CN103404509A - Cell preserving fluid as well as preparation method and application of cell preserving fluid - Google Patents
Cell preserving fluid as well as preparation method and application of cell preserving fluid Download PDFInfo
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- CN103404509A CN103404509A CN2013103415702A CN201310341570A CN103404509A CN 103404509 A CN103404509 A CN 103404509A CN 2013103415702 A CN2013103415702 A CN 2013103415702A CN 201310341570 A CN201310341570 A CN 201310341570A CN 103404509 A CN103404509 A CN 103404509A
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- sodium chloride
- albumin
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- 239000012530 fluid Substances 0.000 title abstract 6
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- 108010088751 Albumins Proteins 0.000 claims abstract description 22
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- 238000004321 preservation Methods 0.000 claims description 79
- 210000004027 cell Anatomy 0.000 claims description 68
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- 108091006905 Human Serum Albumin Proteins 0.000 claims description 14
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Abstract
The invention discloses a cell preserving fluid, as well as a preparation method and application of the cell preserving fluid. The cell preserving fluid is calculated according to 100 ml of sodium chloride injection and/or compound sodium chloride injection. The 100 ml of sodium chloride injection and/or compound sodium chloride injection comprises the following components by weight: 0.01-1 g of vitamins, 0.1-10 g of albumin, 0.1-1 g of saccharides, and 0.001-0.1 g of triphosadenine and/or trinosin. According to the cell preserving fluid, cells are preserved within the temperature range of 2-10 DEG C, the viability and biological characteristics of cells within 24 hours are better maintained, the risks of cell anabiosis and secondary washing are avoided, cell death and change of the biological characteristics of cells caused by the existing preserving method are also avoided, and the territory coverage area of cell application of a central lab is greatly enlarged.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of cell-preservation liquid, its preparation method and application.
Background technology
Stem cell (Stem Cell, SC) is to be present in the cell colony that a class in living individual has height self-renewal capacity and multi-lineage potential.They do not exist only in embryonic development period and in adult, are distributed widely in the privileged site of various histoorgans yet, therefore macroscopic view is upper, it are divided into to embryonic stem cell and adult stem cell.Along with the development of cell replacement therapy, cellular replacement therapy has become the important means for the treatment of clinically some disease.
Immunocyte treatment is a kind of emerging, brand-new antitumour treatments with significant curative effect, made up the drawback of traditional operation, radiation and chemotherapy side effect, being acknowledged as a kind for the treatment of means active, the most rising in 21st century combined therapy of tumour pattern, is also current unique treatment means that is hopeful complete tumors destroyed cell in the world.Immunocyte in clinical middle employing comprises at present: cytokine induced kill cell (CIK), dendritic cell (DC), DC+CIK cell, natural killer cell (NK) and DC-T cell etc.
Cell therapy application prospect has clinically obtained scientist's extensive approval, but still has in actual applications many difficult problems to overcome.At present, cell therapy adopts the mode of drip-feed to carry out mostly.The cell that culture in vitro is good is expelled in the sodium chloride injection of different size, adds or do not add a certain proportion of human serum albumin, and the cell suspension prepared is fed back to sufferer by drip-feed.The cell-preservation liquid adopted is sodium chloride injection or contains groups of people's blood albumin.Adopt this traditional store method, cell is after the preservation of several hours, and Cell viability and biological efficacy have reduction significantly.The routine preservation method is unfavorable for the preservation of cell long period and transportation at a distance.Cell is in the real-life clinical application, because clinical applying unit does not possess corresponding cell culture technology and the specialized cell preparation experiment of high standard chamber, so need to carry out the cell preparation in regional central laboratory, the cell prepared is delivered to clinical unit's application through the transportation of certain hour and distance.Cell needs the transportation holding time of growing before feedback, just require to adopt suitable preservation liquid to maintain motility rate and the biological efficacy of cell in transportation.And frozen cell can adopt the mode of-80 ℃ of dry ice transportations to carry out remote long-time transportation.But the cryopreserving liquid of freeze-stored cell contains serum, medium and DMSO.Frozen cell is applied to before clinical again to be needed to recover and wash, and recovery can cause a certain proportion of cell death, again washs and easily causes cell contamination.
