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 requires that a standardized and reproducible protocol 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 typically frozen with a cryopreservant; either DMSO or glycerol, and at a rate of 1°C/minute to avoid the detrimental effects of ice crystal formation as water within the cell is frozen. BioCision's tools for cell cryopreservation include CoolBox XT ice-free cell preparation workstations and CoolCell alcohol-free cell freezing containers for reproducible controlled-rate freezing.

CELL PREPARATION

Cryogenic vial preparation is typically performed under asceptic 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's 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 cryogenic vials during the process. CoolBox XT does not require ice, electricity or batteries.

CELL FREEZING

Typically, cell cryopreservation requires a controlled rate freezing of -1℃/minute for most types of cells. The freezing can be performed in a step-down programmable ultralow freezer, or in a -80℃ freezer or in a dry ice locker with a controlled-rate passive freezing container. Current methods such as those that use 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 dry ice locker. CoolCell freezing containers provide uniform, consistent and reproducible cell cryopreservation.


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.

Snap freezing in dry ice using a CoolRack
SOLUTION

CoolCell alcohol-free cell freezing containers do not require alcohol or any 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 the heat removal and provide uniform and reproducible freezing to all vials.

Highly insulative closed-cell polyethylene insulation 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 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

Kevin Grady - ATCC
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.

John Gardner - Senior Project Leader, Roslin Cellab
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.

Rohit Gupta - Stanford University
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.

Matt Donne - UCSF Stem Cell Lab
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.

Sam Knight - Ceramisphere (Australia)
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.

 

[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.