CN106554942A - A kind of efficient clinical grade CD56+The preparation method of group's immunocyte - Google Patents

A kind of efficient clinical grade CD56+The preparation method of group's immunocyte Download PDF

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CN106554942A
CN106554942A CN201611028882.8A CN201611028882A CN106554942A CN 106554942 A CN106554942 A CN 106554942A CN 201611028882 A CN201611028882 A CN 201611028882A CN 106554942 A CN106554942 A CN 106554942A
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姜丽君
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Jilin Tuo Hua Biotechnology Co ltd
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Abstract

The invention provides a kind of efficient clinical grade CD56+The preparation method of group's immunocyte, which includes：A) mononuclearcell is isolated from peripheral blood or Cord blood；B) a) the middle mononuclearcell for obtaining is inoculated into in the coated culture vessel of anti-CD16 antibody；C) add rabbit Antithymocyte Globulin, Li Erfan and non-animal derived property cytokine induction in the medium, stimulate the mononuclearcell of the inoculation in b)；D) per 23 days according to cell counts supplementing culture medium and non-animal derived property cell factor；E) obtain CD56+Group's immunocyte.Clinical grade CD56 that the present invention is provided+The preparation method of group's immunocyte not only reduces cost, more meets clinical grade and prepares requirement, shows good killing activity in vivo and in vitro.

Description

A kind of efficient clinical grade CD56+The preparation method of group's immunocyte
Technical field
The present invention relates to biological technical field, and in particular to a kind of efficient clinical grade CD56+The preparation of group's immunocyte Method.
Background technology
Cell therapy is that, after operation, another tumor therapeuticing method after radiation and chemotherapy, it is generally as performing the operation, put The supplementary means of chemotherapy, extends the life-span of patient, removes the remaining cancer cell of internal Post operation.Cell therapy mainly includes nature Kill cell (natural killer cell, NK) treatment, the treatment of NKT cell therapies, CIK cell etc..These cells are with CD8+ CD56+Or CD8-CD56+For main effects cell (Fig. 1).
NK cells are that a class is restricted without MHC (MHC), can direct killing target cell it is special Lymphocyte population, it is considered to be the important effect cell of immunotherapy of tumors.This two class CD56+Lymphocyte is thin to tumour The killing of born of the same parents have be not required to immunological sensitization, the features such as inorganization histocmpatibility (MHC) is restricted.NK is machine The immunocyte that body weight is wanted, it is not only relevant with antitumor, viral infection resisting and immunological regulation, and participate in some cases super Quick reaction and the generation of autoimmune disease.NKT cells are also immunocyte important in vivo, its coexpression φt cell receptor With NK cell receptors, immunological regulation and CDCC are primarily served.The NKT cells of activation can produce substantial amounts of IL-4 and INF- γ, has cell damage to act on tumour cell；On the other hand, there is NK cell-like cell cytotoxic activities after NKT cell activations, passes through Perforin approach dissolves tumour cell.
Clinically using CD56 such as NK, CIK or NKT+During group's immune cell therapy tumour, need the quantity of cell higher, And the limited amount of common cell induction cultural method amplification, the requirement of clinical reinfusion is extremely difficult to, therefore use must be found It is another kind of to strengthen CD56 simultaneously+The proliferation activity of group's immunocyte, and it is thin to strengthen the immunity of the killing ability of cell simultaneously Born of the same parents' cultural method.
The content of the invention
Based on above-mentioned purpose, the invention provides a kind of clinical grade CD56+The preparation method of group's immunocyte, which includes：
A) mononuclearcell is isolated from peripheral blood or Cord blood；
B) a) the middle mononuclearcell for obtaining is inoculated into in the coated culture vessel of anti-CD16 antibody；
C) add rabbit Antithymocyte Globulin, Li Erfan and non-animal derived property cell factor in the medium to lure Lead, stimulate the mononuclearcell of the inoculation in b)；
D) per 2-3 days according to cell counts supplementing culture medium and non-animal derived property cell factor；
E) obtain CD56+Group's immunocyte.
