CN104719282B - Peripheral blood mononuclear cell serum-free freezing medium and freezing method - Google Patents
Peripheral blood mononuclear cell serum-free freezing medium and freezing method Download PDFInfo
- Publication number
- CN104719282B CN104719282B CN201510083493.4A CN201510083493A CN104719282B CN 104719282 B CN104719282 B CN 104719282B CN 201510083493 A CN201510083493 A CN 201510083493A CN 104719282 B CN104719282 B CN 104719282B
- Authority
- CN
- China
- Prior art keywords
- peripheral blood
- blood mononuclear
- mononuclear cell
- cell
- free
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a peripheral blood mononuclear cell serum-free freezing medium. The peripheral blood mononuclear cell serum-free freezing medium is prepared from the following components in percentage by volume: 5 to 20% of dimethyl sulfoxide, 0.5 to 10% of dextran 40, and the balance of plasmalyte A. The invention further discloses a method for freezing peripheral blood mononuclear cells through the peripheral blood mononuclear cell serum-free freezing medium. Compared with the prior art, the freezing medium is free of animal serum, human serum and a cell culture medium, and has a good freezing effect; the cells can be directly applied to the clinic after resuscitation and can be also induced in vitro into immune cells NKT and CIK and have high application value in the clinic.
Description
Technical field
The present invention relates to cells frozen storing liquid technical field, be specifically related to a kind of PERIPHERAL BLOOD MONONUCLEAR CELL serum-free frozen stock solution
And cryopreservation methods.
Background technology
PERIPHERAL BLOOD MONONUCLEAR CELL (peripheral blood mononuclear cell, PBMC), refers to have in peripheral blood
There is the cell of single core, comprise lymphocyte, mononuclear cell (monocyte), dendritic cell and hematopoietic stem cell etc..PBMC
Panimmunity cell can be induced differentiation in vitro, such as cytokine induced kill cell (CIK), natural killer cell
(NK), natural killer T cells (NKT), at the aspect such as antitumor, infection, there is great utility.Therefore, frozen PBMC is carefully
Born of the same parents' treatment aspect tool has very important significance.
Cell cryopreservation is one of main method of cell long-period preservation.Utilize Cryopreservation Technology that cell is placed in-196 DEG C of liquid nitrogen
Middle cryopreservation, can make cell be temporarily disengaged from growth conditions and be saved by its cell characteristics, so needs when
Recovery cell is used for testing again.And moderately preserve a certain amount of cell, it is possible to prevent the cell because cultivating to be contaminated
Or other thunder boltes and make cell lose kind, serve the effect of cell conservation.
At present, the most frequently used technology of cell cryopreservation is liquid nitrogen freezing preservation method, main use add appropriate protectant slowly
Freezing method freeze-stored cell.The most the most frequently used freezing protective agent is dimethyl sulfoxide (DMSO), and this molecular weight of material is little, dissolves
Degree is big, easy penetration cell, can make freeze point depression, reduces intracellular and forms the chance of ice crystal, thus reduces the ice crystal damage to cell
Wound.Owing to high concentration DMSO is toxic to cell, it is necessary to add other liquid component, such as serum, cell culture medium, with fall
The concentration of low DMSO, reduces the injury to cell.
Existing PBMC frozen stock solution, mainly based on DMSO, is combined hyclone (FBS) or human serum (HAS), and is added thin
Born of the same parents' culture medium.Serum can protect PBMC to protect against frozen, the damage of recovery.Such as, Wang Guixi etc. compare DMSO10%+
FBS90%, DMSO10%+ Dextran 40 (Dextran-40) 10%+FBS80%, DMSO10%+ (hetastarch) HES
6%+FBS 84% frozen stock solution formula such as grade is to the protective effect to PBMC, result display DMSO10%+Dextran-4010%+
The frozen effect of FBS80% is best, and after PBMC recovery, motility rate is the highest, is 79.7 ± 1.5%.Robab Nazarpour etc. study
Finding, 10%～15%DMSO+40%FBS frozen PBMC effect is preferable.Additionally, the middle promulgated by the State Council of Publication No. CN102669087A
Bright patent application, discloses a kind of PERIPHERAL BLOOD MONONUCLEAR CELL frozen stock solution, comprises permeability cryoprotective agent, beta glucan, blood
Clear or blood plasma, normal saline.Additionally, cell culture medium is also the usual component of frozen stock solution, such as Qin Chun victory etc. is in order to RPMI
1640 culture medium and DMSO, 10% albumin make freezing protective agent direct frozen procedure for peripheral blood stem cells under the conditions of-80 DEG C.
