CN108271770A - Application of the micron particles in cryopreservation - Google Patents
Application of the micron particles in cryopreservation Download PDFInfo
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- CN108271770A CN108271770A CN201710012559.XA CN201710012559A CN108271770A CN 108271770 A CN108271770 A CN 108271770A CN 201710012559 A CN201710012559 A CN 201710012559A CN 108271770 A CN108271770 A CN 108271770A
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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
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Abstract
Present invention discover that micron particles have the effect of good inhibition ice-crystal growth, the growth of ice crystal in modest recovery temperature-rise period can be dropped with freezing-inhibiting, protects cells from the mechanical damage that ice-crystal growth is brought, improves cell freezing recovery survival rate.On this basis, the application the present invention provides micron particles in cell cryopreservation, application of the micron particles in preparing cell cryopreservation protective agent or protecting liquid, and the method using micron particles progress cell cryopreservation.
Description
Technical field
The application that the present invention relates to micron particles in cryopreservation, especially answering in the protection of the cryopreservation of cell
With belonging to biochemical material field.
Background technology
With social development, the technologies such as regenerative medicine and organ transplant cause the great attention of medical domain, and by
Gradually realize the treatment to many illnesss.Therefore in medical domain, the demand of the cell of different distribution type, tissue and organ is got over
Come more urgent.For example, in Britain, the blood of 6000 units is needed daily, but blood can only be in the case of no freezen protective
Effectively storage 42 days, and there is prodigious Hemolysis Rate in simple cell isotonic solution.Although more and more cell, groups
It knits and is contributed with organ and tissue engineering product is commercialized, but still there are many patient because being lost without suitable ligand
The chance for the treatment of is gone, another side is then that many cells contributed, tissue and organ are damaged because that can not find suitable receptor
It is bad.In the U.S., the waiting list of organ transplant is presently more than 118,000 people (in May, 2013), and the transplanting carried out in 2012
Operation is less than 30,000.Under this overall situation, must be requested that no organ is wasted, and thus urgently requires related field
Worker seeks the method for preserving cell, tissue and organ that can be permanently effective, so that energy is timely when patient needs
Supply.
Cryopreserved cells, tissue and organ method are tradition but effective store method, but in cryopreservation process
In, the formation and growth of ice not only result in the mechanical damage of cell, can also be reduced because of liquid water volume fraction, extracellular solute
The raising of concentration and lead to osmotic shock.Nature provides good model for the research of anti-icing material.For example, cold ground
The fish of the insect in area, arctic regions can also survive under very low temperature environment.1969, the DeVries of Stanford University
It is found that antifreeze protein for the first time in a kind of blood for the cold water fish for moving in the South Pole.After this, scientists successively exist again
Polar region fish, insect, plant etc. are found that a variety of antifreeze proteins in vivo.Antifreeze protein (antifreeze proteins, AFPs)
It is a kind of protein that can inhibit ice-crystal growth, it can be reduced the freezing point of water in the form of non-colligative property and be influenced on its fusing point
It is little, lead to occur difference between the fusing point of water and freezing point, referred to as thermo-lag activity.By molar concentration rate compared with antifreeze protein drop
The efficiency of low freezing point is 200~500 times of colligative property salt, and it is different from colligative property salt that this illustrates that it reduces the mechanism of freezing point.
When antifreeze protein is soluble in water, antifreeze protein molecule can be selectively adsorbed onto on certain crystal faces of ice crystal, to change
The habit of ice causes the change of ice crystal form, and the growth rate of ice crystal is caused to be substantially change compared with pure water.But
Application of the antifreeze protein in cryopreservation has two：One, antifreeze protein can only be isolated out of organism, and produces
It measures very low；Two, the effect of actual use antifreeze protein cryopreserved cells is more far short of what is expected than anticipation, even reduces cell sometimes
Survival rate, research find this mainly caused by the active characteristic of its thermo-lag.
Although application of the antifreeze protein in cryopreservation is limited, many researchers inhibit ice crystal by simulating antifreeze protein
The characteristic of growth has synthesized many macromolecules or glycoprotein to inhibit the recrystallization of ice, and then realizes these new materials in low temperature
Freeze the application in field.For example, Matthew I.Gibson effectively inhibit the knot again of ice by the polyvinyl alcohol (PVA) of synthesis
Crystalline substance, and in cell low-temperature frozen liquid storage cell survival rate can be increased substantially when addition PVA.But from the angle of practical application
It sets out, macromolecule is added in cell-preservation liquid, forms stable solution state, it is difficult to which it is separated from the system.
In addition, certain nano particle graphene oxides, zirconium oxide etc. due to its can with some crystal face of ice generate specific adsorption,
Also the recrystallization process for inhibiting ice can be realized.But nano particle can enter cell even nucleus since size is small
In, the toxicity to cell is unknown.In addition, nano particle, which is easy the phenomenon that aggregation, also limits always it in cell low-temperature frozen
Application in depositing.Therefore, the high molecular material and nano particle of synthesis, biocompatibility and by the ability of organism metabolism at
To limit the main problem of its application.
