CN105211052B - Frozen stock solution of cultured NKT cells and preparation method thereof - Google Patents
Frozen stock solution of cultured NKT cells and preparation method thereof Download PDFInfo
- Publication number
- CN105211052B CN105211052B CN201510727840.2A CN201510727840A CN105211052B CN 105211052 B CN105211052 B CN 105211052B CN 201510727840 A CN201510727840 A CN 201510727840A CN 105211052 B CN105211052 B CN 105211052B
- Authority
- CN
- China
- Prior art keywords
- solution
- autoserum
- nkt cells
- lentinan
- stock solution
- 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
Abstract
The invention relates to the technical field of cell frozen stock solution, in particular to frozen stock solution of cultured NKT cells and a preparation method thereof, the frozen stock solution of the cultured NKT cells comprises lentinan, algal polysaccharide, autologous serum and DMSO, and the autologous serum is adopted, so that the pollution of exogenous animal viruses and the introduction of exogenous proteins are avoided, and the safety is high; the added lentinan and algal polysaccharide can effectively maintain the activity of cells and have no harm to human bodies, and the state of the cells after cryopreservation recovery is basically close to the state before cryopreservation, so that the cryopreservation effect is good.
Description
Technical field
The present invention relates to cells frozen storing liquid technical field, and in particular to a kind of frozen stock solution of the NKT cells after culture and its
Preparation method.
Background technology
Cell cryopreservation is a kind of long-term effective ways for preserving and maintaining cytoactive, its freeze preserved by cryogenic freezing and
In two stages of Ultra-cryofreezing preservation, cryogenic freezing, which is preserved, can reduce the metabolism of cell, and ultralow temperature (- 198 DEG C of liquid nitrogen
In) freezen protective can preserve living cells metabolism and almost completely stop cell growth, in Cryopreservation, regulation and
The activity of the various enzymes of control cell growth metabolism is suppressed, and makes intracellular biochemical reaction very slow, or even is stopped, from
And maintain the activity of cell.
Phenotypic characteristic in recent years in NKT cells, distribution and development, immunology effect and with disease relationship, oncotherapy
Had a great development with the research in terms for the treatment of of autoimmune diseases.Because NKT cells are in tumour and immunity disease
Research in terms for the treatment of, also determines the importance frozen after NKT cell culture, and Extended incubation can be avoided in terms for the treatment of
The cytoactive caused is low, and the cell cryopreservation in optimum state period is got up；In economic aspect, it is to avoid what Extended incubation was caused
Cost increase；In terms of research, the NKT cell cryopreservations of different times are got up, the NKT cells work that its difference freezes the time is studied
Property.
But if simple freezes, cell can be damaged by two aspects during freezing：First, what is frozen
During, extracellular moisture is frozen first so that electrolyte concentration is raised in the solution not frozen, is caused cell death, is belonged to
Permeability is damaged；Second, intracellular structure can be destroyed because low temperature causes cell born of the same parents to be internally formed ice crystal, belongs to mechanical damage
Wound.Therefore 2 reasons more than combining, it is necessary to solve the speed of freezing, and the formula of agent is frozen, the speed of thawing during recovery
Directly affect the activity of cell.
Cells frozen storing liquid refers to the material for protecting cells from freezing loss, generally can be divided into permeability and impermeable
Two classes of property.Permeability frozen stock solution is mainly small-molecule substance, soluble in water, strong with water molecules ability, easily by cell
Film enters intracellular, reduces the freezing point of cell, improves permeability of the cell membrane to water, reduces ice crystal and is formed, so as to reduce ice crystal
Damage.
In the prior art, generally prepared using the hyclone (FBS) and dimethyl sulfoxide (DMSO) (DMSO) of addition different proportion
Frozen stock solution, then carry out different temperatures and the program of time cooling come freeze culture after NKT cells, addition DMSO be intended merely to
Prevented during freezing intracellular ice crystal formation and damaging cells, and it is to protect cell not damaged by DMSO to add FBS
Wound and offer nutrition, but FBS belongs to heterologous material, complicated component, and there is the risk for introducing pollution and anaphylactogen, discomfort
Together in clinical practice.Particularly in cell therapy, the presence of heterologous protein may cause unknown adverse reaction, sternly
Ghost image rings treatment results.