In a specific embodiment, the culture medium is AIM-V serum free mediums.
In a specific embodiment, wherein after isolating mononuclearcell in a), being trained using AIM-V serum-frees Foster base will be the mononuclearcell resuspended, adjustment cell density to 1 × 106Individual cell/ml.
In an optional embodiment, wherein the dosage of the rabbit-anti human thymocyte immunoglobulin (Ig) is 50-500ng/ml, preferably 200ng/ml.
In an optional embodiment, the dosage of Qi Zhong Suo Shu Li Erfan is 1-100 μ g/ml, preferably 10 μ g/ml。
In an optional embodiment, wherein the non-animal derived property cell factor includes：100-1000U/mL's The IL-21 of the IL-15 and 10-100ng/mL of IL-2,10-100ng/mL.
Preferably, the non-animal derived property cell factor includes the IL-15 and 50ng/ of IL-2,50ng/ml of 1000U/ml The IL-21 of ml.
In a specific embodiment, wherein in d), after cell count, supplementing culture medium to cell density is 2 ×106Individual cell/ml；Add IL-2,10-100ng/ of 100-1000U/mL by the cumulative volume of the culture medium after supplementing culture medium The IL-21 of IL-15,10-100ng/mL of mL.
In an optional embodiment, wherein incubation time is -21 days 14 days.
Specifically, the incubation time refers to from separation mononuclearcell in a) and CD56 is obtained in e)+Group's immunocyte The required time.
In an optional embodiment, during coating culture vessel, the final concentration of 50ng- of the anti-CD16 antibody 1mg/ml, preferably 1 μ g/ml.
In some embodiments, peripheral blood and Cord blood are from mammal.Mammal includes but is not limited to monkey, little Mouse, rat, cavy, rabbit, dog, people.In some embodiments, peripheral blood and Cord blood are from people.
Another aspect of the present invention additionally provides a kind of cell product, and which includes：Above-mentioned clinical grade CD56+Group's immunocyte The CD56 of preparation method production+Group's immunocyte；And pharmaceutically acceptable carrier.
The definition of term
CD56+Group's immunocyte：A group surface marker CD56 protein moleculars are positive immunocyte.
Non-animal derived property cell factor is also referred to as AF levels, refers to culture medium or the middle mistake being related in the whole production process of product Journey condition of culture does not all have animal derived cell factor.
Clinical grade refers to and can be applied to clinical disease treatment from vitro culture.
NK cells：NK (Natural killer cell, abbreviation NK) is considered as that body is anti-infective, anti- First natural defence line of tumour, is the important immunocyte of body.
NKT cells：Natural killer T cells (Natural killer T, abbreviation NKT) are the existing T of a group cell surface Cell receptor TCR, the special T cell subgroup for having NK cell receptors again.
CIK cell：Cytokine induced kill cell (Cytokine-induced killer cells, referred to as CIK), it is in vitro with cytokine profiles (such as CD 3-resisting monoclonal antibody, IL-2 and IFN- by human peripheral blood single nucleus cell γ etc.) a group foreign cell that obtains afterwards for a period of time of co-incubation.
In a particular embodiment, it would however also be possible to employ the surface marker of cell representing cell, such as：CD3+CD16+ CD56+：NKT cell major surfaces marks；CD3-CD16+CD56+：NK cell major surfaces marks；CD8+CD56+：This is thin Born of the same parents group is mainly NKT cells and CIK cell；CD8-CD56+：This cell mass is mainly NK cells；CD16+CD56+Group：It is main to wrap Include CD3+CD16+CD56+NKT and CD3-CD16+CD56+NK cells；CD3+CD56+：Generally as the table of Testing and appraisal CIK cell Face mark.
