Cell Cryopreservation

INTRODUCTION

Cryopreservation is the use of low temperatures to preserve structurally intact living cells. Cells are cryopreserved to avoid loss by contamination, to minimize genetic change in continuous lines and to avoid transformation in finite lines. Successful cryopreservation of cells depends on optimal freezing conditions, storage, and proper cell thawing techniques. A standardized and reproducible protocol must be followed, although each protocol may require optimization for a given cell type or line, to achieve maximum viability upon thaw. Mammalian cells that are cryopreserved include immortalized cell lines, primary cells isolated from tissues and stem cells. Mammalian cells are best frozen in the presence of a cryopreservant such as DMSO or glycerol, with a freeze rate of 1°C/minute to avoid detrimental ice crystal formation as water within the cell is frozen. BioCision solutions for for cell cryopreservation include CoolBox® XT ice-free cell preparation workstations for temperature controlled sample preparation, CoolCell® alcohol-free cell freezing containers for reproducible controlled-rate freezing, TruCool® cryogenic vials and hinged cryoboxes for sample storage, and ThawSTAR™ automated thawing system for reproducible cell thawing.

CELL PREPARATION

Cryogenic vial preparation is typically performed under aseptic conditions in a cell culture hood. Cell suspensions are typically kept cold (0.5 to 4.0℃) which requires the use of ice outside the hood or an ice-free cooling unit inside the hood.  BioCision CoolBox® XT ice-free cooling workstations can be used inside a sterile hood environment and will keep up to 48 cryogenic vials cold (0.5 to 4℃) for >16 hours. The thermo-conductive properties of the cooling core inside the CoolBox XT and the cryogenic vial CoolRack holder ensure uniform temperature distribution to all vials during the process. CoolBox XT does not require ice, electricity or batteries.

CELL FREEZING

Optimal cell cryopreservation requires a controlled freezing rate of -1℃/minute for most types of cells. The freezing can be performed in a step-down programmable ultra-low freezer, a -80℃ freezer or with a portable dry ice temperature stability system like BioCision BioT™ ULT Transporter with a controlled-rate passive freezing container. Current methods that utilize alcohol-based freezing containers and styrofoam boxes do not provide uniform freezing rates to all cryogenic vials and/or may not be reproducible. CoolCell® alcohol-free cell freezing containers provide a cost effective means of reproducibly conducting the cell freezing process in a -80℃ freezer or with a portable dry ice temperature stability system. CoolCell freezing containers provide uniform, consistent and reproducible cell cryopreservation.

mr frosty

PROBLEM 
Alcohol-based cell freezing containers require a constant fresh supply of 100% iso-propyl alcohol to maintain an approximate -1ºC/minute freeze rate, resulting in on-going purchase cost and hazardous waste disposal. Concentric rings of cryogenic vials inhibits uniform cryogenic vial freezing as heat from inner vials must pass through outer vials to escape the vessel.

coolcell cell freezing
SOLUTION
CoolCell® alcohol-free cell freezing containers do not require any alcohol or other fluids to control the -1ºC/minute freeze rate. Insulative outer materials and inner alloy core, combined with radially-symmetric vial placement regulate a uniform heat removal rate for all vials. Freeze runs are consistent and highly reproducible.

 

How do CoolCell containers work?

CoolCell freezing containers are passive devices that provide a controlled -1℃/minute freeze rate to all cryogenic vials when placed in a -80℃ freezer. CoolCell containers do not require isopropanol or any fluids - the design and materials regulate heat removal and provide uniform and reproducible freezing to all vials.

  • • Highly insulative cross-linked closed-cell polyethylene foam has superior material properties at cryogenic temperatures

  • • Radially-symmetric vial distribution ensures identical heat removal profiles for each vial

  • • Solid alloy thermal core fine-tunes and balances the freezing profile

  • • Extremely durable single-block base construction provides nearly indefinite product life-cycle without change in performance


CoolCell® consistency. Five consecutive freeze runs in a -80℃ freezer show reproducible freezing profiles.

 

How do CoolCell containers compare to other freezing methods?

coolcell, cryopreservation

CoolCell alcohol-free cell freezing containers
• Consistent -1˚C/minute freeze rate to all vials
• Alcohol-free; no ongoing cost, maintenance or waste
• Reproducible freezing profiles
• 5-10 minute wait period between freeze runs (allows 2 freeze runs per day)
• Optimal for pluripotent stem cell recovery (see above)

 


Styrofoam Boxes

Unstandardized; difficult to document rate of cell freezing as Styrofoam containers can vary greatly in size, geometry, density and structure; not reproducible


Isopropanol-Based Container

Stated freeze rate of -1°C/minute, but varies based on vial position; continuous isopropanol replenishment, cost and waste; isopropanol is a variable in each freeze run hindering consistent reproducibility; long wait periods between freeze runs


Controlled-Rate Freezer

Programmable freeze rates are reproducible and documentable; expensive to purchase; difficult to operate; large bench footprint; prone to malfunction and maintenance*; logistically difficult to ensure at all collection sites

*Controlled-rate freezers require that a minimum of two thermocouples be carefully located in the freezing chamber and on the samples that are to be cooled. If these sensors are not connected appropriately, the control system will no longer receive accurate data and will respond with incorrect inputs of liquid nitrogen and subsequent cooling [1].

