What are freezer boxes?
Cell cryopreservation is one of the main methods of cell preservation. By using lyophilization technology to cryopreserve cells in liquid nitrogen at -196°C, the cells can be temporarily removed from growth and their cellular properties can be preserved so that the cells can be revived for experiments when needed. This prevents the cells from being lost due to contamination of the cells in culture or other unexpected events and also serves as a cell preservation agent. When cells are directly frozen without any additional protective agent, ice crystals are formed from water both inside the cells and in the external environment, which can lead to mechanical damage, elevated electrolytes, changes in osmotic pressure, dehydration, PH changes, protein denaturation, etc., which can cause cell death. Therefore, when freezing and storing, protective agents should be added to the culture solution, which can lower the freezing point. Under slow freezing conditions, it can make the intracellular water permeate out of the cells before freezing. Storage in liquid nitrogen can reduce the formation of ice crystals. At this point, freezer boxes
are a good choice.
Why is it important to freeze immune cells?
Cell storage is the extraction of cells from human tissues or blood through special technical means, directly isolated and purified or after in vitro culture and amplification, stored in liquid nitrogen environment at -196℃. In this ultra-low temperature environment, all biochemical reactions within the cells stop and are temporarily removed from the growth state, and the function and activity of the cells are not significantly affected, and their preservation time can reach more than 20 years.
Currently, the two main types of cells commonly used in cell storage are stem cells and immune cells. Among them, stem cells include cord blood hematopoietic stem cells, umbilical cord stem cells, placental stem cells, adipose stem cells, endometrial stem cells, dental pulp stem cells, etc.; immune cells include peripheral blood immune cells and umbilical cord blood immune cells.
Storing immune cells is of great significance
A. Coping with high cancer incidence
With the high incidence of cancer today, storing immune cells is a life-saving measure to reduce the risk of cancer and prevent it from happening in the first place.
B. Coping with the reality of a declining immune system after puberty
Immune organs decline with age. The most obvious example is the thymus gland, which begins to degenerate after puberty, leading to a decrease in T cells. Professor Ryo Yasube of Tokyo University of Science in Japan, who has long studied immunology, said, "The biggest reason for the decline in immunity is the increase in age." After the body's immunity reaches its peak at age 20, the immune system gradually goes downhill with age; at age 40, immunity is only 1/2 of its peak, and at age 70, it is only 1/10 of its peak. storing immune cells helps intervene in the process of the immune system's decline.
C. Prevention of diseases caused by the decline of the immune system
The development of many diseases is closely related to the decline of the immune system. Due to the decline of the immune system in old age, the elderly are prone to immunodeficiency diseases such as infections, autoimmune diseases, and cancer. Therefore, some scholars consider aging as an "epidemic immune disease". One of the serious consequences of the decline of the immune system is that the "old eyes" mistake normal tissues or cells for the enemy and attack these tissues, causing inflammation and damage, resulting in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Storing immune cells and "refueling" the immune system at all times can help prevent these diseases caused by the decline of the immune system.
D. Counteracts aging of the body due to immune function decline
Scientists have found that the decline of immune function is related to human aging, and thus have proposed the immune theory of aging. The function of the immune system decreases with age, and when it reaches old age, it is unable to recognize subtle changes in cells or molecules in the body, and even if it can, it cannot mobilize the immune response to remove them effectively. As a result, the incidence of malignant cells increases in old age. In addition, the decline in immune function leads to a weakening of the immune system's stimulatory effect on the nervous and endocrine systems, resulting in a decrease in nerve cells, memory loss, slower response, reduced stress function, and atrophy and decreased function of the male testes and female ovaries, among other signs of human aging. It stores immune cells, replenishes them when needed, enhances immune function, kills and removes malignant cells, senescent cells, and dead cells, and thus achieves the purpose of anti-aging.
Why immune cells are stored in freezer boxes?
A. Reasonable allocation
Peripheral blood immune cell storage single collection of blood is generally 150-200ml, after the separation and purification of the components are mainly lymphocytes, a small number of monocytes and granulocytes, etc., the average recovery rate of about 80%, the normal human lymphocytes are generally 1 × 10^6 ~ 2 × 10^6 / ml, so the final immune cells can be isolated about 1.5 × 10^8 ~ 3 × 10^8 8, that is, 1.6×10^7 to 3.3×10^7 cells per tube freezing specification.
The current immune cell technology applications mainly include CIK, NK, NKT, CTL, CAR-T, CAR-NK, etc. The number of cells required for a single application is about 30 million, i.e., the number of application tubes is 1 to 2. 9 tubes can fully meet the cell culture application for 3 to 4 times of a course of treatment.
B. Easy management
The lyophilized tubes containing cells cannot be placed directly into liquid nitrogen and require a spacer and support device in between - a freezer box
. The conventional model of lyophilization cassette
is a 9×9 square box, where 9 tubes of cells correspond to one column for one sample, which is also convenient for future management and use.