In the preservation of mammalian cell, often use the preservation liquid that contains serum.Serum generally derives from cow's serum or people AB serum.But in the clinical practice of cell therapy, the method requirement for preparing cell is free serum culture, if use serum in preserving liquid, will introduce serum and pollute.Composition in serum is not clear simultaneously, not only contains the compositions such as cell factor, growth factor, hormone, also may contain the problem of unknown virus impact.So exist unknown virus to pollute and owing to preserving the multiple shortcomings such as the uncertain quality caused of liquid composition is difficult to stablize.
Therefore, this area is needed badly and a kind ofly can be maintained well in a long time the cell-preservation liquid that Cell viability and biological property and component are clear and definite.
Summary of the invention
The object of the present invention is to provide a kind of cell-preservation liquid, it preserves cell in the temperature range of 2-10 ℃, Cell viability and biological property in 24 hours, have been maintained well, the risk of having avoided cell recovery and again having washed, also avoid cell death and biological characteristics that existing store method causes to sexually revise, greatly enlarged the region coverage of central laboratory's cell application.
For realizing purpose of the present invention, the present invention by the following technical solutions:
In first aspect, the invention provides a kind of cell-preservation liquid, it wherein contains vitamin 0.01～1 gram, albumin 0.1～10 gram, carbohydrate 0.1～1 gram, adenosine triphosphate and/or trinosin 0.001～0.1 gram in 100 milliliters of sodium chloride injections and/or compound sodium chloride injection.
In cell-preservation liquid provided by the invention, described albumin is selected from one or more in human albumin and mammal albumin, one or more in preferred people and mammiferous blood albumin, ovalbumin and lactoalbumin, more preferably one or more in the blood albumin of people, ox, horse and sheep, ovalbumin and lactoalbumin, most preferably human serum albumin.
In cell-preservation liquid provided by the invention, described vitamin is selected from one or more in vitamin A, vitamin B1, vitamin B2, adenine phosphate, vitamin B6, FA, cobalamin, vitamin C, vitamin D, vitamin E, vitamin K, biotin, citrin, nicotinic acid, vitamin(e) M, vitamin(e) T and vitamin, preferred vitamin C.
In cell-preservation liquid provided by the invention, described carbohydrate is selected from one or more in monose, disaccharides, trisaccharide and polysaccharide, preferably one or more in glucose, galactose, fructose, ribose and trehalose, more preferably glucose.
As preferably, in cell-preservation liquid provided by the invention, in 100 milliliters of sodium chloride injections and/or compound sodium chloride injection, wherein contain vitamin 0.05～0.8 gram, preferred 0.1～0.6 gram, more preferably 0.2～0.5 gram, 0.3～0.4 gram most preferably, albumin 0.5～8 gram, preferred 1～6 gram, more preferably 2～5 grams, 3～4 grams most preferably, carbohydrate 0.2～0.9 gram, preferred 0.3～0.8 gram, more preferably 0.4～0.7 gram, 0.5～0.6 gram most preferably, adenosine triphosphate and/or trinosin 0.005～0.08 gram, preferred 0.01～0.06 gram, more preferably 0.02～0.05 gram, 0.03～0.04 gram most preferably.
In cell-preservation liquid provided by the invention, in 100 milliliters of sodium chloride injections and/or compound sodium chloride injection, wherein can contain vitamin 0.02 gram, 0.03 gram, 0.04 gram, 0.06 gram, 0.07 gram, 0.08 gram, 0.09 gram, 0.11 gram, 0.12 gram, 0.15 gram, 0.18 gram, 0.22 gram, 0.25 gram, 0.28 gram, 0.32 gram, 0.4 gram, 0.45 gram, 0.55 gram, 0.65 gram, 0.7 gram, 0.75 gram, 0.85 gram, 0.9 gram, 0.95 gram or 0.99 gram.
