CN105794768A - Immune cell freezing medium and cryopreservation method - Google Patents
Immune cell freezing medium and cryopreservation method Download PDFInfo
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Abstract
The invention relates to an immune cell freezing medium.The immune cell freezing medium comprises dimethyl sulfoxide, human serum and an AIM-V culture medium.According to the immune cell freezing medium, the recovery survival rate of immune cells can be increased, the cell activity is kept, the survival time of the immune cells is prolonged, and losses of surface antigens of the immune cells are reduced.
Description
Technical field
The present invention relates to biological technical field, particularly to a kind of immunocyte frozen stock solution and cryopreservation methods.
Background technology
Cellular immunotherapy be a kind of emerging, there is the brand-new antitumour treatments of significant curative effect,
Compensate for traditional operation, radiotherapy, the drawback of chemotherapy, be acknowledged as 21st century tumor comprehensive
A kind for the treatment of means the most active, the most rising in Therapeutic mode, the Ye Shi world is currently the only to be hopeful
The treatment means of tumors destroyed cell completely.
Immunocyte refers to participate in immunne response or the cell relevant to immunne response, including lymphocyte,
Dendritic cell, Monocytes/Macrophages, granulocyte, mastocyte etc..Immunocyte can be divided into many
Kind, in human body, various immunocytes serve as important role.The single core separated from blood
Cell includes: T cell, B cell, NK cell and DC cell etc., be in immune cell therapy
Initial cell source, through various types of cells factor induced amplification cultivate, obtain a group have Efficient killing effect live
The immunocyte group of property.
Common immune cell therapy kind include NK cell, CIK cell, DC-CIK cell,
Til cell, LAK cell, CART cell etc..All these immunity through induction and amplification cultivation are thin
The initial cell source of born of the same parents is all the mononuclearcell of peripheral blood or umbilical blood, and is to be directly separated cultivation.
With advancing age, immunocyte activity in vivo also can decrease, so freezing in young period
Deposit the immunocyte with greater activity, can play very for suffering from the treatment of tumor-related illness future
Good guarantee, the recovery after therefore immunocyte is frozen and frozen is cultivated has the biggest meaning.Except this
Outside, along with the progressively popularization of immune cell therapy project, the long-distance transport of immunocyte, preserve for a long time
Problem just highlights, and is also that cell prepares the problem that must solve in compartmentalization process.
At present, cell cryopreservation mainly takes the preservation scheme under-196 DEG C of condition of ultralow temperature, at ultralow temperature
Under the conditions of, the metabolic function of cell is stagnated, but not dead.Cell recovery mainly use frozen carefully
Born of the same parents are in the method for 37 DEG C of quick-thawings, to recover activity and the function of cell.But existing immunocyte
Cryopreservation methods is applicable to frozen stem cell and tumor cell, for immunocyte, the easiest during frozen
Forming ice crystal damaging cells, after recovery, Cell viability is low, cell proliferation quantity not enough, cell is the most aging,
There is inefficiency, poor activity and the problem such as survival rate is the highest after frozen of low quality, cell recovery.
Summary of the invention
The technical problem to be solved is, there is frozen effect for existing immunocyte Cryopreservation Technology
Rate variance, and the defect such as immunocyte recovery survival rate is low, it is provided that one can improve the frozen effect of immunocyte
Rate also improves the immunocyte frozen stock solution of immunocyte recovery survival rate.
The technical solution adopted for the present invention to solve the technical problems is: provide a kind of immunocyte frozen stock solution,
Including dimethyl sulfoxide, human serum and AIM-V culture medium.
In the immunocyte frozen stock solution that the present invention provides, in described AIM-V culture medium, dimethyl sulfoxide
Volume fraction be 5～15%, the volume fraction of human serum is 5～15%.
In the immunocyte frozen stock solution that the present invention provides, in described AIM-V culture medium, dimethyl sulfoxide
Volume fraction be 10%, the volume fraction of human serum is 10%.
In the immunocyte frozen stock solution that the present invention provides, in described AIM-V culture medium, dimethyl sulfoxide
Volume fraction be 5%, the volume fraction of human serum is 5%.
