CN1218632C - Method for storing human cell tissue organs used for therapeutic clone - Google Patents
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Abstract
The present invention relates to a preservation technology for living cells, living tissue and living organs, primary cells, subculture cells and cell strains of a human body in vitro. The preservation technology comprises: various tissue cells of the human body are immersed in freezing protection liquid, and the tissue cells are fully and evenly mixed with the freezing protection liquid; the freezing protection liquid effectively penetrates the tissue, and a temperature is reduced to a predeterminate temperature by stages. Before used, the freezing tissue cells can be preserved without day. The present invention can be used for treating donor cells or cell nuclei which are cloned by sexual organs. In addition, after a frozen human organism and cells are unfrozen and resuscitated, culture in vitro can be used as the research of functional genomics, and raw materials of bioactive compounds can be selected by disease-related genes.
Description
The present invention relates to frozen technology, the external frozen technology of human cell tissue organ (as umbilical cord tissue and umbilical cord relevant cell) especially, wherein frozen cell is used to therapeutic cloning and other purposes.By frozen technology of the present invention, frozen tissue and cell can be preserved before recovery is used indefinite duration.After frozen tissue and cell are recovered, can be used for the raw material that therapeutic cloning and transplanting, functional genomics research, disease related gene screening and screening have bioactive compound.
At present, the storage time of biomaterial is determined by the process that biomaterial is cooled to freezing storage temperature.The inside and outside moisture of cell will experience freezing process in the temperature-fall period from the liquid condition to the solid state, comprises the orderly arrangement of hydrone---crystallization or noncrystallineization process.For freezing scholar, its key problem in technology still can make it get back to physiological status and not damage after being to make cell to enter cryogenic temperature.
Two committed steps of cell and tissue freezing are freeze thawing and crystallization.Freeze-thaw technology requires extracellular liquid to freeze, and freezes but adopted a lot of steps to reduce in the cell.Someone advises adopting bulk sample, and this is frozen, but still can't prevent to freeze in the cell, and these ice crystals pair cell in freeze thawing causes fatal damage just.Another technology is a crystallization technique, wherein attempts to adopt high concentration liquid and/or polymer to reduce ice crystal and forms.Yet the infringement that is exposed to pair cell in the crystallization additive of high concentration even toxic level muchly is also very big.
In the various cellular tissure organs of human body, umbilical cord tissue and umbilical cord relevant cell are considered to the biology refuse always, after the fetus birth, discarded,, be generally short-term research although some umbilical cord tissue and umbilical cord relevant cell can be used as the human body cell source and be used in vitro study.That umbilical cord tissue and umbilical cord relevant cell process is not frozen, the report that is used further to study after thawing.
In recent years, development and success along with somatic cell clone technique, therapeutic organ cloning and of the same race or heterograft technology, require the method for a kind of energy long preservation umbilical cord tissue and umbilical cord relevant cell, guarantee that the various cells of umbilical cord tissue and umbilical cord can recover safely after long-term and even indefinite preservation, and keep its physicochemical property constant, so that have the raw material of bioactive compound used for umbilical cord supplier or other people provide organ cloning, transplanting, functional genomics research, disease related gene screening and screening.
Therefore, the object of the present invention is to provide and a kind of the human cell tissue organ that is used for the treatment of sex clone is carried out frozen method, carry out frozen human body cell, tissue and organ with this method, after thawing, can recover safely and keep physicochemical property constant, thereby be used for various uses as the preparation therapeutic cloning.
In one aspect of the invention, provide a kind of human body cell, tissue or the organ that is used for the treatment of sex clone carried out frozen method, this method may further comprise the steps:
(a) human body cell, tissue or the organ that obtains immersed frozen solution, fully the mixing frozen solution makes frozen solution fully infiltrate through this cell, tissue, organ;
(b) initial temperature is made as 20 ± 5 ℃, is cooled to 5-8 ℃ with 5-15 ℃/minute, continues 15-30 minute then, carries out an ice;
(c) after the ice, kept 5 ± 3 minutes so that cryogenic temperature still remains on 5-8 ℃;
(d) adjust cooling rate to-0.5 to-1.5 ℃/minute and cool the temperature to-8 ± 1 ℃, kept 30 ± 20 minutes;
(e) drop to-0.1 to-0.5 ℃/minute speed and be lower than-70 ℃ storage temperature.