There is still a need for the recrystallization that can effectively inhibit ice is developed in this field, for biomaterials cryopreservations such as cells
When can be improved cell survival rate, and there is low cytotoxicity, and be easy to from freezing the material removed in system.
Invention content
In order to solve bio-toxicity existing for current cell cryopreservation protective agent, hardly possible is detached from low-temperature protection system,
It cannot reuse, or prepare the problems such as difficult, it is good to have found that micron particles have by scientific research by the present inventor
The growth of ice crystal in modest recovery temperature-rise period, can drop in the effect of good inhibition ice-crystal growth with freezing-inhibiting, to which protection is thin
The mechanical damage that born of the same parents bring from ice-crystal growth improves cell freezing recovery survival rate.
After micron particles are added in cell-preservation liquid system, the presence of micron particles provides not only cryopreservation and cooled down
Heterogeneous nucleation point in journey, and can effectively inhibit ice-crystal growth in temperature-rise period so that cell cryopreservation can obtain
Higher survival rate.This effect of micron particles can reduce other antifreezes in cell low-temperature frozen liquid storage, such as poly- second two
The usage amount of alcohol, polyvinyl alcohol or DMSO, to reduce toxic effect of these antifreezes to cell.
Inventors discovered through research that it is one and the relevant property of granular size that micron particles, which inhibit the effect of ice-crystal growth,
Matter is not influenced by the material of micron particles, is not also influenced by the chemical group on micron particles surface, this is micron particles and nanometer
The maximum difference of particle.This feature of micron particles can be widely used in all kinds of cryopreservation liquid systems, that is, do not have
There is the selectivity of applicable system；In addition, but also various micron particles can be used in cryopreservation protection, that is, do not granulate
The selectivity studied point.
The present invention is completed based on above-mentioned purpose and discovery.
Term：
" cryopreservation " refers to preserving substance in 0 DEG C of temperature below in the present invention.For example, by using -4 DEG C~-20 DEG C
Low temperature refrigerator, the low temperature of the offers such as refrigerant such as liquid ammonia, dichlorodifluoromethane, monochlorodifluoromethane, Freon 13
Condition；Either -70 DEG C~-80 DEG C of cryogenic refrigerator dry ice (- 78 DEG C), liquid nitrogen (- 196 DEG C) or liquid nitrogen vapor phase (- 140
DEG C) moment icing, substance is preserved for a long time under ultralow temperature.
" cryopreservation protective agent " and " cryoprotectant " in the present invention, " cryoprotector ", " antifreeze ", " antifreeze
Agent ", the meaning of " anti-freezing agent " are identical, and cryopreservation protective agent is in order to prevent in the cooling and temperature-rise period of cryopreservation
Ice crystal or ice crystal is formed to grow up the substance that causes to damage and use.The substance can protect the material preserved under low temperature, such as
Cell resists the damage that is generated to cell of low temperature, such as intracellular ice, dehydration, solute concentration raising and protein denaturation and its
The damage etc. of skeleton structure.
" cryopreservation protection system " and " cryopreservation protection liquid " in the present invention, " frozen stock solution ", " frozen solution "
Meaning is identical, is the cryopreservation liquid system of the liquid containing cryopreservation protective agent and cryopreservation.
" cell " is consistent with art technology term meaning in the present invention.The cell includes that prokaryotic cell and eukaryon are thin
Born of the same parents.Prokaryotic cell includes fungi, bacterium, Richettsia, mycoplasma, Chlamydia, conveyor screw, algae.Eukaryocyte includes animal
Cell and plant cell." animal " including humans in the present invention.
" grain size " of micron particles in the present invention, is exactly particle diameter for spherosome；For the several of other shapes
It is equivalent volume diameter for what body, that is, the diameter of ball identical with practical particle volume may be used as known in the art each
Kind laser method measurement obtains.
The first aspect of the invention is to provide application of the micron particles in cell cryopreservation.
According to the present invention, the particle size range of the micron particles is 0.5-100 μm.Preferably 1 μm -20 μm, more preferably 3
μm‐10μm。
The material of the micron particles is not particularly limited, as long as bio-compatible and be not dissolving in for non-bioactivity
Water may be used to application of the present invention.The material can be：Inorganic material, such as：Dioxy
SiClx, alundum (Al2O3), titanium dioxide, hydroxyapatite, ferroso-ferric oxide, calcium carbonate, barium sulfate etc.；Organic polymer material
Material, for example, it is polystyrene, polymethyl methacrylate, polylactic acid, polypropylene, polyethylene, polyethylene terephthalate, poly-
(ethylene naphthalate), makrolon, polycaprolactam, polyvinyl chloride, polyvinyl alcohol, polyethylene glycol etc..It is obtained from material
Cheap property angle set out, the preferably described micron particles are silica, alundum (Al2O3), titanium dioxide, hydroxyapatite, four
The micron particles of Fe 3 O, calcium carbonate, barium sulfate, polystyrene or polymethyl methacrylate.
