Cryopreservation: Key Principles and Recent Advances

Cryopreservation Key Principles and Recent Advances Cryopreservation has played a critical role in biomedical research for many decades and continues to be an important tool for several current and emerging applications. It involves preserving biological samples by storing them at very low tem peratures. These samples can include materials such as cells, tissues and organelles. The process can be complex and relies on appropriate freez ing, storage and thawing approaches. Progress in fields such as cell therapy and mRNA vaccines, as well as the need for more efficient long-term storage of organs for transplantation, is driving continuous improvements in cryopreservation. This infographic will explore some of the key principles of cryopreservation and highlight recent advances in this field The Benefits of Cryopreservation Cryopreservation keeps cells in “suspended Cryopreservation is a vital technique for animation,” stopping metabolic activity. several applications, including: There are many reasons why researchers may wish to cryopreserve samples, such as: Cell therapy Banking complex cellular models or Fertility treatment rare cells for future research Conservation of endangered species Saving time and money Microbiology Reducing heterogeneity caused by repeated passaging Molecular biology Protecting cell stocks Crop security Vaccines The Cryopreservation Process Traditional cryopreservation approaches involve identifying and harvesting cells, which are then frozen at a controlled rate (typically -1 0C per minute for mammalian cells) to -80 0C. Cells are then transferred to ultralow temperature storage at -196 0C. By contrast, vitrification involves the rapid cooling of When samples are needed for samples, in which the whole use, they are thawed quickly. solution solidifies without any ice crystallization. These freezing and thawing Choosing high-quality starting Care should also be taken processes can damage cells, material can help to increase to choose the most suitable causing cryoinjuries, which can the chances of successful freezing rate for the sample impact recovery and survival post-cryopreservation sample type to reduce potential rates, cellular function and their retrieval. detrimental effects on cells. efficiency as therapeutics. Slow cooling rates can cause ex Rapid cooling rates can cause tracellular ice formation. This can intracellular ice formation. This draw water out of cells, leading to can lead to mechanical damage cell dehydration and shrinkage. to cells during thawing. The Role of Cryoprotectants Damage to cells from freezing and thawing can be reduced by the addition of cryoprotective agents (CPAs). A range of CPAs are available, including permeating agents such as glycerol, dimethyl sulfoxide (DMSO), propylene glycol and ethylene glycol, and non-permeating agents such as polyethylene glycol, polyvinylpyrrolidone, raffinose, sucrose and trehalose. Permeating CPA Non-permeating CPA Permeating CPAs Non-permeating CPAs move into the do cell where they form hydrogen bonds not enter the cell. They dehydrate with water molecules, reducing the cells before freezing, reducing amount of ice that forms. intracellular ice formation. CPAs can help to reduce crystal Despite being one of the most used formation and the chance of cell injury. CPAs, DMSO has been associated with However, they are not without a range of negative effects, including: limitations and a balance must be met between effectiveness and toxicity. Differentiation in embryonic stem cells Epigenetic changes in hepatic Different combinations of CPAs and the addition of fetal bovine serum can be microtissues used to optimize cryoprotective effec tiveness while minimizing toxicity. Toxicity in transplant recipients However, to meet regulatory require Plasticizer leaching from cell ments, cells intended for clinical use storage bags need a xeno-free freezing medium. Advances in Cryopreservation Despite the Objective widespread utility evaluation of of cryopreservation, Improving the impact of efficien cryopreservation several challenges on samples remain to be addressed. Developing Minimizing approaches the negative for cell types effects of not currently cryopreservation amenable to on cells freezing Scaling up Maintaining Reducing issues protocols heterogeneity associated of organoids with thermal gradients, particularly in larger samples As our understanding of the effects of cryopreservation on biological samples increases, many current challenges are beginning to be addressed and methods optimized further. In recent years, progress has been made in several areas, including: Synthetic Polymers Combining CPAs Anti-freeze proteins are produced by several CPAs can be toxic to cells, especially species, including some polar fish. They in concentrations needed for efficie selectively bind ice crystals and help the fis cryopreservation. Using combinations of CPAs survive at low temperatures. and other substances could serve as an alternative approach to reduce the concentrations of CPAs However, their use in cryopreservation is needed and the resulting toxicity. limited due to the formation of needle-shaped ice crystals, alongside high costs and risk of Combining trehalose with glycerol was shown toxicity. Instead, synthetic polymers that mimic to efficiently preserve the viability and cell anti-freeze proteins are being developed. function of cryopreserved human adipose derived stem cells. Engineering Approaches Enhanced Thawing/Cooling In addition to advances in chemical approaches, The warming rate can significantly affect the novel engineering strategies are in development. recovery rate of cryopreserved materials, with thermal gradients causing ice crystal formation. Nanoparticle-mediated intracellular delivery Magnetic warming approaches are being enables trehalose to enter cells and reduce developed to reduce cryoinjury to cells. intracellular ice damage. Nanowarming has been shown to prevent A range of cell encapsulation approaches are cellular apoptosis and oxidative stress in being developed to reduce extracellular ice cryopreserved ovarian tissue in a sheep model. damage, including microfluid, extrusion and emulsion methods. An ultrarapid cooling method – superflas freezing – uses inkjet printing to cryopreserve Natural structures, such as the zona pellucida cells without the use of CPAs. structure of egg cells, are inspiring the design of biomaterial constructs capable of ice inhibition. The Future of Cryopreservation As scientists work towards addressing cryopreservation challenges, future advances could also be seen in the following areas: Increased standardization of protocols Implementation of automated approaches Development of more novel CPAs In silico molecular modeling Sponsored by