The content of the invention
The present invention is high there is provided a kind of clinical safety for problems of the prior art, while can protect well again
The cells frozen storing liquid of freeze-stored cell activity is held, for freezing the NKT cells after culture.
The present invention also provides a kind of preparation method of the frozen stock solution of the NKT cells after culture.
The present invention is achieved through the following technical solutions the purpose：
A kind of frozen stock solution of NKT cells after culture, the frozen stock solution is formulated by solution A and autoserum, solution A
Volume ratio with autoserum is 1~3：1, wherein：
The solution A include lentinan, algal polysaccharides, autoserum and DMSO, wherein lentinan, algal polysaccharides,
Autoserum and DMSO volume ratio are 1~3：1~3：1~5：1.
As preferred scheme, the frozen stock solution is formulated by solution A and autoserum, solution A and autoserum
Volume ratio is 1~2：1, wherein：
The solution A include lentinan, algal polysaccharides, autoserum and DMSO, wherein lentinan, algal polysaccharides,
Autoserum and DMSO volume ratio are 1~2：1~2：1~3：1.
As another preferred scheme, the frozen stock solution is formulated by solution A and autoserum, solution A and autologous blood
Clear volume ratio is 2~3：1, wherein：
The solution A include lentinan, algal polysaccharides, autoserum and DMSO, wherein lentinan, algal polysaccharides,
Autoserum and DMSO volume ratio are 2~3：2~3：3~5：1.
As preferred, the concentration of the lentinan is 1~6mg/mL, the concentration of the algal polysaccharides for 0.05~
0.2mg/mL。
A kind of preparation method of the frozen stock solution of NKT cells after culture, comprises the following steps：
1) it is that 1~6mg/mL lentinan and 0.05~0.2mg/mL algal polysaccharides are separately added into autologous blood by concentration
In clear, then slow addition DMSO, lentinan is added by volume：Algal polysaccharides：Autoserum：DMSO is 1~3：1~3：
1~5：1, the solution A that preparation is obtained is placed in 2~8 DEG C of degree refrigerator precoolings, standby；
2) above-mentioned solution A is taken out again, be slowly added into autoserum, solution A is added by percent by volume：From
Body serum is 1~3：1, it is configured to NKT cells frozen storing liquids.
Relative to prior art, beneficial effects of the present invention are：The frozen stock solution of NKT cells after the culture of the present invention includes
Lentinan, algal polysaccharides, autoserum and DMSO, because it uses autoserum, in the absence of the pollution of exogeneous animal virus
And the introducing of exogenous proteins, it is safe；Lentinan, the algal polysaccharides wherein added can not only effectively keep cell
Activity, to state of the human body without cell after any injury, and cryopreservation resuscitation substantially close to the state before freezing, freeze effect
It is good.
Brief description of the drawings
Fig. 1 is the NKT cellular morphology figures before freezing.
Fig. 2 is the NKT cellular morphology figures of the control group after freezing.
Fig. 3 is the NKT cellular morphology figures of the experimental group 1 after freezing.
Fig. 4 is the NKT cellular morphology figures of the experimental group 2 after freezing.
Fig. 5 is the NKT cellular morphology figures of the experimental group 3 after freezing.
Fig. 6 is to freeze front and rear NKT cell growth curve figures.
Fig. 7 is to freeze front and rear NKT cell killing activity curve maps.
Embodiment
Below in conjunction with drawings and the specific embodiments, the present invention will be described in detail.
Embodiment 1,
The present embodiment provides a kind of preparation method of the frozen stock solution of the NKT cells after culture, comprises the following steps：
1) it is that 1~6mg/mL lentinan and 0.05~0.2mg/mL algal polysaccharides are separately added into autologous blood by concentration
In clear, then slow addition DMSO, lentinan is added by volume：Algal polysaccharides：Autoserum：DMSO is 1~3：1~3：
1~5：1, the solution A that preparation is obtained is placed in 2~8 DEG C of degree refrigerator precoolings, standby；
2) above-mentioned solution A is taken out again, be slowly added into autoserum, solution A is added by percent by volume：From
Body serum is 1~3：1, it is configured to NKT cells frozen storing liquids.