According to the present invention, the surface of the micron particles can further modify different chemical groups, including but not
It is limited to：Hydroxyl, carboxyl, amino, aldehyde radical, amide groups, epoxy group etc. or the macromolecules such as polyethylene glycol, polyvinylpyrrolidone
Chain.The cheap property angle being modified from micron particles surface, preferably to hydroxyl in the surface modification of micron particles, carboxyl and/
Or amino.
According to the present invention, the micron particles are the solids of any shape including spherosome.
In the specific embodiment of the present invention, the micron particles are the titanium dioxides that surface is unmodified or modifies
The group of the micron particles of silicon, polystyrene or polymethyl methacrylate, modification is hydroxyl, carboxyl and/or amino.
According to the present invention, application mode of the micron particles in cell cryopreservation is that micron particles are added to cell is low
In the liquid that temperature freezes.Micron particles can form stable or unstable suspension in a liquid.
The liquid of cell cryopreservation can be can cell dispersion and bio-compatible any liquid, including but not limited to
Water, physiological saline, buffer solution, balanced salt solution, glycerine water solution, cell culture fluid etc..The buffer solution, e.g.：Tris
Buffer solution, HEPES buffer solution, TB buffer solutions, phosphate buffer, acetic acid-acetate buffer, carbonate buffer solution etc.；Institute
Balanced salt solution is stated, e.g.：PBS solution, Hanks solution, D-Hanks solution, Ringer solution, Tyrode solution, Earle
Solution etc.；The glycerine water solution, the e.g. glycerine water solution of 4-50%；The cell culture fluid, e.g.：LB is cultivated
Base, MS culture mediums, B5Culture medium, M199 culture solutions, CMRL1066 culture solutions, Eagle culture solutions, BME culture solutions, MEM cultures
Liquid, Alpha-MEM culture solutions, DMEM culture solutions, McCoy5A culture solutions, PRMI 1640 culture mediums, 109 culture solutions of NCTC,
135 culture solutions of NCTC, F12G culture solutions, DM160 culture solutions, MD752/1 culture solutions, HIWO5 culture solutions etc..
It can contain in cryopreservation protection system containing the liquid and micron particles or without containing except the present invention
Other cryopreservation protective agents except the micron particles.The others cryopreservation protective agent can be that permeability is cold
Freeze protective agent, including but not limited to glycerine, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, acetamide etc.；Can be that impermeability is cold
Freeze protective agent, including but not limited to sucrose, ficoll, hydroxyethyl starch, glucan, bovine serum albumin(BSA), fetal calf serum, poly- second
Glycol, polyvinylpyrrolidone or polyvinyl alcohol etc.；It can also be antifreeze protein.
Content of the micron particles in cell cryopreservation protects liquid is 1 × 10‐15‐1×10‐12M, preferably 1 × 10‐14‐
1×10‐13M, more preferably 2 × 10‐14‐7×10‐14M。
According to the present invention, micron particles, other cryopreservation protective agents are added in the liquid of cell cryopreservation
It can no longer add or additive amount reduces.
According to the present invention, the cell cryopreservation is preferably the cryopreservation of eukaryocyte, more preferably zooblast
Cryopreservation.
According to the present invention, the cryopreservation is preferably Cryopreservation.
The second aspect of the invention is to provide a kind of method of cell cryopreservation.
According to the present invention, the method is to add micron particles in the liquid of cell cryopreservation.
According to the present invention, the particle size range of the micron particles is 0.5-100 μm.Preferably 1 μm -20 μm, more preferably 3
μm‐10μm。
The material of the micron particles is not particularly limited, as long as bio-compatible and be not dissolving in for non-bioactivity
Water may be used to application of the present invention.The material can be：Inorganic material, such as：Dioxy
SiClx, alundum (Al2O3), titanium dioxide, hydroxyapatite, ferroso-ferric oxide, calcium carbonate, barium sulfate etc.；Organic polymer material
Material, for example, it is polystyrene, polymethyl methacrylate, polylactic acid, polypropylene, polyethylene, polyethylene terephthalate, poly-
(ethylene naphthalate), makrolon, polycaprolactam, polyvinyl chloride, polyvinyl alcohol, polyethylene glycol etc..It is obtained from material
Cheap property angle set out, the preferably described micron particles are silica, alundum (Al2O3), titanium dioxide, hydroxyapatite, four
The micron particles of Fe 3 O, calcium carbonate, barium sulfate, polystyrene or polymethyl methacrylate.
According to the present invention, the surface of the micron particles can further modify different chemical groups, including but not
It is limited to：Hydroxyl, carboxyl, amino, aldehyde radical, amide groups, epoxy group etc. or the macromolecules such as polyethylene glycol, polyvinylpyrrolidone
Chain.The cheap property angle being modified from micron particles surface, preferably to hydroxyl in the surface modification of micron particles, carboxyl and/
Or amino.
