CN105211052A - Frozen stock solution of cultured NKT cells and preparation method thereof - Google Patents
Frozen stock solution of cultured NKT cells and preparation method thereof Download PDFInfo
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- CN105211052A CN105211052A CN201510727840.2A CN201510727840A CN105211052A CN 105211052 A CN105211052 A CN 105211052A CN 201510727840 A CN201510727840 A CN 201510727840A CN 105211052 A CN105211052 A CN 105211052A
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Abstract
The invention relates to the technical field of cell frozen stock solution, in particular to frozen stock solution of cultured NKT cells and a preparation method thereof, the frozen stock solution of the cultured NKT cells comprises lentinan, algal polysaccharide, autologous serum and DMSO, and the autologous serum is adopted, so that the pollution of exogenous animal viruses and the introduction of exogenous proteins are avoided, and the safety is high; the added lentinan and algal polysaccharide can effectively maintain the activity of cells and have no harm to human bodies, and the state of the cells after cryopreservation recovery is basically close to the state before cryopreservation, so that the cryopreservation effect is good.
Description
Technical field
The present invention relates to cells frozen storing liquid technical field, be specifically related to cryopreserving liquid of the NKT cell after a kind of cultivation and preparation method thereof.
Background technology
Cell cryopreservation is the long-term a kind of effective ways preserving maintenance cytoactive, it is frozen through cryogenic freezing preservation and two stages of Ultra-cryofreezing preservation, cryogenic freezing preserves the metabolism that can reduce cell, and ultralow temperature (in the liquid nitrogen of-198 DEG C) freezen protective can preserve living cells metabolism and almost stop Growth of Cells completely, when Cryopreservation, the activity of the various enzymes of regulation and control Growth of Cells metabolism is suppressed, make intracellular biochemical reaction very slow, even stop, thus maintain the activity of cell.
In recent years NKT cell phenotypic characteristic, distribution and growth, immunology effect and have a great development with the research in disease relationship, oncotherapy and treatment of autoimmune diseases etc.Due to the research of NKT cell in tumour and immunological disease treatment etc., importance frozen after also determining NKT cell chulture, cytoactive Extended incubation can being avoided to cause in treatment is low, is got up by the cell cryopreservation in optimum state period; At economic aspect, avoid the cost increase that Extended incubation causes; In research, the NKT cell cryopreservation of different times is got up, study the NKT cytoactive of its different frozen time.
If but simple frozen, in frozen process, cell can be subject to the damage in two: the first, and in frozen process, the moisture outside born of the same parents is first icing, electrolyte concentration in the solution do not frozen is raised, causes cell death, belong to permeability damage; The second, intracellular structure can be destroyed because low temperature causes cell born of the same parents inside to form ice crystal, belong to mechanical damage.Therefore combine above 2 reasons, freezing speed must be solved, and the formula of frozen dose, during recovery, the speed of thawing also directly affects the activity of cell.
Cells frozen storing liquid refers to for the protection of the material of cell from freezing loss, generally can be divided into permeability and impermeability two class.Permeability cryopreserving liquid mainly small-molecule substance, soluble in water, strong with water molecules ability, easily enter in cell by cell membrane, reduce the freezing point of cell, improve cell membrane to the permeability of water, reduce ice crystal and formed, thus reduce the damage of ice crystal.
In prior art; the cryopreserving liquid that the hyclone (FBS) of usual employing interpolation different proportion and dimethyl sulfoxide (DMSO) (DMSO) are prepared; carry out the NKT cell after the next frozen cultivation of programmed cooling of different temperatures and time again; add DMSO just in order to prevent the formation of intracellular ice crystal and damaging cells from frozen process; and add FBS be in order to Cell protection by DMSO damage and nutrition is provided; but FBS belongs to heterologous material; complicated component; and there is the risk introducing pollution and anaphylactogen, be not suitable for clinical practice.Particularly in cell therapy, the existence of heterologous protein, may cause unknown bad reaction, have a strong impact on treatment results.
Summary of the invention
The present invention is directed to problems of the prior art, provide a kind of clinical safety high, well can keep again the cells frozen storing liquid of freeze-stored cell activity, for the NKT cell after frozen cultivation simultaneously.
The present invention also provides the preparation method of the cryopreserving liquid of the NKT cell after a kind of cultivation.