In the immunocyte frozen stock solution that the present invention provides, in described AIM-V culture medium, dimethyl sulfoxide
Volume fraction be 15%, the volume fraction of human serum is 15%.
In the immunocyte frozen stock solution that the present invention provides, in described AIM-V culture medium, dimethyl sulfoxide
Volume fraction be 8～10%, the volume fraction of human serum is 8～10%.
In the immunocyte frozen stock solution that the present invention provides, in described cells frozen storing liquid, described AIM-V trains
Supporting in base, the volume fraction of dimethyl sulfoxide is 8%, and the volume fraction of human serum is 8%.
In the immunocyte frozen stock solution that the present invention provides, in described AIM-V culture medium, dimethyl sulfoxide
Volume fraction be 10%, the volume fraction of human serum is 10%.
The present invention protects a kind of immunocyte cryopreservation methods further, it is characterised in that comprise the following steps,
Obtain immunocyte,
Above-mentioned immunocyte frozen stock solution is used to carry out frozen to described immunocyte.
Implement immunocyte frozen stock solution and cryopreservation methods that the present invention provides, can reach following beneficial effect:
Improve the recovery survival rate of immunocyte, and keep its cytoactive, extend immunocyte survival period, subtract
The loss of few immune cell surface antigenic.
Detailed description of the invention
The immunocyte frozen stock solution that the present invention provides, cultivates including dimethyl sulfoxide, human serum and AIM-V
Base.Wherein, in AIM-V culture medium, the volume fraction of dimethyl sulfoxide is 5～15%, human serum
Volume fraction is 5～15%；It is further preferable that in AIM-V culture medium, the volume integral of dimethyl sulfoxide
Number is 8～10%, and the volume fraction of human serum is 8～10%.
AIM-V culture medium is purchased from American I nvitrogen company, wherein mainly contains L-glutaminate, sulfur
Acid streptomycin and gentamycin sulfate etc., in order to keep immunologic cellular activity so that after immunocyte recovery
It is able to maintain that its cytoactive, and extends immunocyte survival period.
Dimethyl sulfoxide is small molecular organic compounds, permeability protective agent, it is possible to reduce cell freezing point,
That reduces ice crystal forms the infringement to immunocyte.
Human serum is nourishment protecting agent, including serum albumin, glucose, inorganic ion, islets of langerhans
Element, adrenocortical hormone, steroid hormone etc., the nutrition needed for can not only providing for immunocyte
Material, and serum albumin defines the viscosity of serum, can protect cells from mechanical damage.
Further, the invention provides a kind of microencapsulation immunocyte cryopreservation methods, comprise the following steps:
S1, acquisition immunocyte；
S2, immunocyte is mixed with the capsule material being used for making microcapsule carry out microencapsulation process, it is thus achieved that microcapsule
Change immunocyte；
S3, by step S2 obtain microencapsulation immunocyte be placed in the invention described above provide cell cryopreservation
Liquid is carried out frozen.
Specifically, in step S1, immunocyte can be NK cell (Natural Killer Cells, from
Natural killer cell), CIK cell (Cytokine-Induced Killer, cytokine induced kill cell),
Deng, derive from peripheral blood, umbilical blood or bone marrow etc., under as a example by NK cell and CIK cell, illustrate that it obtains
Take process.
In the present invention, the acquisition process of NK cell comprises the following steps:
From human peripheral, isolate mononuclearcell, go out CD34 by magnetic bead sorting+Hematopoietic stem cell list
Individual nucleus, and by CD34+Hematopoietic stem cell mononuclearcell is in containing hematopoietic stem cell growth factor
Stem cell media carries out stimulation and is divided into NK cell.
In the present invention, the acquisition process of CIK cell comprises the following steps:
From human peripheral, isolate mononuclearcell, mononuclearcell mixed with fibrin gel,
Add containing PPP (Poor Platelet Plasma, low platelet blood plasma) and IFN-γ (Interferon-γ,
IFN-γ) culture medium culturing, then add growth factor I L-1 α and IL-2 to mononuclearcell induce
Change into CIK cell.
