CN108271770B - Application of micron particles in low-temperature freezing storage - Google Patents
Application of micron particles in low-temperature freezing storage Download PDFInfo
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- CN108271770B CN108271770B CN201710012559.XA CN201710012559A CN108271770B CN 108271770 B CN108271770 B CN 108271770B CN 201710012559 A CN201710012559 A CN 201710012559A CN 108271770 B CN108271770 B CN 108271770B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
Abstract
The invention discovers that the micron particles have good effect of inhibiting the growth of ice crystals, can inhibit the growth of the ice crystals in the processes of freezing, cooling and resuscitation, protects cells from mechanical damage caused by the growth of the ice crystals, and improves the survival rate of cell freezing and resuscitation. On the basis, the invention provides application of the micron particles in low-temperature cell cryopreservation, application of the micron particles in preparation of a cell cryopreservation protective agent or protective solution, and a method for performing low-temperature cell cryopreservation by using the micron particles.
Description
Technical Field
The invention relates to application of micron particles in low-temperature cryopreservation, in particular to application of micron particles in low-temperature cryopreservation protection of cells, and belongs to the field of biochemical materials.
Background
With the development of society, techniques such as regenerative medicine and organ transplantation have attracted high attention in the medical field, and the treatment of many diseases has been gradually achieved. The need for differently matched cells, tissues and organs is therefore increasingly acute in the medical field. For example, in the uk 6000 units of blood are required per day, but blood can only be stored effectively for 42 days without cryopreservation, and has a significant rate of hemolysis in simple cell isotonic solutions. Although more and more cells, tissues and organs are donated and tissue engineering products are commercialized, there are still many patients who lose their chance of treatment because of the absence of a suitable ligand, and many donated cells, tissues and organs are damaged because of the failure to find a suitable recipient. In the united states, the waitlist for organ transplantation currently exceeds 118,000 people (5 months 2013), while fewer than 30,000 transplant procedures were performed in 2012. Under such circumstances, it is required that no organ is wasted, and thus there is a strong demand for workers in the related art to find a method for effectively preserving cells, tissues and organs for a long period of time so as to supply them in time when needed by a patient.
Cryopreservation of cells, tissues and organs is a traditional but effective preservation method, but during cryopreservation, ice formation and growth can lead not only to mechanical damage to the cells, but also to osmotic shock due to a decrease in liquid water volume fraction and an increase in extracellular solute concentration. The nature provides a good model for the research of the anti-icing material. For example, insects in cold regions, and fish in polar regions can survive in very low temperature environments. In 1969, DeVries at Stanford university found anti-freeze proteins in the blood of a cold water fish living in Antarctic. After that, scientists have found a plurality of anti-freeze proteins in polar fishes, insects, plants and the like. Antifreeze proteins (AFPs) are a class of proteins that inhibit ice crystal growth and act to depress the freezing point of water in a non-colligative manner with little effect on its melting point, resulting in a difference between the melting point and the freezing point of water, known as thermal hysteresis activity. Compared with the molar concentration, the freezing point lowering efficiency of the antifreeze protein is 200-500 times that of the colligative salt, which shows that the freezing point lowering mechanism of the antifreeze protein is different from that of the colligative salt. When the antifreeze protein is dissolved in water, molecules of the antifreeze protein can be selectively adsorbed on certain crystal faces of ice crystals, so that the growth habit of the ice crystals is changed, the morphology of the ice crystals is changed, and the growth rate of the ice crystals is obviously changed compared with that of pure water. However, the use of antifreeze proteins in low temperature cryopreservation presents two problems: firstly, the antifreeze protein can only be separated from the organism, and the yield is very low; secondly, the effect of freezing and storing cells at low temperature by using the antifreeze protein is much worse than expected, and sometimes even the survival rate of the cells is reduced, and researches show that the antifreeze protein is mainly caused by the characteristics of thermal hysteresis activity.
Although the application of the antifreeze protein in low-temperature cryopreservation is limited, many researchers synthesize many macromolecules or glycoprotein to inhibit the recrystallization of ice by simulating the characteristic that the antifreeze protein inhibits the growth of ice crystals, and further realize the application of the new materials in the field of low-temperature cryopreservation. For example, Matthew i.gibson effectively inhibits the recrystallization of ice by synthetic polyvinyl alcohol (PVA), and when PVA is added to a cell cryopreservation solution, the cell survival rate can be greatly improved. However, from the viewpoint of practical use, the polymer is added to the cell preservation solution to form a stable solution state, and it is difficult to separate the polymer from the system. In addition, certain nanoparticles such as graphene oxide, zirconium oxide and the like can generate specific adsorption with a certain crystal face of ice, and can also realize the recrystallization process for inhibiting the ice. However, nanoparticles, because of their small size, are able to enter cells and even nuclei, and their toxicity to cells is unknown. In addition, the easy aggregation of nanoparticles has also limited their application in low temperature cryopreservation of cells. Therefore, the biocompatibility and the ability to be metabolized by the body of synthetic polymeric materials and nanoparticles become major problems limiting their applications.
