CN104705291A - Mononuclear cell frozen stock solution of cord blood, application and preparation method - Google Patents
Mononuclear cell frozen stock solution of cord blood, application and preparation method Download PDFInfo
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Abstract
The invention relates to the field of biological technology, and particularly relates to a mononuclear cell frozen stock solution of cord blood, an application and a preparation method. The mononuclear cell frozen stock solution of cord blood provided by the invention comprises dimethyl sulfoxide (DMSO) united plasmalyte force A (compound electrolyte injection) and hydroxyethyl starch (HES). Compared with the traditional formula, the scheme can be used for effectively preventing the pollution of animal derived pathogens and avoiding using of a cell culture medium to serve as a component of the frozen stock solution and guaranteeing a good cell frozen stock effect; after cell frozen for 2 months, the average cell viability of resurgent CBMC cell frozen by the cell frozen solution is 94.73%, which is remarkably (P is smaller than 0.05) higher than the cell frozen effect of the traditional cell frozen solution (10%DMSO+90%FBS). The resurgent cell can be directly applied to clinical transplantation, so the mononuclear cell frozen stock solution of the cord blood has a large practical value in clinical application of cell therapy.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of CBMC cryopreserving liquid, application, preparation method.
Background technology
CBMC (cord blood mononuclear cell, CBMC), refer to the cell in bleeding of the umbilicus with single core, comprise candidate stem cell, lymphocyte, monocyte (monocyte), dendritic cell and other a small amount of cell.CBMC can promote body hematopoiesis in vivo, strengthens immunity of organisms; In vitro can differentiation-inducing one-tenth panimmunity cell, as cytokine induced kill cell (CIK), natural killer cell (NK), natural killer T cells (NKT), in antitumor, anti-infective etc., there is great utility.Therefore, frozen CBMC tool in cell therapy has very important significance.
Cell cryopreservation is one of main method of cell long-period preservation.Utilize Cryopreservation Technology cell to be placed in-196 DEG C of liquid nitrogen Cord blood, cell can be made temporarily to depart from growth conditions and be saved by its cell characteristics, when needs, recovery cell is used for experiment more like this.And moderately preserve a certain amount of cell, can prevent from making cell lose kind because of just contaminated in cultured cells or other accidents, serve the effect of cell conservation.
Cell cryopreservation process understands the thermodynamics, chemistry and the physical environment that significantly change cell, has the danger with causing biological injury.In order to be down to minimum by the damage of cell in cell cryopreservation, resuscitation process, further must optimize chemistry and temperature operation process, but needs add one or more freezing protective agent before frozen, are removed again upon dissolution.Freezing protective agent the most frequently used is at present dimethyl sulfoxide (DMSO) (DMSO), and this molecular weight of material is little, and solvability is large, and easy penetration cell, can make freeze point depression, reduces the chance forming ice crystal in born of the same parents, thus reduces ice crystal to the damage of cell.Because high concentration DMSO is toxic to cell, also must add other liquid component, as serum, cell culture medium, to reduce the concentration of DMSO, reduce the injury to cell.
The cryopreserving liquid formula of existing frozen CBMC adopts DMSO to combine animal blood serum more, has the potential risk of the substance that spreads disease, adopts medium then to limit its direct applied possibility clinically.The hyclone that current stem cell cryopreserving liquid uses often carries animal derived virus, and such as rabid ox disease occurs in European multiple country, containing the virus causing rabid ox disease in its serum, this viroids can be caused to propagate for freeze-stored cell.In addition, there are some researches show that the stem cell contacted with hyclone for a long time can to the hyclone generation endocytosis in solution medium, likely there is the change of some protein expression in the stem cell after endocytosis hyclone, the immune response that heterogenous animal albumen causes likely is there is after being applied to human body, thus the generation of success rate reduction and bad reaction after causing stem cell transplantation.Therefore, " human body cell treatment clinical research and quality control guideline " that Chinese medicine and food quality supervision management board issue for 2003 explicitly points out, and should avoid using animal blood serum cultured cell as far as possible.