Preferably, step (b)-(e) is at CO
2Carry out in the buffer environment; And, form cell exterior point ice with the metal probe point ice of cooled with liquid nitrogen.Preferably, after ice crystal formed, temperature dropped to-8 ℃ approximately with the speed of 0.5 to 1.5 ℃ of per minute (as 1 degree), and kept 10-50 minute to guarantee that freezing tissue enters before the freezing state, reaches physics and biological balance state in frozen solution.
The present invention also provides thaws frozen human body cell, tissue, organ, is used to prepare the method for therapeutic cloning for nuclear, and it comprises above-mentioned frozen human cell tissue organ quick-thawing in 1-3 minute, examines as supplying in culture in vitro then.
In the frozen process of biomaterial, the cryoprotector role is a cell activity in the protection freezing process.Mainly playing a protective role in the following manner of cryoprotector promptly when water is transformed into ice and salt ionic concentration rising, played diluting effect.Icing amount depends on temperature and solution component.In addition, cryoprotector can also reduce intracellular freezing temperature, stabilizing cell membrane and albumen.
All solution can both descend cold and not freeze before forming the ice crystal core in the temperature that is lower than its freezing point.When with freeze-thaw method when frozen, the some ice when the freezing out of extracellular medium crossed cold low spot now.It is icing that non-some ice causes is spontaneous freezing when solution temperature is below the freezing point widely.Because this process is spontaneous, freezing betides under the temperature that can't predict at random, and therefore the viability of cell is widely different under same frozen program.
In addition, easily cause cell and tissue damage when extremely crossing freezing fast of taking place when cold.In addition, if the extracellular freeze to take place extremely to cross when cold, the possibility that ice forms in the cell increases greatly.This phenomenon comes from the cell dehydration that causes that freezes and postpones to take place, and further increases the retention of water in the cell, thereby the possibility of freezing in the cell has been increased again widely.
In case cell exterior point ice is finished, sample is iced encirclement, and sample just can be cooled to frozen state.
Cooling step is the key point in the freeze-thaw method.The extracellular solution concentration that part is frozen is higher than solution in the cell, for reaching thermodynamical equilibrium, and cell loss moisture and dewatering.Along with increasing gradually of extracellular ice, it is serious further to dewater.Three characteristics of cell determine the speed of its dehydration; (1) the water permeability of cell membrane (the water permeability is poorer, and the time of cell dehydration is longer); (2) temperature dependency of cell film water permeability (its water permeability decrease when all cells film reduces in temperature); (3) size of cell (maxicell needs the longer time to dewater than cellule).Because the characteristic of each cell has nothing in common with each other, its frozen optimum condition just has certain difference.
Although, when frozen cellular damage really cutter system do not illustrate fully as yet, yet measure to find that by cell viability very fast and very slow cooling velocity all reduces cell viability widely, medium cooling velocity produces most preferred result.Although for different cells, its preferred cooling velocity and temperature lowering curve are can difference very big, and freezing overall degree is close.Too fast frozen cell does not have the time enough dehydration and forms ice in the cell, and cellular damage is mainly owing to icing in the cell.When crossing slow cooling and freezing, cellular damage is main because after the cell that is exposed to high concentration for a long time in and extracellular salinity and frozen solution, or ice in the extracellular and cell between interaction.
Ice-nucleus must make cell dehydration before forming in cell.When in the freezing preservation liquid of cell in 1M and 2M concentration, this incident of cell dehydration mostly occurs between-50 ℃-40 ℃.What deserves to be mentioned is, intracellular icing amount may not can in cooling pair cell damage, if but thaw soon inadequately, cell can the death because when dissolving, swelling took place.
