CN105230610B - A kind of freezen protective liquid and its preparation method and application - Google Patents
A kind of freezen protective liquid and its preparation method and application Download PDFInfo
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Abstract
The invention provides a kind of freezen protective liquid for containing two-dimensional material-graphene oxide, it prepares and applied, and belongs to biomaterial freezing Techniques of preserving field.In freezen protective liquid, by adding graphene oxide, can freezing-inhibiting preserve the growth of tiny ice crystal in liquid resuscitation process, and then destruction of the ice-crystal growth process to cell is avoided, so that substantially increasing biomaterial freezing preserves survival rate and cell activity after recovery.Because this method is conceived to suppression ice-crystal growth, therefore this freezen protective liquid has higher universality, can be used for the freezen protective of cell, tissue etc., the need for real use is met to greatest extent.
Description
Technical field
The present invention relates to the application of freezen protective, especially graphene oxide in biomaterial freezing preservation, Yi Jiyi
Kind of the freezen protective liquid containing graphene oxide, the preparation method of the preservation liquid, and the application preservation liquid carry out cell, tissue or
The method that organ is preserved, belongs to biomaterial freezing Techniques of preserving field.
Background technology
Low temperature or cryopreservation technology are the one kind developed rapidly 1970s in dry ice or liquid nitrogen
Under conditions of preserve the technology of the biomaterial such as biological tissue, organ and cell.Under cryogenic, biological tissue, which is in, " stops
The state of dormancy ", it is ensured that the heredity of biomaterial and morphological stability, is a kind of effective method for preserving biomaterial.It is near several
In 10 years, the application of this technology concentrates on the animals such as the mammality with economic value, birds, fish and various plants
Research field.Low temperature, cryopreservation not only promote the hair of the basic subjects such as biology, medical science as an emerging technology
Exhibition, has also played great effect in fields such as agricultural, animal husbandry, aquacultures, has generated great economic benefit.
However, in resuscitation process, contained ice crystal meeting continued propagation in freezen protective liquid, and ice crystal damage is freezing guarantor
Deposit in technology to the topmost injury of conservation object, be due to freeze proof preservation liquid or intracellular moisture formation ice crystal, stab thin
Caused by born of the same parents.
Slow-rate freezing method and vitrifying freeze process has had been developed in Refrigeration Technique field at present, and latter of which utilizes high concentration
Cryoprotector solidified under ultra-low temperature surroundings, form irregular glassy solid, ice crystal do not formed, so that cell exists
Protected in violent fast cooling, to overcome ice crystal to damage, still, vitrified sample can still be produced in resuscitation process
Substantial amounts of ice crystal.Presently mainly by adjusting the composition of freezen protective liquid, for example using permeability antifreeze (such as glycerine,
DMSO etc.) and impermeability antifreeze (such as polyethylene glycol, PVP), and regulate and control the mode of frozen cooling formula, subtract to try one's best
The formation of few ice crystal.
Graphene oxide is a kind of to repeat unsaturated six-membered carbon ring as the amphipathic two-dimensional material of skeleton, its surface distributed
Oxygen-containing functional group and complete graphene region.Graphene oxide can generally there are interface such as interfacial agent, and reduce boundary
Energy between face.Current graphene oxide is polymerizeing the application of species composite and inorganic species field of compound material gradually
Get up extensively.
The content of the invention
The present inventor's research finds that graphene oxide can be dispersed stably in liquid, and can freezing-inhibiting
And/or in resuscitation process ice crystal growth.On this basis, present invention protection following technical scheme：
Graphene oxide is used for the growth that freezing-inhibiting preserves ice crystal in liquid freezing and/or resuscitation process.It is preferred that described
Freezing and/or resuscitation process are the freezing and/or recovery of biomaterial.
Application of the graphene oxide in freezen protective liquid is prepared.It is preferred that described freezen protective liquid is to be used for biomaterial
Freezen protective.
A kind of method of freezen protective, it is characterised in that add graphene oxide in freezen protective liquid.It is preferred that 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 claimed technical scheme of the invention.
" biomaterial " available for the present invention includes but is not limited to：Bacterium, fungi, virus, algae, Richettsia, spiral shell
Body is revolved, from human body, the cell of animal and plant, tissue, organ, and the bacterium, true obtained by biotechnology
Bacterium, virus, algae, cell and tissue.In a word in biotechnology and association area it is all can freezen protective, and need freezing
The living material for preserving and recovering all is encompassed within the category of " biomaterial " of the invention.In certain embodiments, biomaterial
Can be naturally occurring bacterium or the engineering bacteria obtained by biotechnology, such as Escherichia coli, salmonella, gold
Yellow staphylococcus, streptococcus, bacillus subtilis etc.；Biomaterial can be derived from human body, animal in certain embodiments
With the cell of plant, or the engineering cell obtained by cell engineering, the liver cell of such as animal or human body, reproduction are thin
Born of the same parents, nerve cell, myofibroblasts, Epithelial cell, root cells, the leaf cell of plant, or various tumour cells or miscellaneous
Oncocyte is handed over, such as Hela cells, SHG-44 glioma cells, A549 cells；Biomaterial can be with certain embodiments
Be derived from human body or the tissue or organ of animal, or the tissue obtained by tissue engineering technique, for example skin histology or
Skin engineered tissue, cartilaginous tissue, bone tissue or bone engineered tissue, connective tissue, musculature, nerve fiber, kidney, liver
Dirty, cornea, heart, lung etc..Biomaterial is candidate stem cell, cartilaginous tissue, kidney device in some embodiments of the invention
The sperm of official, the root cells of pseudo-ginseng, skin engineered tissue, mammal or the mankind.