There is still a need in the art to develop materials that effectively inhibit the recrystallization of ice, improve the cell viability when used in cryopreservation of biological materials such as cells at low temperatures, have low cytotoxicity, and are easily removed from the cryopreservation system.
Disclosure of Invention
In order to solve the problems of biotoxicity, difficulty in separation from a low-temperature protection system, incapability of recycling, difficulty in preparation and the like of the conventional cell low-temperature freezing protective agent, the inventor of the invention finds that the micron particles have a good effect of inhibiting the growth of ice crystals through scientific research, and can inhibit the growth of the ice crystals in the processes of freezing, cooling and resuscitation, so that cells are protected from mechanical damage caused by the growth of the ice crystals, and the cell freezing and resuscitation survival rate is improved.
After the micron particles are added into a cell preservation solution system, the existence of the micron particles not only provides heterogeneous nucleation points in the low-temperature cryopreservation and cooling process, but also can effectively inhibit the growth of ice crystals in the heating process, so that the cells can obtain higher survival rate in the low-temperature cryopreservation. This effect of the microparticles may reduce the amount of other cryoprotectants, such as polyethylene glycol, polyvinyl alcohol or DMSO, used in the cryo-cryopreservation solution of the cells, thereby reducing the toxic effect of these cryoprotectants on the cells.
The inventor finds that the effect of the micron particles on inhibiting the ice crystal growth is a property related to the particle size, which is not influenced by the material of the micron particles and the chemical groups on the surfaces of the micron particles, and the micron particles are the biggest difference from the nanometer particles. The characteristic of the micron particles enables the micron particles to be widely applied to various low-temperature frozen stock solution systems, namely, the micron particles have no selectivity suitable for the systems; in addition, it also allows the use of a variety of microparticles for low temperature cryopreservation, i.e., no selectivity in particle chemistry.
The present invention has been accomplished based on the above objects and findings.
The terms:
in the present invention, "cryopreservation at low temperature" means that a substance is stored at a temperature of 0 ℃ or lower. For example, a low-temperature refrigerator with the temperature of-4 ℃ to-20 ℃ is adopted, and a refrigerant such as liquid ammonia gas, difluorodichloromethane, difluorochloromethane, trifluorochloromethane and the like provides low-temperature conditions; or a cryogenic refrigerator with the temperature of-70 ℃ to-80 ℃, dry ice (-78 ℃), liquid nitrogen (-196 ℃) or liquid nitrogen steam phase (-140 ℃) is frozen instantaneously, and the materials are preserved for a long time at ultralow temperature.
The low-temperature freezing protective agent has the same meaning as a freezing protective agent, an antifreeze and an anti-freezing agent, and is used for preventing ice crystals from being formed or growing to cause damage in the processes of temperature reduction and temperature rise of low-temperature freezing. The substance can protect materials stored at low temperature, such as cells, and resist damage caused by low temperature to cells, such as intracellular freezing, dehydration, increased solute concentration, protein denaturation and damage to the skeleton structure thereof.
The "low-temperature cryopreservation protection system" in the present invention is the same as the "low-temperature cryopreservation protection solution", "cryopreservation solution", and "cryopreservation protection solution", and is a liquid system for low-temperature cryopreservation containing a low-temperature cryopreservation protective agent and a low-temperature cryopreservation liquid.
The term "cell" in the present invention is consistent with the meaning of the term of art. The cells include prokaryotic cells and eukaryotic cells. Prokaryotic cells include fungi, bacteria, rickettsia, mycoplasma, chlamydia, spirochetes, algae. Eukaryotic cells include animal cells and plant cells. In the present invention, "animal" includes human.
The "particle size" of the microparticles in the present invention is the particle diameter for spheroids; equivalent volume diameters for geometries of other shapes, i.e., the diameter of a sphere of the same volume as the actual particle, can be measured using various laser methods known in the art.
In a first aspect the invention provides the use of microparticles for cryopreservation of cells.
According to the invention, the microparticles have a particle size in the range of 0.5 to 100 μm. Preferably 1 μm to 20 μm, more preferably 3 μm to 10 μm.
The material of the microparticles is not particularly limited, and any water-insoluble material that is biocompatible and non-bioactive can be used for the application of the present invention. The materials may include, but are not limited to: inorganic materials, such as: silicon dioxide, aluminum oxide, titanium dioxide, hydroxyapatite, ferroferric oxide, calcium carbonate, barium sulfate and the like; organic polymer materials such as polystyrene, polymethyl methacrylate, polylactic acid, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polycarbonate, polycaprolactam, polyvinyl chloride, polyvinyl alcohol, polyethylene glycol and the like. From the viewpoint of the cheapness of material acquisition, it is preferable that the microparticles are microparticles of silicon dioxide, aluminum oxide, titanium dioxide, hydroxyapatite, ferroferric oxide, calcium carbonate, barium sulfate, polystyrene, or polymethyl methacrylate.