Utilize tissue of the present invention and cell freezing method, can avoid the use of interior ice crystal formation of cell and cytotoxicity chemical reagent at low temperatures, utilize the program frigorimeter suitably carrying out the cryoprotection tissue under the cooling rate.The freezing and storing method of this tissue and cell makes tissue and cell keep its vigor and physiological property indefinite duration, for the application of organ cloning, transplanting, vitro detection and other function aspects.
Provided by the invention the human cell tissue organ that is used for the treatment of sex clone is carried out frozen method, comprises step:
(a) human body cell, tissue or the organ that obtains immersed frozen solution, fully the mixing frozen solution makes frozen solution fully infiltrate through this cell, tissue, organ;
(b) initial temperature is made as 20 ± 5 ℃, is cooled to 5-8 ℃ with 5-15 ℃/minute, continues 15-30 minute then, carries out an ice;
(c) after the ice, kept 5 ± 3 minutes so that cryogenic temperature still remains on 5-8 ℃;
(d) adjust cooling rate to-0.5 to-1.5 ℃/minute and cool the temperature to-8 ± 1 ℃, kept 30 ± 20 minutes;
(e) drop to-0.1 to-0.5 ℃/minute speed and be lower than-70 ℃ storage temperature.
The human cell tissue organ that is applicable to the inventive method can be any human body cell, tissue and organ, comprising (but being not limited to): umbilical cord relevant cell, Skin Cell, mammary glandular cell, oviduct cell, ear cell, gonad cell, epithelial cell, fibroblast, endothelial cell, cumulus cell, muscle cell, nerve cell, Gegenbaur's cell; Umbilical cord tissue, skin, mammary gland etc.
In this application, " umbilical cord relevant cell " refers to derive from the various cells of umbilical cord, comprises the various cells of the umbilical cord tissue of digestion back culture in vitro, as vascular endothelial cell, vascular smooth muscle cell, fibroblast etc." umbilical cord tissue " refers to whole tissues of umbilical cord, comprises segment, section etc.
In the methods of the invention, frozen temperature is generally and is lower than-70 ℃, preferably for being not more than-120 ℃, more preferably for being not more than-140 ℃, best for being not more than-196 ℃.Can place-76 ℃ (temperature of dry ice) as preserving (in transit) for short-term.During long preservation, temperature should be lower, for example adopts liquid nitrogen to preserve (196 ℃).
The frozen solution that is applicable to the inventive method comprises that the cell permeability crystal forms reagent or the infiltrative crystal of acellular forms reagent.The example that suitable cell permeability crystal forms reagent comprises (but being not limited to): propane diols, ethylene glycol, methyl-sulfoxide, glycerine are preferably glycerine.The example that the infiltrative crystal of acellular forms reagent comprises (but being not limited to): the HMW glycoconjugate is such as chondroitin sulfate, polyvinylpyrrolidone, polyethylene glycol or HES etc.
Usually, frozen solution is diluted in the thinner.The suitable diluent example comprises: the mixture of phosphate buffer and cell culture fluid such as DMEM, IDMEM, MEM, M199, RPMI1640, Ham ' s F-10, NCTC109, NCTC135 culture fluid or above thinner.Preferably, this thinner is the DMEM culture fluid.In order to keep best cell viability in frozen-thaw process, frozen solution can be adjusted according to each different cell with thinner.
A kind of preferred cryoprotection solution is to be diluted in the 1.5M of DMEM culture fluid to 2.5M glycerine, preferably the glycerine of 2M.
In an example of the present invention, cryoprotector is diluted in the DMEMF medium for 1.5-2.5M glycerine; Freezing tissue is finished in 20 ℃ when cell immerses frozen solution, then, reduces temperature from 20 ℃ to 6 ℃.Cooling rate is-5 to-15 ℃/minute.