" freezen protective liquid " available for the present invention is for the various molten of suspended biological material in process of cryopreservation
Liquid, for example：10~50% glycerine, the physiological saline added with the antifreeze such as glycerine, DMSO, PVP or polyethylene glycol,
Hank ' s liquid, DMEM nutrient solutions, RPMI-1640 nutrient solutions or all kinds of bacteria culture medias etc. or Hank ' s liquid, DMEM trainings
Nutrient solution, RPMI-1640 nutrient solutions or all kinds of bacteria culture medias etc..
The effect that graphene oxide suppresses ice-crystal growth is based primarily upon containing on its structure and chemical property, graphene oxide
Hydroxyl in oxygen functional group effectively can be combined with each other with ice crystal, hydrone be prevented to the diffusion on ice crystal interface, so as to press down
The brilliant growth of ice making.Based on this, those skilled in the art are it is contemplated that the graphene oxide in various sources can be used for suppressing cold
The growth of ice crystal, for the freezen protective of biomaterial, and is added graphene oxide into various in jelly and/or resuscitation process
In freezen protective liquid, the moisture ice-crystal growth inside liquid and biomaterial can be preserved by freezing-inhibiting, is further improved cold
Freeze the freezen protective efficiency for preserving liquid.
Graphene oxide available for the present invention can be obtained by commercially available mode, such as Sigma-Aldrich
Co.LLC. the production code member sold is 763713,763705 and 777676 graphene oxide aqueous dispersions, it would however also be possible to employ oxygen
The conventional method of graphite alkene is prepared, and peels off to obtain after such as Hummers method graphite oxides.
In the present invention, mol ratio of the carbon oxygen element of graphene oxide than referring to carbon oxygen element.
Be preferred for the present invention graphene oxide be flaky material, its size be 200nm-12 μm, preferably 200~
500nm；The number of plies is 1~3 layer, preferably 1~2 layer；Carbon oxygen element is than 1.5~2.2:1, preferably about 1.9:1.
It is preferred that mass percent concentration of the graphene oxide in freezen protective liquid is 0.001%~2%, it is preferably
0.01%~1%, more preferably 0.05~0.5%, more preferably 0.08~0.3%, most preferably from about 0.1%.
Those skilled in the art can also add according to the characteristics of the biomaterial for freezing or cultivating in freezen protective liquid
Plus other chemical compositions, the functional polypeptide such as protein, growth factor or protein, physiology can such as sugar, seralbumin
Buffer system, inorganic electrolyte of receiving etc..
The present invention still further provides a kind of specific freezen protective liquid, in terms of every liter of water, contain 0.3~0.7g grapes
Sugar, 7~10g lactose, 0.1~0.5g raffinoses, 0.9~1.3g 4- hydroxyethyl piperazineethanesulfonic acids, 2.0~5.0g phosphate delay
Punching to, 1~2g sodium chloride, 2~4g potassium chloride, 0.01~0.04g magnesium sulfate, 0.05~0.1g calcium chloride, 0.5~1g junket eggs
In vain, the pH value of freezen protective liquid is 6.9~7.5, preferably 7.1~7.3.
Described phosphate-buffered phosphatic Conjugate Acid-Base Pairs to being made up of, and the phosphate can be the alkali of phosphoric acid
Metal salt, such as tertiary sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, tripotassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate；Phosphoric acid
Alkali salt, such as calcium phosphate, calcium dihydrogen phosphate, calcium monohydrogen phosphate, tricresyl phosphate magnesium, the magnesium of phosphoric acid one, di(2-ethylhexyl)phosphate magnesium；Phosphoric acid
Ammonium salt, such as diammonium hydrogen phosphate, ammonium dihydrogen phosphate.The phosphate can be the hydrate of anhydride or different hydrautures.
It is preferred that described phosphate-buffered is to composition：Sodium dihydrogen phosphate and disodium hydrogen phosphate, disodium hydrogen phosphate and phosphoric acid
Trisodium, potassium dihydrogen phosphate and dipotassium hydrogen phosphate, dipotassium hydrogen phosphate and tripotassium phosphate, diammonium hydrogen phosphate and ammonium dihydrogen phosphate.It is optimal
Elect sodium dihydrogen phosphate and disodium hydrogen phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate as.
In described freezen protective liquid, containing carbohydrates such as glucose, lactose, raffinoses, it can provide outer for biomaterial
Source energy, forms sugar-coat protection, prevents damage of the external environment to biological material cell film under freezing conditions.Contain HEPES, phosphorus
The materials such as hydrochlorate, sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, for biomaterial preservation provide suitable osmotic pressure environment and
Suitable acid-base value.The freezen protective liquid is applied to the cell and tissue, the freezen protective of organ of all kinds of animal and plants, this
Art personnel can adjust the formula of freezen protective liquid in above-mentioned content range as needed.
It is preferred that, the freezen protective liquid contains following component：In terms of every liter of water, about 0.5g glucose, about 9.0g lactose, about
0.3g raffinoses, about 1.1g 4- hydroxyethyl piperazineethanesulfonic acids, about 1.2g NaH2PO4·2H2O, about 1.6g Na2HPO4·2H2O、
About 1.5g sodium chloride, about 3.0g potassium chloride, about 0.02g magnesium sulfate, about 0.07g calcium chloride, about 0.6g caseins.
The preparation method of the freezen protective liquid is：After each component is weighed according to consumption, it is added in deionized water, fills
Divide after mixed dissolution, using conventional degerming or sterilizing method, such as heat sterilization method is degerming to be positioned over 4 DEG C of refrigerations afterwards
It is kept in dark place.