According to the present invention, the surface of the microparticles may be further modified with different chemical groups, including but not limited to: hydroxyl, carboxyl, amino, aldehyde, amide, epoxy, or the like, or a polymer chain such as polyethylene glycol, polyvinylpyrrolidone, or the like. From the viewpoint of the cheapness of surface modification of microparticles, hydroxyl groups, carboxyl groups and/or amino groups are preferably modified on the surface of microparticles.
According to the invention, the microparticles are geometric bodies of any shape, including spherical bodies.
In one embodiment of the invention, the microparticles are surface unmodified or modified silica, polystyrene or polymethylmethacrylate microparticles and the modified groups are hydroxyl, carboxyl and/or amino groups.
According to the invention, the application mode of the micron particles in the low-temperature cell cryopreservation is to add the micron particles into the liquid for low-temperature cell cryopreservation. The microparticles may form a stable or unstable suspension in the liquid.
The liquid in which the cells are cryopreserved can be any liquid that is capable of dispersing the cells and is biocompatible, including, but not limited to, water, physiological saline, a buffer, a balanced salt solution, an aqueous glycerol solution, a cell culture solution, and the like. The buffer is, for example: tris buffer, HEPES buffer, TB buffer, phosphate buffer, acetate-acetate buffer, carbonate buffer, and the like; the balanced salt solution is, for example: PBS solution, Hanks solution, D-Hanks solution, Ringer solution, Tyrode solution, Earle solution, etc.; the aqueous glycerol solution is, for example, a 4-50% aqueous glycerol solution; the cell culture solution is, for example: LB medium, MS medium, B5Culture medium, M199 culture medium, CMRL1066 culture medium, Eagle culture medium, BME culture medium, MEM culture medium, Alpha-MEM culture medium, DMEM culture medium, McCoy5A culture medium, PRMI 1640 culture medium, NCTC 109 culture medium, NCTC 135 culture medium, F12G culture medium, DM160 culture medium, MD752/1 culture medium, HIWO5 culture medium, etc.
The low-temperature freezing protection system containing the liquid and the microparticles can contain or not contain other low-temperature freezing protection agents except the microparticles. The other cryoprotectant may be an osmotic cryoprotectant, including but not limited to glycerol, dimethyl sulfoxide, propylene glycol, ethylene glycol, acetamide, and the like; may be an impermeable cryoprotectant, including but not limited to sucrose, polysucrose, hydroxyethyl starch, dextran, bovine serum albumin, fetal bovine serum, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, or the like; it may also be an antifreeze protein.
The content of micrometer particles in the protective solution for freezing cells at low temperature is 1 × 10‐15‐1×10‐12M, preferably 1X 10‐14‐1×10‐13M, more preferably 2X 10‐14‐7×10‐14M。
According to the invention, the micron particles are added into the liquid for freezing the cells at low temperature, and other low-temperature freezing protective agents can not be added or the addition amount is reduced.
According to the invention, the low-temperature cryopreservation of cells is preferably the low-temperature cryopreservation of eukaryotic cells, more preferably the low-temperature cryopreservation of animal cells.
According to the invention, the low-temperature cryopreservation is preferably ultra-low-temperature cryopreservation.
In a second aspect of the invention, a method for cryopreserving cells is provided.
According to the invention, the method consists in adding microparticles to the liquid in which the cells are frozen at low temperature.
According to the invention, the microparticles have a particle size in the range of 0.5 to 100 μm. Preferably 1 μm to 20 μm, more preferably 3 μm to 10 μm.
The material of the microparticles is not particularly limited, and any water-insoluble material that is biocompatible and non-bioactive can be used for the application of the present invention. The materials may include, but are not limited to: inorganic materials, such as: silicon dioxide, aluminum oxide, titanium dioxide, hydroxyapatite, ferroferric oxide, calcium carbonate, barium sulfate and the like; organic polymer materials such as polystyrene, polymethyl methacrylate, polylactic acid, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polycarbonate, polycaprolactam, polyvinyl chloride, polyvinyl alcohol, polyethylene glycol and the like. From the viewpoint of the cheapness of material acquisition, it is preferable that the microparticles are microparticles of silicon dioxide, aluminum oxide, titanium dioxide, hydroxyapatite, ferroferric oxide, calcium carbonate, barium sulfate, polystyrene, or polymethyl methacrylate.
According to the present invention, the surface of the microparticles may be further modified with different chemical groups, including but not limited to: hydroxyl, carboxyl, amino, aldehyde, amide, epoxy, or the like, or a polymer chain such as polyethylene glycol, polyvinylpyrrolidone, or the like. From the viewpoint of the cheapness of surface modification of microparticles, hydroxyl groups, carboxyl groups and/or amino groups are preferably modified on the surface of microparticles.
According to the invention, the microparticles are geometric bodies of any shape, including spherical bodies.
In one embodiment of the invention, the microparticles are surface unmodified or modified silica, polystyrene or polymethylmethacrylate microparticles and the modified groups are hydroxyl, carboxyl and/or amino groups.
