CN106754688A - A kind of efficient method for resuscitation for freezing PMNC - Google Patents
A kind of efficient method for resuscitation for freezing PMNC Download PDFInfo
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- CN106754688A CN106754688A CN201611045894.1A CN201611045894A CN106754688A CN 106754688 A CN106754688 A CN 106754688A CN 201611045894 A CN201611045894 A CN 201611045894A CN 106754688 A CN106754688 A CN 106754688A
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- C—CHEMISTRY; METALLURGY
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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Abstract
The present invention relates to a kind of recovery solution for freezing PMNC, its component include AIM V culture mediums, serum,Nuclease ultrapure, the volume fraction of serum is 5 20% in the recovery solution,
Description
Technical field
The present invention relates to cells frozen storing liquid technical field, and in particular to a kind of to freeze the efficiently multiple of PMNC
Soviet side's method.
Background technology
Immunocyte (immune cell) is being commonly called as leucocyte, also special including lymphocyte and various phagocytes etc.
Finger can recognize antigen, the lymphocyte for producing specific immune response etc..Lymphocyte is the basis of immune system, in body
Very extensively, mainly T lymphocytes, bone-marrow-derived lymphocyte are activated (activation) by antigenic stimulus for interior distribution, division, increasing
Grow, specific immune response occurs.T lymphocytes are a multi-functional cell masses.In addition to T lymphocytes and bone-marrow-derived lymphocyte,
Also a kind of lymphocyte is NK (natural killer cells, NK).In addition to lymphocyte, participate in immune
The cell of response also has the cell of thick liquid cell, granulocyte, mast cell, antigen presenting cell and mononuclear phagocyte system.
PMNC (peripheralblood mononuclear cell, PBMNC), i.e., have in peripheral blood
There is the cell of single core, including lymphocyte, BMDC and other a small amount of cells (candidate stem cell etc.).PBMNC be by
Leucocyte further extract, with the part for being divided into more powerful effector cell.By the induction of specificity factor, PBMNC
Panimmunity cell can be divided into vitro, be such as now widely used for the cytokine induced kill cell of biological therapy
(cytokine induced killer, CIK), BMDC (dendritic cells, DCs) and NK
(NK) cell such as.
Malignant tumour is common disease, the frequently-occurring disease of a class serious harm human health.According to WHO Report, every year
Global cancer new cases more than 1,000 ten thousand, more than dead 700 ten thousand, account for the 12% of total death toll.The incidence of disease of China's malignant tumour
Also it is rise year by year, case fatality rate remains high, therefore finds and a kind of effectively treating cancer, improvement patients ' life quality can control
Treatment method is extremely urgent.
Immunotherapy of tumors is a kind of emerging tumor treatment model, is by the novel therapeutic side of autoimmunity anticancer
Method.Immune system is the defense system of human body, on the one hand plays the function of bacteria removal, virus, alien material, on the other hand
Eliminate internal senile cell and the cell undergone mutation.The result that body immune system and cancer cell interact determines cancer
The final differentiation of disease.For the people of health, the powerful cancer cell for being enough to remove mutation in time of its immune system.But for
For tumour patient, generally existing weakened immune system, it is impossible to efficiently identify, kill cancer cell；On the other hand, cancer is thin
Born of the same parents largely breed, and can further suppress the immunologic function of patient, and, cancer cell has number of mechanisms to escape immunocyte
Identification and killing.
The immunization therapy of tumour is exactly, by Protocols in Molecular Biology and cell engineering, to improve the immune of tumor tissues
Originality, sufficient amount of normally functioning immunocyte and correlation molecule are supplemented to body, excite and strengthen body Antitumor immunity
Response, improves tumor tissues to the sensitiveness of antitumor immunity of organism effect, in vivo, outer inducing tumor-specific and non-specific
Property effector cell and molecule, reach the final purpose for removing tumour.Immunotherapy of tumors is after operation, radiation and chemotherapy
4th kind of tumor therapeuticing method.Early in 1985, immunotherapy of tumors was just classified as tumour Synthetic by National Cancer center
The fourth-largest Therapeutic mode treated.
2009, " autoimmune cell treatment " was formally included " the 3rd class medical technology " catalogue by the Ministry of Public Health of China.