The hyclone that in prior art, stem cell cryopreserving liquid is used often carries animal derived virus, the most European multiple
There is bovine spongiform encephalopathy in country, contains the virus causing bovine spongiform encephalopathy, can cause this type of virus disseminating for freeze-stored cell in its serum.This
Outward, there are some researches show that the stem cell contacted with hyclone for a long time can be to the hyclone generation endocytosis in solution medium, endocytosis
Stem cell after hyclone is likely to occur the change of some protein expression, is likely to occur heterogenous animal after being applied to human body
The immunoreation that albumen causes, thus after causing stem cell transplantation, success rate reduces and the generation of untoward reaction.And human serum
Also potential virus, such as hepatitis B virus, HIV (human immunodeficiency virus) etc. can be propagated.Therefore, China's medicine and food quality supervision management board
" human body cell treatment clinical research and the quality control guideline " within 2003, issued explicitly points out, and should avoid using blood as far as possible
Cultivate clearly cell, including animal serum and human serum.
In order to avoid using serum to bring virus infection, some people employs human albumin, but human blood protein is in clinic
On may cause some untoward reaction, such as anaphylactic shock, heating, heart damage, respiratory system damage, kidney damage etc.,
Which has limited the safety the most directly used containing albuminous cells frozen storing liquid.
It addition, containing cell culture medium in existing frozen stock solution formula, cell culture medium can not be injected directly into human body, therefore
If directly transplanting after needing cell recovery, then must pass through to be centrifuged and cell culture medium is got rid of, and be centrifuged and can cause just
The just cell damage of recovery, motility rate reduces, affects cell therapy effect.
Summary of the invention
It is an object of the invention to overcome the defect of above-mentioned prior art, it is provided that a kind of PERIPHERAL BLOOD MONONUCLEAR CELL serum-free
Frozen stock solution, this frozen stock solution is made up of DMSO, Dextran 40, Bomaili A, without animal serum, human serum, cell culture medium,
And frozen respond well, both can be directly used for clinic after cell recovery, immunoblast NKT, CIK can have been induced again in vitro,
Have much using value clinically.The technical scheme that the present invention is concrete is as follows:
A kind of PERIPHERAL BLOOD MONONUCLEAR CELL serum-free frozen stock solution, it is characterised in that by volume mark meter includes following components:
5～the dimethyl sulfoxide of 20%, 0.5～10% Dextran 40, surplus is Bomaili A.
Wherein Bomaili A is Bureau of Drugs Supervision of the China approval Multiple electrolytes injection for clinic, and its component is: every
1000ml sodium chloride-containing 5.26g, sodium gluconate 5.02g, sodium acetate 3.68g, potassium chloride 0.37g, magnesium chloride 0.30g.
The purpose of the present invention can also be realized by below scheme:
A kind of technical scheme realizing the present invention is: described PERIPHERAL BLOOD MONONUCLEAR CELL serum-free frozen stock solution by volume mark
Meter includes following components: the dimethyl sulfoxide of 10%, 1% Dextran 40, surplus is Bomaili A, and described Dextran 40 is
6% dextran-40 glucose injection, it contains glucose, can be that cell provides certain nutritional labeling.
A kind of technical scheme realizing the present invention is: described PERIPHERAL BLOOD MONONUCLEAR CELL serum-free frozen stock solution by volume mark
Meter includes following components: the dimethyl sulfoxide of 20%, 2% Dextran 40, surplus is Bomaili A, and described Dextran 40 is
6% dextran-40 glucose injection.
Present invention also offers and utilize above-mentioned PERIPHERAL BLOOD MONONUCLEAR CELL serum-free frozen stock solution to carry out frozen peripheral blood single core
The method of cell, concrete technical scheme is as follows:
A kind of PERIPHERAL BLOOD MONONUCLEAR CELL serum-free cryopreservation methods, it is characterised in that comprise the following steps:
(1) PERIPHERAL BLOOD MONONUCLEAR CELL is provided；
(2) preparation PERIPHERAL BLOOD MONONUCLEAR CELL serum-free frozen stock solution described in claims 1 to 3 any one；
(3) by the resuspended PERIPHERAL BLOOD MONONUCLEAR CELL of Bomaili A, and it is frozen to add isopyknic serum-free in cell suspension
Liquid, is then sub-packed in cryopreservation tube, is placed in by cryopreservation tube equipped with in the freezing storing box of isopropanol, freezing in-80 DEG C of environment, next day
Move into liquid nitrogen container again.