With cellular tissure sample and cryoprotection solution mixing the time, can use any mechanical agitation mode to carry out, thereby strengthen the permeability of frozen solution.Suitable mixing method example comprises: vibration, level shakes, centrifugal, vavuum pump suction etc.Usually, the abundant infiltration of time enough with the assurance frozen solution be arranged, so mixing time is generally 1-4 hour.It preferably is 2-3 hour.
Protecting the medium of liquid when dilution refrigeration is the sodium bicarbonate buffer system, preferably can adopt CO
2Buffer environment because infiltrate through in the process of osmosis of tissue and cell at frozen solution, needs CO
2Buffer environment is stable with the acid-base value of guaranteeing solution.CO
2CO in the buffer environment
2Concrete content can be adjusted according to different tissues and cell, in the hope of keeping best cell viability.Usually, CO
2Concentration is for containing 5% or 10% or higher.
In the methods of the invention, should earlier sample temperature be reduced to-5 ℃ to-10 ℃ approximately from room temperature.This is can adopt quick-freeze or freeze program slowly, and being preferably cooling rate is 5-12 ℃/minute, and more preferably cooling rate is 8-10 ℃/minute.
When an ice forms to inspire ice crystal; the frozen pipe of tissue or cell is equipped with in the metal probe contact of available cooled with liquid nitrogen; the also direct frozen solution in the contact tube or by the robotic arm manipulation in the refrigerating chamber also can inject freon in refrigerating chamber or dry ice carries out an ice.
In the temperature-fall period after an ice, should employing freeze program slowly, promptly cooling rate is lower than 0.1-0.5 ℃/minute, and more preferably cooling rate is 0.2-0.5 ℃/minute, more preferably cooling rate is 0.2-0.4 ℃/minute, and cooling rate is about 0.2-0.3 ℃/minute best.
When dissolving cryopreserved tissue or cell, should heat up at a relatively high speed, in 1-3 minute, temperature is risen to 37 ℃.A kind of defreezing method is that frozen pipe is directly inserted 37 ℃ of water-baths.Also can adopt other directly and fast heating means.
After thawing, for avoiding the frozen solution toxic action of pair cell at normal temperatures, must thaw back 15 minutes in, preferably after thawing, remove freezing liquid immediately.
Tissue after thawing or cell can be used for preparing therapeutic cloning, and as functional genomics research, disease related gene screening and screening have the raw material of bioactive compound.
Describe the present invention in further detail below in conjunction with embodiment, should be understood that these embodiment only are used for purposes of illustration and are not used in the restriction scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to this area normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
Human umbilical tissue and umbilical cord relevant cell frozen
Umbilical cord tissue is immersed in the frozen protection liquid of DMEM medium of the 2.0M glycerine of the precooling on ice of equivalent at least, continue 1-2 hour.Perhaps, the same frozen protection liquid with the precooling on ice of the suspension of umbilical cord endothelial cell and equivalent continues 0.5 hour.Abundant during this time mixing frozen solution can place CO
2In the buffer environment, shake up with the 50-70rpm shaking table, or in pipe, jolt gently.
Move into frozen pipe and vacuum seal then.Frozen pipe is inserted program frigorimeter (Planer), and initial temperature is made as 20 ℃, is cooled to 6 ℃ with 5-15 ℃/minute, continues 20 minutes then, then carries out an ice.Continuing 20 minutes to guarantee global tissue temperature unanimity (will keep at least 15 minutes), is 6 ℃.The contact point of some ice is below the liquid level of cryovial.
After the some ice, kept 5 minutes so that freezing indoor temperature still remains on 6 ℃.The adjustment cooling rate is to-1 ℃/minute and cool the temperature to-8 ℃, keeps 30 minutes.Then with-0.3 ℃ or more jogging speed drop to predetermined storage temperature, can be-70 ℃, also can be that more low temperature is as under-120 ℃ or-140 ℃, then the damage of pair cell is less.
Cryopreserved tissue (or cell) moves in-196 ℃ of freezers or the refrigerating chamber from the program frigorimeter and preserves, until use.