In addition to above-mentioned each component, for enhancing controlled-rate freezing, mesh can also be added in the freezen protective liquid
The antifreeze that preceding in the market is commonly used such as glycerine, DMSO etc., can also add graphene oxide, further to improve its freezing
The effect of preservation.
The method that graphene oxide is added in freezen protective liquid, can be the graphene oxide for weighing respective amount, physics
Smalls is ground to form after broken, by the smalls ultrasonic disperse of graphene oxide into freezen protective liquid；Or according to the oxygen to be added
The consumption of graphite alkene, the graphene oxide aqueous dispersions of the concentration known of proper volume are added into mixing in freezen protective liquid is
Can.
It is preferred that graphene oxide used is flaky material, its size is 200nm-12 μm, preferably 200~500nm；Layer
Number is 1~3 layer, preferably 1~2 layer；Carbon oxygen element is than 1.5~2.2:1, preferably about 1.9:1.
It is preferred that mass percent concentration of the graphene oxide in freezen protective liquid is 0.001%~2%, it is preferably
0.01%~1%, more preferably 0.05~0.5%, more preferably 0.08~0.3%, most preferably from about 0.1%.
When the freezen protective liquid provided using the present invention carries out Slow-rate freezing to biomaterial, operating procedure can be as follows：
(1) by biomaterial, such as cell suspending liquid, tissue or organ are added in appropriate freezen protective liquid, for
In organ, also biomaterial then can be transferred to insulating box by freezen protective perfusion wherein as needed so that temperature drops
To 3~6 DEG C, preferably 5 DEG C, maintain 60~120 minutes, preferably 90 minutes at such a temperature.
(2) mode of temperature programmed control is used so that preserve liquid system temperature is down to -140 with 20~60 DEG C/min speed
DEG C, the preservation liquid system after cooling is placed in liquid nitrogen container, preserved by liquid nitrogen.It is preferred that being dropped with 40 DEG C/min speed
To -110 DEG C, then -140 DEG C are down to 20 DEG C/min.
Biomaterial after Cord blood, recovers in 37 DEG C of environment, is gently shaken using shaking table in equilibrium process, then
Cryopreservation tube or other biological material culture vessel are placed under room temperature condition, gently shaken to melting completely.
The advantage of the invention is that：
Graphene oxide is sheet two-dimensional material, and the oxygen-containing functional group of its surface distributed causes graphene oxide to be easier in water
In disperse.In water, graphene oxide can be hydrated and then negatively charged, and this electronegative graphene oxide can overcome
Van der Waals force and interaction of hydrogen bond between lamella and lamella, it is ensured that the stability of graphene oxide dispersion.Also, stone
The lattice structure and I of black alkenehThe ice crystal of type has perfect Lattice Matching, and being provided for graphene oxide suppression ice-crystal growth can
Energy property, the effect of this suppression ice-crystal growth is based primarily upon the special structure and chemical property of graphene oxide, graphene oxide
On oxygen-containing functional group formation cluster be distributed on lamella, complete graphene-structured also integrated distribution, therefore whole graphene
Lamella should be the admixture of oxygen-containing functional group and complete graphene sheet layer.And the hydroxyl in oxygen-containing functional group has with hydrone
Very strong interaction, effectively can be combined with each other with ice crystal, and hydrophobic graphene oxide layer is after ice crystal is formed
It can be arranged by hydrone, so as to preferably be combined with ice crystal.Furthermore, complete graphene oxide and IhType ice crystal
There is extraordinary matching effect in basal faces, therefore during biomaterial freezing and/or recovery, after ice crystal is formed,
The graphene oxide of two dimension will be adsorbed firmly on the interface of ice crystal, prevent hydrone to the diffusion on ice crystal interface, from
And suppress the growth of ice crystal.Graphene oxide is effectively reduced the growth due to ice crystal to biology to the inhibitory action of ice crystal
The harm that Materials Cell is punctured.And graphene oxide has higher specific surface area, using very in freezen protective liquid system
Few amount has universality with regard to that can play a part of effectively suppressing ice-crystal growth.
Be not true solution in addition, graphene oxide is dispersed in freezen protective liquid, recovered in the later stage when removing, by from
The heart or the mode of sedimentation are easy to reject, and antifreeze more conventional than DMSO etc. is easy to operate.
Brief description of the drawings
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 thickness, which is shown, is dispersed in freezen protective
Graphene oxide in 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, and right figure is oxygen
Ice-crystal growth figure in graphite alkene aqueous dispersions.
The effect statistical chart that Fig. 5 difference samples are recovered to horse sperm freezing
Fig. 6 contains influence statistical chart of the various concentrations graphene oxide freezen protective liquid to sperm cryopreservation
Embodiment
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 change all exist
Within protection scope of the present invention.
Embodiment 1
The preparation of graphene oxide
1st, graphite is pre-oxidized
2.5g phosphorus pentoxide and 2.5g potassium peroxydisulfate are weighed, is added into 250mL conical flasks.What is be stirred continuously
In the case of add the 20mL concentrated sulfuric acids, be heated to after 80 DEG C, be slowly added to 3g graphite powders into solution, keep the temperature of solution 80
DEG C reach 4.5h after be cooled to room temperature.Deionized water is slowly added to afterwards, ensures that the temperature of solution is no more than 80 DEG C during this,
Mixed liquor is stood overnight, and the excessive concentrated sulfuric acid, 60 DEG C of drying are removed after filtering.