Preferably, the amount of the Bomaili A that step (3) adds is so that the concentration of PERIPHERAL BLOOD MONONUCLEAR CELL is for the most frozen
In liquid, the twice of PERIPHERAL BLOOD MONONUCLEAR CELL concentration is as the criterion.
Preferably, in step (3) cryopreservation tube, the density of PERIPHERAL BLOOD MONONUCLEAR CELL is (0.5～1) × 107/ml。
Compared with prior art, the invention has the beneficial effects as follows:
1, the present invention abandons one of the composition using animal serum, human serum, cell culture medium as frozen stock solution, but adopts
Combine dimethyl sulfoxide with Bomaili A and Dextran 40 and be used as the frozen stock solution of PBMC, it is to avoid propagation potential pathogen
Risk, improve the safety of cell cryopreservation, in turn ensure that the frozen effect of PBMC；It addition, freezing without cell culture medium
Liquid storage, can have great practical value with direct feedback human body after PBMC recovery clinically；The formula of the present invention is not having
In the case of albuminous, also obtain good frozen effect.
2, in the frozen stock solution of the present invention, the easy penetration cell of DMSO, freeze point depression can be made during cell cryopreservation, reduce
The chance of intracellular formation ice crystal, thus reduce the ice crystal damage to cell；Dextran 40 then plays the work of stabilizing cell membrane
With, it is to avoid cell suffers frozen injury；Bomaili A then maintains electrolyte balance, makes cell be in a kind of comparatively gentle more connect
It is bordering in the solution of internal milieu.
3, the present invention selects to be appropriate to the frozen material of PERIPHERAL BLOOD MONONUCLEAR CELL and is configured to frozen stock solution, and simplifies frozen
The component of liquid, but obtain prominent effect, after recovering by the PERIPHERAL BLOOD MONONUCLEAR CELL that frozen stock solution of the present invention is frozen, safe nothing
Poison, can be directly used for clinic, can induce again immunoblast NKT, CIK in vitro, and the activity of cell is high.
Accompanying drawing explanation
Fig. 1 be use the present invention the frozen PBMC of method after recover to the NKT inducing culture cellular figure of the 14th day；
Fig. 2 is that PBMC is to NKT induction 14 days rear surface marker detection figures of differentiation；
Fig. 3 is that PBMC is to CIK induction 14 days rear surface marker detection figures of differentiation
Fig. 4 be use the present invention the frozen PBMC of method after recover to the CIK inducing culture cellular figure of the 14th day.
Detailed description of the invention
Below by being embodied as example, the present invention is described in further detail, in order to be clearly understood that the present invention to protect
The technical scheme protected.
Embodiment one
The separation method of human peripheral blood single nucleus cell, gathers including following step:
1, the separation of PBMC
The 30ml peripheral blood adding sodium citrate anticoagulant is transferred to 50mL centrifuge tube with the disposable pipet of 10mL by 1.1
In, 800g is centrifuged 10min.
Suck upper plasma with pasteur pipet after 1.2 centrifugal end, add 20ml physiology salt with the disposable pipet of 10mL
Water dilutes, mix homogeneously.
1.3 separately take a new 50mL centrifuge tube, add 12mL separation of lymphocytes with 10mL disposable pipet often pipe
Liquid (blood dilution liquid: lymph separation liquid=2:1), is slowly transferred to lymph with the disposable pipet of 10mL by the blood after dilution
The surface of cell separation liquid, makes to form therebetween interface clearly.
1.4 700g is centrifuged 30min, centrifugal elevation rate changes 0 into.
1.5 centrifugal after from pipe end the to liquid level, be divided into 4 layers, be followed successively by erythrocyte and GCL, lymphocyte separation medium layer,
PERIPHERAL BLOOD MONONUCLEAR CELL layer (PBMC layer), plasma layer.With pasteur pipet by PBMC layer sucking-off, it is transferred to another 50mL and is centrifuged
Guan Zhong.
1.6 add normal saline to 40mL with the disposable pipet of 10mL, and resuspended PBMC, 400g are centrifuged 5min.
