CN106665560A - Direct venous re-transfusion immune cell cryopreservation medium and application thereof - Google Patents
Direct venous re-transfusion immune cell cryopreservation medium and application thereof Download PDFInfo
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- CN106665560A CN106665560A CN201710028750.3A CN201710028750A CN106665560A CN 106665560 A CN106665560 A CN 106665560A CN 201710028750 A CN201710028750 A CN 201710028750A CN 106665560 A CN106665560 A CN 106665560A
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- 210000002865 immune cell Anatomy 0.000 title abstract description 7
- 239000012595 freezing medium Substances 0.000 title abstract 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 50
- 210000004027 cell Anatomy 0.000 claims abstract description 50
- 238000000034 method Methods 0.000 claims abstract description 28
- 229920001612 Hydroxyethyl starch Polymers 0.000 claims abstract description 22
- 229920002307 Dextran Polymers 0.000 claims abstract description 19
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 claims abstract description 17
- 229920000669 heparin Polymers 0.000 claims abstract description 17
- 229960001008 heparin sodium Drugs 0.000 claims abstract description 17
- 102000008100 Human Serum Albumin Human genes 0.000 claims abstract description 14
- 108091006905 Human Serum Albumin Proteins 0.000 claims abstract description 14
- 229940050526 hydroxyethylstarch Drugs 0.000 claims abstract description 10
- 239000011550 stock solution Substances 0.000 claims description 63
- 239000007788 liquid Substances 0.000 claims description 50
- 239000000203 mixture Substances 0.000 claims description 21
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 20
- 238000002347 injection Methods 0.000 claims description 18
- 239000007924 injection Substances 0.000 claims description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 15
- 238000010999 medical injection Methods 0.000 claims description 14
- 229940027278 hetastarch Drugs 0.000 claims description 12
- 229910052757 nitrogen Inorganic materials 0.000 claims description 10
- 238000001816 cooling Methods 0.000 claims description 9
- 239000008354 sodium chloride injection Substances 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 8
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 4
- 210000004475 gamma-delta t lymphocyte Anatomy 0.000 claims description 4
- 210000005087 mononuclear cell Anatomy 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 2
- 235000019628 coolness Nutrition 0.000 claims description 2
- 239000003792 electrolyte Substances 0.000 claims description 2
- 238000002844 melting Methods 0.000 claims description 2
- 230000008018 melting Effects 0.000 claims description 2
- 238000010257 thawing Methods 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 239000008215 water for injection Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 11
- 230000008569 process Effects 0.000 abstract description 10
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- 230000004899 motility Effects 0.000 description 15
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- 238000002360 preparation method Methods 0.000 description 11
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- 229940093181 glucose injection Drugs 0.000 description 6
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
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- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 3
- 229940119744 dextran 40 Drugs 0.000 description 3
- 150000002304 glucoses Chemical class 0.000 description 3
- 238000001802 infusion Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
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- 210000002966 serum Anatomy 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 2
- 229930003268 Vitamin C Natural products 0.000 description 2
- 235000009392 Vitis Nutrition 0.000 description 2
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 229920001491 Lentinan Polymers 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 150000008065 acid anhydrides Chemical class 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 229940115286 lentinan Drugs 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
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- 239000002356 single layer Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000008227 sterile water for injection Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000003462 sulfoxides Chemical class 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a direct venous re-transfusion immune cell cryopreservation medium and application thereof. The direct venous re-transfusion immune cell cryopreservation medium is prepared from dimethyl sulfoxide, human serum albumin, dextran, hydroxyethyl starch, heparin sodium and other clinical medicinal level raw materials by optimizing the proportion of the dose of each raw material. In addition, the operation steps are simplified and the cryopreservation effect is improved by optimizing the cryopreservation method. On one hand, the potential risk of introducing an animal source antigenic substance in the cell treatment process is avoided, and on the other hand, the cryopreservation effect of immune cells is better than that of a conventional cryopreservation medium, thereby guaranteeing the safety and validity in clinical use. Two kinds of components are used for preservation, so that the cryopreservation medium can be preserved stably for a long time. The method disclosed by the invention is simple in operation, provides stronger operability for implementation of large-scale cryopreservation, and simultaneously provides a fundamental technical guarantee for implementation of immune cell treatment.