In cell-preservation liquid provided by the invention, in 100 milliliters of sodium chloride injections and/or compound sodium chloride injection, wherein can contain albumin 0.2 gram, 0.3 gram, 0.4 gram, 0.6 gram, 0.7 gram, 0.8 gram, 0.9 gram, 1.1 grams, 1.2 grams, 1.5 grams, 1.8 grams, 2.2 grams, 2.5 grams, 2.8 grams, 3.2 grams, 3.5 grams, 3.8 grams, 4.2 grams, 4.5 grams, 5.5 grams, 6.5 grams, 7 grams, 7.5 grams, 8.5 grams, 9 grams or 9.5 grams.
In cell-preservation liquid provided by the invention, in 100 milliliters of sodium chloride injections and/or compound sodium chloride injection, wherein can contain carbohydrate 0.1 gram, 0.11 gram, 0.12 gram, 0.15 gram, 0.18 gram, 0.22 gram, 0.25 gram, 0.28 gram, 0.3 gram, 0.4 gram, 0.5 gram, 0.6 gram, 0.7 gram, 0.8 gram, 0.9 gram, 0.95 gram or 0.99 gram.
In cell-preservation liquid provided by the invention, in 100 milliliters of sodium chloride injections and/or compound sodium chloride injection, wherein can contain adenosine triphosphate and/or trinosin 0.002 gram, 0.003 gram, 0.004 gram, 0.006 gram, 0.007 gram, 0.008 gram, 0.009 gram, 0.011 gram, 0.015 gram, 0.02 gram, 0.03 gram, 0.04 gram, 0.06 gram, 0.07 gram, 0.08 gram or 0.09 gram.
In second aspect, the invention provides a kind of preparation method as the described cell-preservation liquid of first aspect, by corresponding content, other described vitamin of injection stage, albumin, carbohydrate and adenosine triphosphate and/or trinosin are added in described sodium chloride injection and/or compound sodium chloride injection, thoroughly dissolve, fully mix.
In the preparation method of cell-preservation liquid of the present invention, described vitamin, albumin, carbohydrate and adenosine triphosphate and/or trinosin add in described sodium chloride injection and/or compound sodium chloride injection with the form of parenteral solution and/or freeze-dried powder.
In the third aspect, the invention provides a kind of method of preserving cell, cell is added in cell-preservation liquid as described as first aspect, be placed in 2～10 ℃, preferably 3～8 ℃, more preferably 3～6 ℃, most preferably under 4 ℃ of conditions, preserve.
In the method for the preservation cell provided, temperature can be 2 ℃, 2.5 ℃, 2.8 ℃, 3.2 ℃, 3.8 ℃, 4.2 ℃, 5.5 ℃, 6.5 ℃, 7 ℃, 7.2 ℃, 7.8 ℃, 8.2 ℃, 8.8 ℃, 9.5 ℃ or 9.9 ℃.
In the method for the preservation cell provided, the time of described preservation is 0～48 hour, preferably 6～42 hours, more preferably 12～36 hours, particularly preferably 18～30 hours, most preferably 24 hours, such as can be 1 hour, 2 hours, 5 hours, 8 hours, 10 hours, 14 hours, 15 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 38 hours, 40 hours or 46 hours.
In fourth aspect, the invention provides the application of a kind of cell-preservation liquid as described as first aspect in preserving cell; Preferably, described cell is stem cell or immunocyte, preferred embryo stem cell, adult stem cell, lymphocyte, dendritic cell, Monocytes/Macrophages, granulocyte or mast cell, more preferably fat stem cell, cytokine induced kill cell (CIK), dendritic cell (DC), DC+CIK cell, natural killer cell (NK) or DC-T cell, most preferably fat stem cell or natural killer cell.