In step 2, the capsule material for making microcapsule include alginate, poly-D-lysine, chitosan,
2-methacrylic acid, hydroxyethyl meth acrylate, Methylmalonate, agarose, polypropylene amine and hydroxyl
Methylcellulose etc.；In the present invention, preferential employing chitosan or alginate-polylysine make microcapsule,
Both materials not only convenient sources, and inanimate object toxicity and immunogenicity, the microcapsule being made
Film has good biocompatibility, permeability, and stronger mechanical stability, carries for immunocyte
For a stable three-dimensional attachment space, additionally, at cell after frozen-recovery, immunity isolation can be played
The effect of barrier, it is possible to reduce immunocyte and transplant the immunological rejection caused.
Immunocyte is wrapped in capsule material the microcapsule forming diameter some tens of pm to hundreds of microns by the present invention,
Due to the adherent property of immunocyte, therefore microcapsule is that immunocyte provides a good attaching substratum,
Make immunocyte contact with each other and form a kind of three dimensional structure；Additionally, this microcapsule is prevented from biomacromolecule
Escape from microcapsule with cell, and the nutrient substance of small-molecule substance, culture medium, cells frozen storing liquid and generation
Thank to thing can freely come in and go out microcapsule, reach to cultivate and frozen purpose etc..
Further, in step S2, before being mixed with capsule material by immunocyte, also include adding immunity thin
Born of the same parents' culture medium stands the step of 20～40 minutes, it is preferable that this immune cell media is serum-free culture
Base；Just immunocyte is being kept when this step makes immunocyte mix with capsule material and in capsule material forming process
Normal growth, and keep cytoactive.
Specifically, using alginate-polylysine is the process that capsule material makes microcapsule, also includes following step
Rapid:
By infiltration immunocyte in immune cell media and Sargassum that mass fraction is 0.5%～2.5%
Acid solution mixing and stirring so that immunocyte concentration is (0.5～2) × 107Individual/ml, is added dropwise over
1.0～2.0% in calcium chloride solution；Alginate runs into calcium ion and ion can be occurred rapidly to exchange, and generates gel
Ball, then, then embeds 1～12 minute with the polylysine that concentration is 0.1～1.0g/L, makes gel ball surface
Film forming；The polylysine embedding time is preferably 6 minutes, to increase the thickness of microcapsule membrane, improves microcapsule
Stability.Finally process, with the organic acid soln of 35～75mM, the calcium ion removed in gel ball, so that solidifying
Alginic acid in glueballs is in a liquid state, and immunocyte is suspended wherein.Alginate is formed with calcium ion
Gel has heat irreversible, gelling performance not temperature influence, better mechanical property；Preferably, sea
Alginate are sodium alginate.
Using chitosan is the process that capsule material makes microcapsule, comprises the following steps:
Weighing a certain amount of chitosan, being completely dissolved in volume fraction is in 1%～3% spirit of vinegar, is made into matter
Amount mark is the chitosan-acetic acid solution of 0.1%～0.5%, adds 5-fluorouracil so that it is be completely dissolved,
Adding surfactant, keeping temperature is 40～60 DEG C, stirs, and adds NaOH solution tune pH and is
5～6；
Add infiltration immunocyte in immune cell media, mixing and stirring, make immunity thin
Born of the same parents' concentration is (0.5～2) × 107Individual/ml, adds flocculating agent, after being again stirring for uniformly, and 10～30 minutes
Rear addition volume fraction is the glutaraldehyde solution of 2%～8%, starts to solidify to form microcapsule, and normal saline cleans
?.
Further, after step S3 adds cells frozen storing liquid in microencapsulation immunocyte, need to stand
30～90 minutes, it is beneficial to frozen stock solution and fully permeates.
After standing, after the temperature of microencapsulation immunocyte is down to-60～-100 DEG C, be transferred to freeze liquid nitrogen or
Gas nitrogen is frozen, and frozen temperature is-170～-200 DEG C.
Further, the process of the immunocyte recovery after the present invention is frozen, comprise the following steps:
Take the frozen rear microencapsulation immunocyte obtained in step S3 with the speed of 20～40 DEG C/min by frozen
Temperature is warming up to-100 DEG C, is subsequently placed in the aqueous solution containing 20%～40% dimethyl sulfoxide and is brought rapidly up
To melting.