Cell culture medium is also composition common during current cryopreserving liquid is filled a prescription, its complicated component, human body can not be injected directly into, if therefore directly transplanting after needing cell recovery, then must pass through and centrifugal cell culture medium to be got rid of, and the centrifugal cell damage that can cause just having recovered, motility rate reduces, and affects cell therapy effect.
Summary of the invention
In view of this, the invention provides a kind of CBMC cryopreserving liquid, application, preparation method.CBMC cryopreserving liquid of the present invention does not adopt animal blood serum, avoids the risk that animal derived pathogene propagated by serum.And adopt cryopreserving liquid of the present invention, motility rate is higher after cell recovery, show its better frozen effect.In addition, CBMC cryopreserving liquid formula of the present invention, not containing medium, can be directly used in clinical reinfusion after cell recovery, improves its value in clinical practice.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a kind of CBMC cryopreserving liquid, comprise following component:
DMSO 5 ~ 20 parts by volume
HES 5 ~ 60 parts by volume
Bomaili A 20 ~ 90 parts by volume.
The present invention abandons and adopts animal blood serum, cell culture medium as one of cryopreserving liquid composition, and take the Bomaili A and HES that obtain Chinese Drug Administration lot number, and combine DMSO, for frozen CBMC, both avoided the risk propagating potential pathogen, improve the safety of cell cryopreservation, in turn ensure that the frozen effect of CBMC, and not containing cell culture medium, can direct feedback human body after CBMC recovery, have great practical value clinically.
Wherein the easy penetration cell of DMSO, can make freeze point depression in cell cryopreservation process, reduces the chance forming ice crystal in born of the same parents, thus reduces ice crystal to the damage of cell.HES then plays the effect of stabilizing cell membrane, avoids cell to suffer frozen injury.Bomaili A then maintains electrolyte balance, cell is in a kind of comparatively gentle closer in the solution of vivo environment.
CBMC cryopreserving liquid of the present invention does not adopt animal blood serum, avoids the risk that animal derived pathogene propagated by serum.And adopt cryopreserving liquid of the present invention, motility rate is higher after cell recovery, show its better frozen effect.
In addition, CBMC cryopreserving liquid formula of the present invention, not containing medium, can be directly used in clinical reinfusion after cell recovery, improves its value in clinical practice.
In specific embodiments more of the present invention, CBMC cryopreserving liquid comprises following component:
DMSO 10 parts by volume
HES 30 parts by volume
Bomaili A 60 parts by volume.
In specific embodiments more of the present invention, CBMC cryopreserving liquid comprises following component:
DMSO 5～20v/v％
HES 5 ~ 60v/v%
Bomaili A 20 ~ 90v/v%
The percentage by volume sum of each component is 100%.
In specific embodiments more of the present invention, CBMC cryopreserving liquid comprises following component:
DMSO 10v/v％
HES 30v/v%
Bomaili A 60v/v%.
Present invention also offers the application of described CBMC cryopreserving liquid in frozen CBMC.
Present invention also offers a kind of cryopreservation methods of CBMC, with the frozen described CBMC of described CBMC cryopreserving liquid.
In specific embodiments more of the present invention, cryopreservation methods comprises the steps:
Step 1: obtain CBMC;
Step 2: resuspended described CBMC, obtains CBMC suspension;
Step 3: get described CBMC suspension and mix with described CBMC cryopreserving liquid, packing, frozen.
In specific embodiments more of the present invention, the suspension of CBMC described in cryopreservation methods is 5:1 ~ 1:5 with the volume ratio of the CBMC cryopreserving liquid as described in any one of Claims 1-4.
In specific embodiments more of the present invention, obtain CBMC in cryopreservation methods described in step 1 and be specially the bleeding of the umbilicus obtaining and add sodium citrate anticoagulant, after diluting with RPMI1640 medium 1:1, add on lymphocyte separation medium Ficoll, the centrifugal 30min of 700g, draws CBMC layer, washs 2 times with RPMI1640 medium, the centrifugal 5min of 250g, obtains CBMC.
In specific embodiments more of the present invention, in cryopreservation methods, the resuspended described CBMC of step 2 is specially with the resuspended CBMC of Bomaili A.As preferably, the cell density of CBMC suspension is 1 × 10
7/ mL.
