative option. Many University associated laboratories that work with human PBMCs have IRB approvals in place to collect blood from healthy human donors. You could ask if they would share samples with you.

dear Georg it is highly depends on your goals. I usually work with PMBC in gene expression related experiments and freezing/thawing cycles affect the mRNA. The same about growth factors. I use PBS or RPMI40 depending on my conditions and subsequent analisys type.

I try to avoid thawing/freezing cycles.

If you buy 1 vial of 10 million cells from a supplier, you can recover up to 8 million cells depending on your thawing protocol. Although not recommended, you could try to refreeze them but you need to put at least 4 million cells/vial to have a decent recovery the next time around. Trying to culture PBMCs so you have a substantial yield is also not a good idea as it might affect your experimental objectives/setup (like Andrei S. Babenka mentioned).

Try obtaining a leukopak/whole blood and then isolate PBMCs yourself. Leukopaks can give you >1000 million PBMCs. You could freeze 100s of vials (@10 million/vial) and use them at your pace. The only drawback is that all these cells would be from a single donor. If you would rather have multiple donors, get whole blood samples and a 50-100 mL draw should last you a while (average 1 million cells from 1 mL of whole blood). Many life science suppliers sell these and these options are more cost effective than buying pre-processed PBMCs (unless you are looking for specific PBMC types). PBMC isolation is pretty easy and you can find protocols online.

If you only need a few cells here and there and want to save a few hundred dollars, there is an alternative option. Many University associated laboratories that work with human PBMCs have IRB approvals in place to collect blood from healthy human donors. You could ask if they would share samples with you.

Thanks Andrei and Julie for your very clear answer.

Would I be able to isolate PBMC from whole blood samples or Leukopaks? Or is the cell count of RBC / Platelets in Leukopaks so small that separation is easy?

The reason is for asking is, I read that using standard PBMC isolation techniques only work until 24h after blood sample collection.

I.e.: I cannot freeze the whole blood itself but instead I have to process the entire blood at once and freeze individiual cryo-vials of PBMC -- am I understanding this correctly?

PBMCs can be isolated from both leukopak and whole blood. Although not as much as whole blood, leukopaks still have a significant proportion of RBCs. You need to do Ficoll separation to separate PBMCs from RBC/granulocytes. Duke University has an excellent protocol for PBMC isolation, which I have used for both whole blood and Leukopak (linked below, if you can't open it google 'leukopak processing iqa' and it is the ppt file result). Regardless of your starting material, you need to process it within 24 hours of collection (the earlier the better). PBMC viability and functionality decreases with time.

https://iqa.center.duke.edu/sites/iqa.center.../IQA_LPK_ACTG_complete_web.ppt

Thanks again!

Sorry for being slow witted: If I then order Leukopaks, fresh means actually fresh; i.e. less then a day old? How's this even possible if I would order them through mail? Or can I not extract PBMC from Leukopaks if ordered through mail and I need to get them from a local supplier?

No problem. Yes, both whole blood and leukopaks have to be less than 24 hours old.

I used to order leukopaks from StemCell Technologies (link below). This is how it works: I contact their rep and tell them the size I want and any special requirements I have (eg. BMI< a certain number or CMV+ and so on). They have a donor database that they use to identify potential donors.We decide on a date that works for the donor (for blood draw) and works for me (the day after, for PBMC isolation). This requires a lot of back and forth but SCT is great to work with and very reliable. The collection centers are in the US and the cells are sent to me (usually by air) so I have it within 24 hours of the draw.

https://www.stemcell.com/human-peripheral-blood-leuko-pak-fresh.html

I don't know whether SCT has a collection site in Europe but it doesn't hurt to ask. If not, I am sure you can find other suppliers who do the same.