Embodiment 2
Thawing of the frozen sample of human umbilical tissue and umbilical cord relevant cell
The cryopreserved tissue dissolving requires to finish fast in 1-3 minute.In this embodiment, the frozen pipe that will contain the frozen sample of human umbilical tissue and umbilical cord relevant cell is directly inserted 37 ℃ of water-baths, or other direct and fast heating means.
After thawing, use the centrifugal 10 minutes method of 1000rpm immediately, remove freezing liquid.
Embodiment 3
People's umbilical cord endothelial cell carries out somatic cell clone
1, obtain for people's umbilical cord endothelial cell of examining:
With embodiment 2 or the people's umbilical cord endothelial cell that directly cultivate to obtain, cultivate with culture fluids such as M199 or RM1640 (adding 10% hyclone), go down to posterity genetic analysis, freezing preservation or directly do for examining.
2, the super row of milch goat is synchronous with the foster mother sheep:
The super row of milch goat: every animal intramuscular injection PG, 0.06-0.1mg/ it is inferior, inject after 10-14 days for the second time at interval, after injecting PG 10-14 days for the second time, begin super row, i.e. intramuscular injection FSH at first, consumption divides 6 times by 7-12IU/Kg (the allogenic animal consumption is not quite similar), 2 times/day, each 8-12 hour at interval.Injection LRH when oestrusing in 24 hours at interval 25ug/ time, does suitably to adjust according to the body weight of sheep.
The foster mother sheep synchronously: for synchronous with the confession matter ewe that the egg mother cell kytoplasm is provided, acceptor ewe is 10-14 days intramuscular injection PG at twice at interval, injecting PG preceding 24 hours for the super row of matter ewe, acceptor is also injected PG simultaneously, time and the same donor of dosage of acceptor injection LRH.
3, the recovery of egg mother cell:
Operation recovery egg mother cell after 28-30 hour behind the milch goat injection LRH.By the conventional cropping of operation, sterilize, open the abdominal cavity, pull out uterus and oviduct, with No. 7 syringe needles of 10ml syringe, be the F10 culture fluid that contains 5%BSA towards ovum liquid, go out intratubal ovum and be connected in the cup of garden.Under stereoscopic anatomical lens, egg mother cell detected place the CZB culture fluid, 37 ℃, cultivate in 5% CO2gas incubator.
4, egg mother cell stoning and the somatic cell of injecting the people
Egg mother cell is moved 20min in the CZB culture fluid that contains the 7.5ug/ml cytochalasin B, move to again in CZB (the containing 20mM HEPES) culture fluid, will be digested to spherical umbilical cord endothelial cell or fibroblast simultaneously and also move in this liquid.Routinely with the egg mother cell stoning, again people's umbilical cord endothelial cell is directly injected in the kytoplasm of egg mother cell after the stoning, the cell of formation is referred to as the reconstruct ovum.The reconstruct ovum places CZB liquid to cultivate 30 minutes, activates then.
5, activation and embedding:
The reconstruct ovum was cultivated 5 minutes for 37 ℃ in CZB+ cytochalasin B (7.5 μ g/ml)+ionomycin (10 μ m).Move to CZB+ cytochalasin B (7.5 μ g/ml)+6-DMAP (2 μ m) 370C, sessile drop method was cultivated 2-6 hour.Reconstruct ovum after the activation moves among the CZB, treats embedding.
Double-layer embedment is adopted in embedding.Used embedding liquid is 0.8% and 1.0% agarose (low melting point glue, about 40 ℃) solution.This embedding liquid is like this preparation: in 10ml cone-shaped glass centrifuge tube an amount of agarose mixed with physiological saline and boil, dissolve extremely fully and be placed on 42 ℃, water-bath is stand-by.