As preferably, be packed as and cell suspension is sub-packed in cryopreservation tube, often pipe 1ml.Cryopreservation tube is placed in the freezing storing box that isopropyl alcohol is housed.
As preferably, frozen is that next day moves into liquid nitrogen container in-80 DEG C of refrigerator overnight.
Concrete, the cryopreservation methods of CBMC provided by the invention is specially:
1) separation of CBMC: after the bleeding of the umbilicus RPMI1640 medium 1:1 adding sodium citrate anticoagulant is diluted, add on lymphocyte separation medium Ficoll, the centrifugal 30min of 700g, draw CBMC layer, 2 times are washed with RPMI1640 medium, the centrifugal 5min of 250g, obtains CBMC.
2) CBMC's is frozen: preparation protectant concentration is the cryopreserving liquid (20%DMSO+60% HES+Bomaili A) of final concentration 2 times, final volume half, is placed in 4 DEG C of more than refrigerator 10min.With the resuspended CBMC of Bomaili A, its cell density is made to be 1 × 10
7/ ml.Slowly add and the isopyknic cells frozen storing liquid of pipe inner cell suspension along tube wall in cell suspension, cell suspension is sub-packed in cryopreservation tube, often pipe 1ml.Cryopreservation tube is placed in the freezing storing box that isopropyl alcohol is housed, in-80 DEG C of refrigerator overnight, next day moves into liquid nitrogen container.
The present invention abandons and adopts animal blood serum, cell culture medium as one of cryopreserving liquid composition, and take the Bomaili A and HES that obtain Chinese Drug Administration lot number, and combine DMSO, for frozen CBMC, both avoided the risk propagating potential pathogen, improve the safety of cell cryopreservation, in turn ensure that the frozen effect of CBMC, and not containing cell culture medium, can direct feedback human body after CBMC recovery, have great practical value clinically.
CBMC cryopreserving liquid of the present invention does not adopt animal blood serum, avoids the risk that animal derived pathogene propagated by serum.And adopt cryopreserving liquid of the present invention, motility rate is higher after cell recovery, show its better frozen effect.After frozen 2 months, be 94.73% with Cell viability mean value after the CBMC recovery that cryopreserving liquid of the present invention is frozen, significantly (P < 0.05) is higher than the frozen effect with conventional freeze liquid storage (10%DMSO+90%FBS).
In addition, CBMC cryopreserving liquid formula of the present invention, not containing medium, can be directly used in clinical reinfusion after cell recovery, improves its value in clinical practice.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
Fig. 1 shows the streaming result of firm separation, not frozen CBMC: CD34 expression rate is 1.0%;
Fig. 2 gives instructions in reply the streaming result of CBMC of Soviet Union: CD34 expression rate is 0.9%, with frozen front no significant difference;
Fig. 3 shows the cultivation NKT cell of the 14th day, 100 ×, cell state is good;
Fig. 4 shows the cultivation CIK cell of the 14th day, 100 ×, cell state is good;
Fig. 5 shows that CBMC is to the streaming result of NKT after differentiation-inducing 14 days: CD3+CD56+ cell is 39.0%, and display inducing effect is good;
Fig. 6 shows that CBMC is to the streaming result of CIK after differentiation-inducing 14 days: CD3+CD56+ cell is 32.7%, and display inducing effect is good.
Embodiment
The invention discloses a kind of CBMC cryopreserving liquid, application, preparation method, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
In CBMC cryopreserving liquid provided by the invention, application, preparation method, raw materials used and reagent all can be buied by market.
CBMC can have market to buy, or obtains according to the method for the invention.
Bomaili A (Multiple electrolytes injection, Shanghai Baxter Healthcare Ltd.): its component is: every 1000ml sodium chloride-containing 5.26g, gluconic acid sodium salt 5.02g, sodium acetate 3.68g, potassium chloride 0.37g, magnesium chloride 0.30g.Bomaili A, as a kind of isotonic compound electrolyte solution, has that not calcium ions, pH value are 7.4, capacity antacid is good, composition is similar to extracellular fluid and not containing advantages such as lactates.
HES (6% HES 40 sodium chloride injection, Shandong Cologne Medicine Industry Co., Ltd).