The invention has the beneficial effects as follows：Stimulation reagent Li Erfan, Thymoglobuline (rabbit Antithymocyte Globulin) are Clinical application, it is relative it is safer with experiment level reagent, more meet GMP requirements.Wherein Thymoglobuline is (a kind of to be clinically used for preventing With the immunosuppressive drug for the treatment of kidney transplantation exclusion reaction) can optionally expand immunological effect cell in biological therapy (CD3+CD16+CD56+Cell and CD3-CD16+CD56+Cell), the expression of NK cell activations/suppression acceptor is raised, promotes its point The ripe raising with anti tumor activity in vitro of chemical conversion；Li Erfan main components are α-mannatide, belong to immunomodulator, can live Change macrophage and lymphocyte, this two kinds of drug combinations can greatly increase CD56+The quantity of group's cell, it is thin with interleukin class Intracellular cytokine Co stituation culture immunocyte has the features such as quantity is big, and purity is high, cytotoxicity is strong such that it is able to meet clinical Needs.Toxigenic capacity is substantially reduced compared with immunological magnetic bead sorting method simultaneously, the wind compared with feeder cells stimulus method Danger substantially reduces, and technical operation is simple.The present invention used from clinical practice application feasibility, to stimulant species, Combination of cytokines, preparation cost, cytotoxic activity etc. have carried out comprehensive measurement, to realize CD56+Group's immunocyte exists Large-scale application in clinical adoptive immunotherapy.
Description of the drawings
Fig. 1 is analyzed for healthy human peripheral blood T cell subgroup：The immunocyte in human body with anti-tumor activity is illustrated, Wherein CD8-CD56+Cell mass is mainly NK cells, CD8+CD56+Cell mass mainly includes NKT cells and CIK cell, wherein CIK cell CD8 molecular fluorescence intensity is strong compared with NKT cells, is marked as CD8 as difference in this manual++CD56+。
Fig. 2A to Fig. 2 B is the microscopy figure using method of the present invention cultured cells, and wherein Fig. 2A is the 14th day under 100 times Microscopy figure；Fig. 2 B are the 21st day 100 times of lower microscopy figures.
Fig. 3 A to Fig. 3 C are using method of the present invention cultured cells the 0th day, the 14th day, the streaming phenotypic map of the 21st day.
Specific embodiment
Below in conjunction with accompanying drawing, the present invention is further illustrated by embodiment, but not as limitation of the present invention.Hereinafter carry The concrete material supplied used in embodiment of the present invention and its source.It is to be understood that these are only example Property, it is not intended to the present invention is limited, the type, model, quality, property or function with following reagent and instrument is same or similar Material may be incorporated for implement the present invention.Experimental technique used in following embodiments if no special instructions, is routine Method.In following embodiments, material used, reagent etc., if no special instructions, commercially obtain.
Clinical grade CD56+The preparation method of group's immunocyte：
Embodiment 1：Mononuclearcell is isolated from peripheral blood or Cord blood
1st, (man, 17 one full year of life, acute myeloid leukaemia, Jing patient agree to pick up from aseptic collection patient anticoagulation cirumferential blood 50ml Hematology of hospital of Siping City of Chinese Medical Sciences University)；
2nd, the blood sample of collection is gone to into 50ml centrifuge tubes (healthy and free from worry, article No.：430828), it is centrifuged with Thermo 4KR under room temperature Machine is adjusted to 800g rotating speeds, centrifugation 10min；
3rd, draw upper plasma standby when culture；By 0.9% physiological saline：Residual blood=1：1 dilution proportion blood Liquid, 15ml lymphocyte separation mediums are separately added in taking 2 50ml centrifuge tubes, and (Tianjin Hao oceans biological products science and technology Limited Liability is public Department, article No.：LTS1077006), slowly by diluted blood：Lymphocyte separation medium=2:1 is added on lymphocyte separation medium；
4th, the centrifuge tube for adding good diluted blood is carried out into density gradient centrifugation, Thermo4KR centrifuges is adjusted under room temperature 800g rises 1 drop 1 and 20min is centrifuged, and is layered after centrifugation；
5th, upper plasma is drawn into 50ml centrifuge tubes, 56 DEG C of water-baths inactivate 10min, and centrifuging and taking supernatant is continued to employ；
6th, PMNC is drawn in centrifugation after finishing, with more than 10 times of volumes of PMNC 0.9% physiological saline is fully mixed, is washed, 450g centrifugation 10min, abandon supernatant, in triplicate after, carry out cell count.Take 2 × 106Individual PMNC adds frozen stock solution (DMSO:FBS:AIM-V=1：3：6, wherein DMSO are produced for sigma companies Product, article No.：D5879；FBS be HyClone Products, article No.：SH300.84.03；AIM-V serum free mediums are GIBCO Products, article No.：- 80 DEG C of refrigerators are placed in 0870112-DK) frozen, for streaming phenotype analytical.
