Cryopreservation uses ultra-low freezing temperatures to maintain live leukocyte viability through long storage periods. Because cells cannot be preserved with conventional freezing methods, researchers have collaborated to achieve successful cryopreservation of cells by manipulating cooling rates and introducing cryoprotective agents into the cell samples.
Cryopreservation protects the organelles, cell tissues, and other biological components by carefully cooling them for liquid nitrogen storage, essentially suspending cells in a moment. Successful cryopreservation also allows for seamless thawing and a guarantee of cell viability. Cryopreservation allows for experimental flexibility and lets researchers maximize their resources.
The meticulous temperature control necessary for successful cryopreservation is challenging to achieve with conventional freezing methods. One useful innovation in cryopreservation has been understanding the water-to-ice phase transition stage and the effects these changes have on cell membranes and underlying cell interactions. Passive cooling devices or controlled rate freezers are utilized to achieve stepwise temperature increments that gently move cells through the phase shift. These machines use electricity or liquid nitrogen to maximize temperature control and avoid shocking the cells or causing them to burst.
There are two verified methods of cryopreservation: slow freezing and vitrification. These protocols implement different concentrations of cryoprotective agents (CPAs) and cooling rates. Slow enough cooling can eliminate the supercooling phenomenon which triggers intracellular ice crystallization. Slow freezing is accessible and low cost and avoids major contamination.
Unfortunately, slow freezing does not adequately adjust for extracellular ice formation, which can be significantly damaging during cryopreservation. Vitrification uses cell suspension to move cells from a liquid state to a glass state via deep cryogenic temperatures. This technique can be costly and requires expert handling, although it provides a high cell survival rate.
Damage resulting from incorrect cryopreservation is called cryoinjury. The best way to avoid cryoinjury is by implementing CPAs that are introduced before freezing and removed post-thaw. These CPAs mitigate the osmotic diffusion phenomena across cell membranes that amplify cryoinjury by decreasing ice’s crystallization ability. One of the most common CPAs in use today is DMSO, a valuable chemical buffer due to its low cost, low level of toxicity, and ability to protect even the most fragile mammalian cells.
Cell cryopreservation has many important applications in cell separation research. The biggest advantage of cryopreservation is the prolonged storage of valuable cells for research and the maintenance of experimental viability after thawing. By increasing the available storage times of common cell sources, such as leukopaks, through cryopreservation, researchers can plan their experiments more efficiently; no longer relying on fresh cell deliveries. In addition to more accessible cell sources, successful cryopreservation of stem cells is the promising initial component in tissue engineering and other next-generation biomedical goals.
Freezing cells incorrectly can cause cryoinjury to the cell tissue and membrane viability and can induce osmotic shock. This is caused by the osmotic diffusion across the cell membrane during the water-to-ice phase shift and the formation of ice crystals either within or around the cell, leading to tears and perforation of the membrane.
Many factors can contribute to accrued cryoinjury during cryopreservation, including the rate of cooling, the hardiness of the cell type, and the temperature extremity. Careful considerations must be made regarding the proper CPA usage and temperature regulation processes.
The type of cell being preserved can affect the freezing procedure chosen. Mammalian cells have different requirements for successful cryopreservation than other cell types and can be considerably more fragile. Because of this added fragility, DMSO is the standard CPA used in mammalian cell cryopreservation.
Due to the increasing extracellular osmolarity during the freezing process, water diffuses out of the cell and begins to crystalize around the cell membrane. Prolonging the cooling step can cause a majority of the water to diffuse across the membrane, effectively dehydrating the inner cell plasma. Freezing too rapidly can cause water retention within the cell and introduce ice crystals to the intracellular matrix.
CPAs can prevent both diffusion extremes by inhibiting the amount of ice formed during the phase change state and their ability to diffuse across the cell membrane. By properly regulating the freezing rate, cryopreservation can be accomplished.
The primary step in cryopreservation is mixing the cells to be frozen with cryoprotectants so they can withstand intense temperature fluctuation without damaging the cells. The cells are then cooled using specialized machines. The cells are inundated with liquid nitrogen or exposed to slow incremental temperature drops facilitated by electricity.
Once the cells are needed for application, the solution must be thawed for further use. When the solution has been carefully brought to temperature, the CPAs must be removed from the solution so they do not inhibit any downstream in vivo cell functions.
Cryopreservation is maintained between sub-freezing temperatures, below −80°C and typically below −140°C. This is possible using liquid nitrogen bath storage containers and −80°C electric freezers. Too intense a temperature fluctuation could compromise the cryopreservation of the entire sample.
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A quick and easy leukopak processing protocol facilitated by our microbubble technology yields an untouched and robust cell population ready for downstream analysis. The careful nature of Akadeum’s buoyancy-activated cell sorting (BACS) exposes the sample to much less potential harm, ensuring proper cell culture maintenance.
Akadeum offers cell solutions across research fields, providing kits for both human and murine cell isolation. Ensure proper processing of precious cryopreserved cells by utilizing Akadeum’s cutting-edge isolation techniques.