The present invention is achieved through the following technical solutions this object:
The cryopreserving liquid of NKT cell after cultivating, described cryopreserving liquid by solution A and autoserum formulated, solution A and autoserous volume ratio are 1 ~ 3:1, wherein:
Described solution A comprises lentinan, algal polysaccharides, autoserum and DMSO, and wherein the volume ratio of lentinan, algal polysaccharides, autoserum and DMSO is 1 ~ 3:1 ~ 3:1 ~ 5:1.
As preferred scheme, described cryopreserving liquid by solution A and autoserum formulated, solution A and autoserous volume ratio are 1 ~ 2:1, wherein:
Described solution A comprises lentinan, algal polysaccharides, autoserum and DMSO, and wherein the volume ratio of lentinan, algal polysaccharides, autoserum and DMSO is 1 ~ 2:1 ~ 2:1 ~ 3:1.
As another preferred scheme, described cryopreserving liquid by solution A and autoserum formulated, solution A and autoserous volume ratio are 2 ~ 3:1, wherein:
Described solution A comprises lentinan, algal polysaccharides, autoserum and DMSO, and wherein the volume ratio of lentinan, algal polysaccharides, autoserum and DMSO is 2 ~ 3:2 ~ 3:3 ~ 5:1.
As preferably, the concentration of described lentinan is 1 ~ 6mg/mL, and the concentration of described algal polysaccharides is 0.05 ~ 0.2mg/mL.
A preparation method for the cryopreserving liquid of the NKT cell after cultivating, comprises the following steps:
1) be that the lentinan of 1 ~ 6mg/mL and the algal polysaccharides of 0.05 ~ 0.2mg/mL add in autoserum respectively by concentration, add DMSO slowly again, add lentinan by volume: algal polysaccharides: autoserum: DMSO is 1 ~ 3:1 ~ 3:1 ~ 5:1, the precooling of 2 ~ 8 DEG C of degree refrigerators is placed in by preparing the solution A obtained, for subsequent use;
2) above-mentioned solution A taken out again, it slowly joined in autoserum, by volume percentage adds solution A: autoserum is 1 ~ 3:1, is mixed with NKT cells frozen storing liquid.
Relative to prior art, beneficial effect of the present invention is: the cryopreserving liquid of the NKT cell after cultivation of the present invention comprises lentinan, algal polysaccharides, autoserum and DMSO, because it adopts autoserum, there is not the pollution of exogeneous animal virus and the introducing of exogenous proteins, safety is high; The lentinan wherein added, algal polysaccharides effectively can not only keep the activity of cell, and to human body without any injury, and after cryopreservation resuscitation, the state of cell is basic close to frozen front state, frozen effective.
Accompanying drawing explanation
Fig. 1 is frozen front NKT cellular morphology figure.
Fig. 2 be frozen after the NKT cellular morphology figure of control group.
Fig. 3 be frozen after the NKT cellular morphology figure of experimental group 1.
Fig. 4 be frozen after the NKT cellular morphology figure of experimental group 2.
Fig. 5 be frozen after the NKT cellular morphology figure of experimental group 3.
Fig. 6 is the NKT cell proliferation curve map of frozen front and back.
Fig. 7 is the NKT cell killing activity curve map of frozen front and back.
Embodiment
Describe the present invention below in conjunction with drawings and the specific embodiments.
Embodiment 1,
The present embodiment provides the preparation method of the cryopreserving liquid of the NKT cell after a kind of cultivation, comprises the following steps:
1) be that the lentinan of 1 ~ 6mg/mL and the algal polysaccharides of 0.05 ~ 0.2mg/mL add in autoserum respectively by concentration, add DMSO slowly again, add lentinan by volume: algal polysaccharides: autoserum: DMSO is 1 ~ 3:1 ~ 3:1 ~ 5:1, the precooling of 2 ~ 8 DEG C of degree refrigerators is placed in by preparing the solution A obtained, for subsequent use;
2) above-mentioned solution A taken out again, it slowly joined in autoserum, by volume percentage adds solution A: autoserum is 1 ~ 3:1, is mixed with NKT cells frozen storing liquid.
Embodiment 2,
The present embodiment cultivate according to the method preparation described in embodiment 1 after the cryopreserving liquid of NKT cell, described cryopreserving liquid by solution A and autoserum formulated, solution A and autoserous volume ratio are 1:1, wherein:
Described solution A comprises lentinan, algal polysaccharides, autoserum and DMSO, and wherein the volume ratio of lentinan, algal polysaccharides, autoserum and DMSO is 1:1:1:1, and wherein, lentinan concentration is 1mg/mL, and algal polysaccharides concentration is 0.05mg/mL.