2nd, the preparation of graphite oxide
Pre-oxidation graphite after drying is added in the 120mL concentrated sulfuric acids, in the case where being stirred continuously, is slowly added to 15g
Potassium permanganate, the process is carried out in ice bath, it is ensured that whole process temperature persistently stirs 2h below 80 DEG C.Again will reaction
Container is transferred in ice bath environment, is added in deionized water, whole dilution and is ensured that temperature is less than 80 DEG C, then persistently stirs 2h
700mL deionized waters are added afterwards, 20mL are added dropwise afterwards, 30wt% hydrogen peroxide stirs 1h, fully oxidized graphite at room temperature.
3rd, ultrasound, which is peeled off, is made graphene oxide
The graphite oxide of above-mentioned gained is filtered, and gained solid is distributed in dilute hydrochloric acid solution (concentrated hydrochloric acid:Water
(v/v)=1：9) supernatant, is removed after centrifugal filtration, retains lower floor's solid, this process is repeated 8 times.Afterwards, then deionized water is used
The hydrochloric acid remained in solid is removed, it is same by the way of centrifuging, until the pH value of cleaning fluid is 7.Finally by above-mentioned filter
Cake is dissolved in a small amount of deionized water again, in ultrasound 1h under 100W ultrasonic powers, obtains finely dispersed graphene oxide
Suspension, detected through AFM, obtained graphene oxide Size Distribution is in 200-500nm, 1-2 layers of the number of plies, carbon
Oxygen element ratio is 1.9.
4th, prepared by graphene oxide powder
Above-mentioned graphene oxide is transferred to culture dish, is placed in baking oven, 50 DEG C of vacuum dryings, the graphene oxide of gained
Physics is crushed, and using mortar grind into fine powder, is placed in PE pipes, 4 DEG C are kept in dark place.
Embodiment 2
The preparation of freezen protective liquid of the present invention
1st, weigh 0.5g glucose, 9g lactose, 0.3g raffinoses, 1.1g 4- hydroxyethyl piperazineethanesulfonic acids,
1.2gNaH2PO4·2H2O、1.6gNa2HPO4·2H2O, 1.5g sodium chloride, 3g potassium chloride, 0.02g magnesium sulfate, 0.07g chlorinations
Calcium, 0.6g caseins, are added in 1L deionized waters, are sufficiently mixed after dissolving, standby in the insulating box for being saved in 37 DEG C.For
The convenience of following examples description, referred to as the freezen protective liquid that this step is prepared, freezen protective liquid A.
2nd, the preparation of the freezen protective liquid containing graphene oxide
Weigh physics in graphene oxide prepared by a certain amount of embodiment 1, ultra-clean chamber to crush, ground in agate mortar
Into smalls, 100W ultrasound 30min are distributed in the freezen protective liquid A that the 1st step is prepared so that the quality of graphene oxide
Percent concentration is 0.1%, and resulting solution pH value is 7.2.
The convenience described for following examples, this step is prepared the freezen protective liquid containing graphene oxide obtained, letter
Referred to as freezen protective liquid B.
Using the graphene oxide particle size range in AFM measure dispersion liquid in 200~500nm or so, individual layer
Graphene oxide thickness in 1.5nm or so, be specifically shown in Fig. 1-3.
Embodiment 3
Inhibitory action of the different samples to ice-crystal growth
(Deller R C, Vatish M, Mitchell D A, the et al.Synthetic by the way of document report
polymers enable non-vitreous cellular cryopreservation by reducing ice
Crystal growth during thawing [J] .Nature communications, 2014,5.) evaluate different samples and exist
To the inhibitory action of ice-crystal growth in low temperature environment.
1st, sample preparation：Choose 0.7% physiological saline, the freezen protective liquid A of the embodiment of the present invention 2 and (be free of oxidation stone
Black alkene), 20% glycerine, commercially available graphene oxide aqueous dispersions (Sigma-Aldrich Co.LLC.763713, its graphite oxide
The Size Distribution of alkene is 200nm-12 μm, and 2~3 layers, C/O is the graphene oxide aqueous dispersions 1.9) prepared with embodiment 1
(the aqueous dispersions concentration of two kinds of graphene oxides is set as mass percent concentration 0.01%, 0.1% and 0.5%).Meanwhile, it is right
It is 0.01%, 0.1% and 0.5% that physiological saline, freezen protective liquid A and 20% glycerine, which are also separately added into whole mass percent concentration,
Embodiment 1 prepare graphene oxide.
2nd, 10 μ L samples are transferred to freshly prepared mica surface, it is evenly distributed on mica surface, using cold and hot
Mica fast cooling is warming up to -6 DEG C by platform to -60 DEG C, then with 5 DEG C/min speed, and 60min is kept at such a temperature.
3rd, ice crystal is taken pictures using microscope, counts the size of ice crystal, as a result as follows, the error bar in table is derived from
The statistical result of 150 ice crystals：
Influence of the graphene oxide of table 1 to different liquids system ice-crystal growth
As can be seen from Table 1, the freezen protective liquid A of physiological saline, 20% glycerine and the present invention is not adding graphite oxide
Before alkene, the size of ice-crystal growth is larger, and there being the size of ice crystal after the micro graphene oxide of addition significantly reduces, and
With the increase of graphene oxide addition, Ice crystal size is on a declining curve, illustrates graphene oxide to liquid freezing and recovery
During ice-crystal growth have inhibitory action, and this act on all exists in all kinds of liquid systems, with universality.
And the freezen protective liquid A of the present invention is in the case of graphene oxide is added, the size of ice crystal is just and 20% glycerine
Ice crystal size approach, illustrate that freezen protective liquid of the invention inherently possesses the cryoprotective effect of conventional antifreeze.
Add after graphene oxide, the amplitude that freezen protective liquid A of the invention Ice crystal size reduces is maximum, equally, with oxidation
The increase of graphene addition, Ice crystal size persistently declines to a great extent, and most I is to less than 100 μm.Illustrate that the freezing of the present invention is protected
Liquid storage is especially suitable for coordinating used in freezen protective skill with graphene oxide compared with conventional at present biological culture or preservation system
In art.
