Cryopreservation Procedures

Cryopreservation procedures are those that permit cells to be stored indefinitely by using extremely cold temperatures to suspend metabolic activities. There are two main types of cryopreservation procedures: equilibrium (conventional slow freezing) and non-equilibrium or ultra-rapid freezing (vitrification). The term vitrification comes from the Latin "vitreous" or glassy. Both procedures use cryoprotectants with antifreeze-type properties to prevent cellular damage during the freezing process. Once cells are frozen or vitrified, they can be stored indefinitely by immersing them in liquid nitrogen, an extremely cold fluid with a temperature of -196 C (-321 F). To restore metabolic activity in the cell upon thawing, toxic cryoprotectants must be removed from the cell and the normal water balance gradually restored as the cell is returned to a normal functioning temperature.

Overview

Overview

• Cryopreservation procedures are those that permit cells to be stored indefinitely by using extremely cold temperatures to suspend metabolic activities. There are two main types of cryopreservation procedures: equilibrium (conventional slow freezing) and non-equilibrium or ultra-rapid freezing (vitrification). The term vitrification comes from the Latin "vitreous" or glassy. Both procedures use cryoprotectants with antifreeze-type properties to prevent cellular damage during the freezing process. Once cells are frozen or vitrified, they can be stored indefinitely by immersing them in liquid nitrogen, an extremely cold fluid with a temperature of -196 C (-321 F). To restore metabolic activity in the cell upon thawing, toxic cryoprotectants must be removed from the cell and the normal water balance gradually restored as the cell is returned to a normal functioning temperature.

Cell-Specific Factors

• The procedure used to cryopreserve cells or tissue depends on a number of factors including the size of the cell, the quantity of cellular fluid (cytoplasm) and the complexity of the cell (single cell or tissue). Cells with a large amount of cytoplasm such as eggs are more difficult to cryopreserve than cells with only residual cytoplasm, like sperm cells. Cryopreservation of ovarian tissue is more challenging because at least three different cell types of varying sizes are present in ovarian tissue, each with different optimal freezing needs. Slow-freezing protocols have been used with success with various types of cells. Vitrification currently is most effective with single-cell freezing and less effective with tissues, but research is ongoing to optimize vitrification of tissues.

Role of Cryoprotectants

• The cytoplasm of a cell contains water, which turns to ice crystals at freezing temperature. When ice forms inside a cell, the cell is shredded and dies. Therefore, the fluid in the cell needs to be removed prior to reaching freezing temperatures to avoid cell damage. Various types of antifreeze (cryoprotectant) fluids can be used to dehydrate the cell before freezing, including glycerol, propanediol, dimethyl sulfoxde (DMSO), ethanol and sugars like sucrose and trehalose. The optimal rate of cooling (and thawing) depends on the type of cryoprotectant used and on the characteristic of the cell or tissue to be (or that was) frozen. Cryoprotectants are toxic to metabolizing cells so cell exposures to cryoprotectants at warmer metabolic temperatures must be minimized or avoided. Upon thawing or warming the cells, cryoprotectants must be completely removed from the cell before metabolic activity is restored.

Equilibrium Versus Non-Equilibrium Cryopreservation Procedure

• The rate of freezing depends on whether the freezing protocol is equilibrium- or non-equilbrium-based. For equilibrium type procedures, the optimal freezing rate is achieved when there is a balance between the rate at which the cell is being dehydrated of water and the rate at which water outside the cell is being transformed to the ice stage. These equilibrium or slow-freeze protocols usually take hours to complete and a computer is used to run a programmable rate freezer to ensure that the freezing rates are exactly as required. There is typically a "hold" step in the protocol near the start to allow the manual creation of starter ice crystals or "seeding" in the fluid outside the cells.

  In vitrification, an entirely different approach to freezing that does not rely on ramps and achieving an equilibrium between dehydration and ice crystal formation is used. Vitrification is so ultra-rapid that there is insufficient time for ice formation and the cellular fluid is converted directly into a vitreous or glass phase, without damage to the cell. Programmable rate freezers are not required because the cell is directly plunged into extremely cold liquid nitrogen, achieving an instantaneous glassy state.

Slow-Freeze Procedure

• The first step of the slow-freeze procedure is to expose the cell to cryoprotectant in a gradual stepwise fashion to slowly allow equilibration of the cell with the cryoprotectant while releasing water. Once the cells have been cleared of the majority of cellular water, the cells in the cryoprotectant fluid are placed into a container of some kind, such as a plastic straw, a glass ampule or a plastic vial.The volume of liquid surrounding the cells for slow freezing is typically less than a teaspoon and may only be a few drops. The pre-labeled container is filled, sealed and put in a programmable freezer, which slowly decreases the temperature of the container over a period of minutes or hours to very low temperatures. After a few minutes of cooling, starter ice crystals must be formed inside the container by "seeding" the container. Seeding is performed by using a tool (for example, forceps) which have been pre-chilled in liquid nitrogen to touch the container and cause ice crystal formation. This starter crystal will initiate controlled ice crystal formation in one spot, safely away from the cells. Once seeding is complete, the rest of the cooling ramps can proceed. When the container reaches temperatures between -30 and -85 C, the container holding the cells can be plunged directly into liquid nitrogen to complete the cooling to -196 C.

Vitrification

• Ultra-rapid non-equilibrium chilling procedures such as vitrification use higher concentrations of cryoprotectants coupled with an almost instantaneous freezing rate achieved by plunging cells directly into liquid nitrogen. Vitrification bypasses the ice-crystal formation phase and moves the water directly into a glass-like phase. Vitrification achieves the same endpoint as slow freezing, but at a rate of -3000C/min, compared to 10C/min or more. For vitrification, cells are usually placed on the tip of a straw and excess cryoprotectant is removed, leaving just enough so that the cell clings to the container by surface tension, prior to plunging in liquid nitrogen. Because freezing is so rapid, the duration of exposure to cryoprotectants is much less, and much higher concentrations of cryoprotectants can be tolerated by the cell. Warming of the cell to return it to normal metabolic functioning must also be incredibly rapid. Handling of vitrified samples is much more exacting than slow-frozen samples because even a brief exposure to a temperature above that of liquid nitrogen can start an unplanned warming process and refreezing, which is detrimental to the cell.