The Science Behind Cryopreservation
Preserving living cells for future use requires cryopreservation, which involves holding biological material at ultra-low temperatures to suspend all metabolic activity. This technique is fundamental across modern science, enabling the long-term banking of valuable cell lines for research, stem cells for regenerative medicine, and reproductive materials. Successful preservation ensures that the cells retain their viability and genetic integrity indefinitely, provided the temperature remains sufficiently low.
Simply placing a cell sample in a standard freezer is lethal because water, which makes up about 80% of the cell’s mass, crystallizes. Damage occurs primarily through two mechanisms. First, large, sharp ice crystals form inside the cell, physically puncturing membranes. Second, the solution effect (osmotic shock) occurs: as pure water freezes outside the cell, the remaining liquid becomes highly concentrated with salts and solutes. This extreme osmotic imbalance draws water out of the cell, causing it to dehydrate and shrink, leading to structural injury and death.
The cryopreservation process minimizes this physical and chemical damage by controlling the rate at which water leaves the cell and preventing large ice crystals from forming. The goal is to maximize dehydration before freezing, avoiding the internal crystal growth that is detrimental to survival. Achieving this balance requires careful control over the cooling process and the introduction of specialized protective chemicals.
Essential Ingredient: The Role of Cryoprotective Agents
To successfully counteract the destructive effects of freezing, scientists rely on Cryoprotective Agents (CPAs), specialized compounds mixed with the cells before cooling. These agents interfere with water molecules’ ability to form large crystalline structures. They effectively lower the freezing point of the solution and promote a state known as vitrification, where the water turns into an amorphous, glassy solid instead of destructive ice crystals. This glass-like state prevents mechanical damage to the cell membranes and internal organelles.
The most widely used CPA is Dimethyl Sulfoxide (DMSO), a small molecule that easily penetrates the cell membrane and acts on both the interior and exterior of the cell. Glycerol is another common alternative, particularly for certain cell types like red blood cells and sperm. By increasing the total solute concentration inside and outside the cell, these permeable CPAs reduce the amount of free water available to form ice. DMSO is typically used at a concentration of about 10% in the freezing medium.
A drawback of CPAs is that they are inherently toxic to cells, especially at higher concentrations or prolonged exposure times. This toxicity is dependent on the CPA type, its concentration, and the temperature of exposure. For instance, DMSO toxicity increases significantly at warmer temperatures. Therefore, the entire protocol must be carefully timed to minimize the duration that cells are exposed to the CPA solution, particularly after they are warmed up during thawing.
The Step-by-Step Freezing Protocol
The methodology for cell freezing is a sequence of steps designed to maintain cellular balance during temperature descent. The process begins with cell preparation: cells must be healthy and actively growing, ideally with viability greater than 75%. They are harvested and concentrated, often around one million cells per milliliter, to ensure a high recovery rate upon thawing. The concentrated cell pellet is gently mixed with the cold cryopreservation medium containing the CPA, such as 10% DMSO, and quickly dispensed into specialized cryogenic vials.
The most important step for cell survival is the controlled cooling phase, which must proceed at an optimal rate of approximately \(1^circtext{C}\) per minute. This slow cooling rate represents a careful compromise between two opposing forces of cellular damage. A slow rate allows water to gradually exit the cell, minimizing the risk of lethal intracellular ice crystal formation. If the cooling is too fast, the water inside the cell cannot escape quickly enough and freezes internally, causing cell death.
To achieve this precise cooling rate, laboratories typically use either a programmable controlled-rate freezer or a passive freezing container (often referred to as Mr. Frosty). The controlled-rate freezer provides the most consistent and uniform drop in temperature. The passive container relies on a bath of isopropanol alcohol or a specialized alloy core to insulate the vials. The passive container is placed into a \(-80^circtext{C}\) freezer, and the internal insulation ensures the temperature of the cells drops slowly at the target rate over several hours. Once the vials have reached \(-80^circtext{C}\), they are ready for transfer to long-term cryogenic storage.
Long-Term Storage and Cell Recovery
After controlled cooling to \(-80^circtext{C}\), the vials must be quickly transferred to an ultra-low temperature environment for indefinite storage. The standard method for long-term preservation involves placing the cells in specialized tanks containing liquid nitrogen (\(text{LN}_2\)), which maintains a temperature of \(-196^circtext{C}\). At this extremely low temperature, all cellular metabolic and biochemical activity ceases, effectively halting the aging process. The temperature must be maintained consistently below \(-130^circtext{C}\) to prevent any formation or growth of ice crystals.
For safety and to prevent cross-contamination, cells are generally stored in the vapor phase of liquid nitrogen, where the temperature remains below \(-150^circtext{C}\). Storing vials directly in the liquid phase poses a risk that microbial contaminants from one vial could be transferred to another via the \(text{LN}_2\) medium. Additionally, liquid nitrogen can sometimes seep into the vials, creating a pressure hazard that could cause the vial to explode upon warming, which is avoided by vapor phase storage.
The recovery process, or thawing, is critical and must be performed rapidly. The frozen vial is quickly transferred from the \(text{LN}_2\) tank and submerged in a \(37^circtext{C}\) water bath, where it is gently swirled until only a tiny sliver of ice remains. Rapid thawing is necessary to ensure that the small, non-damaging ice crystals formed during controlled cooling do not have time to grow into larger, destructive ones as the temperature rises. Immediately after thawing, the cells must be gently washed and diluted with fresh culture medium to quickly remove the toxic CPA, minimizing the time the cells spend exposed to the chemical at warmer, more damaging temperatures.