Cryopreservation: The Science of Slowing Life

From KAb 26. October 2023 5 min

The concept of cryo-preservation, which involves preserving biological materials at ultra-low temperatures, has been a subject of both intrigue and scientific exploration. Traditionally, it was well-established that lowering temperatures could induce a state of dormancy in cells, effectively slowing down temperature-dependent biochemical reactions. However, as is often the case in scientific pursuits, there was a notable divergence between theory and experimental findings. Many discovered that the act of freezing and subsequent thawing process poses a significant threat to the viability of cells and tissues and that maybe this was not practically possible.

This paradigm began to shift when Christopher Polge, an English biologist, stumbled upon a pivotal breakthrough. While studying rooster sperm and using glycerol to increase sample viscosity for structural analysis, he inadvertently placed a sample containing glycerol into liquid nitrogen. Upon thawing, Polge was astounded to find that the sperm remained active and viable—a revelation that challenged conventional wisdom. So, what we learn from Polge is that we shouldn’t label our samples to discover a new field. Polge's accidental discovery marked the inception of what we now refer to as cryoprotective agents (CPAs), catalyzing the development of cryopreservation techniques that enable us to store biological materials over extended periods. Cryopreservation techniques can be broadly categorized into four primary methods: slow freezing, vitrification, non-freezing subzero preservation, and dry state preservation. Each of these approaches presents its distinct set of challenges and applications.

Slow Freezing vs. Vitrification which is better?

Slow freezing, as its name implies, is a methodical cooling process where cells or tissues are gradually brought to lower temperatures at a controlled rate. While this approach is relatively straightforward, it requires the use of cryoprotective agents (CPAs) to shield biological materials from freezing-induced damage. However, it carries the risk of ice accumulation outside the cells, which can potentially harm them.
In contrast, vitrification is a rapid procedure that exposes cells or tissues to high concentrations of CPAs and promptly immerses them in liquid nitrogen, instantly transforming them into a glass-like state. Vitrification effectively mitigates ice formation, significantly reducing the risk of freeze damage, and making it the preferred method for preserving delicate biological materials. If you are handling sensitive cells such as embryonic stem cells, iPSCs and want to switch to vitrification you could try out StemCell Keep. While traditional cryoprotective agents like DMSO may pose cytotoxicity concerns, hESCs cryopreserved with StemCell Keep exhibit superior attachment ability, increased proliferation, and pluripotency, as confirmed by alkaline phosphatase staining and the expression of key stem cell markers. Moreover, SCK-cryopreserved hESCs maintained normal karyotypes and successfully differentiated into the three germ layers, to form teratoma.

Why are Cryoprotective Agents (CPAs) important?

CPAs play a pivotal role in cryopreservation by mitigating the damage caused by freezing. These substances must meet specific criteria, including low toxicity, cell permeability, and biocompatibility. Achieving the best results requires precise optimization of CPA concentrations, along with controlled cooling and warming rates tailored to the unique characteristics of different cell and tissue types. CPAs can be categorized into two main groups:
1. Cryoprotectants that permeate cell membranes: This category includes substances such as Dimethyl sulfoxide (DMSO), glycerol, and 1,2-propanediol.
2. Membrane-impermeable cryoprotectants: These encompass polymers like polyethylene glycol, polyvinyl alcohol, and hydroxyethyl starch.

Glycerol and DMSO: Key Players in Cryo-Preservation

Glycerol and DMSO, both discovered in different eras, have emerged as prominent figures in the realm of cryo-preservation. Glycerol, recognized as a cryoprotectant in 1949, operates by diluting electrolyte concentrations in the unfrozen fluid surrounding cells. In a similar vein, DMSO, with its reputation for relatively low cytotoxicity, functions analogously. However, it's crucial to acknowledge that DMSO has encountered concerns related to DNA methylation and histone modification, potentially affecting cell survival and differentiation.
If you aim to preserve your cell expression without the influence of DMSO, we recommend considering our CryoStor® cryopreservation media options, available in with 2%, 5% and 10% USP grade DMSO formulations. These solutions are serum-free, protein-free, and manufactured in compliance with cGMP standards. We acknowledge that relying solely on serum and conventional media may not be the ideal approach for safeguarding specialized cell types. Thus, we encourage you to explore our extensive selection of cell-specific cryopreservation solutions tailored to meet the unique needs of your research.