During embedding, in a plastics plate, add and contain Ca
2+, Mg
2+PBS liquid, add hyclone in PBS liquid bottom with suction pipe, the egg mother cell for the treatment of embedding is moved into the serum layer, it is sunk naturally, diapire to be positioned at, sucking-off.Move into then in another little plate that contains 0.8% agarose of just having poured out, the position of egg mother cell is moved (playing cleaning function) 2-4 time, evenly inhale ovum (certain distance is arranged between ovum and the ovum, but mutually by not being too far away) again, move among the PBS.Allow its natural coagulation, cut apart, change the big slightly suction pipe of a bore, carry out second layer embedding by same procedure, difference is: the agarose concentration of embedding liquid is 1.0%.
6, transplant and reclaim:
Use the method for operation, the reconstruct ovum after the embedding is sucked ovum shifting tube, insert ovum shifting tube from the oviduct horn mouth, in ovum shift-in oviduct.Subsequently in oviduct and the suture ligation of uterus connecting portion.After cultivating 6 days in vivo, cut oviduct, go out the embryo, observe the embryonic development situation with culture fluid.
7, embryonic development check and preservation
Adopt PCR method that the embryo who grows is verified promptly whether this embryo is from human body cell.Method is as follows: get its part cell to the embryo of mulberry fruit or blastaea and carry out PCR and detect from growing, the sequence of used single at random primer (10bp) is: 5 '-ACCCCCGAAG-3 ', the PCR response procedures be earlier 94 ℃ 4 minutes, 94 ℃ 10 seconds → 36 ℃ 15 seconds → 72 ℃ 30 seconds (totally 40 circulations) then, be at last 72 ℃ 4 minutes.
Grow to the embryo's of blastaea preservation: can utilize its inner cell mass cell to carry out culture in vitro, set up the ES cell; Perhaps the embryo liquid chilled nitrogen of growing is kept standby.
The result is as follows:
(1) superovulation:
| Super row milch goat sum | The development of ovary (diameter) | Ovulation is counted | Reclaim the ovum number | |||
| ＞2cm | ＞1.5cm | ＞1cm | Do not grow | |||
| 20 | 4 | 8 | 5 | 3 | 225 | 235 |
1, super row milch goat sum: 20
2, the super row of milch goat effect:
Development of ovary rate is (in ovary diameter＞1cm): 85% (17/20)
Average every milch goat number of eggs ovulated: 11.2 pieces (225/20)
Average every milch goat is reclaimed the ovum number: 11.7 pieces (235/20)
Reclaim ovum efficient: 104% (235/225) (wrong when the meter ovulation is counted, the several ovulation points that have abut against together)
(2) acceptor synchronization of estrus: after the PG processing, the sync rates of sheep is 78% (20/26).
(3) Somatic Cell Culture: set up infant umbilical cord endothelial cell or fibroblast.In the process of confession that goes down to posterity, carried out the chromosome number analysis.Observed 100 division phases, 2 times of bodies (46 chromosome) account for 75%.
The used human body cell of this test is umbilical cord endothelial cell or umbilical cord fibroblast, does not carry out freezing and hungry the processing.
(4) egg mother cell stoning and the somatic cell of injecting the people:
The total ovum number of stoning: 153 pieces, wherein survive 148 pieces, survival rate: 96.7%
Survive the ovum number after the somatic cell injection: 133 pieces
Reconstruct survival rate of ovum: 89.8%
(5) reconstructed embryo developmental state
Reconstructed embryo early development information slip
| Transplant the ovum number | Reclaim the ovum number | Reconstruct egg development rate (%) | |||
| The 2-4 cell | The 32-64 cell | Mulberry fruit | In early days+late period blastaea | ||
| 133 | 98 | 17(17.3％) | 23(23.5％) | 12(12.2％) | 24(24.5％) |
The result shows that the developmental rate of people's infant umbilical cord transplanted endothelial cell being gone into enucleation oocyte reaches 75.5%, and the normal development rate reaches 60.2%..
