Cryopreservation is the process of preserving biological materials, like cells and tissues, by cooling them to very low temperatures to stop all biological activity. While this allows for long-term storage, ice crystal formation during freezing can cause irreversible damage. To counter this, scientists use cryoprotective agents (CPAs), with dimethyl sulfoxide (DMSO) being one of the most widely used to protect cells from these effects.
The Protective Mechanism of DMSO
DMSO’s effectiveness as a cryoprotectant stems from its small size, allowing it to readily cross cell membranes. Once inside, DMSO displaces water, which lowers the freezing point of the intracellular solution and reduces the formation of damaging ice crystals. This process also mitigates the osmotic shock that occurs as the extracellular environment freezes.
As ice forms outside the cell, the remaining liquid becomes highly concentrated with solutes, drawing water out of the cell and causing it to shrink. By replacing intracellular water, DMSO minimizes this effect. It also encourages the remaining water to form a glassy, non-crystalline state called vitrification, which is less harmful than sharp ice crystals.
The interaction of DMSO with the cell membrane is also a factor in its protective capabilities. At a 10% concentration, DMSO increases the permeability of the cell membrane by inducing the formation of small water pores. This increased porosity allows for a more efficient exchange of water and cryoprotectant, which is beneficial during the cooling process and helps preserve cell viability.
The Cryopreservation Protocol
The protocol for cryopreserving cells with DMSO begins with preparing a freezing medium. This solution consists of a base cell culture medium, providing nutrients, supplemented with a protein source like fetal bovine serum. DMSO is added to this mixture to a final concentration of 5-10%, and the serum helps stabilize cell membranes against freezing stress.
Once the medium is chilled, harvested cells are gently suspended in it to ensure even distribution. The cell suspension is then transferred into labeled cryovials. Vials are filled to about half their volume to prevent them from cracking as the liquid expands during freezing.
Controlled-rate cooling is a key phase of the protocol. To maximize survival, the temperature is lowered slowly at a rate of -1°C per minute. This slow pace allows water to move out of the cells gradually, preventing the formation of large intracellular ice crystals. This is achieved using a programmable freezer or an isopropanol-filled container placed in a -80°C freezer.
Once frozen to at least -80°C, the cryovials are transferred to long-term storage. This is the vapor or liquid phase of liquid nitrogen, which maintains a temperature of -196°C. At this temperature, all metabolic processes are halted, preserving the cells in suspended animation for years.
Thawing and Post-Thaw Procedures
Thawing cryopreserved cells is as important as freezing, but it requires speed. Quick warming prevents small ice crystals that may have formed during freezing from recrystallizing into larger, more damaging ones. This is accomplished by agitating the frozen vial in a 37°C water bath until only a small amount of ice remains.
Immediately after thawing, the cell suspension is transferred into a larger volume of pre-warmed culture medium. This step dilutes the DMSO, which is toxic to cells at warmer temperatures. The dilution is performed slowly, sometimes dropwise, to avoid osmotic shock. This gradual dilution allows the cells to re-equilibrate to a normal physiological environment more gently.
The final step is to completely remove the DMSO. This is achieved by centrifuging the diluted cells into a pellet. The supernatant, containing the DMSO, is then discarded, and the cell pellet is resuspended in fresh culture medium, ready for a return to normal culture conditions.
DMSO Toxicity and Mitigation
While effective, DMSO has drawbacks. The same properties that protect cells during freezing also make it toxic at temperatures above 4°C. This cytotoxicity is concentration- and time-dependent, so higher concentrations and longer exposure at warmer temperatures increase cell death. The toxicity is partly due to its ability to dissolve cellular membranes at high concentrations.
To manage these toxic effects, the cryopreservation and thawing protocols are carefully designed. Steps such as keeping cells cold during preparation, using a balanced 5-10% concentration, and performing rapid thawing followed by immediate dilution and washing are all in place to minimize cellular damage. These procedural steps allow for the successful use of DMSO, though research continues to explore less toxic alternatives.