1. Storage temperature
Long-term storage of biological samples usually uses the lowest possible temperature to reduce the biochemical reactions in the sample and improve the stability of various components in the sample. Common storage temperatures for biological macromolecules, cells, tissues and organs are -800C (ultra-low temperature refrigerator), -1400C (liquid nitrogen phase or cryogenic refrigerator) and -1960C (liquid nitrogen liquid phase), the lower the temperature, the stable storage time of the sample The longer. 0 ï½ž -600C is the crystallization temperature of water, which is easy to damage the microstructure of cells and tissues. Generally, this temperature is not used to preserve tissues and cells. Some purified biomacromolecules can be stored stably at 0 ï½ž -600C for a certain period of time, but in tissue samples, biomacromolecules are affected by various factors in the cell tissue, and their stability may be significantly reduced. ~ -600C as storage temperature.
Biological sample storage applications0 ï½ž -600C is the crystallization temperature of water in tissues and cells. When the temperature enters this range, the water in the tissue starts to crystallize and damage the microstructure of cells and tissues. Various freezers can maintain the stability of purified biomacromolecules in the medium and long term, but cannot maintain the stability of the biomacromolecules in the tissue, cell activity, and tissue microstructure.
The ultra-low temperature refrigerator can maintain the stability of biological macromolecules in the tissues in the medium term; it can maintain cell activity and tissue microstructure in the short term. -1360C is the glass transition temperature of water, the crystallization of water no longer has obvious damage to cells and tissues, and the various biochemical reactions in the sample have almost stopped. Liquid nitrogen tank / tank gas phase;
-800C is a safe temperature below the crystallization temperature of water, and the biochemical reaction in the sample is significantly weakened. It is also the lowest temperature that can be used in automated storage devices.
Cryogenic refrigeratorIt can maintain the stability of biological macromolecules, cells and tissue microstructures in tissues in the medium and long term. It is recommended for many sample libraries for long-term frozen storage of tissues. -1960C is the temperature of liquid nitrogen evaporation, which is the lowest temperature that can be achieved by conventional methods. The various biochemical reactions in the sample can be considered to be stopped, and the damage of the crystallization of water to the cells and microstructures can be ignored. The liquid nitrogen tank / tank liquid phase can maintain the stability of biological macromolecules in the tissue, cell activity and tissue microstructure for a long time, but has higher requirements for storage consumables.
-800C sample storage
-800C is lower than the crystallization temperature range of the more harmful water, and it is also the temperature that can be reached by ultra-low temperature refrigerators of common equipment. It is considered based on factors such as ease of operation, storage capacity and cost. Common temperature for activity. However, it is still inconclusive as to how long different biological macromolecules can remain at this temperature. The stability of DNA in tissues can be maintained at -800C for several years or longer. However, for RNA, it is easy to be gradually degraded by RNases widely distributed in cells and various tissues. In different cells and tissues, the length of stable storage of RNA also varies greatly, but generally does not exceed 5 years In some sensitive experiments, the measured degradation of RNA occurred at less than one year at -800C, so for long-term preservation of RNA activity, it is recommended to use a lower temperature, or use a small part of the sample to extract RNA and synchronize with the remaining samples Save. Biomacromolecules such as proteins and lipids in other samples can also be stored in the sample at -800C, but the time varies, and the stability gradually declines. If it is to protect a specific biological macromolecule known in the sample, a stabilizer of the molecule can also be added. If the biomacromolecule to be stored in the sample is undecided, it is recommended to use a lower temperature storage. In addition, the current common large-scale automated sample access equipment can only be used with -800C ultra-low temperature equipment, but not with liquid nitrogen equipment. In the UK, UK Biobank stores a copy of a part of the sample (~ 9.5 million) at -800C as a working sample and uses automated equipment to store it; another part of the copy (~ 5.5 million) is stored in another place in the liquid nitrogen phase for safe backup , Manual access to samples.
-1400C sample storage
-1400C is lower than the glass transition temperature of water (~ -1360C), which is also the temperature that liquid nitrogen phase and cryogenic refrigerator can reach. The biological activity of the sample is greatly reduced within this temperature range, which is to preserve the cell activity in the sample Ideal temperature. Similar to the ice-water mixture that can maintain a phase transition temperature of 00C, the liquid and gaseous nitrogen in an insulated liquid nitrogen container should be maintained at a phase transition temperature of -1960C. But in fact, because the lid of the liquid nitrogen container is not sufficiently sealed, a temperature gradient is formed between the liquid nitrogen liquid surface and the liquid nitrogen container tank mouth. The National Cancer Institute recommends that the temperature at the mouth of the liquid nitrogen container should be kept below -1400C. Samples with undetermined future use should be stored in liquid nitrogen phase mode to protect the cell activity in the tissue.
