CN105230610A - Cryopreservation solution as well as preparation method and application thereof - Google Patents
Cryopreservation solution as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a cryopreservation solution as well as a preparation method and an application thereof and belongs to the technical field of biomaterial cryopreservation. In the cryopreservation solution, graphene oxide is added, so that growth of tiny ice crystals during a recovery process of the cryopreservation solution can be inhibited, and further the destructive effect of ice crystals on cells during a growth process is avoided, so that the survival rate and cell activity of biomaterials subjected to cryopreservation recovery are greatly improved. The method aims at inhibiting growth of the ice crystals, so that the cryopreservation solution has relatively high universality, can be used for cryopreservation of cells, tissues and the like, and can meet the needs of actual use to the greatest extent.
Description
Technical field
The present invention relates to freezen protective, especially the application of graphene oxide in biomaterial freezing is preserved, and a kind of freezen protective liquid containing graphene oxide, the preparation method of this conserving liquid, carry out the method for cell, tissue or organ freezen protective with this conserving liquid of application, belong to biomaterial freezing Techniques of preserving field.
Background technology
Low temperature or Excised Embryos technology are a kind of technology of preserving the biomaterials such as biological tissue, organ and cell under the condition of dry ice or liquid nitrogen that 20 century 70s develop rapidly.Under cryogenic, biological tissue is in " state of dormancy ", ensure that heredity and the morphological stability of biomaterial, is a kind of method of effective preservation biomaterial.In nearly decades, the application of this technology concentrates on the research field of the animal such as mammality, bird, fish and each Plants with economic worth.Low temperature, Excised Embryos, as an emerging technology, not only facilitate the development of the basic subject such as biology, medical science, have also played great effect in fields such as agricultural, livestock breeding, aquacultures, have created great economic benefit.
But in resuscitation process, ice crystal contained in freezen protective liquid can continued propagation, and ice crystal damage is to the topmost injury of conservation object in freezen protective technology, is to form ice crystal due to freeze proof conserving liquid or intracellular moisture, stab that cell causes.
Refrigeration Technique field has developed Slow-rate freezing method and vitrifying freeze process at present; wherein the latter utilizes the cryoprotector of high concentration to solidify under ultra-low temperature surroundings; form irregular glassy solid; do not form ice crystal; thus cell is protected in violent fast cooling; overcome ice crystal damage, but vitrified sample still can produce a large amount of ice crystals in resuscitation process.At present mainly by the composition of adjustment freezen protective liquid, such as use permeability antifreeze (as glycerine, DMSO etc.) and impermeability antifreeze (as polyethylene glycol, PVP etc.), and the mode of regulation and control frozen cooling formula, reduce the formation of ice crystal as far as possible.
Graphene oxide is a kind of to repeat the amphipathic two-dimensional material that unsaturated six-membered carbon ring is skeleton, and its surface distributed oxygen-containing functional group and complete Graphene region.Graphene oxide can as interfacial agent Presence of an interface, and reduce the energy between interface.Current graphene oxide extensively gets up gradually in the application of polymer class composite and inorganic matter class field of compound material.
Summary of the invention
The present inventor studies discovery, and graphene oxide can stably disperse in a liquid, and can the growth of ice crystal in freezing-inhibiting and/or resuscitation process.On this basis, the present invention protects following technical scheme:
Graphene oxide is used for the growth of ice crystal in the freezing and/or resuscitation process of freezing-inhibiting conserving liquid.Preferably described freezing and/or resuscitation process is the freezing of biomaterial and/or recovery.
Graphene oxide is preparing the application in freezen protective liquid.Preferably described freezen protective liquid is the freezen protective for biomaterial.
A method for freezen protective, is characterized in that, in freezen protective liquid, add graphene oxide.Preferably described freezing and storing method is the freezing and storing method of biomaterial.
A kind of freezen protective liquid containing graphene oxide.
The following detailed description of the claimed technical scheme of the present invention.
" biomaterial " used in the present invention includes but not limited to: bacterium, fungi, virus, algae, Richettsia, conveyor screw, derive from human body, the cell of animal and plant, tissue, organ, and the bacterium obtained by biotechnology, fungi, virus, algae, biological cells and tissues.All in biotechnology and association area in a word can freezen protective, and need freezen protective and the living material of recovery is all forgiven in the category of the present invention's " biomaterial ".In certain embodiments, the engineering bacteria that biomaterial can be naturally occurring bacterium or be obtained by biotechnology, such as Escherichia coli, salmonella, Staphylococcus aureus, streptococcus, bacillus subtilis etc.; Biomaterial can be the cell deriving from human body, animal and plant in certain embodiments, or by the engineering cell that cell engineering obtains, the liver cell of such as animal or human's body, reproductive cell, nerve cell, myofibroblasts, Epithelial cell, the root cells of plant, leaf cell, or various tumour cell or hybridoma, such as Hela cell, SHG-44 glioma cell, A549 cell etc.; Biomaterial can be the tissue or the organ that derive from human body or animal in certain embodiments, or by the tissue that tissue engineering technique obtains, such as skin histology or skin engineered tissue, cartilaginous tissue, bone tissue or bone engineered tissue, connective tissue, muscular tissue, nerve fiber, kidney, liver, cornea, heart, lung etc.Biomaterial is candidate stem cell in some embodiments of the invention, cartilaginous tissue, kidney organ, the root cells of pseudo-ginseng, skin engineered tissue, the sperm of mammal or the mankind.
" freezen protective liquid " used in the present invention is the various solution for suspended biological material in process of cryopreservation, such as: the glycerine of 10 ~ 50%, being added with the physiological saline of the antifreezes such as glycerine, DMSO, PVP or polyethylene glycol, Hank ' s liquid, DMEM culture fluid, RPMI-1640 culture fluid or all kinds of bacteria culture medias etc., also can be Hank ' s liquid, DMEM culture fluid, RPMI-1640 culture fluid or all kinds of bacteria culture medias etc.
