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Q1 – What is the minimal volume which can be used? 1 mL is recommend for 10^6 cells, you can reduce the amount of Bambanker for lower numbers of cells. For example, for 5×10^5 use 0.5 mL.
Q2 – Does the media contain DMSO?
Yes, all varieties of Bambanker contain DMSO, except Bambanker DMSO Free (BBF01, BBF02)
Q3 – Can we use thawed cells directly in culture medium, without a washing step?
Yes, you do not need a washing step.
Q4 – Can cells which were frozen in Bambanker be used for RNA isolation?
Yes. Please be aware that the low number of cells in each tube (10^6 cells) may lead to a low amount of RNA isolated.
Q5 – Can we use Bambanker for the cryopreservation of PBMCs?
Yes, please see these recent publications:
https://onlinelibrary.wiley.com/doi/pdf/10.1111/bjh.15995
https://www.sciencedirect.com/science/article/pii/S1051044319302933
Here is an example of what to expect regarding PBMCs:
Protocol: i) PBMC were isolated from healthy person’s blood, ii) PBMC were frozen in Bambanker (standard version) and stored for 12 months at -80°C and -196°C (liquid nitrogen) at 1 × 10^6 cells / １mL Bambanker in 1 mL aliquots, iii) Twelve months after freezing, the cells were rapidly thawed and the number of cells was measured, followed by culturing for 7 days using a 25 flask coated with an anti-CD3 antibody and a medium supplemented with IL-2 (700 U / mL). Cell count was measured on days 5, 6 and 7, and flow cytometry was performed on day 7 of culture.
Results: recovery rate = percentage of intact cells after thawing as per the number of cells originally frozen down; survival rate = after set period of time, percentage of living cells per total population as determined by trypan blue:
Stored at -80°C for 12 months —> recovery rate 59.5%; survival rate 88.9%
Stored in liquid nitrogen (-196°C) / 12 months —> recovery rate 63.9%; survival rate 87.3%
Q6 – How do you recommend handling particularly delicate/sensitive cells?
Tips for freezing: The best way to freeze sensitive cells is to place the cells in Bambanker (appropriate type) and keep them at 4°C during the processing. Place them as fast as possible into a -80°C freezer for 24h. Then, either store the cells at -80°C or transfer the frozen cells to liquid nitrogen.
Tips for thawing: Put the vials in a 37°C water bath until the contents are just thawed. The cells should then immediately be transferred to a large volume of pre-warmed media — for the most sensitive cell types (stem cells, primary cells) being added dropwise to preserve viability. Although some cells may be pelleted by centrifugation before being gently resuspended with a pipette and added to a cell culture flask containing pre-warmed growth media, for the most sensitive cell types it is more gentle to transfer the cells to a flask straight after thawing and to perform a media change the next day to remove any residual cryoprotectant.
Q7 – Can we use Bambanker for the cryopreservation of whole organoids?
Yes, Bambanker and Bambanker HRM are your best choices. Both can provide over 70% recovery rates. We recommended to use a small number of organoids (under 20 mg) per cryotube. We also recommend to first soak the organoids in Bambanker at 4°C for 10 min before freezing. Use normal Bambanker for organoids and use Bambanker HRM for human or on human tissue-derived organoids.