Description
Technical field
The present invention relates to a kind of cells frozen storing liquid, more particularly to a kind of direct venous re-transfusion immunocyte frozen stock solution and its
Application process.The invention belongs to biomedicine technical field.
Background technology
Cancer is to perplex the matter of utmost importance of human health, and cellular immunotherapy provides wide for the prevention and treatment of tumor
Prospect.But during clinical practice application, there is that Partial tumors patient's autoimmune cell is difficult to gather, cell expands
Increase ability, the problems such as the time window of immunocyte and other therapeutic modalities is difficult to coordinate；Additionally, from medicament research and development and
The angle of production, strange land transport, preservation condition and the time-to-live of fresh immunocyte are all subject to a definite limitation.So, immunity
The effective frozen of cell carries out commercial application and provides necessary basic technology guarantee for immunocyte as medicine.
But, current on the market immunocyte frozen stock solution it is many with hyclone, at high proportion DMSO, autologous patient blood plasma,
Non-clinical applications level preparation of reagents is formed, and these frozen stock solutions have problems with：1. frozen effect is poor；2. frozen stock solution composition is failed to understand
Really；3. frozen process and subsequent applications program are relatively cumbersome；4. high cost；5. risk be present in clinical practice；6. it is unsuitable for
Form industrialization.For example：
The patent application of Publication No. CN104026118A discloses a kind of immunocyte frozen stock solution, its preparation method and answers
With this application dimethyl sulfoxide, people's autologous plasma of inactivation and immune cell media composition, by the method for programmed cooling
Frozen immunocyte.Its exist technical problem be this application frozen stock solution use patient inactivation autologous plasma, quantity with
Limited source, is not suitable for extensive frozen after cell amplification culture, and frozen process is loaded down with trivial details, and batch control is more difficult.
The patent application of Publication No. CN105248413A discloses a kind of CIK cell frozen stock solution, and this application uses diformazan
The frozen CIK cell of base sulfoxide, Semen Vitis viniferae extract, cottonseed sugar, Human Albumin, hyclone.Its exist technical problem be
The frozen stock solution of this application contains animal derived serum, the potential risk for having the introducing of animal derived antigenic substance, it is impossible to for people
Body direct feedback.
The patent application of Publication No. CN105211051A discloses a kind of frozen stock solution and its system of the NK cells after culture
Preparation Method, this application dimethyl sulfoxide, people's autologous plasma of inactivation, addition Semen Vitis viniferae extract, tea polyphenols and vitamin C are made
For the composition of frozen stock solution.Its exist technical problem be this application frozen stock solution use autologous patient inactivation blood plasma, quantity with
Limited source, is not suitable for extensive frozen after cell amplification culture.
It is frozen that the patent application of Publication No. CN105123671A discloses a kind of cells frozen storing liquid, application and immunocyte
Method, this application dimethyl sulfoxide, Propylene Glycol, cell culture medium, Human Albumin, addition non essential amino acid, trehalose,
The composition of lentinan, vitamin C as frozen stock solution.It is thin that its technical problem for existing is this application frozen stock solution main component
Born of the same parents' culture medium, the not effect with stabilizer, not directly for clinical reinfusion.
The patent application of Publication No. CN104862275A discloses a kind of method of cell cryopreservation with recovery and its preparation
Cell preparation, this application dimethyl sulfoxide, 1640 Cell Basal Mediums, hyclone, dextran, ddH2O conducts
The composition of frozen stock solution.Its technical problem for existing is that the frozen stock solution of this application contains animal derived serum, there is animal derived antigen
The potential risk of the introducing of material, it is impossible to for human body direct feedback.
In sum, existing frozen stock solution the effective ingredient many cell culture medium containing scientific research rank and hyclone etc. into
Point, it is impossible to human body infusion is directly applied to, and it is expensive.It is therefore desirable to a kind of frozen effect of research more preferably, price it is lower
It is honest and clean, using it is more convenient, using safer immunocyte frozen stock solution.
The content of the invention
The technical problem to be solved be overcome that frozen effect present in prior art is poor, frozen stock solution composition not
Clearly, frozen process and subsequent applications program is relatively cumbersome, high cost, clinical practice have risk, are not suitable for scale
A kind of defects such as production, there is provided the immunocyte frozen stock solution and its application process of direct venous re-transfusion.