Graphene oxide suppresses the effect of ice-crystal growth mainly based on its structure and chemical property, and the hydroxyl in the oxygen-containing functional group on graphene oxide can be combined with each other with ice crystal effectively, stops hydrone to the diffusion on ice crystal interface, thus suppresses the growth of ice crystal.Based on this, those skilled in the art it is expected to, the graphene oxide in various source can be used for the growth of ice crystal in freezing-inhibiting and/or resuscitation process, for the freezen protective of biomaterial, and graphene oxide is joined in various freezen protective liquid, the moisture ice-crystal growth of freezing-inhibiting conserving liquid and biomaterial inside can be passed through, improve the freezen protective efficiency of freezen protective liquid further.
Graphene oxide used in the present invention can be obtained by the mode be purchased, such as Sigma-AldrichCo.LLC. product sold is numbered the graphene oxide aqueous dispersions of 763713,763705 and 777676, also the conventional method of graphene oxide can be adopted to prepare, obtain as peeled off after Hummers method graphite oxide.
In the present invention, the carbon oxygen element of graphene oxide is than the mol ratio referring to carbon oxygen element.
Being preferred for graphene oxide of the present invention is flaky material, and it is of a size of 200nm-12 μm, is preferably 200 ~ 500nm; The number of plies is 1 ~ 3 layer, is preferably 1 ~ 2 layer; Carbon oxygen element, than 1.5 ~ 2.2:1, is preferably about 1.9:1.
The mass percent concentration of preferential oxidation Graphene in freezen protective liquid is 0.001% ~ 2%, is preferably 0.01% ~ 1%, more preferably 0.05 ~ 0.5%, is more preferably 0.08 ~ 0.3%, is most preferably 0.1%.
Those skilled in the art also can according to the frozen or feature of biomaterial of cultivating, other chemical compositions are added, the functional polypeptides such as protein, growth factor such as such as sugar, seralbumin or protein, the acceptable buffer system of physiology, inorganic electrolyte etc. in freezen protective liquid.
The present invention still further provides a kind of concrete freezen protective liquid, in every premium on currency, containing 0.3 ~ 0.7g glucose, 7 ~ 10g lactose, 0.1 ~ 0.5g raffinose, 0.9 ~ 1.3g4-HEPES, 2.0 ~ 5.0g phosphate-buffered to, 1 ~ 2g sodium chloride, 2 ~ 4g potassium chloride, 0.01 ~ 0.04g magnesium sulfate, 0.05 ~ 0.1g calcium chloride, 0.5 ~ 1g casein, the pH value of freezen protective liquid is 6.9 ~ 7.5, is preferably 7.1 ~ 7.3.
Described phosphate-buffered forms by phosphatic Conjugate Acid-Base Pairs, and described phosphate can be the alkali metal salt of phosphoric acid, such as tertiary sodium phosphate, sodium dihydrogen phosphate, sodium hydrogen phosphate, tripotassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate; The alkali salt of phosphoric acid, such as calcium phosphate, calcium dihydrogen phosphate, calcium monohydrogen phosphate, tricresyl phosphate magnesium, phosphoric acid one magnesium, di(2-ethylhexyl)phosphate magnesium; The ammonium salt of phosphoric acid, such as diammonium hydrogen phosphate, ammonium dihydrogen phosphate (ADP) etc.Described phosphate can be the hydrate of anhydride or different hydrauture.
Preferably described phosphate-buffered is to consisting of: sodium dihydrogen phosphate and sodium hydrogen phosphate, sodium hydrogen phosphate and tertiary sodium phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate, dipotassium hydrogen phosphate and tripotassium phosphate, diammonium hydrogen phosphate and ammonium dihydrogen phosphate (ADP).Most preferably be sodium dihydrogen phosphate and sodium hydrogen phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.
In described freezen protective liquid, containing carbohydrates such as glucose, lactose, raffinoses, can provide external source energy for biomaterial, form sugar-coat protection, under preventing freezing conditions, external environment is to the damage of biological material cell film.Containing materials such as HEPES, phosphate, sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, the preservation for biomaterial provides suitable osmotic pressure environment and suitable acid-base value.Described freezen protective liquid is applicable to the biological cells and tissues of all kinds of animal and plant, the freezen protective of organ, and those skilled in the art can adjust the formula of freezen protective liquid as required in above-mentioned content range.
Preferably, described freezen protective liquid is containing, for example lower component: in every premium on currency, about 0.5g glucose, about 9.0g lactose, about 0.3g raffinose, about 1.1g4-HEPES, about 1.2gNaH
2pO
42H
2o, about 1.6gNa
2hPO
42H
2o, about 1.5g sodium chloride, about 3.0g potassium chloride, about 0.02g magnesium sulfate, about 0.07g calcium chloride, about 0.6g casein.
The preparation method of described freezen protective liquid is: after each component being taken according to consumption, join in deionized water, after abundant mixed dissolution, adopts the method for conventional degerming or sterilizing, such as heat sterilization method, is positioned over 4 DEG C of refrigerations and keeps in Dark Place after degerming.
Except above-mentioned each component, for strengthening controlled-rate freezing, the antifreeze that also can add often use in the market in described freezen protective liquid, as glycerine, DMSO etc., also can add graphene oxide, to improve the effect of its freezen protective further.
In freezen protective liquid, add the method for graphene oxide, can be the graphene oxide taking respective amount, after physics fragmentation, grind to form smalls, by the smalls ultrasonic disperse of graphene oxide in freezen protective liquid; Or according to the consumption of the graphene oxide that will add, the graphene oxide aqueous dispersions of the concentration known of proper volume is added in freezen protective liquid and mixes.
Graphene oxide preferably used is flaky material, and it is of a size of 200nm-12 μm, is preferably 200 ~ 500nm; The number of plies is 1 ~ 3 layer, is preferably 1 ~ 2 layer; Carbon oxygen element, than 1.5 ~ 2.2:1, is preferably about 1.9:1.
The mass percent concentration of preferential oxidation Graphene in freezen protective liquid is 0.001% ~ 2%, is preferably 0.01% ~ 1%, more preferably 0.05 ~ 0.5%, is more preferably 0.08 ~ 0.3%, is most preferably 0.1%.
When using freezen protective liquid provided by the invention to carry out Slow-rate freezing to biomaterial, operating procedure can be as follows:
(1) by biomaterial, such as cell suspending liquid, tissue or organ join in appropriate freezen protective liquid, be directed to organ, as required also can by freezen protective perfusion wherein, then biomaterial is transferred to insulating box, makes temperature be down to 3 ~ 6 DEG C, be preferably 5 DEG C, maintain 60 ~ 120 minutes at such a temperature, preferably 90 minutes.