Cryogenic refrigerators are electrically cooled and do not require liquid nitrogen. The stable temperature after filling the sample is usually below -140. Compared with the use of liquid nitrogen, the advantage is that it does not require frequent addition of liquid nitrogen and is easy to maintain. However, the temperature reduction rate of electric refrigeration is lower than that of liquid nitrogen. Once the container is opened to take samples, it is easy to cause a wide range of temperature fluctuations and the temperature recovery time is relatively long. Therefore, it is more suitable for the case of less open samples. In addition, electric refrigerators must guarantee power supply. It is recommended to use liquid nitrogen equipment in case of power failure to provide backup storage.
-1960C sample storage
-1960C is the temperature at which liquid nitrogen volatilizes, so only liquid nitrogen liquid storage technology can reach this temperature. The life activities in the sample basically stop at this temperature, and the stability of the sample can be preserved for a long time. It is the most effective method for long-term preservation of cell activity, complex structure and activity of tissues and organs in samples, and is widely recognized. Compared with other freezing modes at different temperatures, the liquid nitrogen container liquid storage samples need to be further protected against cross contamination between samples. The National Cancer Institute recommends the use of spiral cryovials for packaging samples. However, when the sample was transferred from the ultra-low temperature refrigerator (-800C) to liquid nitrogen during the cooling process, the temperature suddenly dropped, causing the inconsistency of the shrinkage of the cryotube cap and the tube body, which easily caused liquid nitrogen to penetrate into the cryotube and increased the risk of cross contamination between samples . One of the solutions is to use a special cryopreservation tube sleeve to heat-shrink seal each cryopreservation tube, or use a sealing film to wrap around the interface between the cryopreservation tube cap and the tube body and store it. The former method will lengthen the tube body and require a higher freezing storage box. The second method will make the tube body slightly thicker. It is recommended to use a conventional frozen storage box with a 10x10 space at the bottom.
2 Freezing technology
In the 1950s and 1970s, Basil Luyet, James Lovelock, Peter Mazur and other scholars conducted in-depth studies on the effects of freezing on cells and tissues, and found that the damage mainly originated from ice crystal damage and solution osmotic pressure damage. As the temperature drops, the water inside and outside the cells freezes, and the ice crystals that are formed can cause damage to the cell membrane and cell wall, resulting in cell death. This cell damage caused by freezing inside the cell is called Intracellular ice damage. Ice crystal damage is caused by excessive cooling speed. The faster the cooling speed, the greater the ice crystal damage. At the same time, as the temperature drops, the water outside the cell will freeze first, so that the electrolyte concentration in the unfrozen solution will increase, and the lipid on the cell membrane will be damaged by long-term exposure to the high solute solution. The cells are dehydrated and leak, causing a lot of water to penetrate into the cells during rewarming and causing cell death. This type of cell damage caused by the increase in the solute concentration of the storage solution is called solution damage. Solution damage is caused by the slow cooling rate, which causes the cells to be exposed to a high concentration of solution for too long. The slower the cooling rate, the more serious the damage.
Program cooling / stepwise freezing (programmed freezing / stepwise freezing)
By controlling the cooling rate of the sample, ice crystal damage and solution damage can be minimized. The cooling rate can be controlled by stepwise freezing or programmed freezing at different temperatures. Compared with the step-by-step cooling, the program cooling instrument can control the cooling rate of the sample itself more accurately by adjusting the flow rate of liquid nitrogen sprayed into the freezing storage space from time to time. For example, when a liquid sample of 0-50C is transformed into a solid and undergoes a phase change, heat is generated, causing the sample to return to temperature, causing additional damage to the sample. The program cooling instrument can increase the cooling strength during the phase change to avoid this injury. The program cooling device has been widely used in the frozen storage of sperm, eggs, fertilized eggs, stem cells, skin and other samples. This method is used by about 300,000 to 400,000 in vitro fertilized infants worldwide. A freezing procedure usually includes several stages, such as slow freezing and quick freezing. The common freezing speed in the presence of cryoprotectant is slow freezing 0.3-1.50C / min, quick freezing 5-80C / min. In the range of 0 ~ -400C, many programs use slow freezing. However, the specific cooling procedure may vary greatly depending on the frozen samples.
The theory of vitrification was first proposed by Luyet. The solidification of liquids can be divided into two forms: one is crystallization, and the molecules in the solution are arranged in an order; the other is the amorphous state, that is, vitrification, and the molecules in the liquid are in a disordered state, maintaining the state before unsolidification . During normal freezing, water easily forms crystals inside the cell, causing crystal damage, and forming crystals outside the cell causes solution damage. However, under the condition of ultra-fast vitrification and freezing, the cells are vitrified and solidified inside and outside. No ice crystals are formed or only small ice crystals are formed, which will not cause damage to the cell membrane and organelles, and the cells will not be in a high concentration of solute for a long time. Damaged by exposure. However, the ultra-high speed cooling required for vitrification freezing is difficult to achieve under routine experimental conditions. In 1981, Fahy proposed the use of a high-concentration cryoprotectant solution (vitrified solution) to greatly reduce the requirement for cooling rate, and completed the vitrification of rat embryos by Rail and Fahy in 1985 to achieve a breakthrough in the use of this technology. .