After defrosting, at room temperature microencapsulation immunocyte is put in phosphate buffer and dilute, progressively
Appear dimethyl sulfoxide, be centrifuged and abandon supernatant, washing, add serum-free medium and cultivate；The present invention
Preferential employing AIM-V culture medium.
In order to make the purpose of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment,
The present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to
Explain the present invention, be not intended to limit the present invention.
Embodiment 1
The microencapsulation immunocyte cryopreservation methods that the present invention provides, comprises the following steps:
S1a, acquisition immunocyte CIK cell；
S11a, from blood, mononuclearcell is separated；
S12a, by mononuclearcell suspension and 1000mm3/ not solidified fibrin gel with
1×104: after the ratio mixing of 1, it is placed in 37 DEG C of calorstats to fibrin gel and solidifies completely, so
After by solidification and with the fibrin gel of mononuclearcell be placed in 50ng/ml CD3McAb and
In the culture bottle pre-coated for RetroNectin of 10g/ml, and add containing 1% volume fraction PPP and
The GTT551 culture medium of the IFN-γ of 1000IU/ml concentration；
S13a, the 2nd day, add IL-1 α and IL-2, and make its in step S12a GTT551 training
The final concentration supported in base is respectively 100IU/ml, 300IU/ml；
S14a, the 3rd day, GTT551 culture medium to the volume supplemented in step S12a is 100ml,
Add IL-2 simultaneously, maintain its concentration 300IU/ml in the medium constant；
S15a, every 2-3 days repeat step S14a CIK is expanded；
CIK after S16a, results amplification in the 21st day.
Wherein, in step S12a, the preparation method of fibrin gel is:
1) preparation 2000KIU/ml aprotinin, takes the aprotinin 1ml prepared and dissolves polypeptide coupling
Lyophilizing Fibrinogen, makes the solution A of the final concentration of 2.0g/L of Fibrinogen；
2) thrombin is added 40mmol/LCaCl2In solution, making concentration of thrombin is 300
The solution B of U/ml；
3) solution A and solution B are mixed, be 100mm in floor space2Mould makes and becomes round
The complex of cylinder, i.e. obtains not solidified fibrin gel, and 4 DEG C of Refrigerator stores are standby.
S2a, acquisition microencapsulation immunocyte；
The CIK cell taking the acquisition of step S16a is incubated at the high sugar DMEN containing 10% hyclone
In, change liquid every other day, change liquid once freezing the previous day；Trypsinization, centrifugal, add 1.5% alginic acid
Sodium solution to cell concentration is 1 × 107Individual/ml, with high-voltage pulse sight spot droplet generator formed drop by
Instill in 1.5% calcium chloride solution, soak 10 minutes, ultimately form gel ball；Gel ball is put
Enter in normal saline and clean, be subsequently adding the polylysine embedding that concentration is 0.5g/L, react 6 minutes,
After normal saline cleans, adding the sodium citrate solution of 55mM, liquefy microcapsule；
Then, the CIK cell of microencapsulation is placed in DMEM culture medium；Wherein, DMEM training
Containing mass fraction in foster base is 1% penicillin and 1% streptomycin, the HEPES of 25mmol/L, matter
Amount mark is 10% hyclone, at CO237 DEG C of cultivations in incubator.
S3a, microencapsulation immunocyte frozen；
After incubated overnight, the microcapsule quantity adjusted in culture medium is 100/ml, by thin for microencapsulation CIK
Born of the same parents move in aseptic cryopreservation tube, add 1.5ml cells frozen storing liquid, sealing, put into 4 DEG C of refrigerators and stand
Balance 60 minutes, be beneficial to the abundant infiltration of cells frozen storing liquid；Then, Control Program for Microcomputers [Digital Research] fall is used
After the temperature of microencapsulation CIK cell is down to-80 DEG C by Wen Yi, finally it is transferred in liquid nitrogen preserve 24 little
Up to 1 month, frozen temperature was-185 DEG C.
Wherein, cells frozen storing liquid includes AIM-V culture medium, and volume fraction is the dimethyl of 10%
Sulfoxide and the human serum of 10%.