(2) adopt the mode of temperature programmed control to make conserving liquid system temperature be down to-140 DEG C with the speed of 20 ~ 60 DEG C/min, the conserving liquid system after cooling is placed in liquid nitrogen container, is preserved by liquid nitrogen.Preferably be down to-110 DEG C with the speed of 40 DEG C/min, then be down to-140 DEG C with 20 DEG C/min.
Biomaterial after Cord blood, recovers in 37 DEG C of environment, utilizes shaking table to shake gently in equilibrium process, and under then cryopreservation tube or other biological material culture vessel being placed in room temperature condition, vibrations are to melting completely gently.
The invention has the advantages that:
Graphene oxide is sheet two-dimensional material, and the oxygen-containing functional group of its surface distributed makes graphene oxide comparatively disperse in Yi Shui.In water, graphene oxide can carry out hydration and then electronegative, and this electronegative graphene oxide can overcome Van der Waals force between lamella and lamella and interaction of hydrogen bond, ensure that the stability of graphene oxide dispersion.Further, the lattice structure of Graphene and I
hthe ice crystal of type has perfect Lattice Matching, for graphene oxide suppresses ice-crystal growth to provide possibility, the effect of this suppression ice-crystal growth is mainly based on the special structure of graphene oxide and chemical property, oxygen-containing functional group on graphene oxide forms cluster and is distributed on lamella, complete graphene-structured also integrated distribution, therefore whole graphene sheet layer should be the admixture of oxygen-containing functional group and complete graphene sheet layer.And hydroxyl in oxygen-containing functional group and hydrone have very strong interaction, can effectively and ice crystal be combined with each other, and the graphene oxide lamella detesting water can be arranged by hydrone after ice crystal is formed, thus can better be combined with ice crystal.Moreover, complete graphene oxide and I
hthere is extraordinary matching effect in the basal face of type ice crystal, therefore in the process of biomaterial freezing and/or recovery, once after ice crystal formed, the graphene oxide of two dimension will be adsorbed on the interface of ice crystal firmly, stop hydrone to the diffusion on ice crystal interface, thus suppress the growth of ice crystal.Graphene oxide, to the inhibitory action of ice crystal, effectively decreases the harm that the growth due to ice crystal punctures biological material cell.And graphene oxide has higher specific surface area, in freezen protective liquid system, use little amount just can play the effect effectively suppressing ice-crystal growth, and there is universality.
In addition, graphene oxide is dispersed in freezen protective liquid, is not true solution, when the later stage, recovery was removed, is easy to reject by mode that is centrifugal or sedimentation, more easy and simple to handle than conventional antifreezes such as DMSO.
Accompanying drawing explanation
The atom of the graphene oxide disperseed in Fig. 1 freezen protective liquid is tried hard to
The size distribution of the graphene oxide disperseed in Fig. 2 freezen protective liquid
The thickness distribution of the graphene oxide disperseed in Fig. 3 freezen protective liquid.The described thickness display graphene oxide be dispersed in freezen protective liquid is individual layer.
Ice-crystal growth figure in Fig. 4 water and in graphene oxide aqueous dispersions.Left figure is ice-crystal growth figure in water, right figure is ice-crystal growth figure in graphene oxide aqueous dispersions.
The statistical effect figure that the different sample of Fig. 5 is recovered to horse sperm freezing
Fig. 6 contains variable concentrations graphene oxide freezen protective liquid affects statistical chart to sperm cryopreservation
Embodiment
Below in conjunction with embodiment, the present invention is described further.It should be noted that, embodiment can not as limiting the scope of the invention, those skilled in the art will recognize that any improvement of doing on basis of the present invention and change are all within protection scope of the present invention.
Embodiment 1
The preparation of graphene oxide
1, pre-oxidation graphite
Take the phosphorus pentoxide of 2.5g and the potassium peroxydisulfate of 2.5g, be added in 250mL conical flask.Adding the 20mL concentrated sulfuric acid when constantly stirring, after being heated to 80 DEG C, in solution, slowly adding 3g graphite powder, cool to room temperature after keeping the temperature of solution to reach 4.5h at 80 DEG C.Slowly add deionized water afterwards, ensure in this process that the temperature of solution is no more than 80 DEG C, mixed liquor hold over night, the concentrated sulfuric acid that after filtering, removing is excessive, 60 DEG C of oven dry.
2, the preparation of graphite oxide
Pre-oxidation graphite after oven dry joins in the 120mL concentrated sulfuric acid, and when constantly stirring, slowly add 15g potassium permanganate, this process is carried out in ice bath, ensures that whole process temperature is below 80 DEG C, Keep agitation 2h.Again reaction vessel is transferred in ice bath environment, adds deionized water, in whole dilution, ensure that temperature is lower than 80 DEG C, add 700mL deionized water after Keep agitation 2h again, drip 20mL afterwards, the hydrogen peroxide of 30wt%, stirred at ambient temperature 1h, fully oxidized graphite.
3, ultrasonic stripping obtains graphene oxide
Filtered by the graphite oxide of above-mentioned gained, and by gained solid dispersal to (concentrated hydrochloric acid: water (v/v)=1:9) in dilute hydrochloric acid solution, remove supernatant after centrifugal filtration, retain lower floor's solid, this process repeats 8 times.Afterwards, then remove hydrochloric acid residual in solid by deionized water, the same mode adopting centrifugation, until the pH value of cleaning fluid is 7.Finally again be dissolved in a small amount of deionized water by above-mentioned filter cake, ultrasonic 1h under 100W ultrasonic power, obtains the suspension of finely dispersed graphene oxide, detect through AFM, the graphene oxide Size Distribution obtained is at 200-500nm, and number of plies 1-2 layer, carbon oxygen element ratio is 1.9.
4, graphene oxide powder preparation
Above-mentioned graphene oxide is transferred to culture dish, is placed in baking oven, 50 DEG C of vacuum dryings, the graphene oxide physics of gained is broken, and use mortar grind into fine powder, be placed in PE pipe, 4 DEG C keep in Dark Place.