Embodiment 2,
The present embodiment according to described in embodiment 1 method prepare culture after NKT cells frozen stock solution, the frozen stock solution by
Solution A and autoserum are formulated, and the volume ratio of solution A and autoserum is 1：1, wherein：
The solution A include lentinan, algal polysaccharides, autoserum and DMSO, wherein lentinan, algal polysaccharides,
Autoserum and DMSO volume ratio are 1：1：1：1, wherein, lentinan concentration is 1mg/mL, and algal polysaccharides concentration is
0.05mg/mL。
Embodiment 3,
The present embodiment according to described in embodiment 1 method prepare culture after NKT cells frozen stock solution, the frozen stock solution by
Solution A and autoserum are formulated, and the volume ratio of solution A and autoserum is 2：1, wherein：
The solution A include lentinan, algal polysaccharides, autoserum and DMSO, wherein lentinan, algal polysaccharides,
Autoserum and DMSO volume ratio are 2：2：3：1, wherein, lentinan concentration is 6mg/mL, and algal polysaccharides concentration is
0.2mg/mL。
Embodiment 4,
The present embodiment according to described in embodiment 1 method prepare culture after NKT cells frozen stock solution, the frozen stock solution by
Solution A and autoserum are formulated, and the volume ratio of solution A and autoserum is 3：1, wherein：
The solution A include lentinan, algal polysaccharides, autoserum and DMSO, wherein lentinan, algal polysaccharides,
Autoserum and DMSO volume ratio are 3：3：3：1, wherein, lentinan concentration is 3mg/mL, and algal polysaccharides concentration is
0.1mg/mL。
Comparative example 1,
The present embodiment prepares regular growth frozen stock solution, and the regular growth frozen stock solution is the DMSO containing 10%FBS.
Embodiment 5, the amplification cultivation of NKT cells and collection
1st, the separation of peripheral blood mononuclear ball
1) 10~80ml peripheral blood is gathered, by it by volume 1 with physiological saline：1 ratio is mixed, after mixing
Blood mixed liquor and lymphocyte separation medium by volume 2：1 ratio slowly adds to lymphocyte separation medium upper strata,
3000rpm centrifuges 20min；
2) centrifuge tube is seen from top to bottom after centrifuging, respectively blood plasma, tunica albuginea layer, i.e. mononuclearcell layer (PBMC), drench
Bar cell separation liquid, red blood cell layer, extract PBMC layers, after the PBMC being collected into is resuspended with brine, 1500rpm from
Heart 10min, repeated washing twice, is collected into PBMC；
2nd, the amplification cultivation of NKT cells
1) PBMC being collected into above-mentioned 1 is resuspended with RPMI1640 basal mediums, by 1*106Individual/ml density inoculation
In blake bottle, while and add Porcine HGF IL-2500U/ml, IL-1530ng/ml, be placed in 37 DEG C, concentration is 5%
CO2Cultivated in incubator；
2) after inducing 5 days, fluid infusion is carried out, full dose adds cell density before IL-2500U/ml and IL-1530ng/ml, fluid infusion
No more than 3*106Cell density is in 0.5-1.0*10 after individual/ml, fluid infusion6Individual/ml, every fluid infusion in 3 days and adds cell growth factor
Son.
3) until the 14th day collects cell.
Embodiment 6, experiment is frozen to the NKT cells after culture
1) frozen stock solution of the preparation of embodiment 2~4 is utilized respectively as the NKT cells after 1~3 pair of culture of experimental group to freeze
Deposit, the regular growth frozen stock solution prepared using comparative example 1 is frozen to the NKT cells after culture as a control group.
Experimental group 1：NKT cells are resuspended in the NKT cells frozen storing liquids prepared with embodiment 2, and cell density is 106Individual/ml is thin
Born of the same parents' suspension, each cryopreservation tube 1ml/ pipes.
Experimental group 2：NKT cells are resuspended in the NKT cells frozen storing liquids prepared with embodiment 3, and cell density is 106Individual/ml is thin
Born of the same parents' suspension, each cryopreservation tube 1ml/ pipes.
Experimental group 3：NKT cells are resuspended in the NKT cells frozen storing liquids prepared with embodiment 4, and cell density is 106Individual/ml is thin
Born of the same parents' suspension, each cryopreservation tube 1ml/ pipes.