(6) checking of embryonic development
With the cell of embryonic development and goat embryo's (blastaea) DNA, carry out the arbitrarily primed PCR analysis respectively, its testing result is seen Fig. 1, and wherein goat1 and 2 is respectively Saanen goat cell and local goat cell, and human1 and human are the cell from 2 therapeutic cloning preimplantation embryo.
The result shows that two sample rooms of the cell of embryonic development are consistent, also is consistent between two kinds of goat blastaeas; And the difference between goat blastaea and the embryonic development (its genome is from human body cell) is clearly.Therefore, this confirms that embryonic development from the people, promptly contains in the embryonic cell and has plenty of human chromosome and genome.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned instruction content of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. one kind is carried out frozen method to human body cell, tissue or the organ that is used for the treatment of sex clone, it is characterized in that this method may further comprise the steps:
(a) human body cell, tissue or the organ that obtains immersed frozen solution, abundant mixing frozen solution, make frozen solution infiltrate through this cell, tissue, organ, described frozen solution contains the thinner and the 1.5-2.5M glycerine of frozen solution, wherein said thinner is selected from: phosphate buffer, or cell culture fluid DMEM, IDMEM, MEM, M199, RPMI1640, Ham ' s F-10, NCTC109, NCTC135 culture fluid, or the mixture of above thinner;
(b) initial temperature is made as 20 ± 5 ℃, is cooled to 5-8 ℃ with 5-15 ℃/minute, continues 15-30 minute then, carries out an ice;
(c) after the ice, kept 5 ± 3 minutes so that cryogenic temperature still remains on 5-8 ℃;
(d) adjust cooling rate and be 0.5 to 1.5 ℃/minute and cool the temperature to-8 ± 1 ℃, kept 30 ± 20 minutes;
(e) drop to 0.1 to 0.5 ℃/minute speed and be lower than-70 ℃ storage temperature.
2. the method for claim 1 is characterized in that, step (b)-(e) is at CO
2Carry out in the buffer environment; And, form cell exterior point ice with the metal probe point ice of cooled with liquid nitrogen.
3. the method for claim 1; it is characterized in that after ice crystal formed, temperature dropped to-8 ℃ with the speed of per minute 1 degree; and keep 10-50 minute to guarantee that freezing tissue enters before the freezing state, reaches physics and biological balance state in frozen solution.
4. the method for claim 1 is characterized in that, the human cell tissue organ is selected from down group:
Umbilical cord relevant cell, Skin Cell, mammary glandular cell, oviduct cell, ear cell, gonad cell, epithelial cell, fibroblast, endothelial cell, cumulus cell, muscle cell, nerve cell, Gegenbaur's cell; Umbilical cord, skin, mammary gland.
5. the method for claim 1 is characterized in that, the temperature of frozen state is not for being higher than-120 ℃.
6. method as claimed in claim 5 is characterized in that, the temperature of frozen state is not for being higher than-140 ℃.
7. the method for claim 1; it is characterized in that; frozen solution also contains the cell permeability crystal and forms reagent or acellular permeability crystal formation reagent, and is diluted in the medium or medium that is selected from down group: DMEM, IDMEM, MEM, M199, RPMI1640, Ham ' s F12, Ham ' s F10, NCTC109, NCTC135 or phosphate buffer.
8. method as claimed in claim 7, it is characterized in that described cell permeability crystal forms reagent or acellular permeability crystal formation reagent is selected from down group: chondroitin sulfate, polyvinylpyrrolidone, polyethylene glycol, HES, propane diols, ethylene glycol, methyl-sulfoxide.
9. the method for claim 1 is characterized in that, frozen solution is the glycerine that is diluted in the DMEM medium; Freezing tissue is finished in 20 ℃ when cell immerses frozen solution, then, reduces temperature from 20 ℃ to 6 ℃, and cooling rate is 5 to 15 ℃/minute.
10. the method for claim 1 is characterized in that, frozen human cell tissue organ is thawed, and is used to prepare the nuclear that supplies of therapeutic cloning, and its step comprises: quick-thawing in 1-3 minute, examine as supplying in culture in vitro then.
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