In addition, only graphene oxide aqueous dispersions itself, the graphene oxide of separate sources can also suppress ice crystal
Growth, it is and also related to concentration to the inhibitory action of ice-crystal growth.The graphene oxide dispersion that embodiment 1 is prepared
Inhibition will be slightly better than commercially available graphene oxide dispersion.Comparison with commercially available graphene oxide is it is known that size point
The effect that cloth presses down ice in the range of 200~500nm can be more preferable, this mainly due to it is oversized when, lamella hair in the solution
Raw bending, should not be combined with ice crystal.And when undersized, on surface, the complete structure area of pure graphene reduces, and is unfavorable for inhaling
It is attached.Therefore preferred graphene oxide size is in 200~500nm scopes.In addition, single-layer graphene oxide quantity is more, phase
Contrast surface area bigger, be more beneficial for absorption, therefore more preferably the number of plies is 1~2 layer.
Embodiment 4
Application of the freezen protective liquid to horse sperm Cord blood
1. horse seminal fluid is obtained, transports and stored
The age of all kinds of horses, sperm was collected once for every three days, using pseudo- vagina method, collects stage casing sperm at 4 to 15 years old
Dense part seminal fluid, jelly is filtered to remove by special filter paper, is tentatively divided by microscope and Computerized analysis system
Analysis, it is ensured that forward motile sperm accounts for more than the 60% of whole sperms in the fresh semen taken, does not have in the fresh semen taken
The sperm count for occurring cell morphology change accounts for more than the 70% of whole sperms.Satisfactory batch is stored in vacuum flask.
2. sperm storage experimental design
The parallel study of different freezen protective systems has been carried out in experiment, and used freezen protective system is respectively：Glycerine,
Glycerine adds the freezen protective liquid A in graphene oxide, glycerine plus embodiment 2 prepared by embodiment 1, the freezing in embodiment 2 to protect
The aqueous dispersions of the graphene oxide prepared in liquid storage B and embodiment 1.
3. the step of horse sperm cryopreservation
(1) seminal fluid taken is according to volume ratio 1：1 ratio is added in freezen protective system, is transferred to insulating box, is made
Obtain temperature and be down to 5 DEG C from body temperature, 90min is maintained at such a temperature.
(2) after 90min, the mode of temperature programmed control is used so that seminal fluid temperature is down to -110 DEG C with 40 DEG C/min speed,
- 140 DEG C are down to 20 DEG C/min again, the seminal fluid after cooling is placed in liquid nitrogen container, preserved by liquid nitrogen.
Sperm after Cord blood gently shakes cryopreservation tube in recovery 30s, equilibrium process in 37 DEG C of environment using shaking table,
Then cryopreservation tube is placed under room temperature condition, gently shakes 30s to melting completely.
The freezen protective liquid and refrigeration operation step of the present embodiment can also be used in the sperm storage of other mammals.
The properties of sperm are detected and characterized using computer system afterwards.
4. the freezen protective result of sperm quality
The seminal fluid after 10 μ L freeze thawing is taken in sperm attributional analysis instrument (sperm quality image system, model RCZ-200G, SSA-
II sperm automatic detection analysis system) counting chamber in, detect 5 visual field above sperms quality.Horse sperm items quality
Testing result mainly includes：Sperm percentage (motile_pct) living, straight ahead sperm percentage (progressive_pct,
In general, to be considered as fertilizing ability most strong for the part sperm), fast forward through sperm percentage (papid_pct).
Can clearly it find out from accompanying drawing 5, as Cryoprotectant, to the effect order of quality of horse sperm cryopreservation
For：Freezen protective liquid B, glycerine add graphene oxide, freezen protective liquid A glycerol addings, graphene oxide dispersion, glycerine.Compare
In pure seminal fluid, the above, which preserves liquid, certain activity, freezen protective liquid B best results, hence it is evident that better than conventional glycerine, and
By adding graphene oxide dispersion, the preservation effect that glycerine preserves liquid is also significantly improved, and illustrates graphene oxide as cold
Freezing preservative agent has certain universality.In addition, individually graphene oxide as Cryoprotectant effect also superior to glycerine, enter
One step demonstrates graphene oxide as the effect of Cryoprotectant.Meanwhile, by comparing graphene oxide dispersion and Ben Fa
The result of bright freezen protective liquid, illustrates that various electrolyte and carbohydrate contained by the freezen protective liquid A that the present invention is provided can
Help improves the preservation effect of freezen protective liquid.
5. influence of the different graphene oxide concentration to sperm cryopreservation
Using the freezen protective liquid B containing graphene oxide in embodiment 2, by changing the graphene oxide added
The mass percent of graphene oxide is dense in influence of the quantity research graphene oxide concentration to sperm cryopreservation, freezen protective liquid
Degree be respectively 0.001%, 0.010%, 0.100%, 1.000% and 2.000%, using the freezen protective liquid A in embodiment 2 as
Blank control.
Freezed according to the Slow-rate freezing step in foregoing third portion and melt again (5 DEG C balance 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), put into Liquid nitrogen storage.37 DEG C of water after the completion of freezing
Bath is thawed.).
Sperm motility parameters after being thawed with compater assisted sperm analysis evaluation, as a result as shown in Figure 6.
From the figure, it can be seen that three indexs after with the addition of the experimental group cryopreservation resuscitation of graphene oxide are significantly better than
Blank control group, the effect of high concentration is better than low concentration, but with the increase of graphene oxide addition, index is gradually reduced,
This is probably that, because excessive graphene oxide statistics is Necrospermia by analysis system, therefore optimal protecting effect appears in oxygen
When graphite alkene concentration is 0.1%.