Below in conjunction with embodiment, set forth the present invention further:
Embodiment 1
DMSO 15% (volume fraction)
HES 40% (volume fraction)
Bomaili A surplus
Said components is mixed and get final product.
Embodiment 2
DMSO 10% (volume fraction)
HES 30% (volume fraction)
Bomaili A surplus
Said components is mixed and get final product.
Embodiment 3
DMSO 5% (volume fraction)
HES 60% (volume fraction)
Bomaili A surplus
Said components is mixed and get final product.
Embodiment 4
DMSO 5% (volume fraction)
HES 5% (volume fraction)
Bomaili A surplus
Said components is mixed and get final product.
Embodiment 5
DMSO 20% (volume fraction)
HES 60% (volume fraction)
Bomaili A surplus
Said components is mixed and get final product.
Embodiment 6
The separation of 1.CBMC
The 30ml bleeding of the umbilicus adding sodium citrate anticoagulant is transferred in 50mL centrifuge tube with the disposable pipette of 10mL by 1.1, the centrifugal 10min of 800g.
Suck upper plasma with pasteur pipet after 1.2 centrifugal end, add 20ml normal saline dilution with the disposable pipette of 10mL, mix.
1.3 separately get a new 50mL centrifuge tube, with the disposable pipette of 10mL often pipe add 12mL lymphocyte separation medium (blood dilution liquid: lymph parting liquid=2:1), with 10mL disposable pipette, the blood after dilution is slowly transferred to the surface of lymphocyte separation medium, makes to form interface clearly therebetween.
The centrifugal 30min of 1.4 700g, centrifugal elevation rate changes 0 into.
1.5 centrifugal after be divided into 4 layers from the bottom of pipe to liquid level, be followed successively by red blood cell and GCL, lymphocyte separation medium layer, CBMC layer (CBMC layer), plasma layer.With pasteur pipet by the sucking-off of CBMC layer, be transferred in another 50mL centrifuge tube.
1.6 add physiological saline to 40mL with the disposable pipette of 10mL, the centrifugal 5min of resuspended CBMC, 400g.
Abandon supernatant after 1.7 centrifugal end, after adding the abundant re-suspended cell of 40mL physiological saline with disposable pipette, get 20 μ L cell suspensions in 1.5mL EP pipe, count with cell counter, the centrifugal 5min of all the other suspensions 250g.
1.9 remove supernatant, obtain CBMC.
1.10 get 1 × 10
6individual above-mentioned cell, carries out its surface antigen of flow cytometer detection, and result shows, and CD34 expression rate is 1.0% (see Fig. 1).
2.CBMC's is frozen
2.1 CBMC taking the cryopreserving liquid of two kinds of different formulations frozen 1.9 to obtain, with more frozen-resuscitation effect.Be the embodiment of the present invention 5 prepare a cryopreserving liquid, another kind is the cryopreserving liquid of conventional formulation, i.e. 10%DMSO+90%FBS.
First 2.2 prepare cryopreserving liquid, two kinds of cryopreserving liquids are all mixed with the cryopreserving liquid of final concentration twice, final volume half, namely cryopreserving liquid of the present invention is 20%DMSO+60% HES+Bomaili A), the frozen liquid of conventional formulation is 20%DMSO+80%FBS.Two kinds of cryopreserving liquids are all placed in 4 DEG C of more than refrigerator 10min.
The CBMC that 1.9 obtain is divided into A, B two groups by 2.3, and often organizing CBMC total cellular score is 2 × 10
7, in A group, add 2ml Bomaili A, in B group, add 2ml FBS, blow and beat mixing gently.
2.4 slowly add 2ml cells frozen storing liquid along tube wall in cell suspension, and (described CBMC suspension is 5:1 ~ 1:5 with the volume ratio of the CBMC cryopreserving liquid as described in any one of Claims 1-4.), wherein, A group cell adds cryopreserving liquid of the present invention, and B group cell adds conventional formulation cryopreserving liquid 20%DMSO+80%FBS.Blow and beat mixing gently, make consistent (the frozen density range: 0.5 ~ 1 × 10 of cell density and instruction requirement
7/ ml).
Cell suspension is sub-packed in cryopreservation tube by 2.5, often pipe 1ml.