Embodiment 2
The preparation of freezen protective liquid of the present invention
1,0.5g glucose, 9g lactose, 0.3g raffinose, 1.1g4-HEPES, 1.2gNaH is taken
2pO
42H
2o, 1.6gNa
2hPO
42H
2o, 1.5g sodium chloride, 3g potassium chloride, 0.02g magnesium sulfate, 0.07g calcium chloride, 0.6g casein, join in 1L deionized water, after abundant mixed dissolution, is saved in the insulating box of 37 DEG C, for subsequent use.For the convenience that following examples describe, the freezen protective liquid this step prepared, referred to as freezen protective liquid A.
2, containing the preparation of the freezen protective liquid of graphene oxide
Take the graphene oxide of a certain amount of embodiment 1 preparation, in ultra-clean chamber, physics is broken, smalls is ground to form in agate mortar, the ultrasonic 30min of 100W, be distributed in the freezen protective liquid A that the 1st step prepares, make the mass percent concentration of graphene oxide be 0.1%, gained solution ph is 7.2.
For the convenience that following examples describe, by the freezen protective liquid containing graphene oxide that the preparation of this step obtains, referred to as freezen protective liquid B.
Utilize AFM to measure graphene oxide particle size range in dispersion liquid at about 200 ~ 500nm, the graphene oxide thickness of individual layer, at about 1.5nm, is specifically shown in Fig. 1-3.
Embodiment 3
Different sample is to the inhibitory action of ice-crystal growth
Adopt the mode (DellerRC of bibliographical information, VatishM, MitchellDA, etal.Syntheticpolymersenablenon-vitreouscellularcryopres ervationbyreducingicecrystalgrowthduringthawing [J] .Naturecommunications, 2014,5.) evaluate different sample inhibitory action to ice-crystal growth in low temperature environment.
1, sample preparation: choose the physiological saline of 0.7%, the embodiment of the present invention 2 freezen protective liquid A (oxygen-free functionalized graphene), 20% glycerine, be purchased graphene oxide aqueous dispersions (Sigma-AldrichCo.LLC.763713, the Size Distribution of its graphene oxide is 200nm-12 μm, 2 ~ 3 layers, C/O is 1.9) and the graphene oxide aqueous dispersions (the aqueous dispersions concentration of two kinds of graphene oxides is set as mass percent concentration 0.01%, 0.1% and 0.5%) prepared of embodiment 1.Meanwhile, the graphene oxide that whole mass percent concentration is embodiment 1 preparation of 0.01%, 0.1% and 0.5% is also added respectively to physiological saline, freezen protective liquid A and 20% glycerine.
2,10 μ L samples are transferred to the mica surface of fresh preparation, make it be evenly distributed on mica surface, utilize cold and hot by mica fast cooling to-60 DEG C, then be warming up to-6 DEG C with the speed of 5 DEG C/min, keep 60min at such a temperature.
3, utilize microscope to take pictures to ice crystal, the size of statistics ice crystal, result is as follows, and the error bar in table derives from the statistics of 150 ice crystals:
Table 1 graphene oxide is on the impact of different liquids system ice-crystal growth
As can be seen from Table 1, physiological saline, 20% glycerine and freezen protective liquid A of the present invention be not before adding graphene oxide, the size of ice-crystal growth is all larger, after adding the graphene oxide of trace, the size of ice crystal has and significantly reduces, and along with the increase of graphene oxide addition, Ice crystal size is on a declining curve, illustrate that graphene oxide has inhibitory action to the ice-crystal growth in liquid freezing and resuscitation process, and this acting in all kinds of liquid system all exists, and has universality.
And freezen protective liquid A of the present invention is not adding in graphene oxide situation, the size of ice crystal is just close with the Ice crystal size of 20% glycerine, illustrates that freezen protective liquid of the present invention has inherently possessed the cryoprotective effect of conventional antifreeze.After adding graphene oxide, the amplitude that the Ice crystal size of freezen protective liquid A of the present invention reduces is maximum, and equally, along with the increase of graphene oxide addition, Ice crystal size continues to decline to a great extent, and most I is to less than 100 μm.Illustrate that freezen protective liquid of the present invention is compared with biological culture conventional at present or preservation system, be applicable to very much coordinating with graphene oxide being used in freezen protective technology.
In addition, only graphene oxide aqueous dispersions itself, and the graphene oxide of separate sources also can suppress the growth of ice crystal, and also relevant to concentration to the inhibitory action of ice-crystal growth.The inhibition outline of the graphene oxide dispersion that embodiment 1 is prepared is better than the graphene oxide dispersion be purchased.Be purchased comparing of graphene oxide and can learn, Size Distribution presses down the effect of ice within the scope of 200 ~ 500nm can be better, and this is mainly due to time oversize, and lamella in the solution bends, and should not be combined with ice crystal.And time undersized, the complete structure area of pure Graphene reduces on the surface, be unfavorable for absorption.Therefore preferred graphene oxide size is in 200 ~ 500nm scope.In addition, single-layer graphene oxide quantity is more, and the surface area that compares is larger, and be more conducive to absorption, therefore more preferably the number of plies is 1 ~ 2 layer.
Embodiment 4
Freezen protective liquid is to the application of horse sperm Cord blood
1. horse seminal fluid obtains, transports and stores
The age of all kinds of horses was at 4 to 15 years old, sperm is collected once for every three days, adopt pseudo-vagina method, collect the dense part seminal fluid of stage casing sperm, crossed by special filter paper and filter jelly, carry out initial analysis by microscope and Computerized analysis system, ensure that in the fresh semen of getting, forward motile sperm accounts for more than 60% of whole sperm, the number of sperm that cell morphology change does not occur in the fresh semen of getting accounts for more than 70% of whole sperm.Satisfactory batch is kept in vacuum flask.
2. sperm storage experimental design
The parallel study of different freezen protective system has been carried out in experiment, and the freezen protective system used is respectively: glycerine, glycerine add the aqueous dispersions that graphene oxide, glycerine prepared by embodiment 1 add the graphene oxide of preparation in the freezen protective liquid A in embodiment 2, the freezen protective liquid B in embodiment 2 and embodiment 1.
