Precision medicine and fertility preservation are all dependent on adequate cryopreservation of biological materials to achieve their goals. Pre- and post-thaw processing, along with storage conditions, can have a severe impact on sample quality and affect downstream utilization.
In this webinar, Lara Mouttham, Laboratory Coordinator, Cornell Veterinary Biobank (NY, USA), discussed the challenges and opportunities of working with cryopreserved materials and provides guidelines for managing a frozen collection.
Watch the webinar on-demand now or read on for highlights of the Q&A session following Lara’s presentation.
What measures would you take after knowing whether cell death is occurring by necrosis or apoptosis? Is anything known using anti-apoptotic measures, for example caspase inhibitors, during or after cryopreservation process?
Necrosis usually indicates insurmountable damage sustained during cryopreservation, while apoptosis is more indicative of a downstream response to damage. In the case of necrosis, I would consider serious changes to the cryopreservation protocol used. For apoptosis, small adjustments and the inclusion of additives before and after cryopreservation can help rescue the cells. Caspase inhibitors have been successfully used to improve the recovery of hematopoietic cells and hepatocytes, among other cell types.
Are there liquid nitrogen (LN2) cryostorage/cryoshipping solutions available for SBS multiwell-plates (gas phase)?
Yes, there are a number of suppliers who can provide various LN2 freezers and racking supplies. The Society for Biomolecular Screening (SBS)-American National Standards Institute (ANSI) standard has had a tremendous impact on our ability to engineer equipment to support the most efficient workflows. Adoptive therapies could also benefit from similar standards for cryobox and cryo-cassette (for various bags) dimensions.
You mentioned that LN2 could not be sterilized. Have there been issues with the validation of sterilization of liquid nitrogen?
There is some contention as to whether or not LN2 can be adequately sterilized by UV irradiation. Parmegiani et al. (2009) demonstrated decontamination of LN2 following UV irradiation and LN2 evaporation, but this requires precise UV intensity and exposure duration; as I mentioned in the webinar, there would need to be strict procedures in place to ensure sterility. Another aspect to consider is not just the presence of pathogens in LN2, but also the potential presence of other compounds or chemicals that may be carried over from previous uses of the LN2 – can you know with certainty that the LN2 you are using has not been in contact with compounds that could affect your samples?
How often is immersion in LN2 used in cryopreservation? What applications require this?
Immersion in LN2 is a common step in slow freezing, after seeding to reach storage temperatures, and the principle component of vitrification (ultra-rapid cooling). If you plan to store your samples at LN2 temperature, whether in the liquid or vapor phase, you will almost always encounter an immersion step in the cryopreservation protocol.
For cell therapy purpose, we need to use GMP cryopreservative solutions. Do you know alternative, DMSO-free option for long terms storage and human use?
I do not know if there are GMP solutions available for this, but trehalose and propanediol are becoming common alternatives to DMSO. There are a number of organizations working to develop GMP-grade alternatives and methods, but to date, DMSO remains the most common cryoprotective agent (CPA).
What is the difference between vapor phase LN2 and liquid phase LN2 cryopreservation?
There are two parts to this question: cooling rate and storage. Samples will reach -196°C much faster in the liquid phase than in the vapor phase because of convection, so if ultra-rapid cooling is necessary for vitrification or to avoid ice crystal formation, cooling the samples in vapor phase will negatively impact the quality of your samples. As for storage, vapor phase is generally considered safer for a few reasons, including eliminating the chance of cross contamination and user safety. There is also a risk of the labels detaching from the tubes and floating away, which severely impacts the traceability of the samples and collection data rigor. In the vapor phase, even if the label adhesive fails, the label will generally remain around the tube and allow for some traceability. This risk can be decreased by using labels intended for LN2 storage.
How can you recover samples after cryopreservation with minimal damage to the samples?
This is dependent on the cell type, the sample volume, the storage system, cryopreservation protocol and, of course, the intended purpose.
Do you have any comments about control rate freezers (CRFs)?