Embodiment 2：Anti- CD16 antibody is coated with culture vessel
In advance in Tissue Culture Flask (healthy and free from worry, 75cm2, article No.：3275) in add containing anti-CD16 antibody (Biolegend, Article No.：302013) coating buffer (5 μ g antibody are added in 5ml PBS cushioning liquid and obtained), is incubated at room temperature 4h, and antibody is dense eventually Spend for 1 μ g/ml.
Embodiment 3：Inoculation mononuclearcell
The blake bottle of 2 coated antibodies in example 2 is slowly rinsed with 0.9% physiological saline, is trained with AIM-V serum-frees Foster base (GIBCO, article No.：0870112-DK) the cell that the resuspended embodiments 1 of 45ml are obtained, adjustment cell density to 1 × 106It is individual thin Born of the same parents/ml, are inoculated in blake bottle.
Embodiment 4：Addition rabbit Antithymocyte Globulin, Li Erfan and non-animal derived property cell factor culture, Induction, stimulation mononuclearcell
Li Erfan (Jilin A-think Pharmaceutical Co., Ltd., article No. are added in the cell being inoculated with embodiment 3： H10970426) final concentration of 10 μ g/ml, Thymoglobuline (rabbit Antithymocyte Globulin, French Sai Da companies, article No.： S20090067) final concentration 200ng/ml, IL-2 (Shenyang three lives pharmaceutical Co. Ltd, article No.：S10970083) final concentration 1000U/ml, IL-15 (PeproTech, article No.：AF-200-15-10) final concentration 50ng/ml, IL-21 (PeproTech, goods Number：AF-200-21-10) final concentration 50ng/ml, is placed on 37 DEG C, 5%CO2Fiber differentiation CD56 in constant incubator+Group's immunity Cell, daily observation of cell upgrowth situation.
Embodiment 5：Cell count, and supplementing culture medium and non-animal derived property cell factor
Cell was counted every 2-3 days, and maintenance cell number is adjusted for 2 × 106Individual cell/ml simultaneously presses the above-mentioned factor Concentration adds the AIM-V serum free mediums containing IL-2, IL-15, IL-21.
Specifically after cell count, according to cell counts, directly add AIM-V serum free mediums, cell is close Degree is adjusted to 2 × 106Individual cell/ml, the cytokine concentrations of addition are by the total culture volume after adjustment cell density Addition, i.e. IL-2 final concentrations 1000U/ml, IL-15 final concentration 50ng/ml, IL-21 final concentration 50ng/ml.
Embodiment 6：Obtain CD56+Group's immunocyte
Cultivate the 14th day, the 21st day and take pictures under inverted microscope.Culture was harvested by centrifugation CD56 after 21 days+Group's immunocyte.
Embodiment 7：Cell concentration, amplification times and motility rate after cell amplification
Total cell amplification times：To expand after the cell Trypan Blue that the 7th, 14,21 days obtain in above-described embodiment Counted with hemacytometer again, by current total cell divided by the mononuclearcell sum before culture, numerical value is thin The amplification times of born of the same parents, TCS × 100 of Cell viability (%)=undyed cell number/observation.As a result referring to table 1 and figure 2A and Fig. 2 B, Fig. 2A are picture under the 14th day inverted microscope of culture, it can be seen that cell is bright, state is good, and propagation ball is dispersed in, Cell is in irregular long pachyrhizus type；Fig. 2 B are picture under the 21st day inverted microscope of culture, substantially become big compared with 14 days cell densities, Propagation ball is reduced.