3. the step of horse sperm cryopreservation
(1) seminal fluid got joins in freezen protective system according to the ratio of volume ratio 1:1, is transferred to insulating box, makes temperature be down to 5 DEG C from body temperature, maintains 90min at such a temperature.
(2), after 90min, adopt the mode of temperature programmed control to make seminal fluid temperature be down to-110 DEG C with the speed of 40 DEG C/min, then be down to-140 DEG C with 20 DEG C/min, the seminal fluid after cooling is placed in liquid nitrogen container, is preserved by liquid nitrogen.
Sperm after Cord blood is recovery 30s in 37 DEG C of environment, utilizes shaking table to shake cryopreservation tube gently in equilibrium process, under then cryopreservation tube being placed in room temperature condition, shakes 30s gently to melting completely.
Other mammiferous sperm storage also can use freezen protective liquid and the refrigeration operation step of the present embodiment.
The properties of computer system to sperm is utilized to detect and characterize afterwards.
4. the freezen protective result of sperm quality
Get the seminal fluid after 10 μ L freeze thawing in the counting chamber of sperm attributional analysis instrument (sperm quality image system, model RCZ-200G, SSA-II sperm automatic detection analysis system), detect the quality of 5 above sperms in the visual field.The testing result of the every quality of horse sperm mainly comprises: sperm percentage (motile_pct) of living, straight ahead sperm percentage (progressive_pct, generally speaking, it is the strongest that this part sperm is considered to fertilizing ability), quick advance sperm percentage (papid_pct).
From accompanying drawing 5 can be clear and definite find out, as Cryoprotectant, to the effect order of quality of horse sperm cryopreservation be: freezen protective liquid B, glycerine add graphene oxide, freezen protective liquid A glycerol adding, graphene oxide dispersion, glycerine.Compared to pure seminal fluid, above conserving liquid has certain activity, freezen protective liquid B best results, obviously be better than the glycerine commonly used, and by adding graphene oxide dispersion, the preservation effect of glycerine conserving liquid also significantly improves, and illustrates that graphene oxide has certain universality as Cryoprotectant.In addition, independent graphene oxide is also better than glycerine as Cryoprotectant effect, further demonstrates the effect of graphene oxide as Cryoprotectant.Meanwhile, by comparing the result of graphene oxide dispersion and freezen protective liquid of the present invention, illustrate that various electrolyte contained in freezen protective liquid A provided by the invention and carbohydrate can help to improve the preservation effect of freezen protective liquid.
5. different graphene oxide concentration is on the impact of sperm cryopreservation
Use the freezen protective liquid B containing graphene oxide in embodiment 2, by changing the quantity research graphene oxide concentration of the graphene oxide added to the impact of sperm cryopreservation, in freezen protective liquid, the mass percent concentration of graphene oxide is respectively 0.001%, 0.010%, 0.100%, 1.000% and 2.000%, using the freezen protective liquid A in embodiment 2 as blank.
According to the Slow-rate freezing step in aforementioned 3rd part carry out melting freezing and again (5 DEG C of balances 90 minutes, Slow-rate freezing (4 DEG C to-110 DEG C, 40 DEG C/min;-110 DEG C to-140 DEG C, 20 DEG C/min), drop into Liquid nitrogen storage.Freezing complete after 37 DEG C of water-baths thaw.)。
Sperm motility parameters after thawing with compater assisted sperm analysis evaluation, result as shown in Figure 6.
As we can see from the figure; with the addition of three indexs after the experimental group cryopreservation resuscitation of graphene oxide all significantly better than blank group; the effect of high concentration is better than low concentration; but along with the increase of graphene oxide addition; index reduces gradually; this may be due to analytical system by excessive graphene oxide statistics for Necrospermia, therefore optimum protected effect appears at graphene oxide concentration when being 0.1%.
Embodiment 5
The application of external Cord blood candidate stem cell
1. the preparation of candidate stem cell suspension, first candidate stem cell activator is expelled in picker's body, when candidate stem cell activity reaches peak value, adopt the liquid in disposable syringe absorption artery, the liquid collected is at the centrifugal lower centrifugal 10 ~ 15min of 500 ~ 1000g, get the cell of precipitation, precipitate with the ratio re-suspended cell of volume ratio 1:2 with HES (HES) solution, left at room temperature 30 ~ 60min, get 100mL supernatant with the centrifugal 5 ~ 10min of 500 ~ 1000g, precipitation is the individual cells of candidate stem cell, adding 50mLDMEM high glucose medium carries out resuspended, candidate stem cell re-suspension liquid is placed in insulating box and preserves.
2. take out the candidate stem cell suspension after preserving, dilute with the freezen protective liquid B that the ratio of volume ratio 1:1 adds in embodiment 2, and in this freezen protective liquid, the concentration of graphene oxide is 0.1%.Carry out freezen protective according to the step of embodiment 4, with DMEM high glucose medium, stem cell suspension is diluted 2 times after recovery, be placed in 4-6 DEG C of environment, adopt the method evaluation candidate stem cell in-vitro multiplication ability that CFU-GM cultivates.
3. after adopting this freezen protective liquid freezen protective, human hematopoietic stem cell maintains proliferation activity substantially, and after candidate stem cell preserves one week, cell proliferation is obvious, and survival rate is high.Illustrate that freezen protective liquid of the present invention is suitable for the freezen protective of candidate stem cell.
Embodiment 6
The application of cartilaginous tissue freezen protective
1. the bilateral knee articular cartilage tissue of what the present embodiment was chosen is new zealand white rabbit, the articular cartilage of surgical exposure rabbit in aseptic environment, obtains the cartilage on articular surface with hollow boring bit, operation finishing, obtain thickness at 1 ~ 2mm, diameter is the discoid cartilage of 5mm.Rinse with DMEM in high glucose medium, be transferred in the DMEM medium containing 5%FBS (hyclone), 37 DEG C, 5%CO
22 ~ 4h is cultivated under condition.