According to the present invention, the liquid for cryopreserving the cells may be any liquid capable of dispersing the cells and being biocompatible, including, but not limited to, water, physiological saline, buffer solution, balanced salt solution, glycerol aqueous solution, cell culture solution, and the like. The buffer is, for example: tris buffer, HEPES buffer, TB buffer, phosphate buffer, acetate-acetate buffer, carbonate buffer, and the like; the balanced salt solution is, for example: PBS solution, Hanks solution, D-Hanks solution, Ringer solution, Tyrode solution, Earle solution, etc.; the aqueous glycerol solution is, for example, a 4-50% aqueous glycerol solution; the cell culture solution is, for example: LB medium, MS medium, B5Culture medium, M199 culture medium, CMRL1066 culture medium, Eagle culture medium, BME culture medium, MEM culture medium, Alpha-MEM culture medium, DMEM culture medium, McCoy5A culture medium, PRMI 1640 culture medium, NCTC 109 culture medium, NCTC 135 culture medium, F12G culture medium, DM160 culture medium, MD752/1 culture medium, HIWO5 culture medium, etc.
The content of micrometer particles in the protective solution for freezing cell at low temperature comprises 1 × 10‐15‐1×10‐12M, preferably 1X 10‐14‐1×10‐13M, more preferably 2X 10‐14‐7×10‐14M。
According to the invention, the micron particles are added into the liquid for freezing the cells at low temperature, and other low-temperature freezing protective agents can not be added or the addition amount is reduced.
The other cryoprotectant may be an osmotic cryoprotectant, including but not limited to glycerol, dimethyl sulfoxide, propylene glycol, ethylene glycol, acetamide, and the like; may be an impermeable cryoprotectant, including but not limited to sucrose, polysucrose, hydroxyethyl starch, dextran, bovine serum albumin, fetal bovine serum, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, or the like; it may also be an antifreeze protein.
According to the invention, the low-temperature cryopreservation of cells is preferably the low-temperature cryopreservation of eukaryotic cells, more preferably the low-temperature cryopreservation of animal cells.
According to the invention, the low-temperature cryopreservation is preferably ultra-low-temperature cryopreservation.
In one embodiment of the present invention, the method for cryopreserving cells comprises the following steps:
1) adding the micron particles into the liquid for freezing the cells at the low temperature to prepare a protective liquid for freezing the cells at the low temperature; 2) adding cells to be cryopreserved at a low temperature into the low-temperature cryopreservation protective solution prepared in the step 1) to obtain a mixed system;
or, 1') adding the cells to be cryopreserved into the liquid in which the cells are cryopreserved at the low temperature; 2 ') adding the micron particles into the liquid which is prepared in the step 1') and contains the cells and is frozen at a low temperature to obtain a mixed system;
3) freezing and storing the mixed system obtained in the step 2) or the step 2') at a low temperature.
According to the invention, the microparticles can be suspended in the cell cryopreservation protective solution by means including but not limited to ultrasound, oscillation and the like.
Preferably, the microparticles have a particle size of 0.5 to 100 μm. Preferably 1 μm to 20 μm, more preferably 3 μm to 10 μm.
Preferably, the microparticles are microparticles of silica, polystyrene or polymethyl methacrylate whose surface is not modified or is modified with hydroxyl groups.
Preferably, the content of the micro-particles in the protective solution for freezing and storing the cells at low temperature is 1 x 10‐15‐1×10‐12M, preferably 1X 10‐14‐1×10‐13M, more preferably 2X 10‐14‐7×10‐14M。
Preferably, the operation steps of the step 3) low-temperature cryopreservation are as follows: directly placing the mixed system into dry ice, liquid nitrogen or liquid nitrogen vapor phase for freezing and storing.
In a third aspect of the invention, the application of the micron particles in preparing a cell cryopreservation protective agent or a cell cryopreservation protective solution is provided.
The size range of the micron particles is 0.5-100 μm. Preferably 1 μm to 20 μm, more preferably 3 μm to 10 μm.
The material of the microparticles is not particularly limited, and any water-insoluble material that is biocompatible and non-bioactive can be used for the application of the present invention. The materials may include, but are not limited to: silicon dioxide, aluminum oxide, titanium dioxide, hydroxyapatite, ferroferric oxide, calcium carbonate, barium sulfate and the like; organic polymer materials such as polystyrene, polymethyl methacrylate, polylactic acid, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polycarbonate, polycaprolactam, polyvinyl chloride, polyvinyl alcohol, polyethylene glycol and the like. From the viewpoint of the cheapness of material acquisition, it is preferable that the microparticles are microparticles of silicon dioxide, aluminum oxide, titanium dioxide, hydroxyapatite, ferroferric oxide, calcium carbonate, barium sulfate, polystyrene, or polymethyl methacrylate.