Table 1：Cell quantity, amplification times, the motility rate table of comparisons before and after amplification
Embodiment 8：CD8 before and after the amplification of flow cytomery cell+CD56+NKT and CIK cell, CD8-CD56+NK is thin Born of the same parents' ratio
Flow cytometry assays：0th day frozen PMNC and embodiment 6 in Example 1 Middle culture 14 days, the cell sample 5 × 10 obtained after 21 days5Cell/pipe, PBS are washed 2 times.It is separately added into flow cytometer detection to resist Body carries out double labelling streaming Phenotypic examination：(344704) BioLegend, article No. mark the anti-human CD8 antibody of mouse of FITC marks with APC (BioLegend, article No. is 304610) for the anti-human CD56 antibody of mouse of note；20min is incubated under room temperature, PBS is washed 2 times, and cell passes through Flow cytometer is analyzed.
As a result as shown in Fig. 3 A to Fig. 3 C, through the Fiber differentiation CD8 of 14 days+CD56+With CD8-CD56+CD56+Group exempts from Epidemic disease cell (Q1+Q2 areas) is increased to about 66% (Fig. 3 B) from about 9.7% (Fig. 3 A) of the 0th day, flow cytometer detection CD8 after cultivating 21 days+CD56+With CD8-CD56+CD56+Group's immunocyte (Q1+Q2 areas) rises to 78% (Fig. 3 C).
Embodiment 9：Cell killing activity is detected
Take the logarithm growth period K562 cell lines (being purchased from Shanghai Ai Yan bio tech ltd) as target cell, and adjust Whole cell density is 8 × 105Individual/ml, takes 50 μ l per hole and is laid in 96 well culture plates.Obtained in taking the embodiment of the present invention 6 The CD56 of 14 days and the 21st day+Group's immunocyte adjustment density is 8 × 105Individual/ml, 4 × 106Individual/ml, 8 × 106Individual/ml, 1.6 ×107Individual/ml, in adding 96 well culture plates, per 50 μ l of hole, makes effect target ratio respectively 1:1、5:1、10:1、20:1, three are set per group Individual multiple holes.Vaccination ways are as follows：
Blank group (a values)：AIM-V(100μl)
(b values)：CD56+Group immunocyte (50 μ l)+AIM-V (50 μ l)
Control group (c values)：k562(50μl)+AIM-V(50μl)
Experimental group (d values)：k562(50μl)+CD56+Group's immunocyte (50 μ l)
By the cell being inoculated with, 37 DEG C are placed in, 5%CO2Under the conditions of co-culture 24 hours after, per hole add 10 μ lCCK-8 try Agent, shakes up, 37 DEG C, 5%CO2Under the conditions of continue culture 3 hours, ELIASA wavelength 450nm mensuration absorbances.It is calculated as follows and kills Wound activity：Kill knurl efficiency=[1- (d value-b value-a values)/(c value-a values)] × 100%.As a result show the CD56 of present invention preparation+ Group's immunocyte has higher killing activity to tumour K562 cell line, 10:1、20:1 effect target was killed than lower 14th day Rate can reach 100%；5:1 effect target can reach more than 85% than lower 14th day killing rate, and killing rate can reach within the 21st day 100%；1:1 effect target is below the killing rate of the 40%, but the 21st day than the killing rate of lower 14th day and 21 days and is significantly higher than 14th day (as shown in table 2).