Vitrification is a relatively new technology, suitable for samples that are difficult to handle by routine procedures, such as complex tissues and organs. However, this technique has not shown a particular advantage for samples that can be handled well by conventional procedures for cooling (such as cells and small tissues). Normal vitrification of water requires extremely fast speed, which is difficult to achieve in conventional laboratories. In order to reduce the requirement of the vitrification temperature of the tissue, a high concentration of cryoprotectant needs to be added to the sample, and the commonly used concentration is about 40% to 60% (W / V). Even a few minutes can cause significant damage to cells and tissues. Therefore, although the vitrification freezing reduces the ice crystal damage and the solution damage in the freezing process, it has not shown a better effect in some comparative tests, but its complicated operation brought inconvenience. At present, in vitrification freezing, the method of reducing the damage of cryoprotectants is mainly to use a mixture of different cryoprotectants and ice blockers. Through this improvement, Twenty-First Century Medicine successfully transplanted frozen and thawed rabbit kidneys into rabbits and maintained normal function.
The freezing method is usually used to protect purified biological macromolecules or to extract tissue samples of biological macromolecules in the future. The method is to put the sample directly in liquid nitrogen, and after a short period of time, transfer it to an ultra-low temperature refrigerator or a lower temperature environment for storage. A more common example is freezing tissue used for future nucleic acid extraction. This method will damage most cells and fine tissue structures and cannot be used to preserve the activity of cells and tissues in the sample.
Cryoprotectant refers to a substance that can protect the microstructure of cells and tissues from freezing damage, and is usually formulated into a solution with a certain concentration. Adding cryoprotectant to the cell suspension can protect the cells from solution damage and ice crystal damage. The cryoprotectant combines with the water molecules in the solution to cause hydration, which weakens the crystallization process of the water and increases the viscosity of the solution to reduce the formation of ice crystals. At the same time, the cryoprotectant can reduce the concentration of electrolyte in the unfrozen solution inside and outside the cell by maintaining a certain molar concentration inside and outside the cell, so as to protect the cell from solute damage. Usually only red blood cells, most microorganisms, and very few nucleated mammalian cells are suspended in water without a cryoprotectant or a simple saline solution, and frozen at the optimum freezing rate to obtain a live frozen stock. However, for most nucleated mammalian cells, without the addition of cryoprotectants, there is no optimal freezing rate, and live frozen products cannot be obtained. For example, when suspending mouse bone marrow cells in a balanced salt solution without cryoprotectant, and freezing at a freezing rate of 0.3 to 6000C / min, more than 98% of the cells will die; while adding glycerol cryoprotectant for cryopreservation , Over 98% of cells can survive.
Cryoprotectants can be divided into two types, permeable and non-permeable, according to whether they penetrate cell membranes. Osmotic cryoprotectants are mostly small molecules that can penetrate into cells through the cell membrane. Such protective agents mainly include Dimethyl Sulfoxide (DMSO), glycerin, ethylene glycol, propylene glycol, acetamide, methanol, etc. Its protection mechanism is to penetrate into the cell before the cell freezing suspension is completely solidified, produce a certain molar concentration inside and outside the cell, reduce the concentration of electrolyte in the unfrozen solution inside and outside the cell, thereby protecting the cell from damage by high concentration electrolyte In addition, the water in the cells will not be excessively extravasated, avoiding excessive dehydration and shrinkage of the cells. At present, DMSO, glycerin, ethylene glycol and propylene glycol are mostly used. DMSO penetrates cells faster and has better protection during freezing. It is more commonly used and the commonly used concentration is 5% -10%. However, DMSO itself has obvious damage to cells, especially at a temperature of 40C. Therefore, it is not recommended to place the sample at this temperature for a long time after adding DMSO. It is usually used in conjunction with a program cooling instrument or gradient cooling box to achieve a continuous and uniform temperature reduction .
Non-permeable cryoprotectants cannot penetrate into cells, and are generally macromolecular substances, mainly including polyvinylpyrrolidone (PVP), sucrose, polyethylene glycol, dextran, albumin, and hydroxyethyl starch. There are many hypotheses about its protection mechanism. One possibility is that macromolecular substances such as polyvinylpyrrolidone can preferentially combine with water molecules in the solution, reducing the content of free water in the solution, reducing the freezing point and reducing the formation of ice crystals; at the same time, due to its The large molecular weight reduces the electrolyte concentration in the solution, thereby reducing solute damage.
Different cryoprotectants have different advantages and disadvantages. The current trend is to use a combination of more than two cryoprotectants. Because many cryoprotectants protect cells under low temperature conditions, they are harmful to cells at normal temperatures. Therefore, the cryoprotectant should be removed in time after the cells are rewarmed.
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