Cryopreservation is a process used to stabilize biological materials at very low temperatures. Previous attempts to freeze biological materials, such as living cells often results in a significant loss of cell viability and in some cases as much as 80% or more loss of cell activity and viability.
In some cases, cell damage during cryopreservation usually occurs as a result of intracellular ice formation within the living cell during the freezing step or during recrystallization. Rapid cooling often leads to formation of more intracellular ice since water molecules are not fully migrated out of the cell during the short timeframe associated with the rapid cool-down rates. Intercellular ice formation also can arise during recrystallization that occurs during the warming or thawing cycles. If too much water remains inside the living cell, damage due to initial ice crystal formation during the rapid cooling phase and subsequent recrystallization during warming phases can occur and such damage is usually lethal.
On the other hand, slow cooling profiles during cryopreservation often results in an increase in the solute effects where excess water is migrated out of the cells. Excess water migrating out of the cells adversely affects the cells due to an increase in osmotic imbalance. Thus, cell damage occurs as a result of osmotic imbalances which can be detrimental to cell survival and ultimately lead to cell damage and cell viability.
Current cryopreservation techniques involve using either conductive based cryogenic cooling equipment such as a cold shelf or lyophilizer type freezer unit or convective based cryogenic cooling equipment such as controlled rate freezers and cryo-cooler units. Such equipment, however, are only suitable for relatively small volume capacities only and not suitable for commercial scale production and preservation of biological materials such as therapeutic cell lines. For example, the largest commercially available controlled rate freezer suitable for use with biological materials holds only about 8000 or so closely packed vials. Such existing controlled rate freezers also suffer from the non-uniformity in cooling vial to vial due, in part, to the non-uniform flow of cryogen within the freezers and the requirement for close packing of the vials within the freezer.
Many conventional freezing systems utilize internal fans to disperse cryogen around the unit and deliver the refrigeration to the vials via convection. Such convection based cooling or freezing systems cannot achieve temperature uniformity as the vials are often located at various distances from the internal fan or packed in the shadow of other vials or trays. Vials of biological material exposed to high velocity turbulent flow of cryogen are typically cooled at a different rate and often much faster than vials situated further away from the fan.
There are also existing lyophilizer type of control rate freezers that can handle large volume of vials but typically rely on thermal conduction between cold shelves in the lyophilizer unit to the vials. However, it is impossible to make the bottom of glass vials to have uniform conductive surface area since most glass vial bottoms are concave. Therefore, temperature variations during the freezing process from vial to vial are the biggest drawback for these types of equipment. Furthermore, the cooling rate can be painfully slow due to very small conductive surface of the vial that remains in contact with the cold shelves.
Prior attempts to mitigate the loss of cell activity and viability involved the use of cryoprotective additives such as DSMO and glycerol. Use of such cryoprotectives during the cryopreservation process has demonstrated a reduction in cell losses attributable to the freezing and subsequent thawing cycles. However, many cryoprotectants such as DSMO are toxic to human cells and are otherwise not suitable for use in whole cell therapies. Disadvantageously, cryoprotectants also add a degree of complexity and associated cost to the cell production and preservation process. Also, cryoprotectants alone, have not eradicated the problem of loss of cell activity and viability.
What is needed is a method and system to further reduce or minimize cell damage occurring due to ice formation or solute effects during cryopreservation processes with or without the use of cryoprotectives. Moreover, the system and method should be both efficient and readily scaleable to handle commercial scale production and preservation of biological materials and provide rapid and uniform cooling of such biological material.