Table 2：CD56 of the present invention+Group's killing activity of the immunocyte to tumour K562 cell

Claims (10)

1. a kind of clinical grade CD56+The preparation method of group's immunocyte, which includes：
A) mononuclearcell is isolated from peripheral blood or Cord blood；
B) a) the middle mononuclearcell for obtaining is inoculated into in the coated culture vessel of anti-CD16 antibody；
C) add in the medium rabbit Antithymocyte Globulin, Li Erfan and non-animal derived property cell factor culture, Induction, the mononuclearcell for stimulating the inoculation in b)；
D) per cell count in 2-3 days, and supplementing culture medium and non-animal derived property cell factor；
E) obtain CD56+Group's immunocyte.
2. clinical grade CD56 according to claim 1+The preparation method of group's immunocyte, wherein the culture medium is AIM-V Serum free medium.
3. clinical grade CD56 according to claim 1 and 2+The preparation method of group's immunocyte, wherein list is isolated in a) After individual nucleus, will be the mononuclearcell resuspended using AIM-V serum free mediums, adjustment cell density to 1 × 106It is individual thin Born of the same parents/ml.
4. clinical grade CD56 according to any one of claim 1 to 3+The preparation method of group's immunocyte, wherein the rabbit The dosage of Antithymocyte Globulin is 50-500ng/ml.
5. clinical grade CD56 according to any one of claim 1 to 4+The preparation method of group's immunocyte, wherein the power Your all dosages are 1-100 μ g/ml.
6. clinical grade CD56 according to any one of claim 1 to 5+The preparation method of group's immunocyte, wherein the nothing Animal derived cell factor includes：The IL- of the IL-15 and 10-100ng/mL of IL-2,10-100ng/mL of 100-1000U/mL 21。
7. clinical grade CD56 according to any one of claim 1 to 6+The preparation method of group's immunocyte, wherein d) In, after cell count, supplementing culture medium to cell density is 2 × 106Individual cell/ml, by the culture medium after supplementing culture medium The IL-21 of the IL-15 and 10-100ng/mL of IL-2,10-100ng/mL of cumulative volume addition 100-1000U/mL.
8. clinical grade CD56 according to any one of claim 1 to 7+The preparation method of group's immunocyte, wherein when cultivating Between be -21 days 14 days.
9. clinical grade CD56 according to any one of claim 1 to 8+The preparation method of group's immunocyte, wherein described outer All blood and the Cord blood are preferred from people from mammal.
10. a kind of cell product, which includes：
Clinical grade CD56 according to any one of claim 1 to 9+The clinical grade of the preparation method production of group's immunocyte CD56+Group's immunocyte；With
Pharmaceutically acceptable carrier.
CN201611028882.8A 2016-11-18 2016-11-18 A kind of efficient clinical grade CD56+The preparation method of group's immunocyte Pending CN106554942A (en)

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CN108192865A (en) * 2017-12-29 2018-06-22 吉林省拓华生物科技有限公司 NK cell expansion ex vivos method and the kit for this method
CN108192865B (en) * 2017-12-29 2021-09-21 吉林省拓华生物科技有限公司 NK cell in-vitro amplification method and kit used for same
CN113631699A (en) * 2018-10-24 2021-11-09 赫贝细胞股份有限公司 Methods and systems for producing cells of hematopoietic lineage
CN109536444A (en) * 2018-12-11 2019-03-29 吉林省拓华生物科技有限公司 A kind of separant induction method suitable for the tumor-infiltrated T lymphocyte of malignant solid tumor
CN110643573A (en) * 2019-10-23 2020-01-03 武汉济源高科技有限公司 Method for culturing chained NK cells
CN115247148A (en) * 2022-06-14 2022-10-28 吉林省拓华生物科技有限公司 Culture medium for culturing immune cells and cell culture method
CN115247148B (en) * 2022-06-14 2023-08-11 吉林省拓华生物科技有限公司 Culture medium for culturing immune cells and cell culture method
CN115558641A (en) * 2022-11-14 2023-01-03 四川新生命干细胞科技股份有限公司 High-purity effector immune cell population, and culture method, reagent composition and application thereof
CN115558641B (en) * 2022-11-14 2023-05-12 四川新生命干细胞科技股份有限公司 High-purity effector immune cell population, culture method, reagent composition and application thereof

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