Lived using adoptive immunity lymphokine from Steve Rosenberg team of nineteen eighty-two US National cancer research institute
The killing cell therapy (LAK) of change, developed current extensive in China's immunotherapy of tumors by 1991 from Stanford University
Cytokine induced kill cell treatment (CIK) of application, the Clinical Exploration about the immune cell therapy of tumour experienced
Very long process.But treatment of these traditional therapies to most of tumours such as oophoroma, colon cancer, breast cancer is not obtained
Gratifying curative effect.Not enough, curative effect has much room for improvement the targeting that it has the disadvantage treatment.With the development of tumor immunology, tree
Prominent shape cell (Dendritic cell, DC) is of increased attention as oncotherapy means and is sent to great expectations.
DC is the unique form cell with dendritic processes that Steinman and Cohn in 1973 has found in mouse spleen, and it is ordered
Entitled BMDC.DC is the bridge for connecting congenital immunity and acquired immunity, is that most active, function is most strong in human body
Professional antigen presenting cell (antigen presenting cells, APC), offers and activates primary tape T cell with antigen
Function.Research finds that tumour antigen sensitization DC feeds back body energy stimulation of host immune system and produces anti-tumor immune response again,
Therefore the DC vaccines of load tumour antigen are considered as a kind of immunotherapy of tumors means of most potential.Particularly 2010 4
Month U.S. FDA have approved first autogenous cell immunotherapy medicaments Sipuleucel-T, just occur in that therapeutic tumor vaccine
Important breakthrough, the approval of this vaccine greatly encouraged the further exploitation of DC vaccines.
At present, the immunocyte for being extracted after the domestic big multiplex patient of immunological cells treatment, exempting from after patient
Difference when epidemic disease cytoactive and amplification ability affirmative are than health, therapeutic effect certainly will be affected；The immunity of Healthy People by
Year successively decreases, therefore, cytoactive, cell biological function are particularly important after storage immunocyte and guarantee immunocyte recovery.
Cell cryopreservation is one of main method that cell long-period is preserved." exempt from there is provided one for current healthy population
The guarantee of epidemic disease cell life bank ", and during future tumors immunization therapy, the chemicotherapy course for the treatment of that effectively staggers carries out immune thin
Born of the same parents collect, store, recovering and culture.Cell is placed in Cord blood in -196 DEG C of liquid nitrogen using Cryopreservation Technology, cell can be made
It is temporarily disengaged from growth conditions and saves its cell characteristics, recovery cell is used to test again so when needing.And
And moderately preserve a certain amount of cell, can prevent because the cell cultivated is contaminated or other accidents make cell
Kind is lost, the effect of cell conservation is served.
The recovery of immunocyte, directly affects the motility rate and bioactivity of later stage immunocyte.In order to solve existing freezing
In resuscitation process water-bath use, PBMNC recover after motility rate is relatively low, cell amplification ability is not enough after recovery, biological function (such as
Effectively kill that cell colony number is relatively low, Cytotoxicity in vitro effect is relatively low) it is not good etc. that technical problem is special proposes the present invention.
The content of the invention
It is molten present invention firstly provides a kind of recovery for freezing PMNC to overcome above-mentioned prior art defect
Liquid, its component include AIM-V culture mediums, serum,Nuclease ultrapure, blood in the recovery solution
Clear volume fraction is 5-20%,The content of Nuclease ultrapure is 0.1-1U/mL；It is balance of
AIM-V culture mediums.
Preferably, the volume fraction of serum is 10% in the recovery solution, and/or, in the recovery solutionThe content of Nuclease ultrapure is 0.2U/mL.
The AIM-V culture mediums,Nuclease ultrapure are commercial prod, respectively by
GIBCO companies, Sigma companies provide.
The serum is preferably the one kind in autoserum, hyclone, AB serum, serum substitute etc., preferably certainly
Body serum.
When autoserum is selected, the autoserum can be prepared during PBMNC is separated.Generally when preparation freezes periphery
The autoserum is used as offal treatment during blood mononuclear cell.The present invention having as above-mentioned recovery solution using the autoserum
Effect component, both twice laid reduces cost, environmental pollution is reduced again.Thus, the present invention possesses practicality using autoserum
Property.
Another advantage using autoserum is：Avoid using exogeneous animal source property serum, dived in reduction successive treatment
Anaphylactogen presence risk.
Present invention additionally comprises application of the above-mentioned recovery solution in the recovery for freezing PMNC.
The present invention also provides a kind of method for resuscitation for freezing PMNC, comprises the following steps：
1) PMNC that will be frozen is transferred in -80 DEG C of freezing storing box, after balance 0.5-5min, then is transferred to
Melt cell in metal bath, the metal bath temperature is 37-42 DEG C；
2) cell after thawing is transferred to and is washed in above-mentioned recovery solution at least 2 times, cell is collected by centrifugation.