According to the present invention, the surface of the microparticles may be further modified with different chemical groups, including but not limited to: hydroxyl, carboxyl, amino, aldehyde, amide, epoxy, or the like, or a polymer chain such as polyethylene glycol, polyvinylpyrrolidone, or the like. From the viewpoint of the cheapness of surface modification of microparticles, hydroxyl groups, carboxyl groups and/or amino groups are preferably modified on the surface of microparticles.
According to the invention, the microparticles are geometric bodies of any shape, including spherical bodies.
In one embodiment of the invention, the microparticles are surface unmodified or modified silica, polystyrene or polymethylmethacrylate microparticles and the modified groups are hydroxyl, carboxyl and/or amino groups.
According to the present invention, the cell cryopreservation protective solution may further contain the above-mentioned cell cryopreservation solution.
According to the present invention, the cell cryopreservation protective solution may further contain the other cryopreservation protective agent described above.
In a fourth aspect of the invention, a protective solution for cryopreservation of cells is provided, wherein the protective solution contains microparticles.
The size range of the micron particles is 0.5-100 μm. Preferably 1 μm to 20 μm, more preferably 3 μm to 10 μm.
The material of the microparticles is not particularly limited, and any water-insoluble material that is biocompatible and non-bioactive can be used for the application of the present invention. The materials may include, but are not limited to: silicon dioxide, aluminum oxide, titanium dioxide, hydroxyapatite, ferroferric oxide, calcium carbonate, barium sulfate and the like; organic polymer materials such as polystyrene, polymethyl methacrylate, polylactic acid, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polycarbonate, polycaprolactam, polyvinyl chloride, polyvinyl alcohol, polyethylene glycol and the like. From the viewpoint of the cheapness of material acquisition, it is preferable that the microparticles are microparticles of silicon dioxide, aluminum oxide, titanium dioxide, hydroxyapatite, ferroferric oxide, calcium carbonate, barium sulfate, polystyrene, or polymethyl methacrylate.
According to the present invention, the surface of the microparticles may be further modified with different chemical groups, including but not limited to: hydroxyl, carboxyl, amino, aldehyde, amide, epoxy, or the like, or a polymer chain such as polyethylene glycol, polyvinylpyrrolidone, or the like. From the viewpoint of the cheapness of surface modification of microparticles, hydroxyl groups, carboxyl groups and/or amino groups are preferably modified on the surface of microparticles.
According to the invention, the microparticles are geometric bodies of any shape, including spherical bodies.
In one embodiment of the invention, the microparticles are surface unmodified or modified silica, polystyrene or polymethylmethacrylate microparticles and the modified groups are hydroxyl, carboxyl and/or amino groups.
The content of micrometer particles in the protective solution for freezing cells at low temperature is 1 × 10‐15‐1×10‐12M, preferably 1X 10‐14‐1×10‐13M, more preferably 2X 10‐14‐7×10‐14M。
According to the present invention, the cell cryopreservation protective solution may further contain the above-mentioned cell cryopreservation solution.
According to the present invention, the cell cryopreservation protective solution may further contain the other cryopreservation protective agent described above.
According to the invention, the cell cryopreservation protective solution is preferably used for cryopreservation of eukaryotic cells, more preferably for cryopreservation of animal cells, particularly for cryopreservation of animal cells.
THE ADVANTAGES OF THE PRESENT INVENTION
Because the inhibition effect of the micron particles on the ice recrystallization is related to the particle size of the particles and is not influenced by the chemical composition and surface groups of the particles, the method is widely suitable for various liquid systems for freezing and storing cells at low temperature.
Compared with the traditional antifreeze agent, the micron particles can effectively inhibit the recrystallization of ice and can improve the survival rate of the frozen cells at low temperature.
Because the micron particles are suspended in the protective solution for low-temperature cryopreservation of the cells, the micron particles do not have chemical reaction with other components in the protective solution, are non-toxic and do not enter the cells, and can be easily separated from the protective solution for low-temperature cryopreservation of the cells after the low-temperature cryopreservation of the cells is finished, the subsequent culture and utilization of the cells are facilitated, and the recycling of the protective agent for low-temperature cryopreservation can be realized.
The micron particles are used as a low-temperature freezing protective agent, and a system to be frozen at normal temperature can be directly placed at ultralow temperature, so that the complicated cooling procedure of low-temperature freezing of cells is avoided. In addition, the micro-particles are used as a low-temperature cryopreservation protective agent, so that the system after cryopreservation can tolerate higher temperature recovery (37-45 ℃) when being subjected to temperature recovery, and the higher the temperature recovery is, the higher the cell recovery survival rate is.
Compared with nano particles, the preparation process of the micro particles is simple, and the industrial cost is low.