S4a, recover frozen after microencapsulation immunocyte；
Take frozen microcapsule CIK cell in-185 DEG C of programmed cooling instrument, with the speed of 30 DEG C/min from
-185 DEG C are warming up to-100 DEG C, then have room temperature containing the aqueous solution that volume fraction is 30% dimethyl sulfoxide
In be brought rapidly up to thawing.
After defrosting, at room temperature microencapsulation CIK cell is put into and phosphate buffer does doubling dilution,
Progressively appear dimethyl sulfoxide, be centrifuged and abandon supernatant, after washing 3 times, add AIM-V culture medium, put
In 37 DEG C, containing the CO of 5% volume fraction2Incubator in cultivate.
Embodiment 2
Being with the difference of embodiment 1, in this embodiment, the cells frozen storing liquid used includes
AIM-V culture medium, volume fraction be 5% dimethyl sulfoxide and volume fraction be the human serum of 5%.
Embodiment 3
Being with the difference of embodiment 1, in this embodiment, the cells frozen storing liquid used includes
AIM-V culture medium, volume fraction be 15% dimethyl sulfoxide and volume fraction be the human serum of 15%.
Embodiment 4
Being with the difference of embodiment 1, in this embodiment, the cells frozen storing liquid used includes
AIM-V culture medium, volume fraction be 8% dimethyl sulfoxide and volume fraction be the human serum of 8%.
Embodiment 5
Being with the difference of embodiment 1, in this embodiment, the cells frozen storing liquid used includes
AIM-V culture medium, volume fraction be 10% dimethyl sulfoxide and volume fraction be the human serum of 10%.
Microencapsulation immunocyte for checking present invention offer further is frozen and method for resuscitation has significantly
Beneficial effect, arranges following experiment and verifies.
Immunocyte after detection the group 1～5 the most corresponding embodiment of the present invention 1～5 defrosting recovery；
The cell that the coated immunocyte of the unused microcapsule of matched group obtains after cryopreservation resuscitation.
1, mtt assay detection cytoactive
Microencapsulated cell after cultivation has recovery adds the MTT of 20 microlitre 5mg/ml, 37 DEG C of cultivations
4h, normal saline is washed twice, adds the dimethyl sulfoxide of 400 microlitres, cultivates 2.5h for 37 DEG C.570nm
Wavelength measurement absorbance, 630nm wavelength is reference value, and testing result see table 1.
Table 1
| Detection object | Detection group 1 | Detection group 2 | Detection group 3 | Detection group 4 | Detection group 5 | Matched group |
| OD value | 0.8579 | 0.8497 | 0.8643 | 0.8527 | 0.8613 | 0.6652 |
Testing result: OD value is the biggest, represents the content of first hairpin in solution the highest, also illustrates simultaneously, promote
The succinate dehydrogenase making MTT enzymolysis be first hairpin is the most, and the quantity of secretion succinate dehydrogenase living cells is relatively
Many.Thus illustrating, the present invention is by after cryopreservation resuscitation after immunocyte microencapsulation, and immunocyte survival rate has
Improved.
2, external functional examination
Take detection group 1～5 and each 8 groups of matched group respectively, survey basis Secretion of Catecholamine amount and high potassium respectively
With the secretory volume that acetylcholine stimulates lower catecholamine, testing result see table 2.
Table 2
Testing result: from the data in table 2, it can be seen that after matched group cryopreservation resuscitation immunocyte basic with high potassium and
Acetylcholine stimulates lower secretory volume to respectively may be about the 60% of frozen money microcapsule immunocyte secretory volume；
After detection group 1～5 cryopreservation resuscitation, microcapsule immunocyte remains the secretory function of catecholamine, is about
The 80% of frozen front microcapsule immunocyte secretory volume, and higher than matched group immunocyte secretory volume；Thus say
Bright, immune cell function can also be maintained relatively by the present invention after cryopreservation resuscitation after immunocyte microencapsulation
Good.
Above embodiments of the invention are described, but the invention is not limited in above-mentioned concrete
Embodiment, above-mentioned detailed description of the invention is only schematic rather than restrictive, this area
Those of ordinary skill under the enlightenment of the present invention, without departing from present inventive concept and claimed
Ambit under, it may also be made that a lot of form, within these belong to the protection of the present invention.