Above-mentioned method for resuscitation also includes that gained cell detection Cell viability, cell phenotype will be collected by centrifugation (can be flowed with FACS
Formula detector), cell (is resuspended in the culture body containing mass fraction for 5%-10% autoserums by detection cell proliferation times
In system cultivate) etc. Conventional procedures.
The PMNC for freezing of the present invention can be prepared by existing conventional method, and the present invention does not do especially limit
It is fixed.
Specifically, the present invention also provides the preparation method of the above-mentioned PMNC for freezing, and comprises the following steps：
1) PMNC for obtaining is cleaned, is purified；The PMNC can be gathered certainly
Adult human peripheral's venous blood；
2) the resuspended PMNC of frozen stock solution is used, cell suspension is obtained, cell content is reached 2~3 × 107
Individual/mL；
3) by step 2) gained cell suspension packing；For example dispense into 1.8mL cryopreservation tubes, often pipe loading amount 1mL；
4) cell suspension after packing is kept sample to be checked, remaining cell suspension (cell cryopreservation tube) after packing is put into 4
In the program temperature reduction box of DEG C pre-temperature, it is transferred to -80 DEG C of refrigerators and temporarily stores；
5) after the cell suspension detection for keeping sample is qualified, by step 4) temporary transient cell suspension (cell cryopreservation tube) jelly for storing
Storehouse is stored in, it is long-term to preserve.
The Testing index of the cell suspension is including bacterium, fungi, endotoxin and mycoplasma etc..
In the recovery of the PMNC for freezing, if having pollution risk using traditional water bath method.This hair
Bright use metal bath, and strictly control metal bath temperature and time, both can greatly reduce pollution risk, and because of metal microbead
It is good lead temp effect, can accelerate to freeze the Fast-Balance of article and heat up, improve recovery efficiency.
In the resuscitation process of the PMNC for freezing, with the intracellular various enzyme starting rows of recovery of cell
Make function.Present invention applicationNuclease ultrapure can digest, suppress abatement resuscitation process in it is extensive
Multiple all kinds of DNA enzymatics, keep the primitive character of cell, are easy to later stage PBMNC to be applied to the directed differentiation of immunocyte culture.
More than 90% is up to using recovery Solution Cell recovery survival rate of the present invention.Frozen using the inventive method and exceeded
The complete immunocyte of 20000000 species, comprising BMDC, NKT (NK) cell and T cell etc., can there is effect
For panimmunity cell therapy and be available for repeatedly treatment use.There is no any animal sources in storage and later stage recovery incubation
The addition of serum or albumen, has ensured the security and stability of clinical practice, substantially reduces the side reaction after application and feelings occur
Condition.
Brief description of the drawings
Fig. 1 is experimental example adult's PBMNC extraction efficiency figures；
Fig. 2 experimental examples adult's PBMNC recovery motility rate figures；
Fig. 3 is experimental example CIK Fiber differentiation amplification times figures；
Fig. 4 kills knurl lab diagram for experimental example CIK cell in vitro.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.It is unreceipted specific in embodiment
Technology or condition person, according to technology or condition described by document in the art, or are carried out according to product description.It is used
Reagent or the unreceipted production firm person of instrument, are the conventional products that can be commercially available by regular distributor.
AIM-V culture mediums as described below,Nuclease ultrapure are commercial prod, respectively
There is provided by GIBCO companies, Sigma companies.Autoserum described in example 1 below -3 is prepared by experimental example methods described.
Embodiment 1
A kind of recovery solution for freezing PMNC, its component include AIM-V culture mediums, autoserum,Nuclease ultrapure, the volume fraction of serum is 10% in the recovery solution,
The content of Nuclease ultrapure is 0.2U/mL；Balance of AIM-V culture mediums.
Embodiment 2
A kind of recovery solution for freezing PMNC, its component include AIM-V culture mediums, autoserum,Nuclease ultrapure, the volume fraction of serum is 5% in the recovery solution,
The content of Nuclease ultrapure is 0.1U/mL；Balance of AIM-V culture mediums.
Embodiment 3
A kind of recovery solution for freezing PMNC, its component include AIM-V culture mediums, autoserum,Nuclease ultrapure, the volume fraction of serum is 20% in the recovery solution,
The content of Nuclease ultrapure is 1U/mL；Balance of AIM-V culture mediums.