According to the present invention, the micron particles are the solids of any shape including spherosome.
In the specific embodiment of the present invention, the micron particles are the titanium dioxides that surface is unmodified or modifies
The group of the micron particles of silicon, polystyrene or polymethyl methacrylate, modification is hydroxyl, carboxyl and/or amino.
According to the present invention, the liquid of the cell cryopreservation can be energy cell dispersion and any liquid of bio-compatible
Body, including but not limited to water, physiological saline, buffer solution, balanced salt solution, glycerine water solution, cell culture fluid etc..The buffering
Liquid, e.g.：Tris buffer solutions, HEPES buffer solution, TB buffer solutions, phosphate buffer, acetic acid-acetate buffer, carbonic acid
Salt buffer etc.；The balanced salt solution, e.g.：PBS solution, Hanks solution, D-Hanks solution, Ringer solution,
Tyrode solution, Earle solution etc.；The glycerine water solution, the e.g. glycerine water solution of 4-50%；The cell culture
Liquid, e.g.：LB culture mediums, MS culture mediums, B5Culture medium, M199 culture solutions, CMRL1066 culture solutions, Eagle culture solutions,
BME culture solutions, MEM culture solutions, Alpha-MEM culture solutions, DMEM culture solutions, McCoy5A culture solutions, PRMI 1640 culture mediums,
109 culture solutions of NCTC, 135 culture solutions of NCTC, F12G culture solutions, DM160 culture solutions, MD752/1 culture solutions, HIWO5 cultures
Liquid etc..
Content of the micron particles in the cell cryopreservation protection liquid of liquid and micron particles including cryopreservation is 1
×10‐15‐1×10‐12M, preferably 1 × 10‐14‐1×10‐13M, more preferably 2 × 10‐14‐7×10‐14M。
According to the present invention, micron particles, other cryopreservation protective agents are added in the liquid of cell cryopreservation
It can no longer add or additive amount reduces.
The others cryopreservation protective agent can be permeability cryoprotector, including but not limited to glycerine, diformazan
Base sulfoxide, propylene glycol, ethylene glycol, acetamide etc.；Can be impermeability cryoprotector, including but not limited to sucrose, poly- sugarcane
Sugar, hydroxyethyl starch, glucan, bovine serum albumin(BSA), fetal calf serum, polyethylene glycol, polyvinylpyrrolidone or polyvinyl alcohol
Deng；It can also be antifreeze protein.
According to the present invention, the cell cryopreservation is preferably the cryopreservation of eukaryocyte, more preferably zooblast
Cryopreservation.
According to the present invention, the cryopreservation is preferably Cryopreservation.
In the specific embodiment of the present invention, the method for cell cryopreservation includes the following steps：
1) micron particles are added in the liquid of cell cryopreservation, prepare cell cryopreservation and protects liquid；2) it will wait for low
The cell that temperature freezes is added in the cryopreservation protection liquid that step 1) is prepared, and obtains mixed system；
Alternatively, 1 ') it will wait for that the cell of cryopreservation is added in the liquid of cell cryopreservation；2 ') micron particles are added
Step 1 ') prepare cryopreservation containing cell liquid in, obtain mixed system；
3) by step 2) or step 2 ') obtained mixed system cryopreservation.
According to the present invention it is possible to using including but not limited to that micron particles are suspended in cell is low for the modes such as ultrasound, oscillation
Temperature freezes in protection liquid.
It is preferred that the grain size of the micron particles is 0.5-100 μm.Preferably 1 μm -20 μm, more preferably 3 μm -10 μm.
It is preferred that the micron particles are that surface is unmodified or modified the silica of hydroxyl, polystyrene or poly- methyl-prop
The micron particles of e pioic acid methyl ester.
It is preferred that content of the micron particles in cell cryopreservation protects liquid is 1 × 10‐15‐1×10‐12M, preferably
1×10‐14‐1×10‐13M, more preferably 2 × 10‐14‐7×10‐14M。
Preferred steps 3) operating procedure of cryopreservation is：Mixed system is directly placed in dry ice, liquid nitrogen or liquid nitrogen to steam
It is frozen in vapour phase.
The third aspect of the invention provides, and micron particles are preparing cell cryopreservation protective agent or cell cryopreservation
Protect the application in liquid.
The particle size range of the micron particles is 0.5-100 μm.Preferably 1 μm -20 μm, more preferably 3 μm -10 μm.
The material of the micron particles is not particularly limited, as long as bio-compatible and be not dissolving in for non-bioactivity
Water may be used to application of the present invention.The material can be：Silica, three oxidations two
Aluminium, titanium dioxide, hydroxyapatite, ferroso-ferric oxide, calcium carbonate, barium sulfate etc.；High-molecular organic material, such as polyphenyl second
Alkene, polymethyl methacrylate, polylactic acid, polypropylene, polyethylene, polyethylene terephthalate, poly- naphthalenedicarboxylic acid second two
Alcohol ester, makrolon, polycaprolactam, polyvinyl chloride, polyvinyl alcohol, polyethylene glycol etc..The cheap property angle obtained from material
It sets out, the preferably described micron particles are silica, alundum (Al2O3), titanium dioxide, hydroxyapatite, ferroso-ferric oxide, carbon
The micron particles of sour calcium, barium sulfate, polystyrene or polymethyl methacrylate.
According to the present invention, the surface of the micron particles can further modify different chemical groups, including but not
It is limited to：Hydroxyl, carboxyl, amino, aldehyde radical, amide groups, epoxy group etc. or the macromolecules such as polyethylene glycol, polyvinylpyrrolidone
Chain.The cheap property angle being modified from micron particles surface, preferably to hydroxyl in the surface modification of micron particles, carboxyl and/
Or amino.
According to the present invention, the micron particles are the solids of any shape including spherosome.
In the specific embodiment of the present invention, the micron particles are the titanium dioxides that surface is unmodified or modifies
The group of the micron particles of silicon, polystyrene or polymethyl methacrylate, modification is hydroxyl, carboxyl and/or amino.
According to the present invention, the liquid of cell cryopreservation above-mentioned can also be contained in the cell cryopreservation protection liquid
Body.
According to the present invention, other cryopreservation protections above-mentioned can also be contained in the cell cryopreservation protection liquid
Agent.
The fourth aspect of the invention provides, a kind of cell cryopreservation protection liquid, wherein containing micron particles.
The particle size range of the micron particles is 0.5-100 μm.Preferably 1 μm -20 μm, more preferably 3 μm -10 μm.
The material of the micron particles is not particularly limited, as long as bio-compatible and be not dissolving in for non-bioactivity
Water may be used to application of the present invention.The material can be：Silica, three oxidations two
Aluminium, titanium dioxide, hydroxyapatite, ferroso-ferric oxide, calcium carbonate, barium sulfate etc.；High-molecular organic material, such as polyphenyl second
Alkene, polymethyl methacrylate, polylactic acid, polypropylene, polyethylene, polyethylene terephthalate, poly- naphthalenedicarboxylic acid second two
Alcohol ester, makrolon, polycaprolactam, polyvinyl chloride, polyvinyl alcohol, polyethylene glycol etc..The cheap property angle obtained from material
It sets out, the preferably described micron particles are silica, alundum (Al2O3), titanium dioxide, hydroxyapatite, ferroso-ferric oxide, carbon
The micron particles of sour calcium, barium sulfate, polystyrene or polymethyl methacrylate.
According to the present invention, the surface of the micron particles can further modify different chemical groups, including but not
It is limited to：Hydroxyl, carboxyl, amino, aldehyde radical, amide groups, epoxy group etc. or the macromolecules such as polyethylene glycol, polyvinylpyrrolidone
Chain.The cheap property angle being modified from micron particles surface, preferably to hydroxyl in the surface modification of micron particles, carboxyl and/
Or amino.
According to the present invention, the micron particles are the solids of any shape including spherosome.
In the specific embodiment of the present invention, the micron particles are the titanium dioxides that surface is unmodified or modifies
The group of the micron particles of silicon, polystyrene or polymethyl methacrylate, modification is hydroxyl, carboxyl and/or amino.
Content of the micron particles in cell cryopreservation protects liquid is 1 × 10‐15‐1×10‐12M, preferably 1 × 10‐14‐
1×10‐13M, more preferably 2 × 10‐14‐7×10‐14M。
According to the present invention, the liquid of cell cryopreservation above-mentioned can also be contained in the cell cryopreservation protection liquid
Body.
According to the present invention, other cryopreservation protections above-mentioned can also be contained in the cell cryopreservation protection liquid
Agent.
According to the present invention, the cell cryopreservation protection liquid is preferred for the cryopreservation of eukaryocyte, more preferably uses
In the Cryopreservation of the cryopreservation of zooblast, especially zooblast.
Advantages of the present invention
Since the depression effect that micron particles recrystallize ice is related to grain diameter, not by particle chemical composition and surface
The influence of group, therefore it is widely used in the liquid system of various types of cells cryopreservation.
Compared with traditional antifreeze, micron particles can effectively inhibit the recrystallization of ice, can improve cell cryopreservation
Survival rate.
Since micron particles are suspended in cell cryopreservation protection liquid, chemistry does not occur with the other compositions in protection liquid
Reaction, it is nontoxic, it will not enter into the cell, easily liquid can be protected from cell cryopreservation after cell cryopreservation
In isolate, not only contribute to follow-up cultivation and the utilization of cell, and can realize the protectant recycling of cryopreservation.
Using micron particles as cryopreservation protective agent, it can will wait for that jelly system is placed directly under ultralow temperature under room temperature, avoid
The complicated cooling process of cell cryopreservation.In addition, using micron particles as cryopreservation protective agent, the system rewarming after freezing
When, it is resistant to higher rewarming temperature (37-45 DEG C), and the higher rewarming temperature the rapider, cell recovery survival rate is higher.
Opposite with nano particle, the preparation process of micron particles is simple, low industrial cost.
Description of the drawings
Water in Fig. 1 temperature-rise periods, added with the water of 5 μm of microballoons, the petrographic microscope photo of Ice crystal size
Water in Fig. 2 temperature-rise periods, added with the water of 5 μm of microballoons, statistical chart that Ice crystal size changes over time
Fig. 3 silicon dioxide microspheres size represents single ice crystal to the statistical chart of the effect of ice recrystallization inhibition with ordinate
Area
Fig. 4 silicon dioxide microspheres content represents single ice crystal to the statistical chart of the effect of ice recrystallization inhibition with ordinate
Area
Fig. 5 silicon dioxide microspheres, poly (methyl methacrylate) micro-sphere and polystyrene microsphere act on ice recrystallization inhibition
Statistical chart
Fig. 6 microballoons, PVA, microballoon combine effect statistical charts of the PVA to red blood cell cryopreservation
Specific implementation mode
The present invention is described further with reference to embodiments.It should be noted that embodiment cannot function as to this hair
The limitation of bright protection domain, it will be understood by those skilled in the art that, any improvements introduced on the basis of the present invention and variation all exist
Within protection scope of the present invention.
1 microballoon of embodiment inhibits ice recrystallization
The silicon dioxide microsphere that microballoon used is 5 μm in the embodiment.A certain amount of microballoon is added to the water, is made into 6
×10‐14The microballoon water slurry of M.Separately pure water is taken to compare.
On the silicon chip that microsphere suspension liquid or water are dropped to -60 DEG C from 60cm height, its moment is set to freeze, then with 15
DEG C/min rates are warming up to -6 DEG C, keep 30min, polarized light microscopy microscopic observation ice crystal size with the time variation.
As a result as shown in Figure 1 and Figure 2.Attached drawing 1 gives microballoon and ice crystal microphoto when no microballoon, attached drawing
2 give variation of the Ice crystal size with the time.By Fig. 1 and Fig. 2 as it can be seen that the Ice crystal size of microsphere suspension liquid is much smaller than pure water
Ice crystal size illustrates that microballoon be added inhibits ice in temperature-rise period to recrystallize, and the inhibiting effect is highly stable.
The inhibiting effect that the microballoon of 2 different-grain diameter of embodiment recrystallizes ice
The selection of dimension of microballoon is 0.5 μm, and 1 μm, 3 μm, 5 μm, 10 μm, 20 μm, the microballoon is silicon dioxide microsphere, is matched
It is made a concentration of 6.10 × 10‐14The microsphere suspension liquid of M, using the method in embodiment 1, test Microsphere Size recrystallizes ice
Inhibiting effect.
Ice crystal average area size is represented with ordinate, parallel laboratory sample result is counted, as a result such as attached drawing 3
It is shown.As seen from Figure 3, with the increase of Microsphere Size, microballoon inhibits the efficiency of recrystallization to will present the rule for first increasing and reducing afterwards
Rule.
The inhibiting effect that the microballoon of 3 different amounts of embodiment recrystallizes ice
The silicon dioxide microsphere that size is 5 μm is chosen, the number concentration of microsphere suspension liquid is 0,0.20 × 10‐14M、0.65
×10‐14M、1.25×10‐14M、3.60×10‐14M、6.10×10‐14M tests microballoon concentration using the method in embodiment 1
To the inhibiting effect of ice recrystallization.
Ice crystal average area size is represented with ordinate, parallel laboratory sample result is counted, as a result such as attached drawing 4
It is shown.From fig. 4, it can be seen that as microballoon dosage is gradually increased to 0.20 × 10‐14M inhibits the effect of ice recrystallization that can carry rapidly
Height is finally reached balance.
The inhibiting effect that the microballoon of 4 unlike material of embodiment recrystallizes ice
The selection of dimension of microballoon is 5 μm, and material is respectively silica, polymethyl methacrylate and polystyrene, dense
Degree is 6.10 × 10‐14M tests the inhibiting effect that microballoon recrystallizes ice using the method in embodiment 1.
Ice crystal average area size is represented with ordinate, parallel laboratory sample result is counted, as a result such as attached drawing 5
It is shown.As seen from Figure 5, the suppression that silicon dioxide microsphere, poly (methyl methacrylate) micro-sphere and polystyrene microsphere recrystallize ice
It makes of identical, illustrates that the change of microballoon material does not influence the inhibition that ice recrystallizes, the effect and micron
The size of grain is related.
5 microballoon of embodiment is to cell cryopreservation protective effect
PBS solution is prepared, wherein 2/3rds volume is taken out, further adds PVA, is configured to contain 0.02mg/ml
The PBS solution of PVA further takes out the volume of the latter's half, further adds the silicon dioxide microsphere that size is 10 μm, makes micro-
A concentration of the 6.10 × 10 of ball‐14M.In addition, configuration microballoon a concentration of 6.10 × 10‐14The PBS solution of M.
PBS：1L, pH7.4 contain：Potassium dihydrogen phosphate (KH2PO4)：0.27g, disodium hydrogen phosphate (Na2HPO4)：1.42g
Sodium chloride (NaCl)：8g, potassium chloride (KCl)：0.2g.
It is separately added into sheep red blood cell (SRBC) into above-mentioned four kinds of solution so that the mass concentration of red blood cell is 15%.It will mixing
It after solution is sufficiently mixed, is placed in liquid nitrogen rapidly, its moment is made to freeze, keep 20min.It is then taken out and is placed on 45 DEG C
Water bath with thermostatic control in heat 10min.Mixed liquor is centrifuged, supernatant is taken, is diluted, the absorption by ultraviolet test supernatant is strong
Degree, calculates the survival rate of red blood cell.
Parallel laboratory sample result is counted, as a result as shown in Fig. 6.As seen from Figure 6, microballoon energy is used alone
The survival rate of cell cryopreservation is effectively improved, effect is slightly better than PVA；Cell can be greatly improved with the use of microballoon and PVA
The survival rate of cryopreservation, survival rate is far above the survival rate that microballoon or PVA is used alone.Illustrate that microballoon can be with existing freeze proof guarantor
Shield agent is used in combination.
Claims (10)
1. application of the micron particles in cell cryopreservation.It is preferred that the grain size of the micron particles is 0.5-100 μm.Preferably
1μm‐20μm.More preferably 3 μm -10 μm.
2. application as described in claim 1, the micron particles are bio-compatibles and are the not soluble in water of non-bioactivity
's.Preferably surface it is unmodified or modification inorganic or organic material micron particles.Preferably surface it is unmodified or modification
Silica, titanium dioxide, hydroxyapatite, ferroso-ferric oxide, calcium carbonate, barium sulfate, polystyrene, gathers alundum (Al2O3)
Methyl methacrylate, polylactic acid, polypropylene, polyethylene, polyethylene terephthalate, polyethylene naphthalate,
The micron particles of makrolon, polycaprolactam, polyvinyl chloride, polyvinyl alcohol, polyethylene glycol.Preferably surface is unmodified or repaiies
Silica, alundum (Al2O3), titanium dioxide, hydroxyapatite, ferroso-ferric oxide, calcium carbonate, barium sulfate, the polystyrene of decorations
Or the micron particles of polymethyl methacrylate.
It is preferred that the modification group is hydroxyl, carboxyl, amino, aldehyde radical, amide groups, epoxy group, polyethylene glycol, polyvinylpyrrolidine
Ketone.Preferably hydroxyl, carboxyl and/or amino.
3. such as claim 1-2 any one of them applications, it is characterised in that the application process is to be added to micron particles
In the liquid of cell cryopreservation.It is preferred that cell low temperature of the micron particles in liquid and micron particles including cell cryopreservation
It is 1 × 10 to freeze the content in protection liquid‐15‐1×10‐12M, preferably 1 × 10‐14‐1×10‐13M, more preferably 2 × 10‐14‐7
×10‐14M。
It is preferred that the cell is eukaryocyte, further preferably zooblast.
4. a kind of method of cell cryopreservation, which is characterized in that add micron particles in the liquid of cell cryopreservation.
It is preferred that the grain size of micron particles is 0.5-100 μm.Preferably 1 μm -20 μm.More preferably 3 μm -10 μm.
It is preferred that the micron particles are bio-compatibles and are the not soluble in water of non-bioactivity.Preferably surface it is unmodified or
Inorganic or organic material the micron particles of modification.Preferably surface it is unmodified or modification silica, alundum (Al2O3), two
Titanium oxide, hydroxyapatite, ferroso-ferric oxide, calcium carbonate, barium sulfate, polystyrene, polymethyl methacrylate, polylactic acid,
Polypropylene, polyethylene terephthalate, polyethylene naphthalate, makrolon, polycaprolactam, gathers polyethylene
The micron particles of vinyl chloride, polyvinyl alcohol, polyethylene glycol.Preferably surface it is unmodified or modification silica, three oxidation two
Aluminium, titanium dioxide, hydroxyapatite, ferroso-ferric oxide, calcium carbonate, barium sulfate, polystyrene or polymethyl methacrylate
Micron particles.
It is preferred that the modification group is hydroxyl, carboxyl, amino, aldehyde radical, amide groups, epoxy group, polyethylene glycol, polyvinylpyrrolidine
Ketone.Preferably hydroxyl, carboxyl and/or amino.
It is preferred that content of the micron particles in cell cryopreservation protects liquid is 1 × 10‐15‐1×10‐12M, preferably 1 × 10‐14‐
1×10‐13M, more preferably 2 × 10‐14‐7×10‐14M。
It is preferred that the cell is eukaryocyte, further preferably zooblast.
5. method as claimed in claim 4, wherein cell cryopreservation step are：1) cell low-temperature frozen is added in micron particles
In the liquid deposited, prepares cell cryopreservation and protect liquid；2) it will wait for that the low-temperature frozen that step 1) is prepared is added in the cell of cryopreservation
It deposits in protection liquid, obtains mixed system；
Alternatively, 1 ') it will wait for that the cell of cryopreservation is added in the liquid of cell cryopreservation；2 ') step is added in micron particles
1 ') in the liquid for the cryopreservation containing cell prepared, mixed system is obtained；
3) by step 2) or step 2 ') obtained mixed system cryopreservation.
6. the operating procedure of method as claimed in claim 5, the step 3) is：Mixed system is directly placed in dry ice, liquid
It is frozen in nitrogen or liquid nitrogen vapor phase.
7. application of the micron particles in preparing cell cryopreservation protective agent or cell cryopreservation protection liquid.It is preferred that described micro-
The particle size range of rice grain is 0.5-100 μm.Preferably 1 μm -20 μm, more preferably 3 μm -10 μm.
8. the use as claimed in claim 7, the micron particles are bio-compatibles and are the not soluble in water of non-bioactivity
's.Preferably surface it is unmodified or modification inorganic or organic material micron particles.Preferably surface it is unmodified or modification
Silica, titanium dioxide, hydroxyapatite, ferroso-ferric oxide, calcium carbonate, barium sulfate, polystyrene, gathers alundum (Al2O3)
Methyl methacrylate, polylactic acid, polypropylene, polyethylene, polyethylene terephthalate, polyethylene naphthalate,
The micron particles of makrolon, polycaprolactam, polyvinyl chloride, polyvinyl alcohol, polyethylene glycol.Preferably surface is unmodified or repaiies
Silica, alundum (Al2O3), titanium dioxide, hydroxyapatite, ferroso-ferric oxide, calcium carbonate, barium sulfate, the polystyrene of decorations
Or the micron particles of polymethyl methacrylate.
It is preferred that the modification group is hydroxyl, carboxyl, amino, aldehyde radical, amide groups, epoxy group, polyethylene glycol, polyvinylpyrrolidine
Ketone.Preferably hydroxyl, carboxyl and/or amino.
It is preferred that the cell is eukaryocyte, further preferably zooblast.
9. a kind of cell cryopreservation protects liquid, which is characterized in that wherein contain micron particles.It is preferred that the grain of the micron particles
Ranging from 0.5-100 μm of diameter.Preferably 1 μm -20 μm, more preferably 3 μm -10 μm.
10. application as claimed in claim 9, the micron particles are bio-compatibles and are the not soluble in water of non-bioactivity
's.Preferably surface it is unmodified or modification inorganic or organic material micron particles.Preferably surface it is unmodified or modification
Silica, titanium dioxide, hydroxyapatite, ferroso-ferric oxide, calcium carbonate, barium sulfate, polystyrene, gathers alundum (Al2O3)
Methyl methacrylate, polylactic acid, polypropylene, polyethylene, polyethylene terephthalate, polyethylene naphthalate,
The micron particles of makrolon, polycaprolactam, polyvinyl chloride, polyvinyl alcohol, polyethylene glycol.Preferably surface is unmodified or repaiies
Silica, alundum (Al2O3), titanium dioxide, hydroxyapatite, ferroso-ferric oxide, calcium carbonate, barium sulfate, the polystyrene of decorations
Or the micron particles of polymethyl methacrylate.
It is preferred that the modification group is hydroxyl, carboxyl, amino, aldehyde radical, amide groups, epoxy group, polyethylene glycol, polyvinylpyrrolidine
Ketone.Preferably hydroxyl, carboxyl and/or amino.
It is preferred that the content of the micron particles is 1 × 10‐15‐1×10‐12M, preferably 1 × 10‐14‐1×10‐13M, more preferably 2
×10‐14‐7×10‐14M。
It is preferred that the cell is eukaryocyte, further preferably zooblast.
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| CN107232185A (en) * | 2017-07-31 | 2017-10-10 | 青岛德瑞骏发生物科技股份有限公司 | A kind of equus oocyte vitrification freezen protective liquid, freezing and storing method and application |
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| CN110698186A (en) * | 2019-10-30 | 2020-01-17 | 航天特种材料及工艺技术研究所 | Homogenized alumina ceramic and preparation method thereof |
| CN112167246A (en) * | 2020-10-31 | 2021-01-05 | 郑州贝贝生物科技有限公司 | Dental pulp mesenchymal stem cell cryopreservation liquid and cryopreservation method |
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| 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 |
| CN114689874A (en) * | 2022-06-01 | 2022-07-01 | 深圳市帝迈生物技术有限公司 | D-dimer reagents for liquid stabilization and freeze-thaw resistance |
| 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 |
| CN117378598A (en) * | 2023-12-08 | 2024-01-12 | 金宝医学科技(深圳)有限公司 | Oocyte cryopreservation liquid and preparation method thereof |
| 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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