2. the cartilaginous tissue of above-mentioned metabolic stability is positioned in EP pipe, DMEM medium is added in each hole, add isopyknic 0.2% graphene oxide aqueous dispersions simultaneously, ensure that conserving liquid interface exceeds cartilaginous tissue, Cord blood is carried out according to the process of cryopreservation described in embodiment 4, duration is one month, using the DMEM of same volume as blank.After freezen protective, cartilaginous tissue is transferred in the constant incubator of 37 DEG C, passes into the CO of 5%
2gas, constant temperature culture 1 week.
3. adopt ELISA to detect the cartilaginous tissue of the albuminous secretory volume of cartilaginous tissue to freezen protective to evaluate, concrete steps are: joined by the above-mentioned cultivation medium of 1 week in 96 hole ELISA Plate (commercialization), every hole 50 μ L, cultivates 60min for 37 DEG C, takes out ELISA Plate, discard liquid, every hole adds the how anti-solution of 100 μ L enzyme mark, incubated at room temperature 30min, and every hole separately adds substrate solution 50 μ L, under room temperature, lucifuge colour developing 10min, measures the light absorption value at 450nm place.Result shows, and after adding freezen protective liquid of the present invention, cartilaginous tissue albumin secretory volume is 801 ± 115ng/mL, and the secretory volume of control group is only 420 ± 22.3ng/mL, and comparatively control group improves nearly one times.Illustrate that graphene oxide also can improve the controlled-rate freezing of cartilaginous tissue.
Embodiment 7
The Cord blood of kidney organ
1. under gnotobasis, take out the kidney organ of rabbit, inject the freezen protective liquid B described in embodiment 2, and the organ injecting freezen protective liquid is saved in the container filling freezen protective liquid B, carry out freezen protective according to the step described in embodiment 4, duration is one month.
2. the various morphological change of rear organ are melted again by observation by light microscope.
3. observe under an optical microscope, the structures such as kidney organ's mesopodium cell nucleus, mesangial cell are very complete, in kidney skin medullary substance structure, glomerulus, capillary is clear and legible, Bowman's capsule does not find to expand phenomenon, and the above results shows that freezen protective liquid provided by the invention has good Cord blood effect to kidney organ.
4. similarly, after freezen protective recovery, the kidney that this example is recovered for ex vivo perfusion freezen protective liquid freezen protective has carried out PSP test (phenol red excretion test), in order to measure renal tubular function, after testing, the kidney organ's amount of urinating of 1 hour, 2 hours after freezen protective recovery is all higher than normal value.Illustrate that freezen protective liquid of the present invention is suitable for the freezen protective of kidney organ.
Embodiment 8
Roots of Panax Notoginseng apparatus
1. the Roots of Panax Notoginseng clear water of collecting season is cleaned, epidermis interior tissue is exposed with sterilizing scalpel at root in super-clean bench, interior tissue is cut into small pieces, put into solid culture medium, at 25 DEG C, lucifuge cultivates 40 days, this solid culture medium is make (Green by oneself after laboratory reference document, C.E., & Phillips, R.L. (1975) .Plantregenerationfromtissueculturesofmaize.CropScience, 15 (3), 417-421.), main component is containing 1g glucose in 1L medium, 25g sucrose, 4g raffinose, 3mg benzyladenine, 2mg Sodium Benzoate, glycine 2g, 7g agar, by phosphate adjust ph to 5.6, medium prepares latter 120 DEG C, sterilizing 25min under 0.1Mpa.
2. through the Secondary Culture of 40 days, choose fast growth, loosely organized Roots of Panax Notoginseng tissue, (this liquid nutrient medium is identical with above-mentioned solid culture based component to be transferred to liquid nutrient medium according to the concentration of 10g/L ~ 20g/L, not containing agar) the middle cultivation that suspends, cultivate 6 days, within every 3 days, add the fresh culture of 10%.
3. the cell mass choosing cultivation metabolic stability after 6 days of about 1cm is inoculated in 200mL aforesaid liquid medium again, and adds the graphene oxide aqueous dispersions of 200mL0.2%.Meanwhile, the cell mass arranging about 1cm is inoculated in 400mL and dilutes as blank in the liquid nutrient medium of a times, and carry out freezen protective according to the freezen protective step described in embodiment 4, duration is one month.
4. take out the Roots of Panax Notoginseng cell after freezen protective, carry out restoration ecosystem, control cultivation temperature at 25 DEG C, intensity of illumination is 2000lx, and every day cultivates 12h, cultivates 15 days.
5. found by microscopic examination, under identical freezen protective step and renewal cultivation condition, add the Roots of Panax Notoginseng cell proliferation amount of graphene oxide aqueous dispersions and survival rate apparently higher than blank sample.Illustrate that graphene oxide can be used for the freezen protective of plant cell, the efficiency of plant cell freezen protective can be improved.
Embodiment 9
Skin cell tissue's Cord blood
1. be transferred in glass culture dish in the circular skin cell tissue engineering of 10cm at the diameter constructed, add vitamin E and vitamin C that 20mL concentration is 20mmol/L, the mass concentration simultaneously adding 20mL graphene oxide is the graphene oxide aqueous dispersions of 0.2%, and design simultaneously adds vitamin E and vitamin c solution that 40mL concentration is 10mmol/L as blank.Freezen protective 24h is carried out according to the step described in embodiment 4.After freezen protective terminates, detect after rewarming 18h in 37 DEG C of environment.
2. be added with the cryogenic freezing conserving liquid group of graphene oxide after freezen protective and recovery, vigor of skin tissue has remained on higher level, carry out contrast find by adding coloring agent and flesh tissue engineering skin, although through the process of freezen protective and recovery, the institutional framework level of organization engineering skin is still comparatively clear, and cell number reduces not obvious, does not occur cell shrinkage and cavity, illustrate at low temperatures, cryopreservation solution is still fine to the preservation effect of organization engineering skin.All similar to the organization engineering skin of brand-new in organizational vitality, cellular morphology.Illustrate that graphene oxide is also suitable for the freezen protective of skin histology.
Claims (10)
1. graphene oxide is used for the growth of ice crystal in the freezing and/or resuscitation process of freezing-inhibiting conserving liquid.