The preparation method of recovery solution described in embodiment 1-3 includes mixing each component by proportioning.
Embodiment 4
A kind of method for resuscitation for freezing PMNC, comprises the following steps：
First, the adult human peripheral's venous blood for collecting is isolated and purified, obtains PMNC；Collect simultaneously
Serum, i.e. autoserum；
2nd, the PMNC for obtaining is cleaned, is purified；
3rd, it is diluted with the resuspended PMNC of frozen stock solution, cell content reaches 2~3 in making cell suspension
×107Individual/mL；
4th, the cell suspension after dilution is dispensed into 1.8mL cryopreservation tubes, often pipe addition 1mL, and it is to be checked to keep sample, will be thin
Born of the same parents' cryopreservation tube is put into 4 DEG C of program temperature reduction boxes of pre-temperature, is transferred to -80 DEG C of refrigerators and is temporarily stored；
5th, reserved freeze-stored cell suspension carries out the detection of the indexs such as bacterium, fungi, endotoxin and mycoplasma, index inspection
After survey is qualified, freezes storage and do long-term preservation；
6th, during recovery, after being transferred in pre-temperature to -80 DEG C of freezing storing box from the cell for freezing taking-up in storage, balance
5min is transferred to 37 DEG C of metal baths and melts；
7th, the cell after thawing is transferred in recovery solution prepared by embodiment 1 and is washed, detect Cell viability, afterwards
Cell is resuspended in inoculated and cultured in the AIM-V cultivating systems containing the autoserum of mass fraction 10%, that is, completes described length
Phase stores the recovery of adult peripheral mononuclear cell.
Embodiment 5
The present embodiment is as different from Example 4：The serum retained after PMNC is obtained in step one,
56 DEG C of metal baths are inactivated 30 minutes, and plasma supernatant is collected by centrifugation, and one is dispensed per 10mL, and -80 DEG C of preservations are standby；
Embodiment 6
The present embodiment is as different from Example 4：The PMNC for obtaining is cleaned in step 2, it is pure
The concrete operations of change are as follows：Mononuclearcell is washed with AIM-V solution twice, collect the supernatant of last time washing, go forward side by side
The detection of row bacterium, fungi, mycoplasma and endotoxin, after detection is qualified, the cell being collected by centrifugation is stand-by.Other and the phase of embodiment 4
Together.
Embodiment 7
The present embodiment is as different from Example 4：Frozen stock solution in step 3 is formulated as follows：By cell non-serum culture medium
The volume ratio of AIM-V, Human autologous serum and DMSO (dimethyl sulfoxide (DMSO)) is 8:1:1 ratio is mixed.Other and embodiment 4
It is identical.
Embodiment 8
The present embodiment is as different from Example 4：Human autologous serum mass fraction in frozen stock solution is at least 10%.Other
It is identical with specific embodiment 4.
Embodiment 9
The present embodiment is as different from Example 4：Indexs measure is qualified in step 5 refers to：Bacterium, fungi, mycoplasma inspection
Survey is all feminine gender, and endotoxin content is not higher than 0.25EU/mL.Other are same as Example 4.
Embodiment 10
The present embodiment is as different from Example 4：The method agents useful for same is medicinal or GMP ranks.Other and embodiment 4
It is identical.
Embodiment 11
The present embodiment is as different from Example 4：Containing monoclonal antibody and thin in serum free culture system in step 7
Intracellular cytokine；Wherein, the concentration in serum free culture system of monoclonal antibody is 500ng/mL.Other are same as Example 4.
Embodiment 12
The present embodiment is as different from Example 4：Cytokine concentrations in serum free culture system in step 7：400-
1000IU/mlGM-CSF, 100~4000IU/mlIL-2,800-1000IU/mlIL-4,0.2~2ng/mlIL-12,500~
One or more in 2000IU/mlIFN- γ, 100~1000ng/ml anti-CD3-McAb is any than mixing.Other and reality
Apply example 11 identical.
Embodiment 13
The present embodiment is as different from Example 4：Cytokine concentrations in serum free culture system in step 7：100~
1000ng/ml anti-CD3-McAb, 500~2000IU/mlIFN- γ, 0.5~4ng/mlIL-1 α, 1000~4000IU/
MlIL-2 and it is mixed by any ratio.Other are same as Example 4.
Present invention is not limited only to the content of the various embodiments described above, and the combination of one of them or several specific embodiments is same
Sample can also realize the purpose of invention.