Control group：NKT cells are resuspended in the regular growth frozen stock solution prepared with comparative example 1, and cell density is 106Individual/ml is thin
Born of the same parents' suspension, each cryopreservation tube 1ml/ pipes.
2) above-mentioned each cryopreservation tube is first put into freezing storing box, then freezing storing box is put into programmed cooling instrument frozen, so
Cryopreservation tube is rapidly transferred in liquid nitrogen again afterwards and frozen.
The recovery of embodiment 7, NKT cells
1) thermostat water bath is heated to 37 DEG C, the cell cryopreservation that above-mentioned each group has frozen 1 year in liquid nitrogen is taken out rapidly
Pipe, is transferred in water-bath and continuous oscillation, until being completely dissolved, then the cell suspension after dissolving is moved to added with complete culture
Centrifugation is washed in the centrifuge tube of base；
2) culture 24h is resuspended with complete medium in the cell after above-mentioned centrifugation, by 106/ ml cell is inoculated in culture
In bottle, full dose addition IL-2500U/ml and IL-1530ng/ml Porcine HGFs are placed in 37 DEG C, 5%CO2Incubator in
Culture.
Morphologic observation before and after embodiment 8, NKT cell cryopreservations
Each group NKT cells before and after freezing observe cellular morphology under inverted microscope, and form observed result such as Fig. 1~
Shown in 5, observed result shows, NKT cells after the recovery of 3 groups of experimental groups and the cellular morphology before freezing freeze before and after without obvious poor
Different, cell is full, translucent luster, and has a more cell conglomeration, relative comparison group, and cells frozen storing liquid of the invention is to cell
The influence frozen is smaller.
Survival rate test before and after embodiment 9, NKT cell cryopreservations
NKT cells before and after freezing are carried out after Trypan Blue, cell count is carried out with cell counting count board, are calculated thin
Born of the same parents' motility rate, it is as shown in table 1 below：
Cell viability before and after the NKT cell cryopreservations of table 1
| Group | Cell viability |
| NKT cells before freezing | 97.3% |
| Freeze rear NKT cells-experimental group 1 | 93.2% |
| Freeze rear NKT cells-experimental group 2 | 95.4% |
| Freeze rear NKT cells-experimental group 3 | 95.6% |
| Freeze rear NKT cells-control group | 81.7% |
The Cell viability recovered after experimental group freezes as can be seen from the above Table 1 is before freeze, especially experimental group 3
As a result it is optimal, and the Cell viability of experimental group is all higher than control group, cell can be kept by illustrating the cells frozen storing liquid of the present invention
The state of cell freezes effect good substantially close to the state before freezing after activity, cryopreservation resuscitation.
The detection of ability of cell proliferation before and after embodiment 10, NKT cell cryopreservations
Before freezing with freeze after, the NKT cells of recovery culture one week are counted, and 48h is counted once, and drafting cell is given birth to
Long curve, as a result as shown in fig. 6, being cultivated after the NKT cell recoveries preserved from described Fig. 6 frozen stock solutions that can be seen that experimental group
Multiplication capacity is better than the NKT cells that the frozen stock solution of control group is preserved.
Killing viability examination before and after embodiment 11, NKT cell cryopreservations
The detection of the NKT cells progress killing activity after front and rear culture will be frozen, using lactic dehydrogenase (LDH) method,
Using K562 cells as target cell, detect that each group freezes the killing vigor of front and rear NKT cells, i.e. effector cell respectively.Imitate target ratio
Respectively 40：1、20：1、10：1、5：Effector cell and target cell are added 96 orifice plates by 1 cell number ratio, and target cell number is
104Individual/hole, every kind of effect target ratio sets 4 parallel holes, per hole 200ul.It is resuspended using the culture mediums of RPMI 1640.In 37 DEG C, 5%
4h is incubated in CO2 incubators, then with LDH-Cytotoxicity Colorimetric Assay Kit II (U.S.,
BioVision, article No.：K313-500) kit is detected, using light absorption value of the ELIASA Detection wavelength in 490nm
(A).The calculation formula of killing vigor is：NKT cytoactives (%)=(spontaneous-K562 of experiment-NKT cells is spontaneous)/(K562 is most
- K562 is spontaneous greatly) × 100%, testing result is from described Fig. 7 result as shown in fig. 7, can be seen that each group NKT cells pair
The killings of K562 cells it is living with effect target than increase and strengthen, after the NKT cell recoveries of experimental group with freeze before cell killing live
The equal no significant difference of power, and NKT cells after control group recovery different effect targets than when kill before vigor is below freezing
Cell.