2.6 carry out mark on cryopreservation tube, comprise the relevant information such as umbilical cord numbering, cell algebraically, cell cryopreservation lot number, frozen date, operator.
Cryopreservation tube is placed in the freezing storing box that isopropyl alcohol is housed by 2.7, and in-80 DEG C of refrigerator overnight, next day moves into liquid nitrogen container.
The recovery of 3.CBMC
3.1 add aqua sterilisa in water-bath, and temperature is adjusted to 37 DEG C; From liquid nitrogen, take out 2.7 frozen each 3 pipes of cell A, B two groups, drop in 37 DEG C of warm water immediately and rock gently, until cryopreserving liquid dissolves completely;
3.2 with 75% ethanol cryopreservation tube lid, flame cryopreservation tube lid; Open cryopreservation tube lid, in cryopreservation tube, slowly add 4 DEG C of precooling RPMI 1640 medium 1ml, blow and beat mixing gently, cell suspension is transferred in 50ml centrifuge tube; In cryopreservation tube, again add 1ml 4 DEG C of precooling RPMI 1640 medium, 1ml rifle head piping and druming washing, moves in centrifuge tube in the lump;
3.3 slowly add 4 DEG C of precooling RPMI 1640 medium 15ml in centrifuge tube, and the centrifugal 5min of 200g, abandons supernatant;
3.4 add 4 DEG C of precooling RPMI 1640 medium 5ml in centrifuge tube, blow and beat mixing gently, sample 10 μ l and count, and calculate Cell viability.
3.5 result displays, after the CBMC recovery of frozen 2 months, the A group Cell viability mean value of cryopreserving liquid formula of the present invention is 94.73%, the B group Cell viability mean value of conventional freeze liquid storage formula is 85.00%, the frozen Be very effective of cryopreserving liquid of the present invention (P < 0.05) is higher than conventional freeze liquid storage, and this illustrates that cryopreserving liquid of the present invention is filled a prescription frozen better effects if (see table 1).
3.6 get 1 × 10
6cBMC after individual recovery, carries out its surface antigen of flow cytometer detection, and result shows, and CD34 expression rate is 0.9% (see Fig. 2), with frozen front no significant difference.
4.CBMC is to NKT cell induction:
4.1 obtain CBMC after, add X-VIVO15 medium, make cell density be 1 × 10
6/ ml, re-suspended cell precipitates, and is inoculated in T75 Tissue Culture Flask, and adds 20-300U/mL IL-2,5-50ng/mL IL-15.
4.2 after this, within every 3 days, gets 20ul cell suspension and count, when cell density is greater than 2 × 10
6during/ml, add X-VIVO15 medium, make cell density be 1 × 10
6/ ml, adds 20-300U/mLIL-2,5-50ng/mL IL-15.
4.3 cultivate the 14th day, collect NKT cell (Fig. 3 is NKT photo under microscope, 100 times).Carry out cell surface antigen CD3, CD56 to detect, and test for the killing activity of tumour cell.
4.4 flow cytometer detection result displays, NKT cell chulture is after 14 days, and CD3+CD56+ cell is 39.0% (Fig. 5), and display inducing effect is good.NKT is to the killing activity of tumour cell K562, and when NKT is 40:1 to K562 cell proportion, its kill rate can reach 44.7%, shows its killing activity good (table 2).
5.CBMC induces to CIK cell:
5.1 obtain CBMC after, according to 1 × 10
6the density of/ml adds X-VIVO15 medium re-suspended cell precipitation, is inoculated in T75 Tissue Culture Flask, and adds 500-2000U/mL IFN-λ, Fiber differentiation CIK cell according to the medium added.
Within 5.2 the 2nd days, full dose adds 5-50ng/mL OKT-3 and 100-500U/mL IL-2.
5.3 after this, within every 3 days, gets 20ul cell suspension and count, when cell density is greater than 2 × 10
6during/ml, add X-VIVO15 medium, make cell density be 1 × 10
6/ ml, and add 100-500U/mL IL-2.
5.4 the 14th days, collect CIK cell (Fig. 4 is CIK photo under microscope, 100 times).Carry out cell surface antigen CD3, CD56 to detect, and test for the killing activity of tumour cell.