In order to achieve the above object, following technological means be present invention employs：
A kind of immunocyte frozen stock solution of the direct venous re-transfusion of the present invention, it is made up of A liquid and B liquid, wherein, it is described
A liquid contains following composition：Dimethyl sulfoxide 4-16v/v%, hetastarch 0.3-5.5w/v%, dextran 0.3-
5.5w/v%, glucose 0.5-5w/v%, heparin sodium 1-1000U/ml, remaining is medical injection；Described B liquid is containing 2-
The medical injection of 25w/v% Human Albumin.
In the present invention, it is preferred to, the content of each composition is in A liquid：Dimethyl sulfoxide 8.3v/v%, hetastarch
2.75w/v%, dextran 2.75w/v%, glucose 2.29w/v%, heparin sodium 625U/ml, remaining is medical injection；Institute
The B liquid stated is the medical injection containing 20w/v% Human Albumin.
In the present invention, it is preferred to, described medical injection is included but is not limited to：Sterile water for injection, normal saline,
One or more in electrolyte injection, dextran injection or Hydroxyethyl starch sodium chloride injection.
Raw material for preparing frozen stock solution of the present invention can be the medicine that is formulated of crude drug or Jing solution into
Product.Such as hetastarch can be Hydroxyethyl starch sodium chloride injection, and dextran, glucose can be Dextran 40s
Glucose Injection, heparin sodium can be heparin sodium injection.As long as the final concentration of its effective ingredient meets above-mentioned requirements.
The present invention passes through dimethyl sulfoxide, Human Albumin, dextran, hetastarch, the proportion optimizing of heparin sodium,
Using the other raw material of clinical pharmaceutical grade, it is formulated with medical injection.On the one hand animal derived Substances Pollution is avoided
Risk, on the other hand, the more conventional frozen stock solution of the frozen effect of immunocyte is more preferable.So as to ensure that the safety of Clinical practice and having
Effect property.Additionally, this method is simple to operate, be adapted to extensive frozen enforcement, after cell recovery can direct infusion, be easy to clinic
Implement.
Further, the invention allows for application of the frozen stock solution described in any of the above item in frozen immunocyte.
Wherein, it is preferred that described immunocyte is included but is not limited to：T cell, NK cells, mononuclearcell, DC are thin
Born of the same parents, gamma delta T cells, CAR-T cells.
Further, the invention allows for a kind of method of external frozen immunocyte, comprises the following steps：
1) by the A liquid and B liquid of frozen stock solution of the present invention by volume 3:2 mix, and are placed in 4 DEG C of refrigerator pre-coolings；
2) collecting needs frozen immunocyte, washs twice；
3) with the resuspended immunocyte of frozen stock solution of pre-cooling, in being transferred to frozen bag, if being placed in 4 DEG C of refrigerator balances or step 3)
Operating time be longer than 30min and can skip equilibrium step directly carry out step 4)；
4) the frozen bag that will be equipped with cell is placed in and is transferred to liquid nitrogen container after -80 DEG C of refrigerator overnights and preserves for a long time.
In the present invention, it is preferred to, when frozen immunocyte is used, take out frozen bag rapidly from liquid nitrogen container, it is placed in
37 DEG C of water-baths completed venoclysises after defrosting to melting completely in 1 hour.
In the present invention, it is preferred to, the time of 4 DEG C of refrigerator balances is 1-30min,.
In the present invention, it is preferred to, described immunocyte is included but is not limited to：T cell, NK cells, mononuclearcell,
DC cells, gamma delta T cells, CAR-T cells.
In the present invention, it is preferred to, the frozen density of described immunocyte is 1 × 106/ml-1×108/ml。
Compared to prior art, the invention has the beneficial effects as follows：
Instant invention overcomes frozen effect present in prior art is poor, frozen stock solution composition is indefinite, frozen process and after
Continuous application program is relatively cumbersome, high cost, clinical practice have risk, are not suitable for the defects such as large-scale production, there is provided
A kind of immunocyte frozen stock solution and application process of direct venous re-transfusion.