C. Reduce waste and risk
The capacity of a single lyophilization tube is fixed, and too few lyophilization tubes may result in too many cells in a single tube when the number of cells is applied leading to cell waste, and the cells with too much lyophilization density are in a poorer state after resuscitation and the number of cell applications is reduced. Too many lyophilization tubes again make the number of cells in a single tube insufficient for the application, thus requiring multiple resuscitations, and too much lyophilization solution will lead to lower recovery and also cell waste. At the same time, too many lyophilization tubes mean too much contact, which tends to increase the risk of contamination during resuscitation operations. Therefore, it is best to apply 1 to 2 tubes per resuscitation and to make full use of the cells.
Features of freezer boxes
a. Keeping your samples safe
b. Automated sample management
c. Easy grabbing
d. More convenient options
e. Easy to use
f. Free of alcohol and other liquid substances
g. Lower cost compared to alcohol-based lyophilization equipment
h. Higher cell recovery ratio and cell viability
i. Good reproducibility
j. Easy to use, easy to standardize the operation
k. Different materials such as plastic and paper lyophilization boxes for long-term or short-term storage.
l. Location identification on the lid of all lyophilized boxes.
m. Plastic boxes that can be autoclaved and used in the temperature range of -196°C to +121°C.
n. Partitions and ventilation holes at the bottom of the lyophilization box cassette for liquid outflow and air circulation.
o. Provide a variety of specifications, such as 81 holes 2 inches, 100 holes 2 inches, etc.
How to perform cell cryopreservation?
Cell cryopreservation is a technique for storing cells in a low-temperature environment to reduce cell metabolism and achieve long-term storage. In most cell lines, cells accumulate changes as they age and evolve, resulting in phenotypic and genotypic "culture drift", and proper handling and appropriate freezing conditions during the freezing process can reduce the change or loss of cellular characteristics and serve as a cell conservation tool. Thus, proper and successful freezing plays a very important role in the long-term application of cells.
A. Proper culture and gentle cell collection
Cells should be in optimal growth condition (in logarithmic or exponential phase) prior to lyophilization, and ideally, the medium should be changed 24 h prior to harvest. It is recommended that cultures be tested for microbial contaminants, especially mycoplasma, to ensure that the cells are free of any contamination. During the collection of cells, experimental manipulations should be as gentle as possible to avoid cell damage.
B. Appropriate low-temperature protectant
Currently, the most commonly used technique for cell cryopreservation is liquid nitrogen cryopreservation, which mainly uses a slow freezing method with an appropriate amount of protective agent to freeze cells and reduce the damage to cells during the freezing process. When cells are directly frozen without any protective agent, the water inside and outside the cells will quickly form ice crystals, which will cause a series of adverse reactions.
Polyvinylpyrrolidone, ethylene glycol, methanol, and methyl acetamide are all cryoprotectants. The most common in cell cryopreservation is dimethyl sulfoxide (DMSO) and glycerol, both of which have small molecular weights, high solubility, penetrate cells easily, lower the freezing point, improve the permeability of cell membranes to water, and crucially, are not significantly toxic to cells. the usual concentration of DMSO is 5 - 10% (v/v), and the optimal concentration varies with the cell line. The final concentration of glycerol in the freezing medium is 5 - 15%, again, the optimal concentration depends on the cell line. To improve the survival of more difficult to preserve cells, the concentration of serum in the preservation medium can be optionally increased when freezing. For faster and more stable cell cryopreservation, a cell cryopreservation solution would be a good choice. No preparation is required, just add the appropriate amount of cryopreservation solution directly to the cells, and you can have high viability cells with a simple operation.
C. Slow freezing speed
Slow freezing is very important for the recovery of cell viability. For most animal cells, a decrease of -1 to -3°C per minute will allow the cells to better adapt to the frozen state. A common method used in most laboratories today is gradient cooling: 30 min at 4°C, then -20°C for 2h, followed by overnight freezing in a -80°C refrigerator, and transferring the tubes to a liquid nitrogen tank the next day for long-term storage. A reusable cell lyophilization cassette can save the time of repeatedly transferring cells several times by putting the prepared cell lyophilization tubes into the cassette and transferring them to a liquid nitrogen tank directly after overnight at -80°C. Regardless of the lyophilization method used, it is important that the transfer of the storage location must be done quickly, and it is recommended that the lyophilization tubes be placed in dry ice at a constant temperature to avoid the effect of temperature changes on cell viability during the transfer.
D. Continuous low-temperature storage
The cell temperature should always be kept below -130°C for optimal survival. If the storage temperature is not well controlled, the survival rate will decrease with time. It is recommended that cells be stored under liquid nitrogen conditions for a long period of time. The tightness of the liquid nitrogen tank and the adequacy of the liquid nitrogen volume need to be manually checked periodically to avoid damage to the cells due to storage conditions.
How to buy freezer boxes?
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