Drawings
FIG. 1 is a photograph of a polarizing microscope showing water, water containing 5 μm microspheres, and ice crystal size during temperature rise
FIG. 2 is a statistical graph showing the changes of the ice crystal size with time in the course of heating water, water to which 5 μm microspheres are added
FIG. 3 is a statistical plot of the effect of silica microsphere size on ice recrystallization inhibition, with the ordinate representing the area of a single ice crystal
FIG. 4 is a statistical graph of the effect of silica microsphere content on ice recrystallization inhibition, with the ordinate representing the area of a single ice crystal
FIG. 5 is a statistical chart showing the inhibition effect of silica microspheres, polymethyl methacrylate microspheres and polystyrene microspheres on ice recrystallization
FIG. 6 is a statistical chart of the effect of microspheres, PVA and microsphere combined PVA on cryopreservation of erythrocytes at low temperature
Detailed Description
The present invention is further described below with reference to examples. It should be noted that the examples are not intended to limit the scope of the present invention, and those skilled in the art will appreciate that any modifications and variations based on the present invention are within the scope of the present invention.
EXAMPLE 1 microspheres to inhibit Ice recrystallization
The microspheres used in this example were 5 μm silica microspheres. Adding a certain amount of microspheres into water to prepare 6 multiplied by 10‐14M aqueous microsphere suspension. Pure water was also used as a control.
Dropping the microsphere suspension or water onto-60 deg.C silicon wafer from 60cm height, freezing instantly, heating to-6 deg.C at 15 deg.C/min, maintaining for 30min, and observing the change of ice crystal size with time under polarizing microscope.
The results are shown in FIGS. 1 and 2. Figure 1 shows micrographs of ice crystals with and without microspheres and figure 2 shows the change in ice crystal size over time. As can be seen from fig. 1 and 2, the ice crystal size of the microsphere suspension is much smaller than that of pure water, indicating that the added microspheres inhibit the ice recrystallization during the temperature rise and that the inhibition is very stable.
EXAMPLE 2 inhibition of Ice recrystallization by microspheres of different particle size
The size of the microsphere is selected to be 0.5 μm, 1 μm, 3 μm, 5 μm, 10 μm and 20 μm, the microsphere is silicon dioxide microsphere and is prepared into the microsphere with the concentration of 6.10 multiplied by 10‐14M microsphere suspension the microsphere size was tested for inhibition of ice recrystallisation using the method of example 1.
The mean area size of the ice crystals is represented by the ordinate, and the results of the parallel experimental samples are counted, and the results are shown in the attached figure 3. As can be seen from fig. 3, the efficiency of the microspheres in inhibiting recrystallization increases with increasing size of the microspheres, and then decreases.
EXAMPLE 3 inhibition of Ice recrystallisation by different amounts of microspheres
Selecting silica microspheres with the size of 5 mu m, wherein the number concentration of the microsphere suspension is 0, 0.20 multiplied by 10‐14M、0.65×10‐14M、1.25×10‐14M、3.60×10‐14M、6.10×10‐14M, the inhibition of ice recrystallisation by microsphere concentration was tested using the method of example 1.
The mean area size of the ice crystals is represented by the ordinate, and the results of the parallel experimental samples are counted, and the results are shown in the attached figure 4. As can be seen from FIG. 4, the amount of microspheres gradually increased to 0.20X 10‐14M, the effect of which on inhibiting ice recrystallization increases rapidly, eventually reaching equilibrium.
EXAMPLE 4 inhibition of Ice recrystallization by microspheres of different materials
The size of the microsphere is 5 μm, and the material is silicon dioxide, polymethyl methacrylate and polyphenylEthylene at a concentration of 6.10X 10‐14M, microspheres were tested for inhibition of ice recrystallization using the method of example 1.
The mean area size of the ice crystals is represented by the ordinate, and the results of the parallel experimental samples are counted, and the results are shown in the attached FIG. 5. As can be seen from FIG. 5, the silica microspheres, the polymethyl methacrylate microspheres and the polystyrene microspheres have the same inhibition effect on the ice recrystallization, which indicates that the change of the microsphere material has no influence on the inhibition effect on the ice recrystallization, and the effect is only related to the size of the micrometer particles.
Example 5 protective Effect of microspheres on Low temperature cryopreservation of cells
Preparing PBS solution, taking out two-thirds of the volume of the PBS solution, further adding PVA to prepare PBS solution containing 0.02mg/ml PVA, taking out one-half of the volume of the PBS solution, further adding silica microspheres with the size of 10 mu m to ensure that the concentration of the microspheres is 6.10 multiplied by 10‐14And M. In addition, the concentration of the prepared microspheres is 6.10 multiplied by 10‐14M in PBS.
PBS: 1L, pH7.4, containing: potassium dihydrogen phosphate (KH)2PO4): 0.27g, disodium hydrogen phosphate (Na)2HPO4): 1.42g, sodium chloride (NaCl): 8g, potassium chloride (KCl): 0.2 g.
And adding sheep red blood cells into the four solutions respectively to ensure that the mass concentration of the red blood cells is 15%. Mixing the mixed solution, rapidly placing in liquid nitrogen, freezing instantly, and keeping for 20 min. Then, it was taken out and placed in a constant temperature water bath at 45 ℃ to be heated for 10 min. And centrifuging the mixed solution, taking supernatant, diluting, testing the absorption intensity of the supernatant through ultraviolet, and calculating the survival rate of the red blood cells.