Embodiment 12, NKT cells freeze front and rear immunophenotype detection
NKT cells are resuspended in NKT cells PBS before freezing with the collection after recovery, and density is 106Individual/ml, is added
The CD56 antibody of CD3 and the PE mark of FITC marks, room temperature lucifuge is incubated after 30min, and with flow cytomery, detection has
The cell proportion (%) of corresponding immunophenotype, testing result is as shown in table 2：
Immunophenotype testing result before and after the NKT cell cryopreservations of table 2
| Group | CD3+CD56+ |
| NKT cells before freezing | 81.3±0.32 |
| Freeze rear NKT cells-experimental group 1 | 81.1±0.20 |
| Freeze rear NKT cells-experimental group 2 | 81.0±0.12 |
| Freeze rear NKT cells-experimental group 3 | 82.1±0.73 |
| Freeze rear NKT cells-control group | 70.1±1.15 |
From the results shown in Table 2, there is some difference for the immunophenotype of the NK cells before and after freezing, and experimental group is compared
Immunophenotype testing result according to group is high, illustrate cells frozen storing liquid of the invention to freeze effect more preferable.
Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (4)
1. the frozen stock solution of the NKT cells after a kind of culture, it is characterised in that the frozen stock solution is prepared by solution A and autoserum
Form, the volume ratio of solution A and autoserum is 1~3：1, wherein：
The solution A includes lentinan, algal polysaccharides, autoserum and DMSO, wherein lentinan, algal polysaccharides, autologous
Serum and DMSO volume ratio are 1~3：1~3：1~5：1, the initial concentration of the lentinan is 1~6mg/mL, the sea
The initial concentration of polysaccharides is 0.05~0.2mg/mL.
2. the frozen stock solution of the NKT cells after culture according to claim 1, it is characterised in that the frozen stock solution is by solution A
It is formulated with autoserum, the volume ratio of solution A and autoserum is 1~2：1, wherein：
The solution A includes lentinan, algal polysaccharides, autoserum and DMSO, wherein lentinan, algal polysaccharides, autologous
Serum and DMSO volume ratio are 1~2：1~2：1~3：1.
3. the frozen stock solution of the NKT cells after culture according to claim 1, it is characterised in that the frozen stock solution is by solution A
It is formulated with autoserum, the volume ratio of solution A and autoserum is 2~3：1, wherein：
The solution A includes lentinan, algal polysaccharides, autoserum and DMSO, wherein lentinan, algal polysaccharides, autologous
Serum and DMSO volume ratio are 2~3：2~3：3~5：1.
4. the preparation method of the frozen stock solution of the NKT cells after a kind of culture, it is characterised in that comprise the following steps：
1) it is that 1~6mg/mL lentinan and 0.05~0.2mg/mL algal polysaccharides are separately added into autoserum by concentration
In, then slow addition DMSO, lentinan is added by volume：Algal polysaccharides：Autoserum：DMSO is 1~3：1~3：1
~5：1, the solution A that preparation is obtained is placed in 2~8 DEG C of degree refrigerator precoolings, standby；
2) above-mentioned solution A is taken out again, be slowly added into autoserum, solution A is added by percent by volume：Autologous blood
Clear is 1~3：1, it is configured to NKT cells frozen storing liquids.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510727840.2A CN105211052B (en) | 2015-10-29 | 2015-10-29 | Frozen stock solution of cultured NKT cells and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510727840.2A CN105211052B (en) | 2015-10-29 | 2015-10-29 | Frozen stock solution of cultured NKT cells and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105211052A CN105211052A (en) | 2016-01-06 |
| CN105211052B true CN105211052B (en) | 2017-11-07 |
Family
ID=54980673
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510727840.2A Active CN105211052B (en) | 2015-10-29 | 2015-10-29 | Frozen stock solution of cultured NKT cells and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105211052B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108064840B (en) * | 2017-12-28 | 2021-01-19 | 重庆斯德姆生物技术有限公司 | NKT cell cryopreservation solution and preparation method thereof |
