The application and progress of cryopreservation in medicine

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The application and progress of cryopreservation in medicine

The application and progress of cryopreservation in medicine. For many years, cryopreservation has been the main method of preserving biological specimens. It allows researchers to use rare or precious samples decades ago to answer new research questions.

Today, it is used to preserve the latest complex cell models for other scientists to use in the future, and is essential for the storage and supply of biological treatments. In this article, we discuss the challenges of cryopreservation and new developments that can improve these methods.

What is cryopreservation?

Freezing means “frost” in Greek, and cryopreservation actually involves freezing cells, tissues, organs or any other biological material at very low temperatures.

There are several ways to achieve this: the most common method used in research laboratories is to freeze samples using solid carbon dioxide (CO2) at –80°C or liquid nitrogen at –196°C. Another method called vitrification is used to freeze clinical samples, such as sperm, fertilized eggs, or ovarian tissue, for better and longer-term storage.

The main difference is that the traditional cryopreservation method allows ice to be formed during storage, while in vitrification, the entire solution freezes without any ice crystals. In this article, we will focus on the most commonly used routine cryopreservation in laboratories.

Application of cryopreservation

As Sameena Iqbal, the biological resource manager of the Wellcome Sanger Institute, explained, traditional cryopreservation is a very effective method of cell and tissue storage. It works by keeping cells “pseudo-dead”. “By freezing the cell, it can stop metabolic activity and retain the compounds inside the cell, such as enzymes.”

Without cryopreservation, you must keep the cells and tissues alive in continuous culture-which means they grow and divide to produce more Multiple cells (called passaging). But cells will multiply over time, which can cause them to lose important characteristics. By freezing them, the heterogeneity is reduced, which would otherwise be introduced by passing them repeatedly.

“From our perspective, cryopreservation allows us to build new cell models for the research community, and people can return to these models in the next few years,” Dr. Charlotte Beaver, senior science manager at the Wellcome Sanger Institute, which develops complex cell model systems Say. As an organoid. “This means that we can keep the models for a long time, but they are not constantly split to the point where they have mutated into units that can hardly represent your beginning.”

One area where cryopreservation is becoming more and more important is the rapidly emerging field, cell therapy: for example, preservation of mesenchymal stem cells for transplantation, or chimeric antigen receptor T cells (CAR-T cells) for cancer treatment. ). With CAR-T cell therapy, the patient’s T cells are removed, redesigned to recognize antigens (such as tumor-associated antigens), and then returned to them. “You have to process a lot of these cells, you may move the cells between different locations, when you donate them to the laboratory, get them from the patient, and then return to the patient again, these cells are really easy to degrade,” Professor Matthew Gibson explained. “Ideally, treatments like this need to be frozen in a form that allows you to quickly thaw at the bedside before applying them to the patient. Once a lot of treatment has to be done in a hospital environment, it creates obstacles, and All these steps incur a lot of costs. In any case, these are extremely expensive therapies, so any method that makes the process more effective and restores more healthy cells after freezing is very beneficial to the patient.”

Limitations of cryopreservation

One of the main limitations of current cryopreservation methods is the recovery rate of cells after freezing. Although in research projects, you may have time to wait for the number of cells to grow, this is simply impossible for the above-mentioned clinical applications.

During the freezing process, ice crystals will form in the sample and damage the cells, and some cells will never recover. Most laboratory and clinical protocols use cryoprotectants to protect cells from ice crystals or to control quick freezing and thawing to avoid shocks to cells due to sudden changes in temperature.

There are many different cryoprotectants available, but the most common cryoprotectant used in mammalian cells and tissues is dimethyl sulfoxide (DMSO). Other options include glycerol for bacterial cells and red blood cells. But cryoprotectants have their own shortcomings. DMSO is considered the best protective agent, but it is toxic to cells at certain concentrations.

Compared with DMSO, glycerol is more friendly to cells, but it is less effective as a cryoprotectant. One way to alleviate potential toxicity problems is to use different cryoprotectant combinations, such as supplementing lower concentrations of DMSO with glycerin or polypropylene glycol. There are also some primary cell types that do not like freezing in cryoprotectant solutions, which can be a challenge.