The results of the parallel experimental samples were counted and shown in FIG. 6. As can be seen from FIG. 6, the survival rate of the cells frozen at low temperature can be effectively improved by using the microspheres alone, and the effect is slightly better than that of PVA; the survival rate of the cells frozen at low temperature can be greatly improved by matching the microspheres and the PVA, and the survival rate is far higher than that of the microspheres or the PVA which are used alone. Indicating that the microspheres can be used in combination with existing cryoprotectants.
Claims (32)
1. The application of micron particles in the low-temperature cryopreservation of cells, wherein the particle size of the micron particles is 1-10 mu m, the micron particles are round spheres, the micron particles are biocompatible and non-bioactive water-insoluble, and the application method is that the micron particles are added into liquid for the low-temperature cryopreservation of the cells, and the number concentration of the micron particles in the liquid for the low-temperature cryopreservation of the cells and a cell cryopreservation protection liquid comprising the liquid for the low-temperature cryopreservation of the cells and the micron particles is 2 x 10-15-1×10-12M；
The micro-particles are micro-particles of silicon dioxide, aluminum oxide, titanium dioxide, hydroxyapatite, ferroferric oxide, calcium carbonate, barium sulfate, polystyrene, polymethyl methacrylate, polylactic acid, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polycarbonate, polycaprolactam, polyvinyl chloride, polyvinyl alcohol and polyethylene glycol, the surfaces of which are not modified or modified; the modifying group is hydroxyl, carboxyl, amino, aldehyde group, amide group, epoxy group, polyethylene glycol and polyvinylpyrrolidone.
2. Use according to claim 1, wherein the microparticles have a particle size of between 3 μm and 10 μm.
3. Use according to claim 1 or 2, wherein the microparticles are surface-unmodified or surface-modified microparticles of silica, alumina, titanium dioxide, hydroxyapatite, triiron tetroxide, calcium carbonate, barium sulphate, polystyrene or polymethylmethacrylate.
4. Use according to claim 1 or 2, wherein the modifying group is a hydroxyl, carboxyl and/or amino group.
5. The use according to any one of claims 1 to 2, wherein the microparticle is present in a cell cryopreservation protection solution comprising a cell cryopreservation liquid and the microparticle in a quantitative concentration of 1 x 10-14-1×10-13M。
6. The use according to claim 5, wherein the microparticle is present in a cell cryopreservation protection solution comprising a cell cryopreservation liquid and the microparticle in a quantitative concentration of 2 x 10-14-7×10-14M。
7. The use of claim 1 or 2, wherein the cell is a eukaryotic cell.
8. The use of claim 7, wherein the cell is an animal cell.
9. The method for cryopreserving the cells is characterized in that microparticles are added into liquid for cryopreserving the cells, the particle size of the microparticles is 1-10 mu m, the microparticles are round spheres, the microparticles are biocompatible and non-bioactive water-insoluble, and the number concentration of the microparticles in the protective liquid for cryopreserving the cells is 2 x 10-15-1×10-12M；
The micro-particles are micro-particles of silicon dioxide, aluminum oxide, titanium dioxide, hydroxyapatite, ferroferric oxide, calcium carbonate, barium sulfate, polystyrene, polymethyl methacrylate, polylactic acid, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polycarbonate, polycaprolactam, polyvinyl chloride, polyvinyl alcohol and polyethylene glycol, the surfaces of which are not modified or modified; the modifying group is hydroxyl, carboxyl, amino, aldehyde group, amide group, epoxy group, polyethylene glycol and polyvinylpyrrolidone.
10. The method of claim 9, wherein the microparticles have a particle size of 3 μ ι η to 10 μ ι η.
11. The method according to claim 9 or 10, wherein the microparticles are surface-unmodified or surface-modified microparticles of silica, alumina, titanium dioxide, hydroxyapatite, ferroferric oxide, calcium carbonate, barium sulfate, polystyrene, or polymethylmethacrylate.
12. The method of claim 9 or 10, wherein the modifying group is a hydroxyl group, a carboxyl group and/or an amino group.
13. The method of claim 9 or 10, wherein the microparticles are present in the cryopreservation solution at a concentration of 1 x 10-14-1×10-13M。
14. The method of claim 13, wherein the microparticle is present in the cryopreservation solution at a concentration of 2 x 10-14-7×10-14M。
15. The method of claim 9 or 10, wherein the cell is a eukaryotic cell.
16. The method of claim 15, wherein the cell is an animal cell.
17. The method of claim 9 or 10, wherein the step of cryopreserving the cells comprises: 1) adding the micron particles into the liquid for freezing the cells at the low temperature to prepare a protective liquid for freezing the cells at the low temperature; 2) adding cells to be cryopreserved at a low temperature into the low-temperature cryopreservation protective solution prepared in the step 1) to obtain a mixed system;
or, 1') adding the cells to be cryopreserved into the liquid in which the cells are cryopreserved at the low temperature; 2 ') adding the micron particles into the liquid which is prepared in the step 1') and contains the cells and is frozen at a low temperature to obtain a mixed system;
3) freezing and storing the mixed system obtained in the step 2) or the step 2') at a low temperature.