5.5 flow cytometer detection result displays, after CIK cell cultivates 14 days, CD3+CD56+ cell is 32.7%, display inducing effect good (Fig. 6).CIK is to the killing activity of tumour cell HL60, and when CIK is 20:1 to HL60 cell proportion, its kill rate can reach 66.8%, shows its killing activity good (table 3).
Table 1 is after frozen 2 months, with Cell viability after the CBMC recovery that cryopreserving liquid of the present invention is frozen
| CBMC batch | NO.1 | NO.2 | NO.3 | Mean value |
| A group: cryopreserving liquid of the present invention | 93.9％ | 95.7％ | 94.6％ | 94.73％ |
| B group: 10%DMSO+90%FBS | 84.70％ | 86.80％ | 83.50％ | 85.00％ |
Table 1 result shows, after frozen 2 months, the CBMC recovery rear Cell viability mean value frozen with cryopreserving liquid of the present invention is 94.73%, and significantly (P < 0.05) is higher than the frozen effect with conventional freeze liquid storage (10%DMSO+90%FBS).
Table 2 NKT is to the killing activity of tumour cell K562
| NKT/K562 | 40:1 | 20:1 | 10:1 |
| Kill rate | 44.7％ | 42.9％ | 37.3％ |
Table 2 result shows, the killing activity of NKT to tumour cell K562 is better.
Table 3 CIK kills and wounds motility rate to tumour cell HL60's
| CIK/HL60 | 20:1 | 10:1 | 5:1 |
| Kill rate | 66.8％ | 43.8％ | 37.7％ |
Table 3 result shows, CIK to tumour cell HL60 to kill and wound motility rate good.
Fig. 1: streaming result shows, just separation, not frozen CBMC CD34 expression rate are 1.0%;
Fig. 2: streaming result shows, and the CBMC CD34 expression rate of recovery is 0.9%, with frozen front no significant difference;
Fig. 3: 100 × cultivation NKT cell of the 14th day, cell state is good;
Fig. 4: 100 × cultivation CIK cell of the 14th day, cell state is good;
Fig. 5: streaming result shows, and CBMC is to NKT after differentiation-inducing 14 days, and CD3+CD56+ cell is 39.0%, display inducing effect is good;
Fig. 6: streaming result shows, and CBMC is to CIK after differentiation-inducing 14 days, and CD3+CD56+ cell is 32.7%, display inducing effect is good.
Experimental result shows, the serum-free cryopreserving liquid of the CBMC that the embodiment of the present invention 1 provides, frozen respond well, after frozen 2 months, the CBMC recovery rear Cell viability mean value frozen with cryopreserving liquid of the present invention is 94.73%, and significantly (P < 0.05) is higher than the frozen effect with conventional freeze liquid storage (10%DMSO+90%FBS).Both can be directly used in clinical after cell recovery, immunoblast NKT, CIK can have been induced in vitro again.
CBMC cryopreserving liquid embodiment 1 to embodiment 4 prepared carries out above-mentioned experiment, and the result of CBMC cryopreserving liquid prepared by result and embodiment 5 is close, without significant difference (P < 0.05).
Comprehensive above-mentioned experimental result, the serum-free cryopreserving liquid of CBMC provided by the invention, be made up of DMSO, HES, Bomaili A, not containing animal blood serum, cell culture medium, and it is frozen respond well, both can be directly used in clinical after cell recovery, and immunoblast NKT, CIK can have been induced in vitro again, have much using value clinically.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a CBMC cryopreserving liquid, is characterized in that, comprises following component:
DMSO 5 ~ 20 parts by volume
HES 5 ~ 60 parts by volume
Bomaili A 20 ~ 90 parts by volume.
2. CBMC cryopreserving liquid according to claim 1, is characterized in that, comprises following component:
DMSO 10 parts by volume
HES 30 parts by volume
Bomaili A 60 parts by volume.
3. CBMC cryopreserving liquid according to claim 1, is characterized in that, comprises following component:
DMSO 5～20v/v％
HES 5 ~ 60v/v%
Bomaili A 20 ~ 90v/v%
The percentage by volume sum of each component is 100%.