Effect of the invention is verified by following example experimental example：
1.PBMNC is extracted and obtained with autoserum：
Autoserum is obtained：
Adult human peripheral's venous whole is transferred to 50ml centrifuge tubes by 1.1, often pipe 25ml.
1.2 room temperature 1800rpm, are centrifuged 8 minutes.
1.3 take upper plasma to 50ml centrifuge tubes, inactivate (56 DEG C of metal bath 30min).
1.4 4 DEG C of placement 30min.
1.5 2800rpm are centrifuged 8min, and it is serum to collect supernatant, is sub-packed in 3 15ml centrifuge tubes, 4 DEG C of preservations
Stand-by, two -20 DEG C save backup.
PBMNC is extracted, i.e., using the mononuclearcell in lymphocyte separation medium washed corpuscles, and it is thin to collect single core
Born of the same parents, comprise the following steps：
1.6 press physiological saline：Haemocyte=1.5:1 pair of sample for having extracted blood plasma is diluted, and mixes.
1.7 diluted sample is slowly added in (method on Ficoll (lymphocyte separation medium) liquid level：Centrifuge tube is inclined
45 °, at 1cm on Ficoll liquid levels, the haemocyte of dilution is slowly injected into, liquid level interface should not be upset), final volume is not after addition
More than 45ml, room temperature 2000rpm, 20min.1.8 slowly extract Ficoll layers with plasma layer intersection tunica albuginea layer with Pasteur's dropper
Cell, according to the principle that two centrifuge tubes, one centrifuge tube of correspondence are rinsed, the tunica albuginea confluent monolayer cells that will be extracted load 50ml from
In heart pipe.Supplement physiological saline is mixed, 1600rpm, 5min to 45ml.
1.9 abandon supernatant, resuspended with physiological saline, are collected in a 50ml centrifuge tube, supplement physiological saline to 45ml.
1200rpm, 10min.
1.10 abandon supernatant, and addition physiological saline is resuspended to 45mL again, mixes, and take 0.5ml middle levels suspension to 1.5mlEP pipes
Middle counting, 1000rpm, 10min obtain final product PBMNC.
1.11 calculate PBMNC extraction efficiencies, as a result see Fig. 1 (adult's PBMNC extraction efficiencies figure).
2.PBMNC freezes：
2.1 cell cryopreservation formula of liquid：According to cell non-serum culture medium AIM-V culture mediums, Human autologous serum and DMSO (two
Methyl sulfoxide) volume ratio 8:1:1 ratio is mixed；
2.2 cryopreservation steps：The PBMNC of collection is well mixed with the cells frozen storing liquid for having configured, and adjusts cell concentration
It is 2~3 × 107Individual/ml；
Cell suspension is dispensed into 1.8ml cryopreservation tubes, and sampling carries out virus, bacterium, fungi, mycoplasma and endotoxin
Detection, program temperature reduction box is put into by cell cryopreservation tube, and -80 DEG C temporarily preserve.After all monitoring results are qualified, cell is entered
Storehouse, seals up for safekeeping and long-term preservation is done in liquid nitrogen.
3.PBMNC recovers：
It is quick to turn after balance 5min after 3.1 cryopreservation tubes that will be taken out in liquid nitrogen are transferred in pre-temperature to -80 DEG C of freezing storing box
Move to and melt cell in 37 DEG C of -42 DEG C of metal baths；
3.2 are transferred in centrifuge tube the cell suspension after thawing, and add the recovery solution of the preparation of embodiment 1, centrifugation
Cell is collected, is cleaned twice altogether；
Cell after 3.3 recoveries, carries out cell viability detection.Result is shown in Fig. 2 (adult's PBMNC recovery motility rates figure)；
Biological function detection after 4.PBMNC recoveries：It is CIK cell including culture, detects amplification times, CIK cultivates it
Flow cytometer detection effector cell surface marker expression, the detection of cytotoxicity afterwards.
4.1CIK is cultivated：With CIK culture medium re-suspended cells, and blow and beat uniform, make that cell is fully dispersed to be opened, be inoculated in culture
In bottle, and people's autologous plasma supernatant is added, PC is reached 5%, and add interferon gamma, make its final concentration of
4000IU/ml；
CIK medium components：AIM-V culture mediums, " 5%-10% autoserums ", 400-1000IU/mL GM-CSF, 100
~4000IU/mL IL-2,800-1000IU/mL IL-4,0.2~2ng/mL IL-12,500~2000IU/mL IFN-γs,
100~1000ng/mL anti-CD3-McAb.
