CN103404509B - 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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- CN103404509B CN103404509B CN201310341570.2A CN201310341570A CN103404509B CN 103404509 B CN103404509 B CN 103404509B CN 201310341570 A CN201310341570 A CN 201310341570A CN 103404509 B CN103404509 B CN 103404509B
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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 the cell colony that the class be present 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 histoorgan yet, therefore are macroscopically divided into embryonic stem cell and adult stem cell.Along with the development of cell replacement therapy, cellular replacement therapy has become the important means of some disease of clinical treatment.
Immune cell therapy is a kind of emerging, brand-new antitumour treatments with significant curative effect, compensate for 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 the treatment means being uniquely hopeful complete tumors destroyed cell in the world at present.Comprise at the immunocyte of clinical middle employing 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 clinically has obtained the extensive accreditation of scientist, but still has many difficult problems to need to overcome in actual applications.At present, cell therapy adopts the mode of drip-feed to carry out mostly.By cell infusion good for culture in vitro in the sodium chloride injection of different size, add or do not add a certain proportion of human serum albumin, the cell suspension prepared is fed back 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 and reduce significantly.Routine preservation method is unfavorable for preservation and the transport at a distance of cell long period.Cell is in real-life clinical application, because Clinical practice unit does not possess corresponding cell culture technology and high standard specialized cell preparation experiment room, so need to carry out cell preparation in regional center laboratory, the cell prepared delivers to clinical unit's application through the transport of certain hour and distance.Cell needs the longer transport holding time before feedback, adopts suitable conserving liquid to maintain motility rate and the biological efficacy of cell with regard to requiring in transportation.And frozen cell can adopt the mode of-80 DEG C of dry ice transports to carry out remote long-time transport.But the cryopreserving liquid of freeze-stored cell contains serum, medium and DMSO.Frozen cell be applied to again clinical before need to carry out recovering and washing, 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 conserving liquid containing serum.Serum generally derives from cow's serum or people AB serum.But in the clinical practice of cell therapy, the method preparing cell requires it is free serum culture, if use serum in conserving liquid, will introduce serum contamination.Composition simultaneously in serum is failed to understand, not only containing compositions such as cell factor, growth factor, hormones, and the problem that also may affect containing unknown virus.So exist unknown virus pollute and because conserving liquid composition is uncertain, quality that is that cause is difficult to multiple shortcomings such as stablizing.
Therefore, this area is needed badly and a kind ofly can be maintained Cell viability and biological property in a long time well and the clear and definite cell-preservation liquid of component.
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 DEG C, Cell viability and biological property is maintained well in 24 hours, the risk avoiding cell recovery and again wash, it also avoid cell death that existing store method causes and biological characteristics sexually revises, greatly expand the regional coverage of central laboratory's cell application.
For realizing object 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 is in 100 milliliters of sodium chloride injections and/or compound sodium chloride injection, wherein containing 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 cell-preservation liquid provided by the invention, described albumin be selected from human albumin and mammal albumin one or more, 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 be selected from vitamin A, vitamin B1, vitamin B2, adenine phosphate, vitamin B6, FA, cobalamin, vitamin C, vitamin D, vitamin E, vitamin K, biotin, citrin, nicotinic acid, vitamine M, vitamine T and vitamin one or more, preferred vitamin C.
In cell-preservation liquid provided by the invention, described carbohydrate be selected from monose, disaccharides, trisaccharide and polysaccharide one or more, one or more in preferred 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 containing vitamin 0.05 ~ 0.8 gram, preferably 0.1 ~ 0.6 gram, more preferably 0.2 ~ 0.5 gram, most preferably 0.3 ~ 0.4 gram, albumin 0.5 ~ 8 gram, preferably 1 ~ 6 gram, more preferably 2 ~ 5 grams, most preferably 3 ~ 4 grams, carbohydrate 0.2 ~ 0.9 gram, preferably 0.3 ~ 0.8 gram, more preferably 0.4 ~ 0.7 gram, most preferably 0.5 ~ 0.6 gram, adenosine triphosphate and/or trinosin 0.005 ~ 0.08 gram, preferably 0.01 ~ 0.06 gram, more preferably 0.02 ~ 0.05 gram, most preferably 0.03 ~ 0.04 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 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 containing 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 of cell-preservation liquid as described in relation to the first aspect, by corresponding content, injection stage vitamin, albumin, carbohydrate and adenosine triphosphate described in other and/or trinosin are added in described sodium chloride injection and/or compound sodium chloride injection, thorough dissolving, fully mixes.
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 in relation to the first aspect, be placed in 2 ~ 10 DEG C, preserve under preferably 3 ~ 8 DEG C, more preferably 3 ~ 6 DEG C, most preferably 4 DEG C of conditions.
In the method for the preservation cell provided, temperature can be 2 DEG C, 2.5 DEG C, 2.8 DEG C, 3.2 DEG C, 3.8 DEG C, 4.2 DEG C, 5.5 DEG C, 6.5 DEG C, 7 DEG C, 7.2 DEG C, 7.8 DEG C, 8.2 DEG C, 8.8 DEG C, 9.5 DEG C or 9.9 DEG C.
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, can be such as 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 a kind of cell-preservation liquid as described in relation to the first aspect and preserving the application in 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 preserves cell in the temperature range of 2-10 DEG C, Cell viability and biological property is maintained well in 24 hours, experiment confirms: preserve after 24 hours, Cell viability is all more than 90%, and cell sign thing marked change does not occur;
(2) cell-preservation liquid definite ingredients of the present invention, good stability, safety high, 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 that short, the cytoactive of holding time in prior art reduces, conserving liquid does not reach Clinical practice rank, need the problems such as recovery and washing, complicated operation, and a kind of cell-preservation liquid that effectively can maintain cytoactive and biological property is in a long time provided, this conserving liquid can reach clinical criteria, directly cell suspension can be infused into human body without the need to other process, and this cell-preservation liquid formula components is clear and definite, proportioning is simple, processing ease realizes, and is very easy to the clinical practice of cell preparation.
Accompanying drawing explanation
Fig. 1 is the microscopic examination figure using the cell-preservation liquid of the embodiment of the present invention 1 to preserve the adhere-wall culture again of the fat stem cell after 24h, left figure is the fat stem cell adhere-wall culture 40 power microscope observation figure before preserving, right figure is preserve 24h to cultivate the fat stem cell adhere-wall culture 40 power microscope observation figure again after 48h again.
Fig. 2 is the microscopic examination figure that the NK cell after using the cell-preservation liquid of the embodiment of the present invention 1 to preserve 24h is cultivated again, left figure is the NK cell 100 power microscope observation figure before preserving, right figure is preserve 24h to cultivate the NK cell 100 power microscope observation figure after 24h again.
The result that Fig. 3 adopts BD FACSAria to detect for preserving front fat stem cell cell surface marker (CD73, CD90, CD105, CD31 and CD133).
Fig. 4 is the result that the fat stem cell cell surface marker (CD73, CD90, CD105, CD31 and CD133) after using the cell-preservation liquid of the embodiment of the present invention 1 to preserve 24 hours adopts BD FACSAria to detect.
The result that Fig. 5 adopts BD FACSCalibur to detect for preserving front NK cells cell surface mark (CD3, CD56).
Fig. 6 is the result that the NK cells cell surface mark (CD3, CD56) after using the cell-preservation liquid of the embodiment of the present invention 1 to preserve 24 hours adopts BD FACSCalibur to detect.
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 only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the technology described by the document in this area or condition, or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
The preparation of embodiment 1 cell-preservation liquid
In 100ml sodium chloride injection, add human serum albumin 1g, vitamin C 200mg, glucose 1g, trinosin 10mg, above-mentioned whole composition is clinical injection rank, thoroughly dissolves, fully mixes.
The preparation of embodiment 2 cell-preservation liquid
In 100ml sodium chloride injection, add human serum albumin 0.1g, vitamin C 1000mg, glucose 0.1g, trinosin 1mg, above-mentioned whole composition is clinical injection rank, thoroughly dissolves, fully mixes.
The preparation of embodiment 3 cell-preservation liquid
In 100ml compound sodium chloride injection, add human serum albumin 10g, vitamin C 10mg, glucose 0.2g, adenosine triphosphate 100mg, above-mentioned whole composition is clinical injection rank, thoroughly dissolves, fully mixes.
The preparation of embodiment 4 cell-preservation liquid
In 100ml compound sodium chloride injection, add human serum albumin 2g, vitamin C 500mg, glucose 0.5g, trinosin 50mg, above-mentioned whole composition is clinical injection rank, thoroughly dissolves, fully mixes.
The preparation of embodiment 5 cell-preservation liquid
In 100ml sodium chloride injection, add ox blood albumin 1g, vitamin B1 200mg, galactose 1g, trinosin 10mg, above-mentioned whole composition is clinical injection rank, thoroughly dissolves, fully mixes.
The preparation of embodiment 6 cell-preservation liquid
In 100ml sodium chloride injection, add sheep blood albumin 0.1g, vitamin A 1000mg, fructose 0.1g, trinosin 1mg, above-mentioned whole composition is clinical injection rank, thoroughly dissolves, fully mixes.
The preparation of embodiment 7 cell-preservation liquid
In 100ml compound sodium chloride injection, add cow's milk albumin 10g, vitamin D 10mg, ribose 0.2g, adenosine triphosphate 100mg, above-mentioned whole composition is clinical injection rank, thoroughly dissolves, fully mixes.
The preparation of embodiment 8 cell-preservation liquid
In 100ml compound sodium chloride injection, add human serum albumin 2g, vitamin K 500mg, glucose 0.5g, adenosine triphosphate 50mg, above-mentioned whole composition is clinical injection rank, thoroughly dissolves, fully mixes.
The application of embodiment 9 embodiment of the present invention 1 cell-preservation liquid in fat stem cell is preserved
The fat stem cell of adhere-wall culture, with the trypsinization 3min of 0.25%, digestion obtains single cell suspension, through 1000rpm, 10min centrifuge washing three times, fat stem cell after washing is divided into three parts, portion sodium chloride injection is preserved, and a sodium chloride injection with containing 1% human serum albumin is preserved, and the cell-preservation liquid of the portion embodiment of the present invention 1 is preserved.6h, 12h, 18h, 24h is preserved at 2 ~ 10 DEG C, trypan blue (Trypan Blue) dyeing is carried out in sampling, and living cell counting and dead cell number calculate Cell viability, Cell viability=number of viable cells/(number of viable cells+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 liquid in NK cell is preserved
Collect the NK cell suspension of the cultivation that suspends to centrifuge tube, 1000rpm, 10min are centrifugal, with DPBS(Du Shi phosphate buffer) wash three times, NK cell after washing is divided into three parts, portion sodium chloride injection is preserved, the a sodium chloride injection with containing 1% human serum albumin is preserved, and the cell-preservation liquid of the portion embodiment of the present invention 1 is preserved.6h, 12h, 18h, 24h is preserved at 2 ~ 10 DEG C, trypan blue (Trypan Blue) dyeing is carried out in sampling, and living cell counting and dead cell number calculate Cell viability, Cell viability=number of viable cells/(number of viable cells+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 liquid in fat stem cell is preserved
The fat stem cell of adhere-wall culture, with the trypsinization 3min of 0.25%, digestion obtains single cell suspension, through 1000rpm, 10min centrifuge washing three times, fat stem cell after washing is divided into three parts, portion sodium chloride injection is preserved, and a sodium chloride injection with containing 1% human serum albumin is preserved, and the cell-preservation liquid of the portion embodiment of the present invention 3 is preserved.6h, 12h, 18h, 24h is preserved at 2 ~ 10 DEG C, trypan blue (Trypan Blue) dyeing is carried out in sampling, and living cell counting and dead cell number calculate Cell viability, Cell viability=number of viable cells/(number of viable cells+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 liquid in CIK cell is preserved
Collect the CIK cell suspension of the cultivation that suspends to centrifuge tube, 1000rpm, 10min are centrifugal, with DPBS(Du Shi phosphate buffer) wash three times, CIK cell after washing is divided into three parts, portion sodium chloride injection is preserved, the a sodium chloride injection with containing 1% human serum albumin is preserved, and the cell-preservation liquid of the portion embodiment of the present invention 4 is preserved.6h, 12h, 18h, 24h is preserved at 2 ~ 10 DEG C, trypan blue (Trypan Blue) dyeing is carried out in sampling, and living cell counting and dead cell number calculate Cell viability, Cell viability=number of viable cells/(number of viable cells+dead cell number), result is as shown in table 4.
Table 4 CIK cell motility rate experimental result
As can be seen from embodiment 9 ~ 12, after using cell-preservation liquid of the present invention to preserve cell 24h, Cell viability is greater than 90%, meet the clinical requirement to Cell viability, considerably increase the Service coverage of central laboratory, facilitate clinician the time to arrange adoptive therapy flexibly according to patient.Analyze its reason, in the process that cell-preservation liquid of the present invention is preserved at cell, decrease the oxidative damage of superoxide anion to cell wall, the nutriment providing some basic for cell again and energy matter, low-level metabolic rate is maintained, simultaneously again for cell provides the osmotic pressure being applicable to survival in low temperature.
Embodiment 13 preserves fat stem cell adhere-wall culture again after 24h
The fat stem cell of adhere-wall culture, with the trypsinization 3min of 0.25%, digestion obtains single cell suspension, through 1000rpm, 10min centrifuge washing three times, after the cell-preservation liquid of the fat stem cell embodiment of the present invention 1 after washing is preserved 24 hours, by cell 1000rpm, 10min collected by centrifugation, according to 5 × 10
5the concentration of/ml is inoculated into T75 blake bottle, and medium is that GIBCO 1640 medium adds 10%FBS(hyclone).37 DEG C, carry out cultivation 48 hours in the Heraeus HERAcell 240i CO2gas incubator of 5% gas concentration lwevel.With Nikon Ti-s microscope, carry out take pictures (Fig. 1) under 40 times of multiplication factors.
Result shows, and after preservation, cultured cells is in good condition again, and dead cell is less, and before and after preserving, cellular morphology is unchanged.
After embodiment 14 preserves 24 hours, NK cell is cultivated again
Collect the NK cell suspension of the cultivation that suspends to centrifuge tube, 1000rpm, 10min are centrifugal, with DPBS(Du Shi phosphate buffer) wash three times, after the cell-preservation liquid of the NK cell embodiment of the present invention 1 after washing is preserved 24 hours, by cell 1000rpm, 10min collected by centrifugation, according to 1 × 10
6the concentration of/ml is inoculated into T75 blake bottle, and culture fluid is Takara GT-T551 medium, adds 500IU/ml IL-2(interleukin 2); 37 DEG C, the Heraeus HERAcell240i CO2gas incubator of 5% gas concentration lwevel carries out cultivation 24 hours.With Nikon Ti-s microscope, carry out take pictures (Fig. 2) under 100 times of multiplication factors.
Result shows, and after preservation, cultured cells is in good condition again, and dead cell is less, and before and after preserving, cellular morphology is unchanged.
Embodiment 15 preserves the situation of change of front and back fat stem cell cell surface marker
CD73PE, CD90FITC, CD105APC, CD31FITC and CD133APC equal purchased from American BD company.
Fat stem cell before preservation adopts BD FACSAria to detect according to this instrument conventional detection step; Fat stem cell after using the cell-preservation liquid of the embodiment of the present invention 1 to preserve 24 hours adopts BD FACSAria to detect according to this instrument conventional detection step equally, result as shown in Figure 3,4, result display fat stem cell uses cell-preservation liquid of the present invention to preserve positive indication CD73, CD90, CD105 after 24 hours and negative indication CD31, CD133 all without significant change, illustrates that this cell-preservation liquid maintains the biological property of fat stem cell well in the process of preserving fat stem cell.
Embodiment 16 preserves the change of front and back NK cells cell surface mark
CD3FITC and CD56PE equal purchased from American BD company.
NK cell before preservation adopts BD FACSCalibur to detect according to this instrument conventional detection step; NK cell after using the cell-preservation liquid of the embodiment of the present invention 1 to preserve 24 hours adopts BDFACSCalibur to detect according to this instrument conventional detection step equally, result as shown in Figure 5,6, result shows, before preserving, NK cell CD3-CD56+ is 77.77%, after using the cell-preservation liquid of the embodiment of the present invention 1 to preserve 24 hours, CD3-CD56+ is 73.85%.There is a little decline before and after preserving, decline less, illustrate that this cell-preservation liquid maintains the biological property of NK cell well in preserving for a long time.
Applicant states, 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 namely mean that the present invention must rely on above-mentioned method detailed and could implement.Person of ordinary skill in the field should understand, any improvement in the present invention, to equivalence replacement and the interpolation of auxiliary element, the concrete way choice etc. of each raw material of product of the present invention, all drops within protection scope of the present invention and open scope.
Claims (29)
1. a cell-preservation liquid, it is characterized in that, in 100 milliliters of sodium chloride injections and/or compound sodium chloride injection, wherein containing 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;
The preparation method of described cell-preservation liquid comprises: add in described sodium chloride injection and/or compound sodium chloride injection by corresponding content by injection stage vitamin, albumin, carbohydrate and adenosine triphosphate described in other and/or trinosin, thorough dissolving, fully mixes.
2. cell-preservation liquid according to claim 1, is characterized in that, described albumin be selected from human albumin and mammal albumin one or more.
3. cell-preservation liquid according to claim 2, is characterized in that, described albumin be selected from people and mammiferous blood albumin, ovalbumin or lactoalbumin one or more.
4. cell-preservation liquid according to claim 3, is characterized in that, described albumin be selected from people, ox, the blood albumin of horse and sheep, ovalbumin or lactoalbumin one or more.
5. cell-preservation liquid according to claim 4, is characterized in that, described albumin is human serum albumin.