Embodiment 5
The application of external Cord blood candidate stem cell
1. candidate stem cell activator, is expelled in picker's body by the preparation of candidate stem cell suspension first, wait to make
When hemocytoblast activity reaches peak value, the liquid in artery is drawn using disposable syringe, the liquid of collection 500~
1000g centrifugations 10~15min of lower centrifugation, takes the cell of precipitation, with HES (HES) solution with volume ratio 1：2 ratio
Cell precipitation is resuspended, 30~60min is stood at room temperature, takes 100mL supernatants to centrifuge 5~10min with 500~1000g, precipitation is
For the individual cells of candidate stem cell, add 50mLDMEM high glucose mediums and be resuspended, candidate stem cell re-suspension liquid is placed in perseverance
Preserved in incubator.
2. the candidate stem cell suspension after preserving is taken out, with volume ratio 1：The freezen protective that 1 ratio is added in embodiment 2
Liquid B is diluted, and the concentration of graphene oxide is 0.1% in the freezen protective liquid.The step of according to embodiment 4, carries out cold
Freeze and preserve, stem cell suspension is diluted 2 times with DMEM high glucose mediums after recovery, is placed in 4-6 DEG C of environment, using CFU-GM
The method of culture evaluates candidate stem cell in-vitro multiplication ability.
3. using after the freezen protective liquid freezen protective, human hematopoietic stem cell maintains proliferation activity substantially, and Hematopoietic Stem is thin
After born of the same parents preserve one week, cell propagation is obvious, and survival rate is high.Illustrate that the freezen protective liquid of the present invention is suitable to the freezing of candidate stem cell
Preserve.
Embodiment 6
The application of cartilaginous tissue freezen protective
1. what the present embodiment was chosen is the bilateral knee articular cartilage tissue of new zealand white rabbit, performed the operation in sterile environment
The articular cartilage of exposure rabbit, the cartilage on articular surface is obtained with hollow boring bit, and operation finishing obtains thickness in 1~2mm,
A diameter of 5mm discoid cartilage.Rinsed with DMEM in high glucose culture medium, be transferred to the DMEM trainings containing 5%FBS (hyclone)
Support in base, 37 DEG C, 5%CO2Under the conditions of cultivate 2~4h.
2. the cartilaginous tissue of above-mentioned metabolic stability is positioned in EP pipes, DMEM culture mediums are added into each hole, together
When add 0.2% isometric graphene oxide aqueous dispersions, it is ensured that preserve liquid interface and exceed cartilaginous tissue, according to embodiment 4
Described process of cryopreservation carries out Cord blood, when a length of one month, blank control is used as using the DMEM of same volume.Freezing
Cartilaginous tissue is transferred in 37 DEG C of constant incubator after preservation, 5% CO is passed through2Gas, incubated 1 week.
3. the secretory volume for detecting cartilaginous tissue albumin using ELISA is commented the cartilaginous tissue of freezen protective
Valency, is concretely comprised the following steps：The culture medium of above-mentioned culture 1 week is added in 96 hole elisa Plates (commercialization), per the μ L of hole 50,37 DEG C of cultures
60min, takes out ELISA Plate, discards liquid, and the 100 how anti-solution of μ L enzyme marks are added per hole, 30min is incubated at room temperature, separately adds per hole
The μ L of substrate solution 50, lucifuge colour developing 10min, determines the light absorption value at 450nm at room temperature.As a result show, add the cold of the present invention
Freeze and preserve after liquid, cartilaginous tissue albumin secretory volume is 801 ± 115ng/mL, and the secretory volume of control group be only 420 ±
22.3ng/mL, is improved by about one time compared with control group.Illustrate that graphene oxide can also improve the freezen protective effect of cartilaginous tissue
Really.
Embodiment 7
The Cord blood of kidney organ
1. the kidney organ of rabbit is taken out under gnotobasis, the freezen protective liquid B described in injection embodiment 2, and will note
The organ for entering freezen protective liquid is saved in the container for filling freezen protective liquid B, is freezed according to the step described in embodiment 4
Preserve, when a length of one month.
2. melt the various morphological changes of rear organ again by observation by light microscope.
3. observing under an optical microscope, the structure such as kidney organ's mesopodium nucleus, mesangial cell is very complete, kidney
Capillary is clear and legible in skin medullary substance structure, glomerulus, and Bowman's capsule does not find to expand phenomenon, and the above results show this hair
The freezen protective liquid of bright offer has good Cord blood effect to kidney organ.
4. similarly, after freezen protective recovery, this example is directed to the kidney that ex vivo perfusion freezen protective liquid freezen protective is recovered
It is dirty to have carried out PSP experiments (phenol red excretion test), to determine renal tubular function, after testing, the kidney after freezen protective recovery
The organ urination amount of 1 hour, 2 hours is all higher than normal value.Illustrate that the freezen protective liquid of the present invention is suitable to the freezing of kidney organ
Preserve.
Embodiment 8
Pseudo-ginseng root cells Cord blood
1. the Roots of Panax Notoginseng of collecting season is cleaned with clear water, epidermis is exposed in root with sterilizing scalpel in super-clean bench
Interior tissue, interior tissue is cut into small pieces, and is put into solid medium, at 25 DEG C, lucifuge culture 40 days, the solid medium
To make (Green, C.E., ＆Phillips, R.L. (1975) .Plant regeneration after laboratory reference document by oneself
From tissue cultures of maize.Crop Science, 15 (3), 417-421.), main component is 1L culture mediums
In contain 1g glucose, 25g sucrose, 4g gossyposes, 3mg benzyladenines, 2mg sodium benzoates, glycine 2g, 7g agar, use
Phosphate adjusts pH value to 5.6, and culture medium prepares the 25min that sterilized under 120 DEG C afterwards, 0.1Mpa.