Embodiment 3,
The present embodiment cultivate according to the method preparation described in embodiment 1 after the cryopreserving liquid of NKT cell, described cryopreserving liquid by solution A and autoserum formulated, solution A and autoserous volume ratio are 2:1, wherein:
Described solution A comprises lentinan, algal polysaccharides, autoserum and DMSO, and wherein the volume ratio of lentinan, algal polysaccharides, autoserum and DMSO is 2:2:3:1, and wherein, lentinan concentration is 6mg/mL, and algal polysaccharides concentration is 0.2mg/mL.
Embodiment 4,
The present embodiment cultivate according to the method preparation described in embodiment 1 after the cryopreserving liquid of NKT cell, described cryopreserving liquid by solution A and autoserum formulated, solution A and autoserous volume ratio are 3:1, wherein:
Described solution A comprises lentinan, algal polysaccharides, autoserum and DMSO, and wherein the volume ratio of lentinan, algal polysaccharides, autoserum and DMSO is 3:3:3:1, and wherein, lentinan concentration is 3mg/mL, and algal polysaccharides concentration is 0.1mg/mL.
Comparative example 1,
The present embodiment preparation regular growth cryopreserving liquid, described regular growth cryopreserving liquid is the DMSO containing 10%FBS.
The amplification cultivation of embodiment 5, NKT cell and collection
1, the separation of peripheral blood mononuclear ball
1) peripheral blood of 10 ~ 80ml is gathered, the ratio of itself and physiological saline 1:1 is by volume mixed, the ratio of the blood mixed liquor after mixing and lymphocyte separation medium 2:1 is by volume added to lymphocyte separation medium upper strata, the centrifugal 20min of 3000rpm slowly;
2) the seeing from top to bottom of centrifugal rear centrifuge tube, be respectively blood plasma, tunica albuginea layer, i.e. mononuclearcell layer (PBMC), lymphocyte separation medium, red blood cell layer, extract PBMC layer, after the PBMC brine collected is resuspended, the centrifugal 10min of 1500rpm, repeated washing twice, collects PBMC;
2, the amplification cultivation of NKT cell
1) by resuspended for the PBMC RPMI1640 basal medium collected in above-mentioned 1, by 1*10
6individual/ml density is inoculated in blake bottle, and simultaneously and add Porcine HGF IL-2500U/ml, IL-1530ng/ml, be placed in 37 DEG C, concentration is the CO of 5%
2cultivate in incubator;
2) induce after 5 days, carry out fluid infusion, full dose adds IL-2500U/ml and IL-1530ng/ml, and before fluid infusion, cell density is not more than 3*10
6individual/ml, after fluid infusion, cell density is at 0.5-1.0*10
6individual/ml, every fluid infusion in 3 days with add Porcine HGF.
3) until the 14th day collecting cell.
Embodiment 6, to the frozen test of NKT cell after cultivating
1) cryopreserving liquid utilizing embodiment 2 ~ 4 to prepare respectively carries out frozen as experimental group 1 ~ 3 to the NKT cell after cultivation, and the regular growth cryopreserving liquid utilizing comparative example 1 to prepare carries out frozen to the NKT cell after cultivation as a control group.
Experimental group 1: the resuspended NKT cell of NKT cells frozen storing liquid prepared by embodiment 2, cell density is 10
6individual/ml cell suspension, each cryopreservation tube 1ml/ manages.
Experimental group 2: the resuspended NKT cell of NKT cells frozen storing liquid prepared by embodiment 3, cell density is 10
6individual/ml cell suspension, each cryopreservation tube 1ml/ manages.
Experimental group 3: the resuspended NKT cell of NKT cells frozen storing liquid prepared by embodiment 4, cell density is 10
6individual/ml cell suspension, each cryopreservation tube 1ml/ manages.
Control group: the resuspended NKT cell of regular growth cryopreserving liquid prepared by comparative example 1, cell density is 10
6individual/ml cell suspension, each cryopreservation tube 1ml/ manages.
2) above-mentioned each cryopreservation tube is first put into freezing storing box, then freezing storing box is put into programmed cooling instrument and carry out frozen, and then cryopreservation tube is transferred in liquid nitrogen rapidly frozen.
