Cryogenic Storage and Revival of Insect Cells

Cryogenic cell storage in liquid nitrogen is one of the most common methods for the preservation of insect cells over the long term.  Although you may obtain new stocks of cells from organisations such as ATCC (USA) or PHE (UK), they are expensive and entail an unavoidable delay in replacing a defunct culture.  If you can keep your own frozen cell stocks it can make life so much easier.  Also key to the process is the method used for the revival of insect cells from the frozen state.  Both aspects are addressed in this month’s blog on cryogenic cell storage and revival.
As often happens the topic was suggested by the antics of our CEO, in this case when he was trying to bring some cells back to life that had been stored for over 35 years in liquid nitrogen.  However, in that period they had experienced at least two lab moves and a few close encounters with cryostorage failures.  The fact that they were able to recover emphasized the resilience of insect cell lines but also the importance of the original freezing process.
Insect cells are most commonly grown in suspension culture these days in serum-free medium.  These conditions enable quite high cell concentrations to be attained while still preserving them in a healthy state.  We can’t stress enough the importance of only undertaking cryogenic cell storage when they are in a log stage of growth.  Use a reagent such as trypan blue* to monitor cell viability.  This does mean that you can very quickly prepare cells for freezing by simply adding a cryopreservation agent such as DMSO* and dispensing into appropriate vials.  For example, insect cells such as Sf9 can be frozen when they are at a concentration of 4-5 x 10⁶ cells/ml.  The major disadvantage of this method is that when the cells are thawed prior to revival, you will probably have to start them off as monolayer cultures owing to the low concentration.  It is better to harvest a log phase culture and concentrate the cells via low speed centrifugation before resuspension at a higher concentration (>10⁷ cells/ml) and addition of the cryopreservation agent.  After cryogenic cell storage and thawing, these cells can usually be revived directly in a suspension culture.
The freezing process can be accomplished using a variety of equipment.  Our CEO’s favourite is to use an attachment that gradually lowers ampoules into the vapour phase of liquid nitrogen.  This does require regular adjustment and it is hard to see how a consistent rate of temperature drop can be achieved, but it does seem to work.  A more predictable method uses one of several vessels that house a number of vials and are then placed in a -70/80°C freezer overnight to achieve the desired rate of freezing.  The next day the vials are moved to liquid nitrogen for cryogenic cell storage.  When you do this, we strongly advise making a note of where you put them and recording this in a spreadsheet/database that is maintained in the cloud.  This idea initially confused our CEO as he asked whether he should use cirrus, stratus or cumulus!  His face turned rather nimbus when we rectified his mistake.
Many procedures recommend that vials of frozen cells are stored in the vapour phase of liquid nitrogen.  The reason for this is that leakage of the liquid nitrogen into the vial via a faulty seal can result in an exploding vial on subsequent thawing (we address this point again below).  However, adopting this approach means that you have to keep your container only half full and as a consequence use half for your cryogenic cell storage.
Now, while the potential for exploding vials is a serious concern, you can store them safely in liquid nitrogen if you ensure that when they are first removed from storage that you loosen the cap to release pressure build up from evaporation.  You should also wear appropriate safety equipment comprising a face mask, lab coat and protective gloves.  Loosening of the vial cap doesn’t risk contamination of your cells but it will prevent an explosion.
If you do thaw a vial of cells and find that the viability is low and they are slow to recover then the following trick may help with subsequent attempts.  If you have the cells of the same type already growing, when they are in mid log phase, harvest some of them and sediment them using low speed centrifugation.  Remove the medium, filter sterilize it and then mix with an equal volume of fresh medium.  This provides you with a 50:50 stock of conditioned cell culture medium.  This seems to help cells that are at very low density to recover more rapidly. In some cases it can be the difference between success and failure.  It is assumed that conditioned medium provides various growth factors that aid division of other cells.  The method has helped us on occasion to revive cells that were frozen at a sub optimal concentration.
 
We hope that the above is of some help to you.  More detailed methods for maintaining and freezing insect cells can be found in our Insect Cell Culture Guide
*Refer to appropriate documentation for safe handling of any chemical.