Identifying the optimized bioprocess includes understanding and optimizing the freezing process. A general industry starting point for control rate freezing is -1°C per minute, down to a temperature of approximately -80°C before being transitioning into LN2 vapor storage. To accomplish this, users can use a passive cooling device, such as a Coolcell from Brooks Life Sciences (Manchester, UK), or one of many more precise CRFs.
When working with CRF manufacturers to select the best model for your workflows, consider top loading over side loading as well as reliability and performance of the solenoid valves. The typical control rate freeze process can take between one and two hours, but can improve the post thaw cell functionality when combined with the other key elements in bioprocessing.
What is the International Stem Cell Biobanking Initiative (ISCBI)?
The ISCBI is a global network of stem cell biobanks which provides best practices and training to promote the appropriate use of pluripotent stem cells for research, industrial and clinical applications.
What is the difference between a cryovial and a cryotube?
These two terms are usually interchangeable, but for this webinar I intended to differentiate between cryotubes, which have a screw-on cap, and cryovials, which have a hermetically sealed top. In this case, cryovials can be used in GMP situations as the seals have been developed and tested to withstand cryogenic storage temperatures.
Is dry ice better than room temperature for storage?
Placing a storage box directly on dry ice from LN2 temperatures results in more rapid warming rates to glass transition temperatures than when placed directly at room temperature. This is thought to be caused by the sublimation of dry ice into gaseous CO2, which increases the convective motion around the box and speeds up warming. The most important best practice for this situation is to minimize the amount of time the samples are out of LN2 storage, regardless of if they are on dry ice or at room temp, and to minimize the movements made with the box or samples to prevent warming through convective forces.
Find out more in this blog from Brooks Life Science>>
Do you have any experience or insight with cryopreservation using hydrogels?
I do not have direct experience with cryopreservation using hydrogel, but I think it is a promising technology and the published results of this type of cryopreservation seem optimistic. When combined with low concentrations of CPA in slow freezing, the porous nature of the hydrogel seems to promote the favorable formation of extracellular ice crystals, thereby protecting the cells. It seems that it would also facilitate the transition from culture to cryopreservation back to culture. It may also be a good carrier for vitrification since it would enable ultra-rapid cooling rates, but I would have the same concerns for possible contamination from direct contact with LN2 as for any other open system.
Why are screw top tubes are not GMP-compatible? Does this apply to -80°C freezers or just LN2 storage?
Screw top tubes may not be acceptable for GMP purposes that involve storage in liquid phase of LN2, because of the small risk of LN2 leaking into the tube, compared to hermetically sealed vials. If stored in vapor phase or in -80°C freezers, this risk is negated. GMP-approved septum top cryovials have been tested for storage in LN2, though they are more expensive and require additional equipment for filling and sealing.
Given the correct circumstances, however, intermediate products used in cell and gene therapy manufacturing can be appropriately stored in screw top cryotubes. It is important to understand the limitations and validated uses of each type of cryovial or cryotube, and how they can be best be used to achieve the intended purpose of the biological sample.
Which quality control markers you would suggest?
This depends on the cell type and the intended purpose of the sample.
Are there any adverse effects on biological materials if stored at -190°C instead of 4°C or -20°C?
The general rule is that the lower the temperature, the safer the cryopreservation. This is due to the need to halt metabolic activity in the cell. The dangers in cryopreservation are from frequent and/or uncontrolled transient warming events; if those can be prevented, there is no adverse effect of storing at lower temperature. Of course, storage at LN2 temperature is more expensive than at -80°C, even more so than at -20°C. You have to do a cost-benefit analysis to determine the most economical way of adequately storing your samples for their intended purpose.
Are there any automated cryo-freezers that keep samples in the liquid phase throughout their lifecycle, such as automate store and retrieve or automated audit trail?
There have been examples of automated systems trying to operate and keep material submerged in LN2. Various functionality and service concerns have moved the industry towards LN2 vapor because of the temperature and safety benefits.