Beneficial effect of the present invention is:
(1) cell-preservation liquid of the present invention is preserved cell in the temperature range of 2-10 ℃, Cell viability and biological property in 24 hours, have been maintained well, experiment confirm: preserve after 24 hours, Cell viability is all more than 90%, and marked change does not occur the cell sign thing;
(2) cell-preservation liquid definite ingredients of the present invention, good stability, safe, have no side effect, be convenient to the storage and transport of cell, adapt to the clinical extensive use of cell therapy, have a extensive future, provide powerful support for for the expansion of cell therapy clinically provides;
(3) the present invention overcomes in prior art that the holding time is short, cytoactive reduces, preserve liquid does not reach clinical use rank, needs recovery and the problems such as washing, complicated operation, and provide a kind of cell-preservation liquid of cytoactive and biological property that can effectively maintain in a long time, this preservation liquid can reach clinical criteria, without other processing, can directly cell suspension be infused into to human body, and this cell-preservation liquid formula components is clear and definite, proportioning is simple, processing ease is realized, is very easy to the clinical practice of cell preparation.
The accompanying drawing explanation
Fig. 1 preserves the fat stem cell microscopic examination figure of adhere-wall culture again after 24h for the cell-preservation liquid that uses the embodiment of the present invention 1, left figure is that fat stem cell adhere-wall culture 40 power microscopes before preserving are observed figure, and right figure be that preservation 24h cultivates the fat stem cell adhere-wall culture 40 power microscope observation figure again after 48h again.
Fig. 2 preserves for the cell-preservation liquid that uses the embodiment of the present invention 1 the microscopic examination figure that the NK cell after 24h is cultivated again, left figure is that NK cell 100 power microscopes before preserving are observed figure, and right figure is the NK cell 100 power microscope observation figure that preserve after 24h cultivates 24h again.
The result that Fig. 3 adopts BD FACSAria to detect for fat stem cell cell surface marker (CD73, CD90, CD105, CD31 and CD133) before preserving.
The result that Fig. 4 adopts BD FACSAria to detect for the fat stem cell cell surface marker (CD73, CD90, CD105, CD31 and CD133) of cell-preservation liquid preservation after 24 hours that uses the embodiment of the present invention 1.
The result that Fig. 5 adopts BD FACSCalibur to detect for NK cell surface marker (CD3, CD56) before preserving.
The result that Fig. 6 adopts BD FACSCalibur to detect for the NK cell surface marker (CD3, CD56) of cell-preservation liquid preservation after 24 hours that uses the embodiment of the present invention 1.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail.It will be understood to those of skill in the art that the following examples are only be used to the present invention is described, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the described technology of the document in this area or condition, or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
The preparation of embodiment 1 cell-preservation liquid
In the 100ml sodium chloride injection, add human serum albumin 1g, vitamin C 200mg, glucose 1g, trinosin 10mg, above-mentioned whole compositions are the clinical injection rank, thoroughly dissolve, and fully mix.
The preparation of embodiment 2 cell-preservation liquids
In the 100ml sodium chloride injection, add human serum albumin 0.1g, vitamin C 1000mg, glucose 0.1g, trinosin 1mg, above-mentioned whole compositions are the clinical injection rank, thoroughly dissolve, and fully mix.
The preparation of embodiment 3 cell-preservation liquids
In the 100ml compound sodium chloride injection, add human serum albumin 10g, vitamin C 10mg, glucose 0.2g, adenosine triphosphate 100mg, above-mentioned whole compositions are the clinical injection rank, thoroughly dissolve, and fully mix.
The preparation of embodiment 4 cell-preservation liquids
In the 100ml compound sodium chloride injection, add human serum albumin 2g, vitamin C 500mg, glucose 0.5g, trinosin 50mg, above-mentioned whole compositions are the clinical injection rank, thoroughly dissolve, and fully mix.
The preparation of embodiment 5 cell-preservation liquids
In the 100ml sodium chloride injection, add ox blood albumin 1g, vitamin B1 200mg, galactose 1g, trinosin 10mg, above-mentioned whole compositions are the clinical injection rank, thoroughly dissolve, and fully mix.
The preparation of embodiment 6 cell-preservation liquids
In the 100ml sodium chloride injection, add sheep blood albumin 0.1g, vitamin A 1000mg, fructose 0.1g, trinosin 1mg, above-mentioned whole compositions are the clinical injection rank, thoroughly dissolve, and fully mix.