“Acute myeloid leukemia peripheral mononuclear cells are easier to freeze and recover than lymphoma cells, so if you don’t know what the malignant tumor is when you freeze the sample, you need to treat them very carefully,” Iqbal explained. “Similarly, different types of healthy blood cells freeze in different ways-T cells may recover very well, although their function may be affected, while granulocytes will not recover at all.”

“With new complex models, such as organs extracted from patient samples, we are in danger of losing the model,” Beaver explained. “One advantage of using organoids is that they are composed of different cell populations, which represent the natural heterogeneity you see in tissues or tumors. Freezing makes them encounter bottlenecks, if it is too harsh for certain cells , You will lose heterogeneity, and it will no longer be a good model.”

Another consideration is the end use of the sample. For example, compared to genomic DNA, RNA is much more sensitive to temperature changes, as Beaver said: “People have been digging out bones many years ago and still try to sequence all DNA, and if RNA is at –80°C It will degrade in the freezer!” If you don’t know how to use the sample, this can cause problems for frozen samples.

“If your organization is very limited, as analytical methods improve, you must decide what is the best way to preserve this material,” Iqbal said. “I remember when I started about 18 years ago, we would get very large lymph node biopsies. There was more tissue than we might use, so it could be preserved in a variety of ways. Now core biopsies are commonly used for diagnosis, so The materials you have used for research purposes are very limited.”

Progress in cryopreservation

Beaver says that there are two main advances that can improve cryopreservation. “There is a lack of mid-range automation in the market. Even if you only freeze 30 to 50 vials, and not necessarily every day, this is still a challenge for people in the laboratory.” In addition to robots, there is another area that needs improvement. It is looking for an alternative to DMSO which is toxic to cells. Fetal bovine serum is usually used together with DMSO to provide proteins that further support the cells. But these are not ideal for freeze-dried cells, for example. “If living cells differentiate in a way you don’t want, it’s meaningless to have living cells. If you inject these cells back into these people, for example for cell transplantation, then you will need a non-animal-derived serum substitute. .”

“The cryopreservation based on DMSO is a good method, but we do need to consider the efficiency of things,” Gibson agreed. “For many cell types that we usually freeze using standard methods, we may only be able to recover 20% to 60% of the cells after freezing. If we can get a higher number, then we can take out smaller batches from the refrigerator. , Or if you use rare cells or primary cells that are difficult to cultivate and expand, the more you recover, the faster you can conduct research, or you can do more experiments per batch.”

To meet this need, Gibson is studying biomimetic materials, and a protein they have been imitating is called antifreeze protein, which is an ice-binding protein. “These proteins somehow manage to selectively bind to ice. They can tell the difference between liquid water and frozen water, which is very obvious.” These ice-binding proteins are produced by many species, the most famous These are fishes found at the poles of the earth.

Protein binds to ice crystals to prevent them from growing, so this fish can survive at lower temperatures than fish without this protein. “It turns out that there are quite a lot of proteins and polysaccharides that can make or prevent the formation of ice, or control how and when the ice is formed. If we can simulate this with synthetic polymers, then we can change the way we freeze cells, inspiration It comes from the way nature protects itself during the cold.” The team has shown that they can use polymers to reduce the amount of cryoprotectants while freezing bacterial cells.

Gibson says another advancement is the ability to freeze cells that have already been attached to tissue culture plates. “For suspended cells, you have to put them on a plate and let them grow before you can use them. We are interested in how to freeze the cells on these plates so that you can take them out of the refrigerator and prepare them for use .” Gibson’s laboratory has developed some materials whose role is not by affecting ice, but by effectively protecting cells during the freezing process. “We found that the recovery level of these cells is very high.

One of our most exciting results is the addition of ampholytes to the monolayer of cells, attached to the tissue culture plastic, and we have seen a dramatic increase in the number of recovered cells from <20% to> 80%.” This research on freezing science is not limited to life sciences-it is applied to everything from making ice cream creamier and less fat to avoiding concrete damage caused by freezing and thawing.

“If you can make the research more efficient, that’s a good result, but such research can also help solve basic questions about how these low temperatures affect our lives and how we can store things better and more efficiently.”

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