The recovery of embodiment 7, NKT cell
1) thermostat water bath is heated to 37 DEG C, the above-mentioned each group of frozen cell cryopreservation tube of 1 year in liquid nitrogen of rapid taking-up, to be transferred in water-bath and continuous oscillation, until dissolve completely, then the cell suspension after dissolving is moved to wash in the centrifuge tube being added with perfect medium centrifugal;
2) by above-mentioned centrifugal after cell perfect medium resuspended cultivation 24h, by 10
6/ ml cell is inoculated in blake bottle, and full dose adds IL-2500U/ml and IL-1530ng/ml Porcine HGF, is placed in 37 DEG C, 5%CO
2incubator in cultivate.
Morphologic observation before and after embodiment 8, NKT cell cryopreservation
By each group of NKT cell observation of cell form under inverted microscope of frozen front and back, form observed result is as shown in Fig. 1 ~ 5, observed result shows, NKT cell after 3 groups of experimental group recoveries and frozen front cellular morphology frozen front and back no significant difference, cell is full, translucent luster, and has more cell conglomeration, relative comparison group, the impact of cells frozen storing liquid of the present invention on cell cryopreservation is less.
Survival rate test before and after embodiment 9, NKT cell cryopreservation
After the NKT cell of frozen front and back is carried out Trypan Blue, carry out cell counting with cell counting count board, calculate Cell viability, as shown in table 1 below:
Cell viability before and after table 1NKT cell cryopreservation
| Group | Cell viability |
| Frozen front NKT cell | 97.3％ |
| Frozen rear NKT cell-experimental group 1 | 93.2％ |
| Frozen rear NKT cell-experimental group 2 | 95.4％ |
| Frozen rear NKT cell-experimental group 3 | 95.6％ |
| Frozen rear NKT cell-control group | 81.7％ |
The Cell viability of the frozen rear recovery of experimental group is close to before frozen as can be seen from the above Table 1, especially the result of experimental group 3 is best, and the Cell viability of experimental group is all higher than control group, illustrate that cells frozen storing liquid of the present invention can keep the activity of cell, after cryopreservation resuscitation, the state of cell is substantially close to frozen front state, frozen effective.
The detection of ability of cell proliferation before and after embodiment 10, NKT cell cryopreservation
By before frozen with frozen after, the recovery cultivation NKT cell of a week counts, 48h counting once, draw cell growth curve, result as shown in Figure 6, as can be seen from described Fig. 6, after the NKT cell recovery of the cryopreserving liquid preservation of experimental group, cultivate the NKT cell that multiplication capacity is better than the cryopreserving liquid preservation of control group.
Viability examination is killed and wounded before and after embodiment 11, NKT cell cryopreservation
NKT cell after the cultivation of frozen front and back is carried out the detection of killing activity, adopt lactate dehydrogenase (LDH) method, with K562 cell for target cell, detect the NKT cell of each group of frozen front and back respectively, namely effector cell kill and wound vigor.Effector cell and target cell are added 96 orifice plates than the cell number ratio being respectively 40:1,20:1,10:1,5:1 by effect target, and target cell number is 10
4individual/hole, often kind of effect target ratio establishes 4 parallel holes, every hole 200ul.All adopt RPMI1640 medium resuspended.In 37 DEG C, hatch 4h in 5%CO2 incubator, then use LDH-CytotoxicityColorimetricAssayKitII (U.S., BioVision, article No.: K313-500) kit detects, and adopts the light absorption value of microplate reader determined wavelength when 490nm (A).The computing formula of killing and wounding vigor is: NKT cytoactive (%)=(spontaneous-K562 is spontaneous for experiment-NKT cell)/(maximum-K562 of K562 is spontaneous) × 100%, testing result as shown in Figure 7, can find out that each group of NKT cell kills and wounds to K562 cell the increasing and strengthen with effect target ratio that live from the result of described Fig. 7, with the equal no significant difference of frozen front cell killing vigor after the NKT cell recovery of experimental group, and NKT cell after control group recovery different effect targets than time kill and wound vigor all lower than the cell before frozen.
