“C80EZ’’ is the abbreviation for “Easy for -80 Celsius’’
The development of C80EZ® media is based on an invention (international patent filing) that applies unique biocompatible polymer combinations to promote nanoscale cubic ice formation. The unique approach minimizes the size of hexagonal ice crystals (the dominating ice structure during freezing of normal aqueous solutions) during freezing and significantly improves the thermal stability of the frozen samples by preventing hexagonal ice regrowth or secondary growth (i.e., recrystallization) during storage and warming. Consequently, all C80EZ® products significantly reduce mechanical damage of tissue structures and fragile cell types, which are majorly associated with ice formation and regrowth during cryopreservation procedures. The anti-apoptosis agents and antioxidants in the C80EZ® media also reduce cell loss due to the use of cell permeating cryoprotectants (e.g., DMSO and glycerol) and excessive intracellular water loss during freezing. Use of C80EZ® hence significantly improves the post-thaw viability and functionality of multiple cell and tissue types that are traditionally difficult to cryopreserve (see examples in Applications).
With the unique ice structural modification mechanism, application of C80EZ® products also realize long-term storage of cells and tissues in regular laboratory deep freezers (approx. -70 to -80°C), and do not require liquid nitrogen facilities (approximately -120°C in the vapor phase and -196°C in the liquid phase for storage). If the C80EZ® products are used to store cells and tissues in liquid nitrogen facilities, the samples can be safely transported by dry ice boxes (at -78°C on dry ice surface) instead of using highly expensive and heavy liquid nitrogen dry shippers or dewars. Also, when liquid nitrogen is accidentally depleted, the C80EZ® products can minimize the possibility for loss of valuable biological samples by providing extended protection before the liquid nitrogen is refilled. For short-term storage of cells and tissues, C80EZ® products allow users to freeze cells and tissues at -18°C or -20°C (i.e., in regular lab freezers) for approximately 10 days without losing viabilities or functionalities, which outperforms most traditional hibernation or hypothermia media.
Novel Cryopreservation Mechanisms:
- Reducing ice crystal size: The relatively large ice crystals generated by traditional slow cooling processes, especially when a low concentration of cryoprotectant is used, are typically on the order of several hundred microns in size. Deploying a unique macromolecular crowding mechanism, C80EZ® significantly reduces the hexagonal ice crystal size by two orders of magnitude through promotion of nanoscale cubic ice formation at non-cryogenic temperatures, and thus significantly reduces mechanical damage to tissue structure and multiple fragile primary cell types.
A comparison between the typical morphology of visible hexagonal ice crystals (white particles in various shapes) formed in frozen traditional cryopreservation medium (left) and those in C80EZ® medium (right) through cryomicroscopic observation at -80°C (bar = 100 µm). The hexagonal ice crystals inside the C80EZ® medium are significantly smaller than those in normal medium (with 10% DMSO and 10% fetal bovine serum).
A comparison between the ice formation (white streaks) in frozen rat myocardium tissues treated by regular cryopreservation medium (left) and C80EZ® medium (right) through microscopic cryo-section observation without sample coating (bar = 1 mm). The tissue frozen with the regular medium presents large holes (indicating large ice crystal formation) in the tissue structures, while the tissue frozen with C80EZ® present a smooth sectional surface (minimal ice formation).
- Preventing recrystallization: The ice recrystallization process typically causes gradual cell loss during storage or temperature fluctuation. Traditionally, the only practical approach to completely prevent recrystallization and associated cell damaging effects is to maintain biological samples at cryogenic temperatures by using expensive liquid nitrogen facilities (typically between -120°C and -196°C). However, an extensive recrystallization process still occurs during warming, that is highly damaging to tissues and also to some fragile cell types. The innovation based on the use of C80EZ® prevents recrystallization during storage in regular laboratory deep freezers (-70°C to -80°C), and thereby enables long-term storage (at least several years) in that temperature range and does not require liquid nitrogen facilities anymore. For tissue cryopreservation, the damages to tissue structures due to recrystallization are minimized when C80EZ® medium is used for both -80°C storage and liquid nitrogen storage.
A comparison between the thermal stability from the observation of exothermal graphs of frozen regular medium (dotted line) and C80EZ® medium (solid line) based on differential scanning calorimetry. The measurement of the devitrification temperatures (Td), an approximation of recrystallization temperature, demonstrates that the C80EZ® medium prevents any recrystallization at temperature below -70°C or -80°C, which provides the basis for its achieving long-term storage in regular deep freezers or minimize tissue damage during warming.
Advantages and Benefits:
- Significantly improving post-thaw viability of cells and tissues traditionally difficult to preserve: Use of C80EZ® products significantly improves the viability of primary neurons, primary chromaffin cells, primary cornea tissues, myocardium tissues, blood vessels and ovarian tissues. See APPLICATIONS for details.
- Facilitating storage and shipping of biological samples: Long-term storage of cells and tissues in normal deep freezers using C80EZ® products offer numerous advantages over traditional liquid nitrogen storage methods, especially in operation, safety, and cost savings in labor and facilities. When C80EZ® is used for storage of samples in liquid nitrogen, it facilitates long-distance transportation in dry ice boxes (instead of using expensive liquid nitrogen dry shippers) by minimizing cell loss due to recrystallization during transfer and warming processes from liquid nitrogen facilities to dry ice boxes. The use of C80EZ® also prevents cell loss due to accidental temperature increase resulting from depletion of liquid nitrogen supplies or power outrage. For short-term storage, the cells and tissues can be safely stored at -20°C for more than one week by using C80EZ® media, which overcomes the limits of hibernation or hypothermia media.
- Convenient to use: C80EZ® does not contain any animal serum, its replacement (e.g., serum replacement), or protein (e.g., albumin) that are popularly used in “homebrew” cryopreservation media. As such, it also removes the need of those compounds for cryopreservation. C80EZ® prevents cell aggregation and maintains cell suspension by itself. C80EZ® requires users to add a low concentration (~ 5% v/v) of cell culture grade dimethyl sulfoxide (DMSO) at time of use. All C80EZ® products are sterile filtered (0.22 µm). C80EZ® has a long shelf life, and can be stored at 2 - 8°C for more than two years, with no need to freeze.
- Versatile production line for various species and specific cell types: In addition to standard C80EZ® products for general mammalian cells (it is a 1x version, i.e. directly adding to cell pellets), CryoCrate has developed a product line to facilitate use by a variety of different users and in various fields of research and development, as demonstrated by the following examples:
C80EZ®-2X: designed for cell suspensions. Different from standard C80EZ®, which is a 1x medium for cell pellets, the C80EZ®-2X is designed for cell suspensions and added to cell suspension at a 1:1 ratio.
C80EZ® -TISSUE: specifically designed for mammalian tissues. It enables highly efficient cryopreservation of tissues in regular lab deep freezers (see APPLICATIONS).
C80EZ®-INSECT: specifically designed for insect cells and tissues, and the first commercially available cryopreservation medium product for insect cells. It also completely removes the need for high serum use for insect cells.
C80EZ®-COMPETENT: specifically designed for competent cell types and other bacteria. It removes the need for high glycerol use for competent cells, etc.
C80EZ®-CORNEA: specifically designed for corneal cryopreservation, and to be used in our IcyEye® kit (see details in Technology – IcyEye®) for medical applications.