Cryopreservation, the freezing of living biological samples at subzero temperatures, evokes scenes from science fiction movies of long journeys across space and resuscitation of terminally ill patients in the future with available cures. Preserving clearly remains the realm of science fiction, but also mammalian cell cultures, primary cells, tissues, microorganisms, plant cell lines and seeds, and even whole organisms such as: Nematode Worms are currently acceptable or in the cryopreservation pipeline. Although currently unfeasible, cryopreservation of whole mammalian organs is an active and expanding area of ​​research that may increase the availability of materials for organ transplantation.

In addition to the wide range of possible biosamples for cryopreservation, there are also numerous future applications. Cryopreservation is a potential technique in reproductive medicine for freezing oocytes and is also being investigated for archiving biological samples for biodiversity and conservation efforts. In biomedical and biological research, cryopreservation is already widely used in laboratories to back up valuable non-commercial cell lines and preserve cultures that are not actively used in ongoing experiments. I’m here. To make this technology more widely available, cryopreservation research has made significant strides, overcoming the challenges inherent in freezing cells, tissues and organs for long-term storage at freezing temperatures. .

basic challenge

“Cryopreservation of biological samples is a complex process for several reasons,” he explained. Jonathan Lakey, Professor Emeritus of the School of Surgery and the Henry Samuel School of Engineering at the University of California, Irvine. “Freezing induces freeze damage Damage caused to cells by the growth of intracellular ice crystals that mechanically disrupt cell structures. In addition, freezing can alter local cellular salt gradients, leading to osmotic shock. These damaged, cryodamaged cells are less likely to survive upon thawing, defeating the purpose of cryopreservation,” Lakey elaborated. “Of course, freezing damage can be mitigated by adding cryoprotectants to the freezing medium, substances that protect cells during the freezing process. Cell-specific considerations, which must be determined empirically to devise the optimal approach for a particular cell or tissue type. For example, pancreatic islets and oocytes do well with rapid cooling, whereas hepatocytes and hematopoietic stem cells require relatively slow cooling. ”

It is these cell-specific considerations that make cryopreservation of complex assemblies more challenging than single-cell samples. “There are two main reasons why cryopreservation of tissues and organs is difficult for him,” explains Saffron Bryant, a research fellow in his STEM science department at RMIT in Australia. “First, tissues and organs are composed of several cell types of varying sizes, which absorb cryoprotectants and freeze at different rates. Second, cryoprotectants, which are essential components of the cooling medium but must permeate the cells to help them survive freezing, also require different cryopreservation protocols for the cells within. Freezing an organ requires exposing multiple layers of tissue and all layers of the organ to a cryoprotectant, except that the organ can be placed directly into a vat of cryoprotectant such as dimethylsulfoxide. put in [DMSO]is the current standard.

Keep it Cool: Cell Cryopreservation for Clinical Applications

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two is better than one

Many studies have focused on developing less toxic or preferably non-toxic cryoprotectants to improve cryopreservation success. Lakey and his team DMSO and ethylene glycol Cryoprotection of pancreatic islet beta cells could potentially treat patients. type 1 diabetes“While the process of transplanting beta cells is still suboptimal, Edmonton Protocol Relatively better graft survival than previous methods. Transplant recipients undergo immunosuppressive therapy to prevent graft rejection, but these immunosuppressive drugs are toxic to the transplanted beta cells, which are in a vulnerable state after thawing. However, if a better cryopreservation method is developed that can make beta cells more viable upon thawing, it may improve the chances of post-transplant survival and immunosuppressive regimens,” Lakey said. I explained about medical treatment.