Preferably described freezing and/or resuscitation process is the freezing of biomaterial and/or recovery; Further preferred described biomaterial is selected from bacterium, fungi, virus, algae, Richettsia, conveyor screw, derive from human body, the cell of animal or plant, tissue or organ, and the bacterium obtained by biotechnology, fungi, virus, algae, cell or tissue; The cell or tissue that further preferably described biomaterial is selected from human body, the cell of animal or plant, tissue or organ and is obtained by biotechnology; Be more preferably candidate stem cell, cartilaginous tissue, kidney organ, the root cells of pseudo-ginseng, skin engineered tissue, the sperm of mammal or the mankind.
2. graphene oxide is preparing the application in freezen protective liquid.
Preferably described freezen protective liquid is the freezen protective for biomaterial; Further preferred described biomaterial is selected from bacterium, fungi, virus, algae, Richettsia, conveyor screw, derive from human body, the cell of animal or plant, tissue or organ, and the bacterium obtained by biotechnology, fungi, virus, algae, cell or tissue; The cell or tissue that further preferably described biomaterial is selected from human body, the cell of animal or plant, tissue or organ and is obtained by biotechnology; Be more preferably candidate stem cell, cartilaginous tissue, kidney organ, the root cells of pseudo-ginseng, skin engineered tissue, the sperm of mammal or the mankind.
3. a method for freezen protective, is characterized in that, in freezen protective liquid, add graphene oxide.
Preferably described freezing and storing method is the freezing and storing method of biomaterial; Further preferred described biomaterial is selected from bacterium, fungi, virus, algae, Richettsia, conveyor screw, derive from human body, the cell of animal or plant, tissue or organ, and the bacterium obtained by biotechnology, fungi, virus, algae, cell or tissue; The cell or tissue that further preferably described biomaterial is selected from human body, the cell of animal or plant, tissue or organ and is obtained by biotechnology; Be more preferably candidate stem cell, cartilaginous tissue, kidney organ, the root cells of pseudo-ginseng, skin engineered tissue, the sperm of mammal or the mankind.
4. the application described in any one of claims 1 to 3 or method, is characterized in that, described graphene oxide is sheet, is of a size of 200nm-12 μm, is preferably 200 ~ 500nm; The number of plies is 1 ~ 3 layer, is preferably 1 ~ 2 layer; Carbon oxygen element ratio is 1.5 ~ 2.2:1, is preferably about 1.9:1.
The mass percent concentration of preferential oxidation Graphene in freezen protective liquid is 0.001% ~ 2%, is preferably 0.01%-1%, more preferably 0.05 ~ 0.5%, is more preferably 0.08 ~ 0.3%, is most preferably 0.1%.
5. the application described in any one of Claims 1 to 4 or method, it is characterized in that, described freezen protective liquid is containing, for example lower component: in every premium on currency, containing 0.3 ~ 0.7g glucose, 7 ~ 10g lactose, 0.1 ~ 0.5g raffinose, 0.9 ~ 1.3g4-HEPES, 2 ~ 5g phosphate-buffered to, 1 ~ 2g sodium chloride, 2 ~ 4g potassium chloride, 0.01 ~ 0.04g magnesium sulfate, 0.05 ~ 0.1g calcium chloride, 0.5 ~ 1g casein, pH value is 6.9 ~ 7.5.
Preferably described phosphate-buffered is to consisting of: sodium dihydrogen phosphate and sodium hydrogen phosphate, sodium hydrogen phosphate and tertiary sodium phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate, dipotassium hydrogen phosphate and tripotassium phosphate, or diammonium hydrogen phosphate and ammonium dihydrogen phosphate (ADP).Most preferably be sodium dihydrogen phosphate and sodium hydrogen phosphate, or potassium dihydrogen phosphate and dipotassium hydrogen phosphate.
Preferable ph is 7.1 ~ 7.3.
Preferably, described freezen protective liquid is containing, for example lower component: in every premium on currency, about 0.5g glucose, about 9.0g lactose, about 0.3g raffinose, about 1.1g4-HEPES, about 1.2gNaH
2pO
42H
2o, about 1.6gNa
2hPO
42H
2o, about 1.5g sodium chloride, about 3.0g potassium chloride, about 0.02g magnesium sulfate, about 0.07g calcium chloride, about 0.6g casein.
6. the application described in any one of Claims 1 to 5 or method, is characterized in that, freezing step comprises:
(1) biomaterial is joined in freezen protective liquid, be transferred to insulating box, make temperature be down to 3 ~ 6 DEG C, be preferably about 5 DEG C, maintain 60 ~ 120 minutes at such a temperature, preferably about 90 minutes; With
(2) adopt the mode of temperature programmed control to make conserving liquid system temperature be down to about-140 DEG C with the speed of 20 ~ 60 DEG C/min, the conserving liquid system after cooling is placed in liquid nitrogen container and preserves; Preferably be down to about-110 DEG C with the speed of about 40 DEG C/min, then be down to about-140 DEG C with about 20 DEG C/min.
7. a freezen protective liquid, is characterized in that, containing graphene oxide, described graphene oxide is sheet, is of a size of 200nm-12 μm, is preferably 200 ~ 500nm; The number of plies is 1 ~ 3 layer, is preferably 1 ~ 2 layer; Carbon oxygen element ratio is 1.5 ~ 2.2:1, is preferably about 1.9:1.
The mass percent concentration of preferential oxidation Graphene in freezen protective liquid is 0.001% ~ 2%, is preferably 0.01%-1%, more preferably 0.05 ~ 0.5%, is more preferably 0.08 ~ 0.3%, is most preferably 0.1%.
8. freezen protective liquid according to claim 7, it is characterized in that, further containing, for example lower component: in every premium on currency, containing 0.3 ~ 0.7g glucose, 7 ~ 10g lactose, 0.1 ~ 0.5g raffinose, 0.9 ~ 1.3g4-HEPES, 2 ~ 5g phosphate-buffered to, 1 ~ 2g sodium chloride, 2 ~ 4g potassium chloride, 0.01 ~ 0.04g magnesium sulfate, 0.05 ~ 0.1g calcium chloride, 0.5 ~ 1g casein, pH value is 6.9 ~ 7.5.