2. by the Secondary Culture of 40 days, choose fast growth, loosely organized pseudo-ginseng root tissue, according to 10g/L~
20g/L concentration is transferred in fluid nutrient medium (fluid nutrient medium is identical with above-mentioned solid culture based component, without agar)
Suspend culture, cultivates 6 days, the fresh culture for adding 10% in every 3 days.
3. the cell mass for choosing metabolic stability after 1cm or so culture 6 days is inoculated in 200mL aforesaid liquid cultures again
Base, and add 200mL0.2% graphene oxide aqueous dispersions.Meanwhile, set 1cm or so cell mass to be inoculated in 400mL
As blank control in the fluid nutrient medium of one times of dilution, freezen protective is carried out according to the freezen protective step described in embodiment 4,
Shi Changwei mono- month.
4. taking out the pseudo-ginseng root cells after freezen protective, restoration ecosystem is carried out, control cultivation temperature is in 25 DEG C, intensity of illumination
For 2000lx, 12h is cultivated daily, is cultivated 15 days.
5. by microscope it has been observed that under the conditions of identical freezen protective step and renewal cultivation, adding graphite oxide
The pseudo-ginseng root cells proliferative amount and survival rate of alkene aqueous dispersions are apparently higher than blank sample.Illustrate that graphene oxide can be used for plant
The freezen protective of cell, it is possible to increase the efficiency of plant cell freezen protective.
Embodiment 9
Skin cell tissue's Cord blood
1. being transferred in a diameter of 10cm constructed circular skin cell tissue's engineering in glass culture dish, add
20mL concentration is 20mmol/L vitamin E and vitamin C, while the mass concentration for adding 20mL graphene oxides is
0.2% graphene oxide aqueous dispersions, while it is molten for 10mmol/L vitamin E and vitamin C to design addition 40mL concentration
Liquid is used as blank control.Freezen protective 24h is carried out according to the step described in embodiment 4.After freezen protective terminates, in 37 DEG C of environment
Detected after middle rewarming 18h.
2. the cryogenic freezing added with graphene oxide preserves liquid group after freezen protective and recovery, vigor of skin tissue is protected
Hold in higher level, by adding coloring agent and the progress contrast discovery of flesh tissue engineering skin, although protected by freezing
The process deposited and recovered, the institutional framework level of organization engineering skin is still more clear, and cell number reduction is not obvious, does not occur
Cell shrinkage and cavity, illustrate at low temperatures, and cryopreservation solution is still fine to the preservation effect of organization engineering skin.Group
Knit vigor, it is similar all to the organization engineering skin of brand-new in cellular morphology.Illustrate that graphene oxide is also suitable for the cold of skin histology
Freeze and preserve.
Claims (43)
1. graphene oxide is applied to the growth that freezing-inhibiting preserves ice crystal in liquid freezen protective and/or resuscitation process, the oxygen
Graphite alkene is sheet.
2. application as claimed in claim 1, described freezen protective and/or resuscitation process is the freezen protective of biomaterial
And/or recovery.
3. application of the graphene oxide in freezen protective liquid is prepared, the graphene oxide is sheet.
4. application as claimed in claim 3, described freezen protective liquid is the freezen protective for biomaterial.
5. a kind of method of freezen protective, it is characterised in that graphene oxide, the graphite oxide are added in freezen protective liquid
Alkene is sheet.
6. method as claimed in claim 5, described freezing and storing method is the freezing and storing method of biomaterial.
7. application or method as described in any one of claim 1~6, it is characterised in that the graphene oxide size is
200nm-12μm。
8. application as claimed in claim 7 or method, the size of the graphene oxide is 200~500nm.
9. application as claimed in claim 7 or method, the number of plies of the graphene oxide is 1~3 layer.
10. application as claimed in claim 9 or method, the number of plies of the graphene oxide is 1~2 layer.
11. application as claimed in claim 7 or method, the carbon oxygen element ratio of the graphene oxide is 1.5~2.2:1.
12. application as claimed in claim 11 or method, the carbon oxygen element ratio of the graphene oxide is 1.9:1.
13. application as claimed in claim 7 or method, mass percent of the graphene oxide in freezen protective liquid are dense
Spend for 0.001%~2%.
14. application as claimed in claim 13 or method, mass percent of the graphene oxide in freezen protective liquid
Concentration is 0.01%-1%.
15. application as claimed in claim 14 or method, mass percent of the graphene oxide in freezen protective liquid
Concentration is 0.05~0.5%.
16. application as claimed in claim 15 or method, mass percent of the graphene oxide in freezen protective liquid
Concentration is 0.08~0.3%.
17. application as claimed in claim 16 or method, mass percent of the graphene oxide in freezen protective liquid
Concentration is 0.1%.
18. application or method as described in any one of claim 1~6, it is characterised in that the freezen protective liquid contains as follows
Component：In terms of every liter of water, contain 0.3~0.7g glucose, 7~10g lactose, 0.1~0.5g raffinoses, 0.9~1.3g 4- hydroxyls
Ethyl piperazidine ethyl sulfonic acid, 2~5g phosphate-buffereds to, 1~2g sodium chloride, 2~4g potassium chloride, 0.01~0.04g magnesium sulfate,
0.05~0.1g calcium chloride, 0.5~1g caseins, pH value are 6.9~7.5.
19. as claimed in claim 18 apply or method, described phosphate-buffered is to composition：Sodium dihydrogen phosphate and phosphoric acid
Disodium hydrogen, disodium 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.