| CN110301432A (en) * | 2019-07-25 | 2019-10-08 | 上海轩锋生物科技有限公司 | A kind of new cell freezing method |
| CN111602648B (en) * | 2020-04-26 | 2021-04-06 | 河南侨创生命科技有限公司 | Immune cell serum-free cryopreservation liquid and cryopreservation method |
| CN114027294B (en) * | 2021-12-12 | 2022-11-29 | 杭州中赢生物医疗科技有限公司 | Immune cell cryopreservation liquid and immune cell cryopreservation method |
| CN115039766B (en) * | 2022-08-13 | 2022-11-18 | 中国农业科学院北京畜牧兽医研究所 | Normal-temperature boar semen diluent |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6037175A (en) * | 1996-03-12 | 2000-03-14 | The University Of South Florida | Method and media for enhancing cryopreservation of cells |
| CN101983562B (en) * | 2010-05-26 | 2013-04-17 | 赛业（广州）生物科技有限公司 | Cell freezing solution without complicated procedures for freezing |
| CN104082277B (en) * | 2014-07-25 | 2016-04-13 | 成都清科生物科技有限公司 | A kind of freezing protective agent of human peripheral blood single nucleus cell and store method |
| CN104920340A (en) * | 2015-07-15 | 2015-09-23 | 广州赛莱拉干细胞科技股份有限公司 | Immune cell preserving fluid and application thereof |
-
2015
- 2015-10-29 CN CN201510727840.2A patent/CN105211052B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN105211052A (en) | 2016-01-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104719282B (en) | Peripheral blood mononuclear cell serum-free freezing medium and freezing method | |
| CN105211051B (en) | Cultured NK cell freezing medium and preparation method thereof | |
| CN105211052B (en) | Frozen stock solution of cultured NKT cells and preparation method thereof | |
| CN105685016B (en) | Cell cryopreservation protection composition, the composition purposes and cell cryopreservation method | |
| Mazur | Freezing of living cells: mechanisms and implications | |
| CN104145943B (en) | A kind of frozen protection liquid of people's umbilical cord China Tong Shi glue tissue and preparation and application thereof | |
| CN106982821A (en) | Umbilical cord mesenchymal stem cells clinic freezes protection liquid composition and application thereof | |
| CN106818710A (en) | A kind of cells frozen storing liquid and its preparation method and application | |
| CN110050782A (en) | A kind of stem cell cryopreserving liquid and preparation method thereof and cryopreservation methods | |
| CN108207930A (en) | A kind of cocktail type cryoprotector and its application | |
| CN106687152A (en) | Human liver scaffolds | |
| CN104026118A (en) | Immunization cell frozen stock solution, preparation method and application | |
| CN102669087A (en) | Freeze-storage liquid of peripheral blood mononuclear cells and freeze-storage method | |
| CN102640745B (en) | Ultralow-temperature cryopreservation and recovery method for embryonic materials | |
| Visch et al. | Underpinning the development of seaweed biotechnology: cryopreservation of brown algae (Saccharina latissima) gametophytes | |
| CN105532650B (en) | A kind of cells frozen storing liquid | |
| CN102907416A (en) | Tissue cryopreservation liquid capable of maintaining cell activity | |
| CN106237313A (en) | A kind of umbilical cord mesenchymal stem cells injection and its preparation method and application | |
| CN107787959A (en) | A kind of immunocyte frozen stock solution, its preparation method, cryopreservation methods and application | |
| CN108064840A (en) | A kind of NKT cells frozen storing liquids and preparation method thereof | |
| CN106665559A (en) | Immune cell cryopreservation liquid and application thereof | |
| CN111602648B (en) | Immune cell serum-free cryopreservation liquid and cryopreservation method | |
| CN107306940A (en) | The spermary Cryoprotectant and the preparation method of spermatogonium of a kind of seawater fish | |
| CN102763642B (en) | Cryoprotectant and method for cryopreserving placenta amnion and chorion | |
| CN109619088A (en) | A kind of preservation liquid of fat mesenchymal stem cell |
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 |