Claims (9)
1. an immunocyte frozen stock solution, it is characterised in that include dimethyl sulfoxide, human serum and AIM-V
Culture medium.
Immunocyte frozen stock solution the most according to claim 1, it is characterised in that described AIM-V
In culture medium, the volume fraction of dimethyl sulfoxide is 5～15%, and the volume fraction of human serum is 5～15%.
Immunocyte frozen stock solution the most according to claim 2, it is characterised in that described AIM-V
In culture medium, the volume fraction of dimethyl sulfoxide is 10%, and the volume fraction of human serum is 10%.
Immunocyte frozen stock solution the most according to claim 2, it is characterised in that described AIM-V
In culture medium, the volume fraction of dimethyl sulfoxide is 5%, and the volume fraction of human serum is 5%.
Immunocyte frozen stock solution the most according to claim 2, it is characterised in that described AIM-V
In culture medium, the volume fraction of dimethyl sulfoxide is 15%, and the volume fraction of human serum is 15%.
Immunocyte frozen stock solution the most according to claim 1, it is characterised in that described AIM-V
In culture medium, the volume fraction of dimethyl sulfoxide is 8～10%, and the volume fraction of human serum is 8～10%.
Immunocyte frozen stock solution the most according to claim 6, it is characterised in that described AIM-V
In culture medium, the volume fraction of dimethyl sulfoxide is 8%, and the volume fraction of human serum is 8%.
Immunocyte frozen stock solution the most according to claim 6, it is characterised in that described AIM-V
In culture medium, the volume fraction of dimethyl sulfoxide is 10%, and the volume fraction of human serum is 10%.
9. the method that an immunocyte is frozen, it is characterised in that comprise the following steps,
Obtain immunocyte,
Use immunocyte frozen stock solution as described in any one of claim 1～8 to as described in immunocyte carry out
Frozen.
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| CN106689117A (en) * | 2017-01-03 | 2017-05-24 | 郑州汉东科技有限公司 | Application and method of isodon excisoides ketone in preparation of cell freezing medium |
| CN106754688A (en) * | 2016-11-21 | 2017-05-31 | 深圳市衍生生物科技有限公司 | A kind of efficient method for resuscitation for freezing PMNC |
| 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 |
| CN108990964A (en) * | 2018-08-09 | 2018-12-14 | 华东理工大学 | Cells frozen storing liquid |
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| CN102839153A (en) * | 2012-09-13 | 2012-12-26 | 济南泰生生物技术有限公司 | Amplifying, freezing and storing and recovering method of activated lymphocyte with CD3+CD8+as major |
| CN104026118A (en) * | 2013-11-13 | 2014-09-10 | 杭州易文赛生物技术有限公司 | Immunization cell frozen stock solution, preparation method and application |
| CN104938477A (en) * | 2015-04-10 | 2015-09-30 | 杭州阿德莱诺泰制药技术有限公司 | CIK (cytokine-induced killer) frozen stock solution and frozen preservation method |
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| CN102839153A (en) * | 2012-09-13 | 2012-12-26 | 济南泰生生物技术有限公司 | Amplifying, freezing and storing and recovering method of activated lymphocyte with CD3+CD8+as major |
| CN104026118A (en) * | 2013-11-13 | 2014-09-10 | 杭州易文赛生物技术有限公司 | Immunization cell frozen stock solution, preparation method and application |
| CN104938477A (en) * | 2015-04-10 | 2015-09-30 | 杭州阿德莱诺泰制药技术有限公司 | CIK (cytokine-induced killer) frozen stock solution and frozen preservation method |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754688A (en) * | 2016-11-21 | 2017-05-31 | 深圳市衍生生物科技有限公司 | A kind of efficient method for resuscitation for freezing PMNC |
| CN106689117A (en) * | 2017-01-03 | 2017-05-24 | 郑州汉东科技有限公司 | Application and method of isodon excisoides ketone in preparation of cell freezing medium |
| 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 |
| CN108990964A (en) * | 2018-08-09 | 2018-12-14 | 华东理工大学 | Cells frozen storing liquid |
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