The preparation of embodiment 7 cell-preservation liquids
In the 100ml compound sodium chloride injection, add cow's milk albumin 10g, vitamin D 10mg, ribose 0.2g, adenosine triphosphate 100mg, above-mentioned whole compositions are the clinical injection rank, thoroughly dissolve, and fully mix.
The preparation of embodiment 8 cell-preservation liquids
In the 100ml compound sodium chloride injection, add human serum albumin 2g, vitamin K 500mg, glucose 0.5g, adenosine triphosphate 50mg, above-mentioned whole compositions are the clinical injection rank, thoroughly dissolve, and fully mix.
The application of embodiment 9 embodiment of the present invention 1 cell-preservation liquids in fat stem cell is preserved
The fat stem cell of adhere-wall culture, trypsinization 3min with 0.25%, digestion obtains single cell suspension, process 1000rpm, 10min centrifuge washing three times, fat stem cell after washing is divided into to three parts, portion is preserved with sodium chloride injection, and a sodium chloride injection that contains 1% human serum albumin of using is preserved, and portion is preserved with the cell-preservation liquid of the embodiment of the present invention 1.At 2～10 ℃, preserve 6h, 12h, 18h, 24h, trypan blue (Trypan Blue) dyeing is carried out in sampling, and living cell counting and dead cell number calculate Cell viability, Cell viability=living cells number/(living cells number+dead cell number), result is as shown in table 1.
Table 1 fat stem cell motility rate experimental result
The application of embodiment 10 embodiment of the present invention 1 cell-preservation liquids in the NK cell is preserved
The NK cell suspension that collecting suspends cultivates is to centrifuge tube, 1000rpm, 10min are centrifugal, with DPBS(Du Shi phosphate buffer) wash three times, NK cell after washing is divided into to three parts, portion is preserved with sodium chloride injection, the a sodium chloride injection that contains 1% human serum albumin of using is preserved, and portion is preserved with the cell-preservation liquid of the embodiment of the present invention 1.At 2～10 ℃, preserve 6h, 12h, 18h, 24h, trypan blue (Trypan Blue) dyeing is carried out in sampling, and living cell counting and dead cell number calculate Cell viability, Cell viability=living cells number/(living cells number+dead cell number), result is as shown in table 2.
Table 2 NK Cell viability experimental result
The application of embodiment 11 embodiment of the present invention 3 cell-preservation liquids in fat stem cell is preserved
The fat stem cell of adhere-wall culture, trypsinization 3min with 0.25%, digestion obtains single cell suspension, process 1000rpm, 10min centrifuge washing three times, fat stem cell after washing is divided into to three parts, portion is preserved with sodium chloride injection, and a sodium chloride injection that contains 1% human serum albumin of using is preserved, and portion is preserved with the cell-preservation liquid of the embodiment of the present invention 3.At 2～10 ℃, preserve 6h, 12h, 18h, 24h, trypan blue (Trypan Blue) dyeing is carried out in sampling, and living cell counting and dead cell number calculate Cell viability, Cell viability=living cells number/(living cells number+dead cell number), result is as shown in table 3.
Table 3 fat stem cell motility rate experimental result
The application of embodiment 12 embodiment of the present invention 4 cell-preservation liquids in the CIK cell is preserved
The CIK cell suspension that collecting suspends cultivates is to centrifuge tube, 1000rpm, 10min are centrifugal, with DPBS(Du Shi phosphate buffer) wash three times, CIK cell after washing is divided into to three parts, portion is preserved with sodium chloride injection, the a sodium chloride injection that contains 1% human serum albumin of using is preserved, and portion is preserved with the cell-preservation liquid of the embodiment of the present invention 4.At 2～10 ℃, preserve 6h, 12h, 18h, 24h, trypan blue (Trypan Blue) dyeing is carried out in sampling, and living cell counting and dead cell number calculate Cell viability, Cell viability=living cells number/(living cells number+dead cell number), result is as shown in table 4.
Table 4 CIK Cell viability experimental result
By embodiment 9～12, can be found out, after using cell-preservation liquid of the present invention to preserve cell 24h, Cell viability is greater than 90%, meet clinical requirement to Cell viability, greatly increased the Service coverage of central laboratory, facilitate the clinician to arrange flexibly adoptive therapy according to patient's time.Analyze its reason, cell-preservation liquid of the present invention is in the process that cell is preserved, reduced the oxidative damage of superoxide anion to cell wall, for cell, provide some basic nutriment and energy matters again, in low temperature, maintain low-level metabolic rate, the osmotic pressure of applicable survival is provided for cell again simultaneously.
Embodiment 13 preserves after 24h fat stem cell adhere-wall culture again
The fat stem cell of adhere-wall culture, trypsinization 3min with 0.25%, digestion obtains single cell suspension, process 1000rpm, 10min centrifuge washing three times, after fat stem cell after washing is preserved to 24 hours with the cell-preservation liquid of the embodiment of the present invention 1, by the centrifugal collection of cell 1000rpm, 10min, according to 5 * 10
5The concentration of/ml is inoculated into the T75 blake bottle, and medium is that GIBCO 1640 medium add the 10%FBS(hyclone).In 37 ℃, the Heraeus HERAcell 240i CO2gas incubator of 5% gas concentration lwevel, cultivated 48 hours.With Nikon Ti-s microscope, under 40 times of multiplication factors, take pictures (Fig. 1).
The result demonstration, after preservation, cultured cells is in good condition again, and dead cell is less, and before and after preserving, cellular morphology is unchanged.
Embodiment 14 preserves NK cell after 24 hours and again cultivates
The NK cell suspension that collecting suspends cultivates is to centrifuge tube, 1000rpm, 10min are centrifugal, with DPBS(Du Shi phosphate buffer) wash three times, after the NK cell after washing is preserved to 24 hours with the cell-preservation liquid of the embodiment of the present invention 1, by the centrifugal collection of cell 1000rpm, 10min, according to 1 * 10
6The concentration of/ml is inoculated into the T75 blake bottle, and culture fluid is Takara GT-T551 medium, adds 500IU/ml IL-2(interleukin 2); At 37 ℃, the Heraeus HERAcell240i CO2gas incubator of 5% gas concentration lwevel, cultivated 24 hours.With Nikon Ti-s microscope, under 100 times of multiplication factors, take pictures (Fig. 2).
The result demonstration, after preservation, cultured cells is in good condition again, and dead cell is less, and before and after preserving, cellular morphology is unchanged.
The situation of change of fat stem cell cell surface marker before and after embodiment 15 preserves
CD73PE, CD90FITC, CD105APC, CD31FITC and CD133APC are all purchased from U.S. company BD.
Fat stem cell before preserving adopts BD FACSAria to detect according to this instrument conventional detection step; Use the fat stem cell of cell-preservation liquid preservation after 24 hours of the embodiment of the present invention 1 to adopt equally BD FACSAria to detect according to this instrument conventional detection step, result as shown in Figure 3,4, result shows that fat stem cell is used positive index CD73, CD90, CD105 and negative index CD31, the CD133 of cell-preservation liquid preservation of the present invention after 24 hours all without significant variation, illustrates that this cell-preservation liquid has maintained the biological property of fat stem cell well in the process of preserving fat stem cell.
The variation of NK cell surface marker before and after embodiment 16 preserves
CD3FITC and CD56PE are all purchased from U.S. company BD.
NK cell before preserving adopts BD FACSCalibur to detect according to this instrument conventional detection step; Use the NK cell of cell-preservation liquid preservation after 24 hours of the embodiment of the present invention 1 to adopt equally BDFACSCalibur to detect according to this instrument conventional detection step, result as shown in Figure 5,6, result shows, before preserving, NK cell CD3-CD56+ is 77.77%, use the cell-preservation liquid of the embodiment of the present invention 1 to preserve after 24 hours, CD3-CD56+ is 73.85%.Before and after preserving, a little decline is arranged, descend less, illustrate that this cell-preservation liquid has maintained the biological property of NK cell well in preserving for a long time.