Compare with conventional cryopreservation methods：1. the method recovery after immunocyte compared with the immunocyte of fresh cultured,
(killing to target cell is imitated for its immunobiology characteristic (cell viability, level of apoptosis, immunophenotype etc.) and biological function
Really, levels of cytokine secretion etc.) without significant difference；2. the frozen stock solution raw material of the present invention is clear and definite, non-animal derived composition, and is
Clinical pharmaceutical grade, possesses clinical practice safety；3. autologous patient blood plasma, raw material quantity and unrestricted and cost of originating are not used
It is low, suitably form industrialization；4. frozen stock solution is by component storing mode, and storage stability is good；5.DMSO contents are relatively low, and
The cytotoxicity of DMSO in frozen stock solution is reduced by the pre-cooling of frozen stock solution；6. the frozen process of immunocyte drops without the need for long-time program
Temperature；7. without the need for conventional centrifugal cleaning step after recovering, venous re-transfusion can be directly carried out, greatly facilitate clinical practice.
To sum up, the present invention has abandoned the composition that clinic is not suitable in conventional cryopreservation liquid, from clinical conventional medicine Jing
The proportioning for crossing science makes frozen stock solution, and simplifies frozen stock solution formulation operations with the storing mode of science, then by the frozen of optimization
Method simplifies operating procedure, lifts frozen effect, and the enforcement for immune cell therapy provides basic guarantee.With existing skill
Art is compared, and the present invention is not only safe, does not contain the animal derived serum of potential pathogenic risk, can be grown by two kinds of components
Phase stably preserves.And the cryopreservation methods are simple and easy to do, the requirement of clinical large-scale operation can be applied to, and frozen effect is excellent
Middle loaded down with trivial details processing links and washing can be reduced to cell with direct infusion after conventional cryopreservation methods, cell recovery
Damage.And cost of material is low, it is adapted to industrialization production, huge economic benefit can be produced.
Specific embodiment
Below by specific embodiment, the invention will be further described, and following examples are descriptive, is not limit
Qualitatively, it is impossible to which protection scope of the present invention is limited with this.
Embodiment 1：The directly preparation and application of the immunocyte frozen stock solution of venous re-transfusion
1st, raw materials used and specification
Hydroxyethyl starch sodium chloride injection：500ml:Hetastarch (200/0.5) 30g
Dextran 40 glucose injection：500ml:30g Dextran 40s and 25g glucoses
Heparin sodium injection：2ml:1.25 ten thousand units
Human Albumin Injection：50ml：10g total proteins
2nd, the preparation and application of the immunocyte frozen stock solution of direct venous re-transfusion
1) A liquid and B liquid are prepared respectively
A liquid (100ml)：8.3ml dimethyl sulfoxide+45.8ml Hydroxyethyl starch sodium chloride injection+45.8ml dextroses
Glucose Injection+0.1ml the heparin sodium injections of acid anhydride 40.
The content of each composition is in A liquid：Dimethyl sulfoxide 8.3v/v%, hetastarch 2.75w/v%, dextran
2.75w/v%, glucose 2.29w/v%, heparin sodium 625U/ml, remaining is medical injection.
B liquid (100ml)：The injection of 20w/v% Human Albumin.
2) by A liquid and B liquid by volume 3:2 mix, and are placed in 4 DEG C of refrigerators stand-by.
3) healthy volunteer peripheral blood 50ml is extracted, in being slowly added to the centrifuge tube equipped with lymphocyte separation medium, 500g
Centrifugation 20 minutes, draws tunica albuginea confluent monolayer cells.Washed with PBS twice, counted.
4) according to count results, prepare in using 2), the resuspended PBMC cells of frozen stock solution of pre-cooling so that cell density is 1
×107/ ml, by cell frozen bag is transferred to.
5) the frozen bag that will be equipped with cell places 4 DEG C of refrigerators balance 15min.
6) the frozen bag that will be equipped with cell places -80 DEG C of refrigerator overnights.
6) the frozen bag that will be equipped with cell places Liquid nitrogen storage.
7) cell is taken out from liquid nitrogen container after 1,6,12 months, is placed in 37 DEG C of water-baths and thaws, take a small amount of cell trypan blue
Staining detects Cell viability.
Simultaneously using the PBMC cells of control frozen stock solution (50%FBS+40%RPMI1640+10%DMSO) process as right
According in addition to frozen stock solution, other steps are ibid.