6. cell-preservation liquid according to claim 1, it is characterized in that, described vitamin be selected from vitamin A, vitamin B1, vitamin B2, adenine phosphate, vitamin B6, FA, cobalamin, vitamin C, vitamin D, vitamin E, vitamin K, biotin, citrin, nicotinic acid, vitamine M, vitamine T or vitamin one or more.
7. cell-preservation liquid according to claim 6, is characterized in that, described vitamin is vitamin C.
8. cell-preservation liquid according to claim 1, is characterized in that, described carbohydrate be selected from monose, disaccharides, trisaccharide or polysaccharide one or more.
9. cell-preservation liquid according to claim 8, is characterized in that, described carbohydrate be selected from glucose, galactose, fructose, ribose or trehalose one or more.
10. cell-preservation liquid according to claim 9, is characterized in that, described carbohydrate is glucose.
11. cell-preservation liquids according to claim 1, it is characterized in that, in 100 milliliters of sodium chloride injections and/or compound sodium chloride injection, wherein containing vitamin 0.05 ~ 0.8 gram, albumin 0.5 ~ 8 gram, carbohydrate 0.2 ~ 0.9 gram, adenosine triphosphate and/or trinosin 0.005 ~ 0.08 gram.
12. cell-preservation liquids according to claim 11, it is characterized in that, in 100 milliliters of sodium chloride injections and/or compound sodium chloride injection, wherein containing vitamin 0.1 ~ 0.6 gram, albumin 1 ~ 6 gram, carbohydrate 0.3 ~ 0.8 gram, adenosine triphosphate and/or trinosin 0.01 ~ 0.06 gram.
13. cell-preservation liquids according to claim 12, it is characterized in that, in 100 milliliters of sodium chloride injections and/or compound sodium chloride injection, wherein containing vitamin 0.2 ~ 0.5 gram, albumin 2 ~ 5 grams, carbohydrate 0.4 ~ 0.7 gram, adenosine triphosphate and/or trinosin 0.02 ~ 0.05 gram.
14. cell-preservation liquids according to claim 13, it is characterized in that, in 100 milliliters of sodium chloride injections and/or compound sodium chloride injection, wherein containing vitamin 0.3 ~ 0.4 gram, albumin 3 ~ 4 grams, carbohydrate 0.5 ~ 0.6 gram, adenosine triphosphate and/or trinosin 0.03 ~ 0.04 gram.
15. cell-preservation liquids according to claim 1, 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.
16. 1 kinds of methods of preserving cell, is characterized in that, added by cell in the cell-preservation liquid as described in any one of claim 1 to 15, preserve under being placed in 2 ~ 10 DEG C of conditions.
17. methods according to claim 16, is characterized in that, preserve under cell being placed in 3 ~ 8 DEG C of conditions.
18. methods according to claim 17, is characterized in that, preserve under cell being placed in 3 ~ 6 DEG C of conditions.
19. methods according to claim 18, is characterized in that, preserve under cell being placed in 4 DEG C of conditions.
20. methods according to claim 16, is characterized in that, the time of described preservation is 0 ~ 48 hour.
21. methods according to claim 20, is characterized in that, the time of described preservation is 6 ~ 42 hours.
22. methods according to claim 21, is characterized in that, the time of described preservation is 12 ~ 36 hours.
23. methods according to claim 22, is characterized in that, the time of described preservation is 18 ~ 30 hours.
24. methods according to claim 23, is characterized in that, the described holding time is 24 hours.
25. cell-preservation liquids as described in any one of claim 1 to 15 are preserving the application in cell.
26. application according to claim 25, is characterized in that, described cell is stem cell or immunocyte.
27. application according to claim 26, is characterized in that, described cell is embryonic stem cell, adult stem cell, lymphocyte, dendritic cell, Monocytes/Macrophages, granulocyte or mast cell.
28. application according to claim 27, is characterized in that, described cell is fat stem cell, cytokine induced kill cell, dendritic cell, DC+CIK cell, natural killer cell or DC-T cell.
29. application according to claim 28, is characterized in that, described cell is fat stem cell or natural killer cell.
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| CN104542578B (en) * | 2015-02-05 | 2017-01-18 | 广州赛莱拉干细胞科技股份有限公司 | Cell preservation solution and preparation method and applications thereof |
| CN104928241B (en) * | 2015-03-27 | 2018-04-13 | 北京康爱瑞浩生物科技股份有限公司 | A kind of activation method and cell preparation method of enhanced NK cells |
| CN104920340A (en) * | 2015-07-15 | 2015-09-23 | 广州赛莱拉干细胞科技股份有限公司 | Immune cell preserving fluid and application thereof |
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