Applicant's statement, the present invention illustrates method detailed of the present invention by above-described embodiment, but the present invention is not limited to above-mentioned method detailed, does not mean that namely the present invention must rely on above-mentioned method detailed and could implement.The person of ordinary skill in the field should understand, any improvement in the present invention, to the interpolation of the equivalence replacement of each raw material of product of the present invention and auxiliary element, the selection of concrete mode etc., within all dropping on protection scope of the present invention and open scope.
Claims (10)
1. cell-preservation liquid, it is characterized in that, in 100 milliliters of sodium chloride injections and/or compound sodium chloride injection, wherein contain vitamin 0.01～1 gram, albumin 0.1～10 gram, carbohydrate 0.1～1 gram, adenosine triphosphate and/or trinosin 0.001～0.1 gram.
2. cell-preservation liquid according to claim 1, it is characterized in that, described albumin is selected from one or more in human albumin and mammal albumin, one or more in preferred people and mammiferous blood albumin, ovalbumin and lactoalbumin, more preferably one or more in the blood albumin of people, ox, horse and sheep, ovalbumin and lactoalbumin, most preferably human serum albumin.
3. cell-preservation liquid according to claim 1 and 2, it is characterized in that, described vitamin is selected from one or more in vitamin A, vitamin B1, vitamin B2, adenine phosphate, vitamin B6, FA, cobalamin, vitamin C, vitamin D, vitamin E, vitamin K, biotin, citrin, nicotinic acid, vitamin(e) M, vitamin(e) T and vitamin, preferred vitamin C.
4. according to the described cell-preservation liquid of claims 1 to 3 any one, it is characterized in that, described carbohydrate is selected from one or more in monose, disaccharides, trisaccharide and polysaccharide, preferably one or more in glucose, galactose, fructose, ribose and trehalose, more preferably glucose.
5. according to the described cell-preservation liquid of claim 1 to 4 any one, it is characterized in that, in 100 milliliters of sodium chloride injections and/or compound sodium chloride injection, wherein contain vitamin 0.05～0.8 gram, preferred 0.1～0.6 gram, more preferably 0.2～0.5 gram, 0.3～0.4 gram most preferably, albumin 0.5～8 gram, preferred 1～6 gram, more preferably 2～5 grams, 3～4 grams most preferably, carbohydrate 0.2～0.9 gram, preferred 0.3～0.8 gram, more preferably 0.4～0.7 gram, 0.5～0.6 gram most preferably, adenosine triphosphate and/or trinosin 0.005～0.08 gram, preferred 0.01～0.06 gram, more preferably 0.02～0.05 gram, 0.03～0.04 gram most preferably.
6. as the preparation method of the described cell-preservation liquid of claim 1 to 5 any one, it is characterized in that, by corresponding content, other described vitamin of injection stage, albumin, carbohydrate and adenosine triphosphate and/or trinosin are added in described sodium chloride injection and/or compound sodium chloride injection, thoroughly dissolve, fully mix.
7. preparation method according to claim 6, it is characterized in that, described vitamin, albumin, carbohydrate and adenosine triphosphate and/or trinosin add in described sodium chloride injection and/or compound sodium chloride injection with the form of parenteral solution and/or freeze-dried powder.
8. a method of preserving cell, is characterized in that, cell is added in cell-preservation liquid as described as claim 1 to 5 any one, is placed in 2～10 ℃, preferably 3～8 ℃, more preferably 3～6 ℃, most preferably under 4 ℃ of conditions, preserves.
9. method according to claim 8, is characterized in that, the time of described preservation is 0～48 hour, preferably 6～42 hours, more preferably 12～36 hours, particularly preferably 18～30 hours, most preferably 24 hours.
10. the application of cell-preservation liquid as described as claim 1 to 5 any one in preserving cell; Preferably, described cell is stem cell or immunocyte, preferred embryo stem cell, adult stem cell, lymphocyte, dendritic cell, Monocytes/Macrophages, granulocyte or mast cell, more preferably fat stem cell, cytokine induced kill cell, dendritic cell, DC+CIK cell, natural killer cell or DC-T cell, most preferably fat stem cell or natural killer cell.
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