Cell banking is the process of storing living cells under specific frozen conditions to create a standardized and stable supply for future use. This repository ensures that experiments can be performed over time with a consistent cell population, a foundational element of reliable scientific study. By preserving cells at very low temperatures, their biological processes are paused, allowing them to be revived later for use.
The Core Concept of Cell Banking
A reliable cell banking system uses a two-tiered structure to protect the integrity of the cell line over the long term. This system involves creating a Master Cell Bank (MCB) and a Working Cell Bank (WCB). The MCB is the original, authenticated, and highly characterized collection of cell vials, derived from a single culture of a selected cell clone. This bank is the definitive source and is accessed infrequently to maintain its pristine condition.
From a single vial of the MCB, a much larger WCB is produced. The WCB serves as the routine source of cells for day-to-day experiments in the laboratory. This hierarchical arrangement is a strategic measure to safeguard the original cell stock from potential issues like contamination, genetic changes from repeated culturing, or simply running out of the cell line. This strategy ensures a consistent and renewable supply for research without repeatedly going back to the original source, preserving the cell line’s genetic and phenotypic stability for reproducible results.
The Cryopreservation Process
The first step in cryopreservation is preparing the cells, which should be harvested when they are in their logarithmic, or “log,” phase of growth, as this is when they are healthiest. After harvesting, the cells are counted to determine their concentration.
A cryoprotective agent (CPA) is then added to the cell suspension. Agents like dimethyl sulfoxide (DMSO) or glycerol are used because they penetrate the cells and prevent the formation of large ice crystals, which can rupture and kill them. The CPA concentration, typically 5-10% for DMSO, must be carefully controlled, as too much can be toxic, and the mixture is then aliquoted into cryogenic vials.
The rate of cooling is a primary factor for survival. For most mammalian cells, a slow and controlled cooling rate of approximately -1°C per minute is optimal. This gradual temperature reduction allows water to move out of the cells before it freezes, minimizing internal ice crystal formation. This rate can be achieved using controlled-rate freezers or isopropanol-based freezing containers placed in a -80°C freezer.
Once the vials have reached approximately -80°C, they are transferred for long-term storage. The final step is to move the vials to an ultra-low temperature environment, typically the vapor or liquid phase of liquid nitrogen, which maintains a temperature of around -196°C. At this temperature, all metabolic activity ceases, preserving the cells in a state of suspended animation.
Cell Bank Maintenance and Quality Control
Maintaining the integrity of a cell bank requires diligent storage practices and a quality control program. Vials are stored in liquid nitrogen, and a distinction is made between storage in the liquid phase versus the vapor phase. Many laboratories prefer the vapor phase, where vials are suspended above the liquid nitrogen, to reduce the risk of cross-contamination if a vial were to leak.
A representative vial from each newly created cell bank undergoes a series of quality control tests. These tests confirm the bank is free from contaminants and that the cells are viable and authentic. Key tests include:
- Sterility testing to check for common microbial contaminants like bacteria and fungi.
- Mycoplasma testing for a specific type of bacteria that can compromise cell cultures without being visible under a microscope.
- Viability assessment, often done after thawing a test vial, to ensure a high percentage of cells survived the freeze-thaw cycle using methods like the trypan blue dye exclusion test.
- Identity verification using techniques like Short Tandem Repeat (STR) profiling to generate a unique DNA fingerprint for human cell lines, which confirms the identity of the cells and prevents cross-contamination.
Thawing and Recovery of Cells
The process of reviving cells from a frozen state is as important as the freezing process, but it follows the opposite principle: thawing must be performed as rapidly as possible. This is to minimize the damage that can be caused by the recrystallization of small ice crystals into larger ones as the cells warm.
The procedure begins by removing a vial from liquid nitrogen storage and immediately placing it into a 37°C water bath, where it is gently swirled. The vial should be removed from the water bath when only a small speck of ice remains. To prevent contamination, avoid complete immersion of the vial’s cap and wipe the outside with 70% alcohol before opening it in a sterile biological safety cabinet.
Once thawed, the cell suspension must be promptly transferred into pre-warmed culture media. This step is important for diluting the cryoprotectant, such as DMSO, which is toxic to cells at room temperature. To completely remove the cryoprotectant, the diluted cell suspension is centrifuged, causing the cells to form a pellet at the bottom of the tube, and the supernatant is then removed.
After the removal of the cryoprotectant, the cell pellet is gently resuspended in fresh, pre-warmed culture medium. The revived cells are then transferred to an appropriate culture vessel and placed in an incubator set to the optimal temperature and CO2 levels for that specific cell line. Plating the cells at a higher density is often recommended to help them recover after the stress of the freeze-thaw cycle.