18. The method as claimed in claim 17, wherein the operation of step 3) comprises: directly placing the mixed system into dry ice, liquid nitrogen or liquid nitrogen vapor phase for freezing and storing.
19. The application of the micron particles in the preparation of the cell cryopreservation protective agent or the cell cryopreservation protective solution, wherein the particle size range of the micron particles is 1-10 mu m, the micron particles are round spheres, the micron particles are biocompatible and non-bioactive water-insoluble, and the number concentration of the micron particles in the cell cryopreservation protective solution or the cell cryopreservation protective solution is 2 x 10-15-1×10-12M；
The micro-particles are micro-particles of silicon dioxide, aluminum oxide, titanium dioxide, hydroxyapatite, ferroferric oxide, calcium carbonate, barium sulfate, polystyrene, polymethyl methacrylate, polylactic acid, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polycarbonate, polycaprolactam, polyvinyl chloride, polyvinyl alcohol and polyethylene glycol, the surfaces of which are not modified or modified; the modifying group is hydroxyl, carboxyl, amino, aldehyde group, amide group, epoxy group, polyethylene glycol and polyvinylpyrrolidone.
20. The use according to claim 19, wherein the microparticles have a particle size in the range of 3 μm to 10 μm.
21. The use according to claim 19 or 20, wherein the microparticles are surface unmodified or modified microparticles of silica, alumina, titanium dioxide, hydroxyapatite, ferroferric oxide, calcium carbonate, barium sulfate, polystyrene or polymethylmethacrylate.
22. Use according to claim 19 or 20, wherein the modifying group is a hydroxyl, carboxyl and/or amino group.
23. The use of claim 19 or 20, wherein the cell is a eukaryotic cell.
24. The use of claim 23, wherein the cell is an animal cell.
25. A cell cryopreservation protection solution, which comprises microparticles, wherein the microparticles have a particle size range of 1-10 μm, are round spheres, are biocompatible and non-bioactive water-insoluble, and have a number concentration of 2 x 10-15-1×10-12M；
The micro-particles are micro-particles of silicon dioxide, aluminum oxide, titanium dioxide, hydroxyapatite, ferroferric oxide, calcium carbonate, barium sulfate, polystyrene, polymethyl methacrylate, polylactic acid, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polycarbonate, polycaprolactam, polyvinyl chloride, polyvinyl alcohol and polyethylene glycol, the surfaces of which are not modified or modified; the modifying group is hydroxyl, carboxyl, amino, aldehyde group, amide group, epoxy group, polyethylene glycol and polyvinylpyrrolidone.
26. The protective solution for cryopreservation of cells on cells of claim 25 wherein the microparticles have a particle size ranging from 3 μm to 10 μm.
27. The protective solution for cryopreservation of cells on the basis of claim 25 or 26, wherein the microparticles are surface-unmodified or surface-modified microparticles of silicon dioxide, aluminum oxide, titanium dioxide, hydroxyapatite, ferroferric oxide, calcium carbonate, barium sulfate, polystyrene or polymethyl methacrylate.
28. The protective solution for cryopreservation of cells on cells of claim 25 or 26 wherein the modifying group is hydroxyl, carboxyl and/or amino.
29. The cell of claim 25 or 26 being cryogenically cooledThe cryopreservation protective solution is characterized in that the number concentration of the microparticles is 1 x 10-14-1×10-13M。
30. The protective solution for cryopreservation of cells of claim 29 wherein the number concentration of microparticles is 2 x 10-14-7×10-14M。
31. The protective solution for cryopreservation of cells of claim 25 or 26 wherein the cells are eukaryotic cells.
32. The protective solution for cryopreservation of cells of claim 31 wherein said cells are animal cells.
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| CN110698186B (en) * | 2019-10-30 | 2022-08-09 | 航天特种材料及工艺技术研究所 | Homogenized alumina ceramic and preparation method thereof |
| CN112167246B (en) * | 2020-10-31 | 2021-07-27 | 北京泰盛生物科技有限公司 | Dental pulp mesenchymal stem cell cryopreservation liquid and cryopreservation method |
| CN113331174B (en) * | 2021-05-13 | 2022-08-26 | 合肥工业大学 | Nanoparticle antifreeze agent containing small molecule monolayer and preparation method thereof |
| CN114689874B (en) * | 2022-06-01 | 2022-11-08 | 深圳市帝迈生物技术有限公司 | D-dimer reagents for liquid stabilization and freeze-thaw resistance |
| CN117378598B (en) * | 2023-12-08 | 2024-03-19 | 金宝医学科技(深圳)有限公司 | Oocyte cryopreservation liquid and preparation method thereof |
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