Cryopreservation is fundamental to modern biological research, allowing scientists to store living cells and tissues for extended periods. Freezing media (cryopreservation media) is a specialized solution designed to protect cells from damage during the freezing process. This media mitigates the physical and chemical stresses imposed by extremely low temperatures. Its primary function is to suspend the biological activity of the cells while preserving their viability and genetic integrity until they are thawed for later use.
Key Ingredients and Their Purpose
The formulation of an effective freezing medium relies on a combination of three main components. The foundation is typically a standard basal cell culture medium, such as Dulbecco’s Modified Eagle Medium (DMEM) or RPMI-1640. This base provides a balanced environment, supplying essential nutrients, salts, and buffers. These components maintain the correct osmotic pressure and \(\text{pH}\).
A protein source is included to stabilize the cells. Fetal Bovine Serum (FBS) is a common additive, often used at concentrations between 10% and 20%. Its proteins help coat the cell membranes, shielding them from damage during the formation of extracellular ice. Specialized alternatives, such as synthetic proteins or polymer supplements, exist for researchers requiring a chemically defined or serum-free system.
The most important component is the cryoprotective agent (CPA), usually Dimethyl Sulfoxide (DMSO) or Glycerol. These agents are typically added to the final mixture at a concentration of about 10% by volume. The CPA lowers the freezing point of the solution and prevents the destructive effects of ice formation and solute concentration.
How Cryoprotectants Prevent Cell Damage
The physical act of freezing introduces several biological challenges. One major threat is the mechanical damage caused by the formation of ice crystals, which can lacerate the delicate membranes and internal structures of the cell. Intracellular ice formation is often lethal.
As pure water freezes into ice, the remaining unfrozen liquid becomes increasingly concentrated with salts and solutes, known as the solution effect. This high solute concentration outside the cell creates a severe osmotic imbalance. Water rapidly rushes out of the cell, causing dehydration and shrinkage that can denature proteins and disrupt cell membranes.
Cryoprotective agents like DMSO are penetrating CPAs, meaning they pass through the cell membrane and enter the cell interior. Once inside, DMSO increases the total solute concentration within the cell, which helps lower the freezing point of the intracellular water. This action reduces the chance of damaging ice crystals forming inside and mitigates osmotic shock by balancing the concentration gradient. Protection is maximized when freezing occurs slowly, typically at a controlled rate of about \(1^\circ\text{C}\) per minute, allowing CPAs sufficient time to penetrate the cells and work their protective function.
Laboratory Protocol for Media Preparation and Storage
Preparing freezing media requires a sterile environment, typically within a biosafety cabinet, to prevent microbial contamination. The process begins with the basal medium, supplemented with the desired protein source, such as Fetal Bovine Serum. The media should be kept chilled at \(4^\circ\text{C}\) during preparation to slow down degradation and reduce the risk of thermal shock to the cells.
The cryoprotective agent, such as DMSO, is the final component added. DMSO is often stored at room temperature but must be added slowly to the chilled basal medium, as its dilution generates heat. The final concentration of the CPA is commonly standardized at 10%.
If any component was not pre-sterilized, the final mixture must be passed through a \(0.22\text{ }\mu\text{m}\) filter to remove bacteria and particulate matter. DMSO is a strong solvent that can rapidly penetrate the skin, so gloves and appropriate personal protective equipment must be worn when handling it. The prepared freezing medium should be stored at \(4^\circ\text{C}\) for short-term use (up to a week), as components like L-glutamine can degrade. For longer storage, the media can be aliquoted and kept at \(-20^\circ\text{C}\), avoiding repeated freeze-thaw cycles.