Embodiment 12, NKT cell frozen front and back immunophenotype detect
By before frozen with recovery after the resuspended NKT cell of NKT cell PBS of collection, density is 10
6individual/ml, add the CD56 antibody of CD3 and the PE mark of FITC mark, after room temperature lucifuge hatches 30min, with flow cytomery, detect the cell proportion (%) with corresponding immunophenotype, testing result is as shown in table 2:
Immunophenotype testing result before and after table 2NKT cell cryopreservation
| Group | CD3 +CD56 + |
| Frozen front NKT cell | 81.3±0.32 |
| Frozen rear NKT cell-experimental group 1 | 81.1±0.20 |
| Frozen rear NKT cell-experimental group 2 | 81.0±0.12 |
| Frozen rear NKT cell-experimental group 3 | 82.1±0.73 |
| Frozen rear NKT cell-control group | 70.1±1.15 |
From the results shown in Table 2, there is some difference for the immunophenotype of the NK cell of frozen front and back, and experimental group is higher than the immunophenotype testing result of control group, and the frozen better effects if of cells frozen storing liquid of the present invention is described.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (5)
1. the cryopreserving liquid of NKT cell after cultivating, is characterized in that, described cryopreserving liquid by solution A and autoserum formulated, solution A and autoserous volume ratio are 1 ~ 3:1, wherein:
Described solution A comprises lentinan, algal polysaccharides, autoserum and DMSO, and wherein the volume ratio of lentinan, algal polysaccharides, autoserum and DMSO is 1 ~ 3:1 ~ 3:1 ~ 5:1.
2. the cryopreserving liquid of the NKT cell after cultivation according to claim 1, is characterized in that, described cryopreserving liquid by solution A and autoserum formulated, solution A and autoserous volume ratio are 1 ~ 2:1, wherein:
Described solution A comprises lentinan, algal polysaccharides, autoserum and DMSO, and wherein the volume ratio of lentinan, algal polysaccharides, autoserum and DMSO is 1 ~ 2:1 ~ 2:1 ~ 3:1.
3. the cryopreserving liquid of the NKT cell after cultivation according to claim 1, is characterized in that, described cryopreserving liquid by solution A and autoserum formulated, solution A and autoserous volume ratio are 2 ~ 3:1, wherein:
Described solution A comprises lentinan, algal polysaccharides, autoserum and DMSO, and wherein the volume ratio of lentinan, algal polysaccharides, autoserum and DMSO is 2 ~ 3:2 ~ 3:3 ~ 5:1.
4. the cryopreserving liquid of the NKT cell after the cultivation according to claims 1 to 3 any one, is characterized in that, the concentration of described lentinan is 1 ~ 6mg/mL, and the concentration of described algal polysaccharides is 0.05 ~ 0.2mg/mL.
5. a preparation method for the cryopreserving liquid of the NKT cell after cultivating, is characterized in that, comprise the following steps:
1) be that the lentinan of 1 ~ 6mg/mL and the algal polysaccharides of 0.05 ~ 0.2mg/mL add in autoserum respectively by concentration, add DMSO slowly again, add lentinan by volume: algal polysaccharides: autoserum: DMSO is 1 ~ 3:1 ~ 3:1 ~ 5:1, the precooling of 2 ~ 8 DEG C of degree refrigerators is placed in by preparing the solution A obtained, for subsequent use;
2) above-mentioned solution A taken out again, it slowly joined in autoserum, by volume percentage adds solution A: autoserum is 1 ~ 3:1, is mixed with NKT cells frozen storing liquid.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108064840A (en) * | 2017-12-28 | 2018-05-25 | 重庆斯德姆生物技术有限公司 | A kind of NKT cells frozen storing liquids and preparation method thereof |
| CN110301432A (en) * | 2019-07-25 | 2019-10-08 | 上海轩锋生物科技有限公司 | A kind of new cell freezing method |
| CN111602648A (en) * | 2020-04-26 | 2020-09-01 | 河南侨创生命科技有限公司 | Immune cell serum-free cryopreservation liquid and cryopreservation method |
| CN114027294A (en) * | 2021-12-12 | 2022-02-11 | 杭州中赢生物医疗科技有限公司 | Immune cell cryopreservation liquid and immune cell cryopreservation method |
| CN115039766A (en) * | 2022-08-13 | 2022-09-13 | 中国农业科学院北京畜牧兽医研究所 | Normal-temperature boar semen diluent |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108064840A (en) * | 2017-12-28 | 2018-05-25 | 重庆斯德姆生物技术有限公司 | A kind of NKT cells frozen storing liquids and preparation method thereof |
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| CN115039766A (en) * | 2022-08-13 | 2022-09-13 | 中国农业科学院北京畜牧兽医研究所 | Normal-temperature boar semen diluent |
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