 

CoolCell cell freezing containers are available for 1.0 mL through 5.0 mL cryogenic vials, and 2.0 mL though 10.0 mL injectable serum vials for cell therapy applications. High-capacity CoolCell FTS30 accommodates 30 x 1.0 mL or 2.0 mL cryogenic vials in one freeze run. See all CoolCell models here.

 

TESTIMONIALS

The device is elegant in its simplicity and ease of use and offers researchers a method to cryopreserve cells in a standardized fashion with great reproducibility and little variability in performance.

- Kevin Grady, ATCC


The technology provided by these systems allows the operator to work in an ice or liquid nitrogen non contact environment, which greatly benefits the sterility status, when handling vials for tissue culture procedures. There is of course the added bonus of safety when using the system for snap freezing with liquid nitrogen, as there is no direct contact with liquid nitrogen for either the operator or the cryogenic vial. The additional option of using the system at 37°C, in a water bath, again without direct contact, aids the sterile thawing of cryogenic vials, and is performed in a consistent and reproducible manner. The combination of controlled freezing with the CoolCell and controlled temperature for thawing greatly increase the reproducibility of the freeze thaw process, with increased cell viability and cell growth post thaw. The versatility of the system is simply brilliant.versatility of the system is simply brilliant.

- John Gardner, Senior Project Leader, Roslin Cellab


We run a registry, in which large amounts of PBMCs are processed for long-term cyropreservation. After testing the CoolCell out, we found slightly better cell viability (>90%) than our current cell freezing containers, and there is no Isopropanol waste generated. Overall, the CoolCell has proven to us to set a new bar in cryopreservation.

- Rohit Gupta, Stanford University


The CoolCell is more efficient and easier to use than the Mr. Frosty. Not needing to add isopropyl, the lack of a screw top, and not having downtime after removing it from the -80° makes freezing cells a lot easier. I plan on only purchasing the CoolCell in the future.

- Matt Donne, UCSF Stem Cell Lab


Purely altruistically, I wanted to mention that one big advantage of the CoolCell is that you can use dry ice to do your freezing if you don't have a -80. I spent nearly a year getting terrible viability-recovery on cell freezing with dry ice (in an alcohol device) followed by storage in LN2, and this on HeLa cells which aren't exactly hard to store. I then bought a CoolCell and now I can just use standard cryo-preservation techniques (DMSO, 20% NBS) and get nearly 100% viability on thawing.

- Sam Knight, Ceramisphere (Australia)

 

CELL THAWING

Current methods of cell thawing include swirling the frozen vial in a water bath with subjective endpoint, rolling the vial between hands, or even warming the vial in the armpits. All of these methods are not standardized, and introduce variability into thaw temperature and rate.

Cell recovery and viability is affected as much by the thawing methodology as freezing. Minimizing the sources of variance in cell thawing standardizes the thaw process into a reproducible one. ThawSTAR™ automated cryogenic vial cell thawing system addresses this last gap in the cell cryopreservation workflow. By replacing inadequate methods of cell thawing, the ThawSTAR system enables rapid, consistent and reproducible thawing.

   

PROBLEM
Cell thawing in communal water baths introduces multiple sources of variance, including method of thawing, subjective determination of thaw endpoint, and potential for contamination.

SOLUTION
ThawSTAR™ automated cell thawing system does not require any water or operator input or interaction to successfully complete the thaw cycle. Patent-pending adaptive sensing technology automates the thaw process for optimized and reproducible thaw profiles.

 

How does the ThawSTAR™ system work?

ThawSTAR™ system is an automated cell thawing system that is powered by patent-pending adaptive sensing technology. Multiple detection parameters automatically sense the starting vial temperature, phase change initiation and thaw completion, resulting in highly reproducible thaw profiles and runs.

How does ThawSTAR™ thawing system compare to other thawing methods?

ThawSTAR™ automated cell thawing system

  • • No water eliminates potential contamination risk from communal water baths
  • • Fully automated sensing technology eliminates operator input and subjective interpretation of phase change and thaw completion
  • • Highly reproducible thaw performance and profiles that are equivalent to water baths, without the water
  • • High unit-to-unit and run-to-run reproducibility

 

ThawSTAR™ automated cell thawing systems are available for 1.8 mL and 2.0 mL cryogenic vials. For other formats, please contact us at thawstar@biocision.com

 

TESTIMONIALS

…Our GMP protocols call for thawing of cells every 60 days from a Master Bank. Use of a water bath within a GMP facility is highly discouraged due to contamination concerns. If one is employed, it must be thoroughly emptied and cleaned after every single use. Incorporation of a ThawSTAR™ thawing system into our protocols allows us to streamline our workflow, recognize time savings, and enforce sterile operating conditions.

- Helen Huls, Laboratory Manager of Pediatric Research, MD Anderson Cancer Center

 

…BioCision is a very innovative company and once we heard about the new cell thawing system, we were really eager to try it out for thawing PBMCs from our viral repository. When using a water bath to thaw there is always a risk of contamination, and we were keen to have a more reproducible cell thawing method. The ThawSTAR™ system solves all of our concerns and we can even use it in the hood which improves our cryopreservation workflow.

- Mars Stone, Viral Reference Lab and Repository Core, Blood Systems Research Institute 

 

 

 

[1]  Shu Z, Kang X, Chen H, Zhou X, Purtteman J, Yadock D, Heimfeld S, and Gao DDevelopment of a Reliable, Low-cost, Controlled Cooling Rate Instrument for the Cryopreservation of Hematopoietic Stem Cells. Cytotherapy. 2010.