Cell cryopreservation Laboratory cryopreservation equipment includes: liquid nitrogen storage tank, liquid nitrogen tank, -140 ° C / -150 ° C cryogenic cryopreservation tank, -86 ° C ultra-low temperature refrigerator, program-controlled cooling instrument, low temperature refrigerator (-20 ° C, -30 ° C, -40 ° C), drug storage box, vaccine storage box, blood bank refrigerator, chromatography cabinet, explosion-proof refrigerator, fire-resistant refrigerator, etc. Methods for verifying the reliability and cooling capacity of the refrigerator: 1. Place the thermometer in a conventional refrigerator; 2. Place the heater in a cryogenic refrigerator, take a large number of samples, or open the door repeatedly. The need to cryopreserve tissues and whole organs has facilitated the development of cryopreservation methods, and the new methods have also improved the recovery of cryopreserved cells and organisms. These methods include varying the concentration of cryoprotectant and adding additives to avoid apoptosis or programmed cell death caused by the cryopreservation process. It has been thought for many years that it is the physical change or damage in the cells produced during the cryopreservation process, resulting in the death of cells after cryopreservation. Recent studies have found that some of the more elaborate intracellular activities may lead to cell death, and these activities can be controlled to some extent by appropriate cryopreservation additives. During the cryopreservation process, the cryoprotectant has multiple functions. For example, DMSO can lower the freezing point and promote more complete dehydration of cells before intracellular ice crystal formation. It is generally believed that the cryopreservation effect is best when the cryoprotectant can effectively penetrate the cells, delay the formation of intracellular ice crystals, and reduce the solution effect. In addition, it is necessary to select a cryoprotectant depending on the type of cell to be frozen. Glycerin is a better choice for most cells because it is less toxic than DMSO. However, DMSO has better permeability and is usually frozen as larger, more complex cells, such as protists. The cryoprotectant should be diluted to a suitable concentration with fresh medium prior to addition to the cell suspension, which minimizes potential chemical damage and ensures that the cells are uniformly exposed to the protective agent, reducing toxic effects. DMSO and glycerol are typically used in concentrations ranging from 5-10% (v/v). In addition to being used in plant cells, they are generally not used in combination. The optimal concentration of cryoprotectant depends on the cell type and the highest concentration that the cell can tolerate. For some materials, the sensitivity of the cells is measured by increasing the concentration of the protective agent, which helps to select the optimal working concentration of the protective agent. The Quick- Reference Chart (page 4) lists the recommended concentrations of protectants for different types of cells and can be used as a reference for selecting suitable protectants. 2. Preparation of Biomaterials Preparations prior to cryopreservation of biological materials may have an impact on the results of cryopreservation. For non-replicating materials, such as tissues, nucleic acids, and proteins, the preparation process involves confirming that the biological material is in a suitable solution or cryopreservation medium. The purpose of this step is to maximize the intended use during resuscitation. However, the stability and revitalization of living cells and microorganisms are affected by growth conditions and preparations prior to cryopreservation. A variety of factors need to be considered before cryopreservation, including cell type, viability, growth conditions, physiological status, number, and cell pretreatment. When establishing a primary seed reserve for a new isolate or cell line, the culture must be tested and disinfected to ensure minimal microbial contamination. Repeat this step each time you prepare for the work, and each time you prepare a new batch of culture. 2, 1 Microbial cells grown under aerated culture conditions, particularly bacteria and yeast, exhibit greater resistance to cooling and cryopreservation than cells grown under non-ventilated conditions. T. Nei believes that under aeration culture conditions, cell permeability is better, and cells cultured under open conditions lose water faster during cooling than cells cultured under non-ventilated conditions. In addition, for microbial cells, cells obtained in the late logarithmic growth phase and early in the stationary phase showed stronger freezing tolerance. Microbial cells collected from late growth and early static culture showed stronger freezing tolerance than cells in early growth and late growth. In general, the greater the number of cells initially frozen, the higher the rate of recovery. For most bacteria and yeast, a cell count of approximately 107/ml is guaranteed to ensure a sufficient number of cell resuscitation. Since these cells can be conveniently collected from agar plates or slanted surfaces, if a larger number of cells are required, the cells can be grown in a liquid medium and collected by centrifugation. In either case, the cells are usually suspended in a cryopreservation. Protectant in fresh medium. Protozoa can be concentrated by centrifugation, but usually need to be suspended in the medium used and then diluted with an equal volume of fresh medium containing cryoprotectant. For cryopreservation of the spores, the spores need to be collected and resuspended in fresh medium containing the cryoprotectant. Before cryopreservation, the operating time must be shortened and the spores are not germinated. For non-spore cryopreservation, special procedures are required to collect mycelium before cryopreservation. For some fungi with hard mycelium, agar blocks containing mycelia are cut out from the medium and placed in fresh medium containing cryoprotectant. The hard mycelium does not adhere well to the agar medium. When growing in the broth medium, the mycelial mass is mixed before cryopreservation. The viability and recovery rate of frozen material is determined by the culture and growth state before and after cryopreservation. Vitality is an indicator of the growth and reproduction of cultures. For example, certain materials such as probiotic materials need to be passaged several times to ensure their stability. There are several methods for estimating the number of resuscitated cells, including serial dilution, plate count or direct cell count. By comparing the difference in the number of cells before and after cryopreservation, the degree of cell resuscitation or the success of the cryopreservation process can be demonstrated. 