4. CBMC cryopreserving liquid according to claim 1, is characterized in that, comprises following component:
DMSO 10v/v％
HES 30v/v%
Bomaili A 60v/v%.
5. the application of CBMC cryopreserving liquid in frozen CBMC according to any one of Claims 1-4.
6. a cryopreservation methods for CBMC, is characterized in that, CBMC as described in frozen with the CBMC cryopreserving liquid as described in any one of Claims 1-4.
7. cryopreservation methods according to claim 6, is characterized in that, comprises the steps:
Step 1: obtain CBMC;
Step 2: resuspended described CBMC, obtains CBMC suspension;
Step 3: get described CBMC suspension and mix with the CBMC cryopreserving liquid as described in any one of Claims 1-4, packing, frozen.
8. cryopreservation methods according to claim 7, is characterized in that, described CBMC suspension is 5:1 ~ 1:5 with the volume ratio of the CBMC cryopreserving liquid as described in any one of Claims 1-4.
9. the cryopreservation methods according to claim 7 or 8, it is characterized in that, obtain CBMC described in step 1 and be specially the bleeding of the umbilicus obtaining and add sodium citrate anticoagulant, after diluting with RPMI1640 medium 1:1, add on lymphocyte separation medium Ficoll, the centrifugal 30min of 700g, draw CBMC layer, wash 2 times with RPMI1640 medium, the centrifugal 5min of 250g, obtain CBMC.
10. the cryopreservation methods according to any one of claim 7 to 9, is characterized in that, the resuspended described CBMC of step 2 is specially with the resuspended CBMC of Bomaili A.
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| CN108882698A (en) * | 2015-10-29 | 2018-11-23 | 阿西姆普托特有限公司 | Method for cryo-conservation |
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| CN108882698A (en) * | 2015-10-29 | 2018-11-23 | 阿西姆普托特有限公司 | Method for cryo-conservation |
| CN105394028A (en) * | 2015-11-16 | 2016-03-16 | 黄林海 | Method for cryopreserving umbilical cord blood stem cells with low cryo-damage |
| CN107148967A (en) * | 2016-11-09 | 2017-09-12 | 深圳宾德生物技术有限公司 | A kind of antigenspecific T lymphocyte frozen stock solution and its preparation method and application |
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| CN107372469A (en) * | 2017-09-06 | 2017-11-24 | 广州赛莱拉干细胞科技股份有限公司 | The frozen stock solution and cryopreservation methods of a kind of endothelial progenitor cells |
| CN107912419A (en) * | 2017-10-11 | 2018-04-17 | 重庆金时代生物技术有限公司 | The frozen stock solution and cryopreservation methods of a kind of human peripheral blood single nucleus cell |
| CN107711823A (en) * | 2017-11-23 | 2018-02-23 | 深圳市北科生物科技有限公司 | The cells frozen storing liquid and its application that a kind of normal temperature preserves |
| CN107711823B (en) * | 2017-11-23 | 2021-05-04 | 深圳市北科生物科技有限公司 | Cell cryopreservation liquid stored at normal temperature and application thereof |
| CN108192867A (en) * | 2017-12-27 | 2018-06-22 | 重庆斯德姆生物技术有限公司 | A kind of preparation method of clinic cord blood monocyte-macrophage |
| CN108617640A (en) * | 2018-06-26 | 2018-10-09 | 吉林省太阳鸟再生医学工程有限责任公司 | A kind of cord blood mononuclear cells frozen stock solution and its application |
| RU2707921C1 (en) * | 2018-12-10 | 2019-12-02 | Федеральное государственное бюджетное учреждение науки "Кировский научно-исследовательский институт гематологии и переливания крови Федерального медико-биологического агентства" | Method for unrestricted freezing peripheral blood precursor cells at minus 80 °c under protection of dimethylsulphoxid |
| WO2021108389A1 (en) * | 2019-11-29 | 2021-06-03 | Nkmax Co., Ltd. | Method of producing natural killer cells and compositions thereof |
| CN112063585A (en) * | 2020-11-16 | 2020-12-11 | 广州杜德生物科技有限公司 | Culture method of cord blood NK-like T lymphocytes through multi-factor stimulation |
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