Preferably described phosphate-buffered is to consisting of: sodium dihydrogen phosphate and sodium hydrogen phosphate, sodium hydrogen phosphate and tertiary sodium phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate, dipotassium hydrogen phosphate and tripotassium phosphate, or diammonium hydrogen phosphate and ammonium dihydrogen phosphate (ADP).Most preferably be sodium dihydrogen phosphate and sodium hydrogen phosphate, or potassium dihydrogen phosphate and dipotassium hydrogen phosphate.
The pH value of preferred freezen protective liquid is 7.1 ~ 7.3.
Preferably, described freezen protective liquid is containing, for example lower component: in every premium on currency, about 0.5g glucose, about 9.0g lactose, about 0.3g raffinose, about 1.1g4-HEPES, about 1.2gNaH
2pO
42H
2o, about 1.6gNa
2hPO
42H
2o, about 1.5g sodium chloride, about 3.0g potassium chloride, about 0.02g magnesium sulfate, about 0.07g calcium chloride, about 0.6g casein.
9. a freezen protective liquid, it is containing, for example lower component: in every premium on currency, containing 0.3 ~ 0.7g glucose, 7 ~ 10g lactose, 0.1 ~ 0.5g raffinose, 0.9 ~ 1.3g4-HEPES, 2 ~ 5g phosphate-buffered to, 1 ~ 2g sodium chloride, 2 ~ 4g potassium chloride, 0.01 ~ 0.04g magnesium sulfate, 0.05 ~ 0.1g calcium chloride, 0.5 ~ 1g casein, pH value is 6.9 ~ 7.5.
Preferably described phosphate-buffered is to consisting of: sodium dihydrogen phosphate and sodium hydrogen phosphate, sodium hydrogen phosphate and tertiary sodium phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate, dipotassium hydrogen phosphate and tripotassium phosphate, or diammonium hydrogen phosphate and ammonium dihydrogen phosphate (ADP).Most preferably be sodium dihydrogen phosphate and sodium hydrogen phosphate, or potassium dihydrogen phosphate and dipotassium hydrogen phosphate.
The pH value of preferred freezen protective liquid is 7.1 ~ 7.3.
Preferably, described freezen protective liquid is containing, for example lower component: in every premium on currency, about 0.5g glucose, about 9.0g lactose, about 0.3g raffinose, about 1.1g4-HEPES, about 1.2gNaH
2pO
42H
2o, about 1.6gNa
2hPO
42H
2o, about 1.5g sodium chloride, about 3.0g potassium chloride, about 0.02g magnesium sulfate, about 0.07g calcium chloride, about 0.6g casein.
Preferably further containing glycerine, DMSO and/or graphene oxide.
10. freezen protective liquid according to claim 9, described graphene oxide is sheet, is of a size of 200nm-12 μm, is preferably 200 ~ 500nm; The number of plies is 1 ~ 3 layer, is preferably 1 ~ 2 layer; Carbon oxygen element ratio is 1.5 ~ 2.2:1, is preferably about 1.9:1.
Preferably, the mass percent concentration of selected graphene oxide in freezen protective liquid is 0.001% ~ 2%, is preferably 0.01% ~ 1%, more preferably 0.05 ~ 0.5%, is more preferably 0.08 ~ 0.3%, is most preferably 0.1%.
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Cited By (8)
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| CN106857498A (en) * | 2016-12-27 | 2017-06-20 | 科兴（大连）疫苗技术有限公司 | A kind of cell cryopreservation protection liquid and its application without dimethyl sulfoxide (DMSO) |
| CN107980766A (en) * | 2017-12-19 | 2018-05-04 | 天津农学院 | A kind of method of donkey testicular fluid freezen protective |
| CN108184817A (en) * | 2018-01-11 | 2018-06-22 | 南京三生生物技术股份有限公司 | A kind of umbilical cord blood hematopoietic stem cell frozen stock solution and cryopreservation methods |
| CN108271770A (en) * | 2017-01-06 | 2018-07-13 | 中国科学院化学研究所 | Application of the micron particles in cryopreservation |
| CN109497043A (en) * | 2018-12-28 | 2019-03-22 | 中国医学科学院整形外科医院 | Serum-free nano material freezes agent and preparation method thereof, application method, frozen stock solution |
| CN111657267A (en) * | 2020-06-17 | 2020-09-15 | 科瑞百奥泰州生物技术有限公司 | Ice-free crystal frozen preservation solution and freezing method for preservation of cartilage, tendon and meniscus |
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| CN106857498A (en) * | 2016-12-27 | 2017-06-20 | 科兴（大连）疫苗技术有限公司 | A kind of cell cryopreservation protection liquid and its application without dimethyl sulfoxide (DMSO) |
| CN108271770A (en) * | 2017-01-06 | 2018-07-13 | 中国科学院化学研究所 | Application of the micron particles in cryopreservation |
| CN108271770B (en) * | 2017-01-06 | 2021-06-22 | 中国科学院化学研究所 | Application of micron particles in low-temperature freezing storage |
| CN107980766A (en) * | 2017-12-19 | 2018-05-04 | 天津农学院 | A kind of method of donkey testicular fluid freezen protective |
| CN108184817A (en) * | 2018-01-11 | 2018-06-22 | 南京三生生物技术股份有限公司 | A kind of umbilical cord blood hematopoietic stem cell frozen stock solution and cryopreservation methods |
| CN109497043A (en) * | 2018-12-28 | 2019-03-22 | 中国医学科学院整形外科医院 | Serum-free nano material freezes agent and preparation method thereof, application method, frozen stock solution |
| CN111793108A (en) * | 2019-04-09 | 2020-10-20 | 北京大学第三医院（北京大学第三临床医学院） | Application of cryopreservation liquid containing peptide compounds in organ and tissue cryopreservation |
| WO2020227843A1 (en) * | 2019-05-10 | 2020-11-19 | 友康恒业生物科技（北京）有限公司 | Formulation and use of preservation solution for clinical cell preparations |
| CN111657267A (en) * | 2020-06-17 | 2020-09-15 | 科瑞百奥泰州生物技术有限公司 | Ice-free crystal frozen preservation solution and freezing method for preservation of cartilage, tendon and meniscus |
| CN111657267B (en) * | 2020-06-17 | 2021-02-02 | 科瑞百奥泰州生物技术有限公司 | Ice-free crystal frozen preservation solution and freezing method for preservation of cartilage, tendon and meniscus |
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