2, 2 Mammalian cells When mammalian cells are ready for cryopreservation, the cells need to be adjusted to a suitable growth state, ensuring that sufficient resuscitated cells are obtained without a large number of cells that are not essential for growth. For most mammalian cells, the optimal number of starting cells is around 106-107/ml. To facilitate the addition of an equal volume of cryoprotectant (2 x cryoprotectant + medium), the cell suspension concentration should be prepared at twice the initial concentration. Alternatively, the pelleted cells can be resuspended in a cryoprotectant (1 x cryoprotectant + medium) to achieve the desired number of cells. Cell manipulation should be gentle and careful to ensure that the cells are healthy before cryopreservation. Try to avoid severe blows and high speed centrifugation. If appropriate, 5% or 10% CO2 can be injected to adjust the pH. Mammalian cell culture is particularly sensitive to contamination by other cells and microorganisms, such as Hela cells. Due to this property, the initial cell line can be detected by isozyme analysis, karyotyping, immunoassay or genomic analysis. The above detection should be performed before and after cryopreservation. Of particular concern is the contamination of viruses and mycoplasma. A complete and robust system of mammalian cell line identification procedures should include the detection of contamination by bacteria, fungi, viruses, mycoplasmas, and even protist cells. 2, 3 Stem cell stem cells are cryopreserved in a manner similar to mammalian cells, except for steps that partially enhance resuscitation and clonal growth activity. Generally, DMSO is used as a cryoprotectant, and sometimes serum is used. It is recommended to freeze it by slow cooling. Trehalose is used in combination with other cryoprotectants to reduce potential damage to cells. At the same time, it is recommended to recover quickly by rapid heating. Cell viability of resuscitation may vary from cell to cell depending on cell type. Vitrification can also be used to cryopreserve stem cells. The protocol includes suspending cells in a concentrated mixture comprising a plurality of cryoprotectants. In addition, the operations required for cooling and cryopreservation and recovery are very fast, avoiding the formation of ice crystals. Some studies have shown that vitrification leads to higher cell viability. DMSO, glycerol and propylene glycol are commonly used protectants for effective cryopreserved stem cells. 2, 4 Plant cells Plant cells cryopreserved similar to other cells. The growth phase of the cells affects the resuscitation effect, and the most suitable growth phase is the late logarithmic growth phase. In addition, cell density also greatly affects the resuscitation effect, and the most suitable cell density depends on the cell type. The stress-resistant culture of plants makes them more resistant to stressful environments, such as cryopreservation processes. Plants produce a number of compounds, such as sugars or glycerol, which protect cells from damage caused by changes in osmotic pressure. Undifferentiated callus cryopreservation is usually in order to obtain stability, and these traits are affected by continuous culture. Seed preservation is also a method of preserving stable germplasm, the most common method being preservation at low humidity and low temperature. However, some seeds can tolerate dehydration caused by cryopreservation, and such seeds can be frozen in liquid nitrogen temperature. 2, 5 viruses Most viruses can be directly frozen in the unprepared phase, and no cooling control is required. However, viruses that are cultured in the same manner as the host cells require cooling control during the cryopreservation process. In the case of a host cell virus, appropriate cryopreservation should be performed to ensure the vitality of the host cell. When a virus is obtained from an egg cell, the high protein material in the allantoic fluid or yolk sac can provide protection during cryopreservation. Plant viruses can be preserved either by infecting plant tissues or as pure viral preparations. Virus preparation is suspended in DMSO or other cryoprotectant prior to cryopreservation. Although most plant viruses can tolerate rapid cooling, proper control of the rate of cooling can provide the best recovery. The plant virus can be resuscitated by soaking it directly in a warm water bath, and then the resuscitation virus can be inoculated to a suitable plant host. 2, 6 Embryo embryos can be preserved by controlling the rate of cooling and vitrification. Resuscitation depends on the stage of embryonic development and is measured by successful implantation and into fetal development. 2,7 Non-replicating materials Non-replicating materials, such as whole blood, serum, tissue, nucleic acids and proteins, have no special requirements for the cryopreservation of materials. These materials are generally directly frozen, without the presence of a cryoprotectant, and with rapid cooling. However, the choice of the actual cryopreservation mode for these materials depends on the ultimate purpose of the material. To successfully retain the characteristics of whole blood after resuscitation, cryopreservation and cooling control are required, and the quality of the frozen tissue can be suspended by using materials such as optimal cutting temperature (OCT) composite materials. Palmitoyl Pentapeptide-4,argireline,GHK-cu,Acetyl,Hexapeptide-8 PYSON Co. ,Ltd. , https://www.pysonbio.com
1. Cryoprotective agents Many compounds can be used as cryoprotectants. They can be used alone or in combination, such as sugars, serums and detergents. Although there is no absolute guideline for cryopreservation, it is generally accepted that dimethyl sulfoxide (DMSO) and glycerol are the most widely used and most effective cryoprotectants for protecting living cells and tissues. Other cryoprotectants may be used according to the situation, and may be used alone or in combination, including: polyethylene glycol, propylene glycol, glycerine, polyvinylpyrolidone. , sorbitol, dextran, trehalose.
Factors affecting mammalian cell resuscitation include: (a) cell type, (b) growth phase, (c) which stage of the cell cycle the cell is in, and (d) the number and concentration of cells in the final suspension. The viability of the resuscitation cells can be improved by optimizing the above factors. In addition, the characteristics of the cryopreservation agent and the specific operation of the cryopreservation process need to be considered.
The combined use of cryoprotectants works better in most cases than in a single use. The cooling rate is very important. In many cases, the two-step cooling method has a significant effect, that is, the cells are first frozen at -30 ° C -40 ° C for a while and then cooled to liquid nitrogen temperature. This method enhances cytoplasmic dehydration prior to cryopreservation. A quick-recovery mode of resuscitation is usually recommended, but in some cases a slow rewarming mode is better. Plant cells can also be cryopreserved using vitrification by using concentrated cell suspensions and rapid cooling.