Abandon supernatant after 1.7 centrifugal end, after adding the abundant re-suspended cell of 40mL normal saline with disposable pipet, take 20
μ L cell suspension, in 1.5mL EP pipe, counts with cell counter, and remaining suspension 250g is centrifuged 5min.
1.9 remove supernatant, obtain PBMC.
Embodiment two
The preparation of human peripheral blood single nucleus cell serum-free frozen stock solution, by volume percent meter is by 5～the dimethyl of 20%
Sulfoxide, 0.5～10% Dextran 40, surplus is Bomaili A mix homogeneously.
Bomaili A component is: every 1000ml sodium chloride-containing 5.26g, sodium gluconate 5.02g, sodium acetate 3.68g, chlorination
Potassium 0.37g, magnesium chloride 0.30g.
The most concrete scheme, can be configured to above-mentioned human peripheral blood single nucleus cell serum-free frozen stock solution by volume
Percent meter, comprises 20%DMSO, 2% Dextran 40, and surplus is Bomaili A.Can also be by above-mentioned human peripheral single core
Cell non-serum frozen stock solution is configured to by volume percent meter, comprises 10%DMSO, 1% Dextran 40, and surplus is vigorous arteries and veins power
A。
Embodiment three
Human peripheral blood single nucleus cell serum-free cryopreservation methods, concrete step is:
1, the cells frozen storing liquid (20%DMSO+2% Dextran 40+Bomaili A) 10～20ml of Example two preparation.
2, the PBMC cell precipitation of Example one isolated.
4, adding Bomaili A in PBMC cell precipitates makes cell density be 1～2 × 107/ ml, blows and beats mixing gently.
5, it is slowly added into along tube wall in above-mentioned PBMC cell suspension and is prepared with the pipe isopyknic step 1 of inner cell suspension
Cells frozen storing liquid, blow and beat mixing gently, making cell density is (0.5～1) × 107/ml。
6, cell suspension is sub-packed in cryopreservation tube, often pipe 1ml.
7, on cryopreservation tube, labelling is carried out, including umbilical cord numbering, cell algebraically, cell cryopreservation lot number, frozen date, operation
The relevant informations such as person.
8, being placed in by cryopreservation tube equipped with in the freezing storing box of isopropanol, in-80 DEG C of refrigerator overnight, next day moves into liquid nitrogen container.
Embodiment four
The recovery of PBMC, concrete grammar is as follows:
1, the temperature of constant water bath box regulates to 37 DEG C；From liquid nitrogen, take out cell cryopreservation tube, put into 37 DEG C of warm water immediately
In rock gently, until frozen stock solution is completely dissolved.
2, with 75% ethanol cryopreservation tube lid, flame cryopreservation tube lid；Open cryopreservation tube lid, toward cryopreservation tube
In be slowly added into 4 DEG C of pre-cooling RPMI 1640 culture medium 1ml, blow and beat mixing gently, cell suspension transferred to 50ml centrifuge tube
In；Again in cryopreservation tube, add 4 DEG C of pre-cooling RPMI 1640 culture medium of 1ml, the piping and druming washing of 1ml rifle head, move into centrifugal in the lump
Guan Zhong.
3, being slowly added into 4 DEG C of pre-cooling RPMI 1640 culture medium 15ml in centrifuge tube, 200g is centrifuged 5min, abandons supernatant；
4, in centrifuge tube, add 4 DEG C of pre-cooling RPMI 1640 culture medium 5ml, blow and beat mixing gently, sample 10 μ l and count
Number, and calculate Cell viability.
5, result shows, after 3 batch PBMC recoveries of frozen 2 months, Cell viability meansigma methods is 84.03%, frozen effect
Fruit is good, is specifically shown in Table 1.
Table 1
| PBMC batch | NO.1 | NO.2 | NO.3 | Meansigma methods |
| PBMC recovery motility rate | 83.50% | 85.70% | 82.90% | 84.03% |
Embodiment five
PBMC tests to NKT cell induction, and concrete grammar is as follows:
1, after obtaining PBMC, adding a certain amount of X-VIVO15 culture medium (Lonza, Switzerland), making cell density is 1 × 106/
Ml, re-suspended cell precipitates, is inoculated in T75 Tissue Culture Flask, and adds 20-300U/mL IL-2,5-50ng/mL IL-15.
2, hereafter, within every 3 days, take 20ul cell suspension and count, when cell density is more than 2 × 106During/ml, add X-
VIVO15 culture medium, making cell density is 1 × 106/ ml, adds 20-300U/mL IL-2,5-50ng/mL IL-15.