Usual concentrations of DMSO for freezing cells are added stepwise up to 2 M. However, by combining 1 M DMSO with his 0.5 M ethylene glycol, Lakey and his team were able to lower the total concentration of cryoprotectant needed, reducing the overall toxicity of this cryoprotectant. I was. mixture. They then frozen pancreatic beta cells in a DMSO-ethylene glycol cryoprotectant mixture and 2 M DMSO to resuscitate the cells and transplanted them into diabetic mice lacking beta cells. If so, they initiate the production of insulin, a glucose-regulating molecule, returning diabetic mice to a normoglycemic state, or normal blood sugar levels. “The results were very encouraging,” Lakey recalled of the study. “Diabetic mice fed beta cells frozen in a DMSO-ethylene glycol mixture were able to return to normoglycemia 12 days earlier than mice fed the same number of beta cells frozen in DMSO alone. These findings indicate that beta cells were more viable upon thawing when they were cryoprotected in the DMSO-ethylene glycol mixture than in DMSO alone. It increases the potential for long-term beta-cell banking.”

Bryant is also investigating the use of binary mixtures called deep eutectic solvents as cryoprotectants. The components of a deep eutectic solvent form a complex network of interactions called hydrogen bonds that lower the freezing point of the solution. In addition, deep eutectic solvents are “glass formers” and when frozen form amorphous solids rather than sharp crystals like water, so the solvents do much less damage to the cells. “We tested six deep eutectic solvents for cryopreserving mammalian cell lines. Cells were then thawed and cell health was assessed using atomic force microscopy and confocal microscopy.” We have identified one deep eutectic solvent that is significantly less toxic than DMSO,” Bryant said of her work. “The candidate cryoprotectant mixture is less toxic than its components, highlighting the advantage of a multi-component system. Furthermore, the deep eutectic solvent is much less toxic than DMSO, which is why cell survival during resuscitation may be compromised. We were able to incubate the cells at 37 °C for several hours without significantly reducing the rate.”

Due to such low toxicity, deep eutectic solvents may have potential applications for freezing organs prior to transplantation. Unfortunately, a significant number of consented organs from deceased individuals are not currently used for transplantation. The availability of non-toxic cryoprotectants may address this problem by freezing organs until needed for transplantation and preventing wastage of donated samples. “Theoretically, culturing an organ in a deep eutectic solvent does not kill the outer layer of cells. So it can wait to penetrate deeper layers. It increases the success rate of freezing organs that are well-infiltrated with .

Cryopreservation of animal cell cultures

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encapsulate and protect

Another approach tested by Lakey and his team to improve cryopreservation was Encapsulate beta cells in a layer of alginatea water-absorbent gelling polymer isolated from brown seaweed. “The rationale behind this project is that the alginate biomaterial can act as a protective coat around islet-beta cells, altering the dynamics of cryoprotectant penetration and minimizing toxicity and exposure.” Alginate membranes can also protect cells by providing another layer of material to hold them together. Indeed, encapsulation enhances beta-cell survival and function after thawing. When transplanted, beta cells that had been encapsulated in alginate prior to cryopreservation returned diabetic mice to normoglycemia approximately two weeks earlier than beta cells that were not encapsulated prior to freezing. This appears to be a promising approach that could improve islet cryopreservation protocols,” concluded Lakey.

Frozen future?

When asked about future directions, Lakey replied that there is a need for customized protocols for specific cell types. We have dealt with beta cells, which are highly potent, but there are many different cell types, such as stem cells and immune cells, that may be useful as therapeutics for certain medical conditions, because each cell type has a different permeation rate. , would require a tailored cytoprotective agent, and by reducing the toxicity of the cytoprotective agent, we might even be able to achieve tissue and organ freezing.”

Bryant agrees, and believes that organ freezing may one day become a reality with less toxic cytoprotective agents and customized freezing protocols. “There are two key aspects that she thinks will move the field forward,” she said. “New cryoprotectants that are less toxic and act on more cell types. Also new techniques for more uniform cooling and heating. Microwave and magnetic techniques, for example, are being investigated by other groups.” , which facilitates better freezing and thawing of larger samples, which is very promising.” Bryant himself has also tested protic ionic liquids that form glasses like deep eutectic solvents. “By tuning the specific properties of these solvents, we can optimize them for cryopreserving different types of cells, and eventually organs,” she says.