After inoculation 24 hours, anti-cd 3 antibodies are added, make its final concentration of 5 μ g/ml, add interleukin-22, make its final concentration
It is 2000IU/ml；
Culture medium (interleukin-22,1000IU/ml, autologous plasma 1%) is added according to culture situation at any time；
Harvesting carries out the detection of streaming, bacterium, fungi, gram, mycoplasma and endotoxin respectively after cultivating 14 days.
CIK amplification times results are shown in Fig. 3 (CIK Fiber differentiation amplification times figure)；
CIK flow cytometer detection effector cells the results are shown in Table 1；
The adult's PBMNC recoveries CIK culture flow cytometer detection results of table 1
4.2CIK cells in vitro kills effect detection (mtt assay detection is external to kill knurl experiment)：
The external target cell for killing knurl experiment is HepG2.The condition of culture of HepG2 is cultivated for the RPMI-1640 of 10%FBS
Base, 37 DEG C, 5%CO2.10 groups thin are randomly selected in four samples of batch frozen from this experimental example " 2.PBMNC freezes "
Born of the same parents, the Fiber differentiation of recovery and CIK, group are carried out according to experimental example " 3.PBMNC recoveries ", " 4.1CIK cultures " described experimental technique
1-10 is not respectively labeled as it.HepG2 is inoculated in 96 orifice plates, per hole inoculating cell 5 × 103It is individual.Treat that HepG2 is completely adherent
Afterwards, the ripe CIK of culture is taken, and acquisition cell is centrifuged, the RPMI-1640 culture mediums using 10%FBS are resuspended by cell precipitation,
And it is 5 × 10 to adjust cell density4(effector cell：Target cell is 20：1) individual/ml, each group selects 5 holes, and carries out mark
Note, discards culture medium therein, adds resuspended good CIK cell suspension, and 200 μ l are added per hole.And its suction is surveyed using MTT in 6h
Light value (OD570nm), and calculate it according to the following equation and kill knurl：
Killing rate=[(blank group OD values-experimental group OD values)/blank group OD values] × 100%.
Killing knurl result sees Fig. 4 in vitro (CIK cell in vitro kills knurl lab diagram).
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (6)
1. a kind of recovery solution for freezing PMNC, its component include AIM-V culture mediums, serum,Nuclease ultrapure, the volume fraction of serum is 5-20% in the recovery solution,The content of Nuclease ultrapure is 0.1-1U/mL；Balance of AIM-V culture mediums.
2. recovery solution according to claim 1, it is characterised in that the volume fraction of serum is in the recovery solution
10%, and/or, in the recovery solutionThe content of Nuclease ultrapure is 0.2U/mL.
3. recovery solution according to claim 1 and 2, it is characterised in that the serum is selected from autoserum, tire ox blood
Clearly, AB serum, serum substitute, in one kind.
4. application of the recovery solution described in any one of claim 1-3 in the recovery for freezing PMNC.
5. a kind of method for resuscitation for freezing PMNC, it is characterised in that comprise the following steps：
1) PMNC that will be frozen is transferred in -80 DEG C of freezing storing box, after balance 0.5-5min, then is transferred to metal
Melt cell in bath, the metal bath temperature is 37-42 DEG C；
2) cell after thawing is transferred to and is washed in recovery solution described in claim any one of 1-3 at least 2 times, be collected by centrifugation
Cell.
6. method according to claim 5, it is characterised in that the preparation side of the PMNC for freezing
Method, comprises the following steps：
1) PMNC for obtaining is cleaned, is purified；
2) the resuspended PMNC of frozen stock solution is used, cell suspension is obtained, cell content is reached 2~3 × 107Individual/mL；
3) by step 2) gained cell suspension packing；
4) cell suspension after packing is kept sample to be checked, remaining cell suspension after packing is put into 4 DEG C of programmed coolings of pre-temperature
In box, it is transferred to -80 DEG C of refrigerators and temporarily stores；
5) after the cell suspension detection for keeping sample is qualified, by step 4) cell suspension of temporarily storage freezes storage, long-term preservation.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108300691A (en) * | 2018-02-07 | 2018-07-20 | 安徽古生物科技有限公司 | A kind of method for resuscitation of freezing peripheral blood mononuclear cells |
| CN110129267A (en) * | 2018-02-08 | 2019-08-16 | 上海细胞治疗集团有限公司 | Freeze the resuscitation fluid and method of human peripheral blood single nucleus cell |
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