3, cultivating the 14th day, collect NKT cell, observe NKT cell, result is shown in that (Fig. 1 is that under microscope, NKT shines to accompanying drawing 1
Sheet, 100 times), carry out cell surface antigen CD3, CD56 detection, and the killing activity for tumor cell tested.
4, flow cytometer detection result shows, after NKT cell is cultivated 14 days, CD3+CD56+ cell is 32.7%, display induction effect
Fruit is good (see Fig. 2).The NKT killing activity to tumor cell K562, when NKT is 40:1 to K562 cell proportion, it kills
Rate can reach 45.4%, shows that the testing result that its killing activity is good is shown in Table 2.
Table 2
| NKT/K562 | 40:1 | 20:1 | 10:1 |
| Killing rate | 45.4% | 43.5% | 38.3% |
Embodiment six
PBMC is to CIK cell Induction experiments, and concrete grammar is as follows:
1, after obtaining PBMC, according to 1 × 106The density of/ml adds a certain amount of X-VIVO15 culture medium re-suspended cell precipitation,
Being inoculated in T75 Tissue Culture Flask, and add 500-2000U/mL IFN-λ according to the culture medium added, inducing culture CIK is thin
Born of the same parents.
2, the 2nd day full dose adds 5-50ng/mL OKT-3 and 100-500U/mL IL-2.
3, hereafter, within every 3 days, take 20ul cell suspension and count, when cell density is more than 2 × 106During/ml, add X-
VIVO15 culture medium, making cell density is 1 × 106/ ml, and add 100-500U/mL IL-2.
4, the 14th day, collect CIK cell, observation of cell state, result see accompanying drawing 4 (Fig. 4 is CIK photo under microscope,
100 times).Carry out cell surface antigen CD3, CD56 detection, and the killing activity for tumor cell is tested.
5, flow cytometer detection result shows, after CIK cell is cultivated 14 days, CD3+CD56+ cell is 24.9%, display induction effect
Fruit is good (Fig. 3).The CIK killing activity to tumor cell HL60, when CIK is 20:1 to HL60 cell proportion, its killing rate
Can reach 67.6%, show that its killing activity is good, the results are shown in Table 3.
Table 3
| CIK/HL60 | 20:1 | 10:1 | 5:1 |
| Killing rate | 67.6% | 45.8% | 38.1% |
The announcement of book and teaching according to the above description, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula changes and revises.Therefore, the invention is not limited in detailed description of the invention disclosed and described above, to the present invention's
Some modifications and changes should also be as falling in the scope of the claims of the present invention.Although additionally, this specification using
Some specific terms, but these terms are merely for convenience of description, the present invention does not constitute any restriction.
Claims (6)
1. a PERIPHERAL BLOOD MONONUCLEAR CELL serum-free frozen stock solution, it is characterised in that by volume mark meter includes following components: 5
～the dimethyl sulfoxide of 20%, 0.5～10% Dextran 40, surplus is Bomaili A.
2. PERIPHERAL BLOOD MONONUCLEAR CELL serum-free frozen stock solution as claimed in claim 1, it is characterised in that by volume mark meter bag
Including following components: the dimethyl sulfoxide of 10%, 1% Dextran 40, surplus is Bomaili A, and described Dextran 40 is 6% right
Rotation sugar acid anhydride-40 glucose injection.
3. PERIPHERAL BLOOD MONONUCLEAR CELL serum-free frozen stock solution as claimed in claim 1, it is characterised in that by volume mark meter bag
Including following components: the dimethyl sulfoxide of 20%, 2% Dextran 40, surplus is Bomaili A, and described Dextran 40 is 6% right
Rotation sugar acid anhydride-40 glucose injection.
4. a PERIPHERAL BLOOD MONONUCLEAR CELL serum-free cryopreservation methods, it is characterised in that comprise the following steps:
(1) PERIPHERAL BLOOD MONONUCLEAR CELL is provided；
(2) preparation PERIPHERAL BLOOD MONONUCLEAR CELL serum-free frozen stock solution described in claims 1 to 3 any one；
(3) by the resuspended PERIPHERAL BLOOD MONONUCLEAR CELL of Bomaili A, and in cell suspension, isopyknic serum-free frozen stock solution is added,
Then being sub-packed in cryopreservation tube, be placed in by cryopreservation tube equipped with in the freezing storing box of isopropanol, freezing in-80 DEG C of environment, next day is again
Move into liquid nitrogen container.