3rd, result
PBMC recovery motility rates using different frozen stock solutions are more as shown in table 1：
Table 1 is compared using the PBMC recovery motility rates of different frozen stock solutions
| The frozen stock solution of the present invention | Control frozen stock solution | |
| 1 month motility rate | 98.5% | 87.1% |
| 6 months motility rates | 96.2% | 85.4% |
| 12 months motility rates | 93.7% | 80.7% |
Embodiment 2:The directly preparation and application of the immunocyte frozen stock solution of venous re-transfusion
1st, raw materials used and specification
Hydroxyethyl starch sodium chloride injection：500ml:Hetastarch (200/0.5) 30g
Dextran 40 glucose injection：500ml:30g Dextran 40s and 25g glucoses
Heparin sodium injection：2ml:1.25 ten thousand units
Human Albumin Injection：50ml：5g total proteins
2nd, the preparation and application of the immunocyte frozen stock solution of direct venous re-transfusion
1) A liquid and B liquid are prepared respectively
A liquid (100ml)：10ml dimethyl sulfoxide+70ml Hydroxyethyl starch sodium chloride injection+19.9ml Dextran 40s
Glucose Injection+0.1ml heparin sodium injections
The content of each composition is in A liquid：Dimethyl sulfoxide 10v/v%, hetastarch 4.2w/v%, dextran
1.19w/v%, glucose 1.0w/v%, heparin sodium 625U/ml, remaining is medical injection；
B liquid (100ml)：The Human Albumin Injection of 10w/v%
2) by A liquid and B liquid according to volume ratio 3:2 mix, and are placed in 4 DEG C of refrigerators stand-by.
3) the NK cells after culture amplification are taken into centrifuge tube, 300g is centrifuged 10min.Washed with PBS twice, counted.
4) according to count results, prepare in using 2), the resuspended NK cells of frozen stock solution of pre-cooling so that cell density 1 ×
108/ ml, by cell frozen bag is transferred to.
5) the frozen bag that will be equipped with cell places 4 degree of refrigerators balance 15min.
6) the frozen bag that will be equipped with cell places -80 DEG C of refrigerator overnights.
6) the frozen bag that will be equipped with cell places Liquid nitrogen storage.
7) cell is taken out from liquid nitrogen container after 1,6,12 months, is placed in 37 DEG C of water-baths and thaws, take a small amount of cell trypan blue
Staining detects Cell viability.
Simultaneously NK cells are processed as control using control frozen stock solution (50%FBS+40%RPMI1640+10%DMSO),
In addition to frozen stock solution, other steps are ibid.
3rd, result
NK recovery motility rates using different frozen stock solutions are more as shown in table 2：
Table 2 is compared using the NK cell recovery motility rates of different frozen stock solutions
| Embodiment 2 | Embodiment 2 is compareed | |
| 1 month motility rate | 96.4% | 82.6% |
| 6 months motility rates | 92.8% | 75.3% |
| 12 months motility rates | 90.5% | 60.2% |
Embodiment 3:The directly preparation and application of the immunocyte frozen stock solution of venous re-transfusion
1st, raw materials used and specification
Hydroxyethyl starch sodium chloride injection：500ml:Hetastarch (200/0.5) 30g
Dextran 40 glucose injection：500ml:30g Dextran 40s and 25g glucoses
Heparin sodium injection：2ml:1.25 ten thousand units.
Human Albumin Injection：50ml：5g total proteins
2nd, the preparation and application of the immunocyte frozen stock solution of direct venous re-transfusion
1) A liquid and B liquid are prepared respectively
A liquid (100ml)：9ml dimethyl sulfoxide+33ml Hydroxyethyl starch sodium chloride injection+57.9ml dextrans Portugal
Grape sugar injection+0.1ml heparin sodium injections.
The content of each composition is in A liquid：Dimethyl sulfoxide 9v/v%, hetastarch 1.98w/v%, dextran
3.47w/v%, glucose 2.9w/v%, heparin sodium 625U/ml, remaining is medical injection；
B liquid (100ml)：The Human Albumin Injection of 10w/v%.
2) A liquid and B liquid are pressed into 3:2 mix, and are placed in 4 DEG C of refrigerators stand-by.
3) the CAR-T cells after culture amplification are taken into centrifuge tube, 300g is centrifuged 10min.Washed with PBS twice, counted.
4) according to count results, prepare in using 2), the resuspended CAR-T cells of frozen stock solution of pre-cooling so that cell density is 5
×107/ ml, by cell frozen bag is transferred to.
5) the frozen bag that will be equipped with cell places 4 DEG C of refrigerators balance 15min.
6) the frozen bag that will be equipped with cell places -80 DEG C of refrigerator overnights.
6) the frozen bag that will be equipped with cell places Liquid nitrogen storage.
7) cell is taken out from liquid nitrogen container after 1,6,12 months, is placed in 37 DEG C of water-baths and thaws, take a small amount of cell trypan blue
Staining detects Cell viability.
The CAR-T cell conducts for being processed using control frozen stock solution (50%FBS+40%RPMI1640+10%DMSO) simultaneously
Control, in addition to frozen stock solution, other steps are ibid.
3rd, result
CAR-T recovery motility rates using different frozen stock solutions are more as shown in table 3：
Table 3 is compared using the CAR-T cell recovery motility rates of different frozen stock solutions
| Embodiment 3 | Embodiment 3 is compareed | |
| 1 month motility rate | 98.1% | 85.5% |
| 6 months motility rates | 96.7% | 80.5% |
| 12 months motility rates | 93.3% | 72.4% |
Above example content only to illustrate the invention, but should not be construed as limiting the invention.Without departing substantially from this
In the case of spirit and essence, the modification made to the inventive method, step or condition or replacement belong to the present invention's
Scope.
If not specializing, the conventional meanses that technological means used are well known to those skilled in the art in embodiment.
Claims (10)
1. a kind of immunocyte frozen stock solution of direct venous re-transfusion, it is characterised in that be made up of A liquid and B liquid, wherein, described A
Liquid contains following composition：Dimethyl sulfoxide 4-16v/v%, hetastarch 0.3-5.5w/v%, dextran 0.3-5.5w/
V%, glucose 0.5-5w/v%, heparin sodium 1-1000U/ml, remaining is medical injection；Described B liquid is containing 2-25w/
The medical injection of v% Human Albumin.
2. frozen stock solution as claimed in claim 1, it is characterised in that the content of each composition is in A liquid：Dimethyl sulfoxide 8.3v/
V%, hetastarch 2.75w/v%, dextran 2.75w/v%, glucose 2.29w/v%, heparin sodium 625U/ml, remaining
For medical injection；Described B liquid is the medical injection containing 20w/v% Human Albumin.
3. frozen stock solution as claimed in claim 1 or 2, it is characterised in that described medical injection is included but is not limited to：It is aseptic
One kind in water for injection, normal saline, electrolyte injection, dextran injection or Hydroxyethyl starch sodium chloride injection
Or it is two or more.
4. application of the frozen stock solution described in any one of claim 1-3 in frozen immunocyte.
5. application as claimed in claim 4, it is characterised in that described immunocyte is included but is not limited to：T cell, NK are thin
Born of the same parents, mononuclearcell, DC cells, gamma delta T cells, CAR-T cells.
6. a kind of method of external frozen immunocyte, it is characterised in that comprise the following steps：
1) by the A liquid and B liquid of the frozen stock solution described in claim 1 or 2 according to volume ratio 3:2 mix, and are placed in 4 DEG C of refrigerator pre-coolings；
2) collecting needs frozen immunocyte, washs twice；
3) with the resuspended immunocyte of frozen stock solution of pre-cooling, in being transferred to frozen bag, if being placed in 4 DEG C of refrigerators balance or step 3) behaviour
Make the time be longer than 30min and can skip equilibrium step directly carry out step 4)；
4) the frozen bag that will be equipped with cell is placed in and is transferred to liquid nitrogen container after -80 DEG C of refrigerator overnights and preserves for a long time.
7. method as claimed in claim 6, it is characterised in that when frozen immunocyte is used, take rapidly from liquid nitrogen container
Go out frozen bag, be placed in 37 DEG C of water-baths to melting completely, venoclysises were completed in 1 hour after defrosting.
8. method as claimed in claim 6, it is characterised in that the time of 4 DEG C of refrigerator balances is 1-30min,.
9. method as claimed in claim 6, it is characterised in that described immunocyte is included but is not limited to：T cell, NK are thin
Born of the same parents, mononuclearcell, DC cells, gamma delta T cells, CAR-T cells.
10. method as claimed in claim 6, it is characterised in that the frozen density of described immunocyte is 1 × 106/ml-1×
108/ml。
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