20. application as claimed in claim 19 or method, described phosphate-buffered is to for sodium dihydrogen phosphate and phosphoric acid hydrogen two
Sodium, or potassium dihydrogen phosphate and dipotassium hydrogen phosphate.
21. application as claimed in claim 18 or method, the pH value of the freezen protective liquid is 7.1~7.3.
22. application as claimed in claim 18 or method, the freezen protective liquid contain following component：In terms of every liter of water,
0.5g glucose, 9.0g lactose, 0.3g raffinoses, 1.1g 4- hydroxyethyl piperazineethanesulfonic acids, 1.2gNaH2PO4·2H2O、
1.6gNa2HPO4·2H2O, 1.5g sodium chloride, 3.0g potassium chloride, 0.02g magnesium sulfate, 0.07g calcium chloride, 0.6g caseins.
23. application or method as described in any one of claim 1~6, it is characterised in that freezing step includes：
(1) biomaterial is added in freezen protective liquid, is transferred to insulating box so that temperature is down to 3~6 DEG C, in the temperature
It is lower to maintain 60~120 minutes；With
(2) mode of temperature programmed control is used so that preserve liquid system temperature is down to -140 DEG C, general with 20~60 DEG C/min speed
Preservation liquid system after cooling, which is placed in liquid nitrogen container, to be preserved.
24. -110 DEG C are down to 40 DEG C/min speed in application as claimed in claim 23 or method, the step (2),
Again -140 DEG C are down to 20 DEG C/min.
25. application as claimed in claim 7 or method, the biomaterial are selected from bacterium, fungi, virus, algae, rickettsia
Body, conveyor screw, from human body, the cell of animal or plant, tissue or organ, and by biotechnology obtain it is thin
Bacterium, fungi, virus, algae, cell or tissue.
26. application as claimed in claim 25 or method, the biomaterial are selected from human body, the cell of animal or plant, group
Knit or organ and the cell or tissue that is obtained by biotechnology.
27. application as claimed in claim 26 or method, the biomaterial are candidate stem cell, cartilaginous tissue, kidney device
The sperm of official, the root cells of pseudo-ginseng, skin engineered tissue, mammal or the mankind.
28. a kind of freezen protective liquid, it is characterised in that containing graphene oxide, the graphene oxide is sheet, size is
200nm-12μm。
29. freezen protective liquid as claimed in claim 28, the size of the graphene oxide is 200~500nm.
30. freezen protective liquid as claimed in claim 28, the number of plies of the graphene oxide is 1~3 layer.
31. freezen protective liquid as claimed in claim 30, the number of plies of the graphene oxide is 1~2 layer.
32. freezen protective liquid as claimed in claim 28, the carbon oxygen element ratio of the graphene oxide is 1.5~2.2:1.
33. freezen protective liquid as claimed in claim 32, the carbon oxygen element ratio of the graphene oxide is 1.9:1.
34. the freezen protective liquid as described in any one of claim 28~33, the graphene oxide is in freezen protective liquid
Mass percent concentration is 0.001%~2%.
35. freezen protective liquid as claimed in claim 34, mass percent of the graphene oxide in freezen protective liquid
Concentration is 0.01%-1%.
36. freezen protective liquid as claimed in claim 35, mass percent of the graphene oxide in freezen protective liquid
Concentration is 0.05~0.5%.
37. freezen protective liquid as claimed in claim 36, mass percent of the graphene oxide in freezen protective liquid
Concentration is 0.08~0.3%.
38. freezen protective liquid as claimed in claim 37, mass percent of the graphene oxide in freezen protective liquid
Concentration is 0.1%.
39. the freezen protective liquid as described in any one of claim 28~33, it is characterised in that further containing following component：
In terms of every liter of water, contain 0.3~0.7g glucose, 7~10g lactose, 0.1~0.5g raffinoses, 0.9~1.3g 4- ethoxys
Piperazine ethanesulfonic acid, 2~5g phosphate-buffereds to, 1~2g sodium chloride, 2~4g potassium chloride, 0.01~0.04g magnesium sulfate, 0.05~
0.1g calcium chloride, 0.5~1g caseins, pH value are 6.9~7.5.
40. freezen protective liquid as claimed in claim 39, described phosphate-buffered is to composition：Sodium dihydrogen phosphate and phosphoric acid
Disodium hydrogen, disodium 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.
41. freezen protective liquid as claimed in claim 40, described phosphate-buffered is to for sodium dihydrogen phosphate and phosphoric acid hydrogen two
Sodium, or potassium dihydrogen phosphate and dipotassium hydrogen phosphate.
42. freezen protective liquid as claimed in claim 39, the pH value of the freezen protective liquid is 7.1~7.3.
43. freezen protective liquid as claimed in claim 39, the freezen protective liquid contains following component：In terms of every liter of water,
0.5g glucose, 9.0g lactose, 0.3g raffinoses, 1.1g 4- hydroxyethyl piperazineethanesulfonic acids, 1.2gNaH2PO4·2H2O、
1.6gNa2HPO4·2H2O, 1.5g sodium chloride, 3.0g potassium chloride, 0.02g magnesium sulfate, 0.07g calcium chloride, 0.6g caseins.
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| CN106857498B (en) * | 2016-12-27 | 2021-05-14 | 科兴（大连）疫苗技术有限公司 | Cell cryopreservation protective solution without dimethyl sulfoxide and application thereof |
| 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 |
| CN111793109B (en) * | 2019-04-09 | 2022-05-27 | 中国科学院化学研究所 | Peptide compound and cryopreservation liquid containing same |
| 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 |
| 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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