5. PERIPHERAL BLOOD MONONUCLEAR CELL serum-free cryopreservation methods as claimed in claim 4, it is characterised in that: step (3) adds
The amount of Bomaili A so that the concentration of PERIPHERAL BLOOD MONONUCLEAR CELL is PERIPHERAL BLOOD MONONUCLEAR CELL concentration in final frozen stock solution
Twice is as the criterion.
6. PERIPHERAL BLOOD MONONUCLEAR CELL serum-free cryopreservation methods as claimed in claim 4, it is characterised in that: step (3) is frozen
Guan Zhong, the density of PERIPHERAL BLOOD MONONUCLEAR CELL is (0.5～1) × 107/ml。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510083493.4A CN104719282B (en) | 2015-02-13 | 2015-02-13 | Peripheral blood mononuclear cell serum-free freezing medium and freezing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510083493.4A CN104719282B (en) | 2015-02-13 | 2015-02-13 | Peripheral blood mononuclear cell serum-free freezing medium and freezing method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104719282A CN104719282A (en) | 2015-06-24 |
| CN104719282B true CN104719282B (en) | 2017-01-11 |
Family
ID=53444157
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510083493.4A Active CN104719282B (en) | 2015-02-13 | 2015-02-13 | Peripheral blood mononuclear cell serum-free freezing medium and freezing method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104719282B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108029679A (en) * | 2018-01-29 | 2018-05-15 | 山东省齐鲁细胞治疗工程技术有限公司 | A kind of frozen stock solution for being used to freeze mononuclearcell |
Families Citing this family (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105076113B (en) * | 2015-08-20 | 2017-12-05 | 广州赛莱拉干细胞科技股份有限公司 | The cryopreservation methods of immunocyte |
| CN105602897B (en) * | 2015-12-15 | 2018-09-04 | 吉林省银丰生物工程技术有限公司 | The method induced after human peripheral blood mononuclear cell cryopreservation and recovery |
| CN105900973B (en) * | 2016-05-20 | 2018-11-30 | 广州赛莱拉干细胞科技股份有限公司 | Macrophage cryopreservation liquid and macrophage cryopreservation method |
| CN105994254A (en) * | 2016-07-28 | 2016-10-12 | 广州赛莱拉干细胞科技股份有限公司 | Cryopreservation solution and cryopreservation method of DC cell |
| CN107148967B (en) * | 2016-11-09 | 2020-11-17 | 深圳宾德生物技术有限公司 | Antigen-specific T lymphocyte cryopreservation solution and preparation method and application thereof |
| CN106719604A (en) * | 2017-02-04 | 2017-05-31 | 上海尚维生物科技有限公司 | Pet peripheral blood directly freezes reagent and method |
| CN106942202A (en) * | 2017-04-27 | 2017-07-14 | 黑龙江八农垦大学 | A kind of serum-free freezing media for MDBK cells |
| CN107047541A (en) * | 2017-05-31 | 2017-08-18 | 东莞市保莱生物科技有限公司 | A kind of immunocyte frozen stock solution and immunocyte cryopreservation methods |
| CN107012119B (en) * | 2017-05-31 | 2020-03-17 | 东莞市保莱生物科技有限公司 | Method for extracting immune cells from bone marrow |
| CN107306939B (en) * | 2017-07-31 | 2020-09-15 | 山东水发生命科学研究有限公司 | Cell cryopreservation liquid for dendritic 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 |
| 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 |
| CN108378021A (en) * | 2018-03-19 | 2018-08-10 | 英普乐孚生物技术(上海)有限公司 | A kind of stored refrigerated system of lymphocyte |
| CN108450458A (en) * | 2018-03-27 | 2018-08-28 | 成都菱祐生物科技有限公司 | A kind of serum-free cell frozen stock solution |
| 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 |
| CN109497043A (en) * | 2018-12-28 | 2019-03-22 | 中国医学科学院整形外科医院 | Serum-free nano material freezes agent and preparation method thereof, application method, frozen stock solution |
| CN110150267A (en) * | 2019-06-24 | 2019-08-23 | 南京沐赛生物技术有限公司 | A kind of low DMSO of clinical grade TCR-T cell freezes agent and cryopreservation methods |
| CN110447636A (en) * | 2019-08-07 | 2019-11-15 | 广东万海细胞生物科技有限公司 | A kind of frozen stock solution of peripheral blood mononuclear cells, cryopreservation methods and method for resuscitation |
| CN112568219B (en) * | 2020-12-29 | 2024-04-05 | 中博瑞康(上海)生物技术有限公司 | Low-temperature preservation solution for stem cells and umbilical cord blood and preparation method thereof |