The choice of cryoprotectant depends on the type of cells being cryo-preserved and the specific requirements of the experiment or application. The effectiveness of cryo-preservation techniques depends not only on the choice of cryoprotectant but also on the freezing and thawing techniques employed. The most effective techniques for achieving successful cryopreservation depend on the specific cell type and the desired outcome. Some commonly used techniques include slow freezing, vitrification, and controlled-rate freezing. Slow freezing involves gradually reducing the temperature of the cell suspension to allow for the gradual removal of water and the formation of small ice crystals. This technique is often used for larger cell types and is compatible with a wide range of cryoprotectants. Vitrification is a technique that involves the rapid cooling of the cell suspension to prevent ice crystal formation. It typically requires higher concentrations of cryoprotectants and is commonly used for smaller cell types or embryos. Controlled-rate freezing involves using specialized equipment to freeze the cell suspension at a controlled rate. This technique allows for precise control of the cooling process and can be tailored to the specific requirements of the cells being cryopreserved. It is important to note that the choice of cryoprotectant and the cryopreservation technique should be optimized for each specific cell type to achieve the best results. Factors such as cell size, membrane permeability, and tolerance to cryoprotectants should be considered when designing a cryopreservation protocol.

Cryo-preservation holds diverse applications across various domains, each marked by its unique significance. It offers a means of fertility preservation for individuals undergoing medical treatments that may compromise their reproductive capabilities. In the fields of research, medical procedures, and organ transplantation, cryopreserved tissues and cells assume pivotal roles, contributing to scientific advancements and healthcare practices. Stem cells, drawn from various sources, find longevity through cryopreservation, supporting endeavors in regenerative medicine, gene therapy, and cell transplantation. Hepatocytes, critical elements in scientific and medical research, also benefit from cryo-preservation and cryobanking. Furthermore, cryopreserved skin grafts emerge as potential alternatives to human cadaveric allografts in burn treatments, representing a notable development in this area.

What are the Current Challenges and Ongoing Research in Cryo-Preservation?

While cryo-preservation stands as a revolutionary technology, it is not without its challenges. The metabolic activity of cells at low temperatures is nearly nonexistent, and excessive use of CPAs can potentially harm cells. Moreover, cryopreservation may introduce genetic shifts, impacting cellular activity and structure.

How should Cells be properly Thawed after Freezing when you intend to use them?

Thawing cells is a critical step in the process of cryo-preservation, and the method used can have a significant impact on the viability and functionality of the cells. Different studies have investigated the effects of non-standardized thawing methods on cell survival and have found that the thawing temperature plays a crucial role in cell viability. For example, one study compared the viability of cryopreserved fat tissue at different thawing temperatures and found that the recovery of antioxidant capacity increased with an increase in the thawing temperature. Similarly, it was observed that oxygen radicals, which are capable of damaging cells, could be generated during the thawing process. However, these radicals could be effectively neutralized if the cells were thawed at a high temperature.

Furthermore, the quality and functionality of thawed cells can also be affected by the freezing and thawing process itself. Another study evaluated the immunomodulatory and therapeutic properties of cryopreserved mesenchymal stromal cells (MSCs) and found that fresh cells and cells of low passage demonstrated improved clinical outcomes compared to freeze-thawed cells at higher passage. The researchers concluded that cryobanked MSCs have reduced immunomodulatory and blood-regulatory properties directly after thawing, which can result in faster elimination after blood exposure. Similarly, another study assessed the architecture and integrity of frozen-thawed testicular tissue and found that while there was a significant increase in nuclear and epithelial score alterations after thawing, the overall lesional score remained comparable to fresh tissue. The number of intratubular spermatogonia and the expression of DNA replication and repair markers did not vary significantly after thawing.

The freezing and thawing process can also induce changes in cell nuclei, which can serve as indicators of the processes occurring during freezing and thawing. Researchers have highlighted the importance of understanding the freeze/thaw mechanisms and the complex states of cell nuclei during the process to comprehend the potential risks associated with cryopreservation and to design more efficient cryoprotective materials and protocols. Thawing can significantly impact these factors; thus, uniform thawing is essential for achieving optimal functionality and differentiation. ThawStar® is an automated thawing system that offers consistent and precise thawing through algorithm-driven processes with temperature-sensing technology. Operating it is user-friendly, with an intuitive software interface that allows access control, quality control procedure enforcement, and optimized thawing algorithms. The system is compatible with standard cryo bag formats from major manufacturers. To enhance biomaterial safety, a single-use Barrier Bag minimizes the risk of biomaterial loss or contamination while optimizing thaw performance. The patent-pending heating technology, coupled with real-time temperature-sensing analytics, ensures reliable and well-documented thaws.


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