| CN113106064A (en) * | 2021-02-24 | 2021-07-13 | 河南省银丰生物工程技术有限公司 | Frozen stock solution of immune cell CIK and activity research method thereof |
| CN115644168B (en) * | 2022-11-09 | 2024-03-01 | 湖南开启时代生物科技有限责任公司 | Frozen stock solution of finished immune cells, preparation method thereof and frozen stock method |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100953400B1 (en) * | 2007-12-06 | 2010-04-20 | 주식회사 바이넥스 | The preservation method of peripheral blood mononuclear cells at high density condition, for manufacturing anticancer material |
| CN103583511B (en) * | 2012-09-03 | 2015-07-29 | 四川新生命干细胞科技股份有限公司 | A kind of mesenchymal stem cell cryopreserving liquid and parenteral solution |
| CN103563888B (en) * | 2013-10-31 | 2015-11-25 | 北京永泰生物制品有限公司 | Cells frozen storing liquid |
-
2015
- 2015-02-13 CN CN201510083493.4A patent/CN104719282B/en active Active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108029679A (en) * | 2018-01-29 | 2018-05-15 | 山东省齐鲁细胞治疗工程技术有限公司 | A kind of frozen stock solution for being used to freeze mononuclearcell |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104719282A (en) | 2015-06-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104719282B (en) | Peripheral blood mononuclear cell serum-free freezing medium and freezing method | |
| CN110050782B (en) | Stem cell cryopreservation solution and preparation method and cryopreservation method thereof | |
| Li et al. | Preservation of cell-based immunotherapies for clinical trials | |
| Berz et al. | Cryopreservation of hematopoietic stem cells | |
| ES2588438T3 (en) | Materials and methods for hypothermic whole blood collection | |
| CN104145943B (en) | A kind of frozen protection liquid of people's umbilical cord China Tong Shi glue tissue and preparation and application thereof | |
| CN103298926B (en) | Stem cell suspension | |
| CN104542578A (en) | Cell preservation solution and preparation method and applications thereof | |
| CN105211051B (en) | Cultured NK cell freezing medium and preparation method thereof | |
| CN104873542A (en) | Umbilical cord mesenchymal stem cell injection as well as preparation method and application thereof | |
| Salehi et al. | Focus: Medical Technology: Advances in Perfusion Systems for Solid Organ Preservation | |
| CN104705291A (en) | Mononuclear cell frozen stock solution of cord blood, application and preparation method | |
| CN105211052B (en) | Frozen stock solution of cultured NKT cells and preparation method thereof | |
| CN112998009A (en) | NK cell cryopreservation liquid and preparation method and application thereof | |
| CN102669087A (en) | Freeze-storage liquid of peripheral blood mononuclear cells and freeze-storage method | |
| CN106620681A (en) | Cell lysis solution, kit and application of cell lysis solution to preparation of tumor whole cell antigen loaded DC tumor vaccine | |
| JP2015174823A (en) | Preservation method of biomaterial, production procedure of biomaterial, biomaterial, transplant material, transplant method and preservation apparatus of biomaterial | |
| CN114181901B (en) | Method for in vitro induction expansion and cryopreservation of immune cells | |
| CN109511650B (en) | Normal-temperature mechanical perfusate capable of enlarging liver supply source | |
| JPWO2019230973A1 (en) | Biomaterial Preservation Compositions, Biomaterial Preservation Methods, Biomaterial Production Methods, Transplant Materials, and Transplant Methods | |
| Galmeas et al. | A Simplified Method for Cryopreservation of Hematopoietic Stem Cells with-80dGC Mechanical Freezer with Dimethyl Sulfoxide as the Sole Cryoprotectant | |
| Berz et al. | Cryopreservation of hematopoietic and non-hematopoietic stem cells–a review for the clinician | |
| Fleming et al. | Cryopreservation of hematopoietic stem cells: emerging science, technology and issues | |
| RU2303631C1 (en) | Method for production of mesenchymal stem cells and biotransplant using the same | |
| Gao et al. | Research progress on hepatic machine perfusion |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |