CN109266607A - A kind of optimization it is clinical with TCM sorting, Fiber differentiation and quality control identification kit and application - Google Patents

A kind of optimization it is clinical with TCM sorting, Fiber differentiation and quality control identification kit and application Download PDF

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CN109266607A
CN109266607A CN201811194036.2A CN201811194036A CN109266607A CN 109266607 A CN109266607 A CN 109266607A CN 201811194036 A CN201811194036 A CN 201811194036A CN 109266607 A CN109266607 A CN 109266607A
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路春光
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Abstract

The present invention relates to immunocyte targeted therapy technical fields, in particular to memory immune cell (TCM) or CD8T+ cell reagent box, the kit system and application of TCM induced amplification kit and control of product quality identification are isolated and purified in the slave human peripheral blood or peripheral blood mononuclear cells of a kind of optimization.In clinical application, the reagent used in TCM cell cultivation process is more, and standard is inconsistent, heavy workload in incubation, incubation time is long, and technique is cumbersome, it is unstable, microbiological contamination risk is big, and cell quality standard is difficult to control, and many technical staff are difficult to cultivate the uniform TCM cell of mass in a short time, there is an urgent need to develop kits that is simple, formatting, the present invention saves raw material, reduces production cost by advanced optimizing inducible factor composition TCM-III simultaneously；2-3 times is improved with TCM-II coating culture plate TCM cell Proliferation multiple before Fiber differentiation, apoptotic index reduces, and activity improves 10%-20%, and the absolute quantity of effective cell improves 2-3 times；Before tcr gene transfection, transfection efficiency can be improved with TCM-II coating culture plate.Present invention improves over techniques, this removes residual, prevent the recurrence of tumour and transfer from having great importance to oncotherapy curative effect is clinically improved.

Description

A kind of the clinical of optimization controls identification kit with TCM sorting, Fiber differentiation and quality And application
Technical field
The present invention relates to immunocyte targeted therapy technical field, in particular to the slave human peripheral blood of a kind of optimization or outer Memory immune cell (TCM) or CD8T+ cell reagent box, TCM induced amplification reagent are isolated and purified in all blood mononuclear cells The kit system and application of box and control of product quality identification.
Background technique
Adoptive cell therapy technology is antitumor to break tumor patient maincenter and peripheral immune tolerance, restoring or enhancing its Immune function provides a kind of effective method.However there is also the solutions of many problems demands at present, comprising: 1) how to be filled The tumor-reactive T cells of dosis refracta are to determine the most critical factor of adoptive cell therapy effect；2) what is fed back at present is immune Cell especially cloning CD8+T cell is difficult to long-term surviving in vivo, the strong influence clinic of adoptive cell therapy Curative effect.
Memory t cell in vivo again meet with same antigen when can prompt activation play immunological effect, while have from My updating ability, is capable of providing more long-term immunoprotection, may provide for adoptive cell therapy more particularly suitable immune Effector cell.It is expected to establish the antineoplastic immune of longer-term after feeding back in vivo.Separation memory t cell simultaneously passes through tcr gene Transfection is expected to provide the tumor-reactive T cells with stronger survival ability and effector function.
Invention in the present inventor 2017 " the memory immune cell of peripheral blood mononuclear cells induction and application " (patent Number: 201710712565.6), in clinical application, the reagent that in TCM cell cultivation process uses more, standard inconsistent, training Heavy workload during supporting, incubation time is long, and technique is cumbersome, and unstable, microbiological contamination risk is big, and cell quality standard is difficult to control, Many technical staff are difficult to cultivate the uniform TCM cell of mass in a short time, and there is an urgent need to develop examinations that is simple, formatting Agent box, while the present invention saves raw material, reduces and be produced by advanced optimizing inducible factor composition TCM-III This；2-3 times is improved with TCM-II coating culture plate TCM cell Proliferation multiple before Fiber differentiation, apoptotic index reduces, and activity mentions High 10%-20%, the absolute quantity of effective cell improve 2-3 times；It, can with TCM-II coating culture plate before tcr gene transfection Improve transfection efficiency.Present invention improves over techniques, this removes residual, prevent from swelling to oncotherapy curative effect is clinically improved The recurrence and transfer of tumor have great importance.
Summary of the invention
It is difficult to long-term surviving in vivo in order to solve immunocyte in adoptive cell therapy technology, lacks asking for targeting Topic；This application provides a kind of to isolate and purify note from the peripheral blood mononuclear cells (PBMC) of human peripheral blood or cryopreservation The property recalled T cell (TCM) or CD8T+ cell, the high kit of TCM induced activation rate and application, then by tomour specific or virus Specificity TCR gene transfection into autologous TCM makes immune cell therapy have long-term effect and targeting.
What the present invention was obtained through the following steps:
Sorting, stimulation and the gene of 1.TCM or CD8T+ cell transfect
(1) present invention provides a kind of human peripheral blood TCM or CD8T+ cell sorting kit and qualified products kit.
(2) the human body TCM or CD8T+ cell sorting kit include CD8 gravity column, Reagent1:CD8 purifying examination Agent is made of CD8Fab-Strep and Buffer CI.Reagent2:CD62L+ purified reagent, by CD62L+Fab-Strep and Buffer IS is constituted.Reagent3:CD45RA- purified reagent is made of CD45RA-Fab-Strep and Buffer IS. Reagent4:Strep-Tactin Magnetic Microbeads.Reagent5: eluent, by D-biotin and Buffer IS is constituted.
(3) application method of the TCM or CD8T+ cell sorting kit
1) CD8 gravity column is rinsed with 8ml Buffer CI.
2) plus in the gravity column in 1mlReagent1 to step 1.
3) add the gravity column after 2 loading of 2mlBuffer CI rinsing step.
4) PBMC is according to density 1x106-1x107/ml upper prop or peripheral blood according to volume 66ml upper prop.
5) it is rinsed pillar four times with 40ml Buffer CI, each 10ml.
6) cell is eluted with Reagent5,400g is centrifuged 6-10min and collects cell, obtains CD8+T cell.
7) remove supernatant, be resuspended with suitable Buffer IS, carry out the sorting of next step.
8) after taking 50-100ul Reagent2 and 150-300ulReagent4 to mix, shaking table shakes 30min.
9) 1-2x108 PBMC 900-1500ul Buffer IS gravity treatment is taken, is added in above-mentioned steps 8.It is shaken in shaking table 20min。
10) plus 5mlBuffer IS is into step 9, puts on magnetic pole, is horizontally arranged, after static 3min, removes supernatant, then With 5mlBuffer IS gravity treatment.
11) step 10 is repeated.
12) cell of positive mark is resuspended in Reagent5.The static 10min of room temperature places static 3min on magnetic pole, will contain The supernatant of positive cell is transferred in new reaction tube.
13) it is washed again once with 5ml Reagent5, repeats step 12.
14) supernatant of mixing twice, 400g are centrifuged 6-10min.
15) supernatant is removed, cell is resuspended with 5mlBuffer IS, shakes 10min on shaking table.
16) it is placed on static 3min on magnetic pole.
17) supernatant is transferred in a new centrifuge tube, 400g is centrifuged 6-10min.
18) step 8 and 9 is repeated, 400g is centrifuged 6-10min and collects cell.
19) remove supernatant, be resuspended with suitable Buffer IS, carry out the sorting of next step.Pass through positive choosing twice in succession The method with the choosing of CD45RA- yin is selected, CD8+CD62L+CD45RO+ cell is obtained, endotoxin detection and phenotypic evaluation is carried out to it.
(4) present invention provides a kind of CD8+T cell sorting kit or TCM Fiber differentiation kit, including container and dress Enter the clinic in container with TCM cell non-serum amplification complete culture solution TCM-I and TCM cell amplification coating buffer TCM-II and lures Lead factor composition TCM-III:
(5) present invention provides a kind of clinical serum-free TCM cell amplification complete culture solution TCM-I, contains human body cell and grows Necessary carbohydrate, somatomedin and hormone, vitamin, human serum albumin, amino acid, inorganic ion, micro member Element, water, human interleukin 2 and human interleukin 15.The present invention is not excluded for can be using the training except above-mentioned Serum-free complete medium It supports base and implements culture.
(6) present invention provides a kind of TCM cell amplification coating buffer TCM-II, it contain recombined human fibrin fragment and PBS, the working concentration of recombined human fibrin fragment are 12.5ug/ml-50ug/ml.
(7) present invention provides a kind of TCM cell inducible factors composition TCM-III, serum-free TCM cell amplification training completely It is mono- that 300-1000u/mL human interferon gamma, 50-200ng/mLCD3 monoclonal antibody, 50-200ng/mLCD28 are added in nutrient solution TCM-I Anti-, 50-200u/mL human interleukin-11 α, 30IU/mL human interleukin 7,30IU/mL human interleukin 15,500-2000u/mL IL-2.
TCM is according to cell density 1 × 10 in preferred steps (7)6/ mL is resuspended with TCM-III, wherein adding 500u/mL IFN-γ, 120u/mL IL-1 α, 1000u/mL IL-2,30IU/mL IL-7,30IU/mL human interleukin 15,80ng/mL CD3 Monoclonal antibody, 80ng/mL CD28.Induced amplification culture is carried out to TCM or CD8T+ cell with TCM-I culture solution after second day, is passed through 12-14 days amplification cultivations are crossed, harvest reaches the TCM cell of clinical application standard, and carries out endotoxin, activity to cell preparation And Phenotypic examination, cellular identification the result shows that, cell activity reaches 95% or more, simultaneously containing the CD8-PE/ of higher proportion CD62L+FITC/CD45RO-ECD, CD62L+FITC/CD45RO-ECD/CD44PE cell；
(8) the TCM cell of quality testing qualification is fitted into 50-100ml physiological saline, and immunocyte preparation is made, and cooperates tumour Clinical treatment needs venous re-transfusion.
(9) serum free medium and inducible factor or the raw material of TCM cell separating liquid that the present invention refers to can be from cities It is bought on, and long-term frozen saves cell and the technology of small molecular protein is highly developed, those skilled in the art are complete It can use above-mentioned technology entirely and save the culture medium and composition for forming kit of the present invention.
(10) complete the invention also includes above-mentioned TCM or CD8T+ cell sorting kit, serum-free TCM cell is expanded Culture solution TCM-I and TCM cell amplification coating liquid TCM-II and TCM-III are packed into special cleansing container and are prepared into kit.
For the present invention by coating TCM-II working solution, TCM cell Proliferation multiple improves 2-3 times, and apoptotic index reduces, living Property improve 10%-20%, this to clinically improve oncotherapy curative effect, remove residual, prevent the recurrence and transfer of tumour Have great importance.
(11) TCR Retroviral Transfer (after culture the 3rd day) TCM cell is recombinated.
According to objective gene sequence, design primer constructs recombined human reverse transcription disease by pcr clone target gene Malicious PLXPXSN-TCRa12-2-IRES-V β 7.1 is turned with after HEK293 cell Packing Intact virion with the ratio of MOI=100 Contaminate TCM.TCM-II is made into 50ug/ml working solution before transfecting, and is coated with into six orifice plates.
(12) tumour antigen is carried out again with DC load hepatic carcinoma antigen A FP and is stimulated within 5 days after cultivating.
(13) growth curve is (in vitro culture to 26 after trypan blue staining obtains CD8+T cell and the activation of TCM stimulated in vitro It).
(14) another aspect of the present invention also provides identification kit after a kind of TCM sorting and induction, the clinic TCM Cell streaming phenotype and activity identification kit include five groups of double-colored antibody compositions, are IgG-FITC/IgG-PE/IgG- respectively ECD、CD3-FITC/CD8-PE、CD8-PE/CD62L+FITC/CD45RO-ECD、CD62L+FITC/CD45RO-ECD/CD44PE And Annexin-FINKTC/PI, cleaning solution 50ml mono- pipe, fixer 50ml mono- are managed；Every two kinds of the antibody mixed according to volume 1:1 It attaches together in 1 brown EP pipe, every 3-8ul is detectable primary, amounts to 10-50 set, the cleaning solution is phosphate buffer；Institute Stating fixer is 1% paraformaldehyde.
(15) quality in the kit preparation controls identification method, requires according to GMP, correlative is sealed and is packed into Kit carries out sterile and endotoxin according to " Chinese Pharmacopoeia " and detects；
(16) quality of the cell preparation of the preparation controls identification method, including according to state food pharmaceuticals administration general bureau In " drug registration management method (the revised draft) " opinion announced " biological products registration classification and declaration material require (tentative) " Quality control is carried out, guarantees safety when clinical application.
(17) in the above method, before obtaining TCM of the present invention, with brine and the TCM cell 3-4 is resuspended It is secondary, until meet " Chinese Pharmacopoeia " require sterile, endotoxic Testing index, guarantee TCM cell of the invention feedback to Patient is that remaining nutrient media components of the present invention and inducible factor composition will not cause patient's discomfort to be reacted.It is described TCM cell composition includes 1x105-3x107TCM/ml of the invention, preferably 1x106-2x107/ml.The composition also wraps Include human serum albumin；Wherein, based on the composition, the quality volume content of the human serum albumin is 1-5%, preferably It is 5%.
(18) the present invention also provides a kind of clinical application of TCM cell composition of the present invention and cosmetic effect evaluating methods.
The present invention provides a kind of application method of TCM in clinical tumor complex treatment, including allowing patient to know The therapeutic effect of TCM of the present invention and after signing informed consent form, unrestricted choice TCM cell of the present invention is as supplementary means in tumour Use in conjunction in complex treatment.
(19) mode that preferably the TCM composition uses is instillation.Specifically, using the TCM cell composition Mode is using No. 6 syringe needles or No. 7 syringe needles, infusion apparatuses, intravenous injection or intravenous drip.It is preferable to use TCM cell combinations The mode of object is to carry out intravenous drip using No. 7 syringe needle transfusion systems in arm or lower limb.
(20) the TCM cell composition preferably comprises 3x106-2x107The TCM cell of the present invention of/ml, effective quantity are The physiological saline of 50ml-100ml/ individual combines object.
(21) the above TCM cell composition is all the dosage of an intravenous drip, three-four vein drops of a course for the treatment of Note treatment, period is continuous since for the first time or feeds back again in each interval of one day next time.After one course for the treatment of, curative effect situation is fitted Continue to be administered after two to three moons can be spaced.If it is necessary, interval time is that can also continue to re-treatment after 1 year or 2 years.
(22) standard of the therapeutic evaluation has:
1) including playing the cell factor such as interleukin-22, interleukin 12, interferon gamma and tumor necrosis factor a of immunological enhancement Th1 type cytokines change level, general increase is effective；
2) cell factor such as interleukin 6, IL-10, transforming growth factor β and the epidermal growth of immunosuppressive action are played The change level of the factor is generally reduced to effectively.
3) T lymphocyte subgroup changes: CD3, CD4, CD8, CD8-PE/CD62L+FITC/CD45RO-ECD, CD62L+ The raising or improvement of FITC/CD45RO-ECD/CD44PE positive rate, the raising of CD16/CD56 positive rate and CD4+FOX3+ Reduction.
4) other index of assessment of curative effect are also: patient tumors marker detection, such as alpha-fetoprotein, squamous cell carcinoma antigen, trip From PSA and t-PSA ratio, carcinomebryonic antigen, glycogen chain antigen 125, carbohydrate antigen CA242, cytokeratin 19 fragment CYFRA21- 1, Free beta-hCG β-HCG, neuritis specificity olefinic alcohol enzyme (NSE), carbohydrate antigen 199, total prostate are special The expression of tumour specific antigen including Specific Antigen TPSA, free prostate gland specificity antigen TPSA, carbohydrate antigen CA724 It reduces, is mainly detected by streaming fluorescence radiation method；Iconography detection, as nuclear magnetic resonance, CT and B ultrasound detect；And patient is raw Deposit phase statistics.
5) Karnofsky point system and life quality scores method.
(23) TCM cell preferably of the invention is used for self-treating, immunological rejection can be avoided to cause to greatest extent Side effect.
(25) statistical analysis
Experimental data is indicated with mean scholar standard deviation.It is for statistical analysis using SPSS16.0 statistical software.Each index is advanced Row normality and homogeneity test of variance.Two factors, two horizontal data compares (different to organize carefully using the variance analysis of 2x2 Factorial Design Born of the same parents' anti-tumor activity compares, and antibody inhibits test, the cytokine secretion of group of cells.Using the variance analysis of duplicate measurements Complete the analysis of group of cells different time place cell quantity.Using one-way analysis of variance to different groups of cell telomere lengths into Row analysis.As P < 0.05, it is considered difference with statistical significance.
Disease of the present invention includes but is not limited to kidney, malignant mela noma, lung cancer, liver cancer, colorectal cancer, gastric cancer, leaching Bar tumor and disease of viral infection include but unlimited virus hepatitis, tuberculosis and AIDS etc..
Beneficial effects of the present invention:
1) the present invention provides it is a kind of it is clinical use TCM cell high-efficient, fast preparation method, compositions related and kit and its answer Use method.The present invention is optimized the induction, culture and control of product quality of TCM cell and perfect, thin by a set of TCM The kit form of born of the same parents' induction, culture and control of product quality identification, specification TCM cell clinical application are that TCM cell high-efficient is fast Speed preparation and clinical application quality standard uniformly provide mode standard.
2) cell survival rate 93% ± 3% that PBMC recovers in the present invention, TCM yield is 21% ± 3%, CD8+ after recovery T cell yield: 58% ± 2%.
3) proliferative capacity of TCM can be improved before Fiber differentiation with TCM-II coating culture plate.
4) before tcr gene transfection, transfection efficiency can be improved with TCM-II coating culture plate.
Specific embodiment
Invention is further explained combined with specific embodiments below:
Embodiment 1
One, peripheral blood acquisition and dilution:
Sodium citrate anticoagulation 100ml is acquired, is divided into two parts, after adding 16mlbuffer CI to dilute, volume about 66ml, Preparation is added in CD8 gravity column.
Two, it recovers after PBMC freezing: taking out the PBMC after freezing 6 months, put into 37 DEG C of water-baths rapidly, and is constantly light Jog is dynamic, guarantees liquid in pipe 80% dissolution in 1 min, cell is drawn onto a certain amount of DMEM cleaning solution, and wash jelly It deposits pipe 1 time, moves into 50 mL centrifuge tubes, 233 g are centrifuged 5 min.Supernatant is abandoned, appropriate culture medium is added, mixes gently, stays It takes 500 microlitres to do counting and surveys cell activity.
Three, cell count and trypan exclusion stain survey cell activity
Countstar detects cell sample, reads the cell average value numerical value shown on screen.According to formula cell number T=cell Number/ml*V calculates total number of cells.
Four, the PBMC rate of recovery
The PBMC rate of recovery is calculated, using formula: PBMC cell recoveries=(cell number before cell number/separation after separation) × 100%.
Embodiment 2
On that basis of example 1, it is sorted with CD8+T cell or TCM sorting kit:
The preparation of T cell subgroup magnetic bead sorting kit and use
It includes that Reagent1:CD8 purified reagent is made of CD8Fab-Strep and Buffer IS that human body TCM, which sorts kit,. Reagent2:CD62L+ purified reagent is made of CD62L+Fab-Strep and Buffer IS.Reagent3:CD45RA- purifying Reagent is made of CD45RA-Fab-Strep and Buffer IS.Reagent4:Strep-Tactin Magnetic Microbeads.Reagent5: eluent is made of D-biotin and Buffer IS.
1) CD8 is rinsed with 8ml Buffer CI+T gravity column.
2) plus in the gravity column in 1mlReagent1 to step 1.
3) add the gravity column after 2 loading of 2mlBuffer CI rinsing step.
4) PBMC is according to density 1x106-1x107/ ml upper prop；Peripheral blood is according to volume 66ml upper prop.
5) it is rinsed pillar four times with 40ml Buffer CI, each 10ml.
6) cell is eluted with Reagent5,400g is centrifuged 6-10min and collects cell, obtains CD8+T cell.
7) remove supernatant, be resuspended with suitable Buffer IS, carry out the sorting of next step.
8) after taking 50-100ul Reagent2 and 150-300ulReagent4 to mix, shaking table shakes 30min.
9) 1x108 CD8+T 900-1500ul Buffer IS gravity treatment is taken, is added in above-mentioned steps 8.It is shaken in shaking table 20min。
10) plus 5mlBuffer IS is into step 9, puts and is horizontally arranged on magnetic pole, after static 3min, removes supernatant, then With 5mlBuffer IS gravity treatment.
11) step 10 is repeated.
12) cell of positive mark is resuspended in Reagent5.The static 10min of room temperature places static 3min on magnetic pole, will contain The supernatant of positive cell is transferred in new reaction tube.
13) it is washed again once with 5ml Reagent5, repeats step 12.
14) supernatant of mixing twice, 400g are centrifuged 6-10min.
15) supernatant is removed, cell is resuspended with 5mlBuffer IS, shakes 10min on shaking table.
16) it is placed on static 3min on magnetic pole.
17) supernatant is transferred in a new centrifuge tube, 400g is centrifuged 6-10min.
18) it repeats step 8 and 9,400g centrifugation 6-10min collects cell.
19) remove supernatant, be resuspended with suitable Buffer IS, carry out the sorting of next step.Pass through positive choosing twice in succession The method with the choosing of CD45RA- yin is selected, CD8+CD62L+CD45RO+ cell is obtained, endotoxin detection and phenotypic evaluation is carried out to it.
The TCM rate of recovery is calculated, using formula: TCM cell recoveries=(PBMC cell before cell number/separation after TCM sorting Number * TCM%) × 100%.See Fig. 1 and Fig. 2
Embodiment 3
The induction agent box of TCM is prepared and is used
The present invention provides a kind of TCM Fiber differentiation kit, including container and the clinic being fitted into container with TCM cell without blood Clear amplification complete culture solution TCM-I and TCM cell expands coating buffer TCM-II and inducible factor composition TCM-III:
1) present invention provides a kind of clinical serum-free TCM cell amplification complete culture solution TCM-I, must containing human body cell growth Must carbohydrate, somatomedin and hormone, vitamin, human serum albumin, amino acid, inorganic ion, microelement, Water and human interleukin 2 and human interleukin 15.The present invention is not excluded for can be using the culture except above-mentioned Serum-free complete medium Base implements culture.
2) present invention provides a kind of TCM cell amplification coating buffer TCM-II, it contain recombined human fibrin fragment and PBS, the working concentration of recombined human fibrin fragment are 12.5ug/ml-50ug/ml.
3) present invention provides a kind of TCM cell inducible factors composition TCM-III, serum-free TCM cell amplification training completely It is mono- that 300-2000u/mL human interferon gamma, 50-200ng/mLCD3 monoclonal antibody, 50-200ng/mLCD28 are added in nutrient solution TCM-I Anti-, 50-200u/mL human interleukin-11 α, 30IU/mL human interleukin 7,30IU/mL human interleukin 15,500-2000u/mL IL-2.
TCM-II was made into 12.5ug/ml working solution in D0 days, be coated with into six orifice plates, lain against big in 37 DEG C of incubators In 2 hours.TCM is according to cell density 1 × 106/ mL is resuspended with TCM-III, wherein addition 500u/mL IFN-γ, 120u/mL IL-1 α, 1000u/mL IL-2,30IU/mL IL-7,30IU/mLIL-15,80ng/mL CD3 monoclonal antibody, 80ng/mL CD28.The Amplification cultivation is carried out to TCM cell with TCM-I culture solution after two days, by 12-15 days amplification cultivations, harvest, which reaches, faced Bed applies the TCM cell of standard, and carries out endotoxin, activity and Phenotypic examination to cell preparation, cellular identification the result shows that, carefully Cytoactive reaches 95% or more, simultaneously containing CD8-PE/CD62L+FITC/CD45RO-ECD, CD62L+FITC/ of higher proportion CD45RO-ECD/CD44PE cell；See Fig. 2 and Fig. 3
Embodiment 4
Recombinate TCR Retroviral Transfer (after culture the 3rd day) CIK cell and TCM cell.
According to 7.1 sequence of liver cancer specific gene TCRa12-2 and V β, design primer, by pcr clone target gene, Recombined human retrovirus PLXPXSN-TCRa12-2-IRES-V β 7.1 is constructed, with HEK293 cell Packing Intact virus Grain, -80 DEG C of refrigerators save virion, transfect TCM with the ratio of MOI=100.
1) TCM-II is made into 50ug/ml working solution, be coated with into six orifice plates, lain against small greater than 2 in 37 DEG C of incubators When.
2) RetroNectin solution is sucked out, 0.5 ml of PBS terminate liquid is added in every hole.
3) 30 minutes are placed at room temperature for.
4) appropriate PBS cleaning orifice is added.
5) cleaning solution is sucked out, obtains the coated orifice plate of RetroNectin.
6) retrovirus saves liquid (- 80 DEG C) and melts in 37 DEG C of water-baths.
7) virus liquid is added to six orifice plates after coating, every hole 1ml.
8) it places 4 hours for 32 DEG C.
9) virus is sucked out and saves liquid.
10) cell suspension to be transfected is added in every hole.
It is stimulated again with DC load hepatic carcinoma antigen A FP tumour antigen within 5 days after culture.
Trypan blue staining obtains growth curve after the activation of TCM stimulated in vitro (in vitro culture to 26 days).
The preparation of TCM composition should all be required in GMP in above-mentioned cell cultivation process and in following embodiment hundred grades are clean Clean room or station carry out, the quality safety of strict guarantee cell and its preparation.
Above-described embodiment includes culture bag culture, the additive being designed into the embodiment of the present invention or factor additional amount Only expressed wherein some proportion, TCM cell culture that all kits are related to identification, and addition associated additives or because Sub- ratio, which is able to satisfy the concentration range in the claims, all should belong to claimed column.
Embodiment 5
Inducible factor composition TCM-III formula advanced optimizes.
Experiment is divided into 4 groups,
A group :+800 u/mL IL-7+1000IU/ml IL-2+800 u/mL of 1000IU/ml IFN-γ+120u/mL IL-1 α CD3+110ng/mL CD28+800 u/mL IL-15；
B group: 500IU/ml IFN-γ+100u/mL IL-1 α+1000IU/ml IL-2+80ng/mL CD3+80ng/mL CD28 + 30IU/mL IL-15+ 30IU/mL IL-7；
C group: 500IU/ml IFN-γ+100u/mL IL-1 α+1000IU/ml IL-2+80ng/mL CD3+80ng/mL CD28 + 30IU/mL IL-15；
D group: 500IU/ml IFN-γ+100u/mL IL-1 α+1000IU/ml IL-2+80ng/mL CD3+ 30IU/mL IL- 15；
Treatment Purity of Percentage of Percentage of
CD8+T cells CD8+TCM CD8+TEM
A group 93.56 ± 0.6 64.2 ± 2.0 29 ± 2.8
B group 93.18 ± 0.3 62.65 ± 1.51 23.53 ± 3.68
C group 92.8 ± 0.25 52.17 ± 1.91 44.59 ± 2.5
D group 90.6 ± 0.4 50.56 ± 2.11 39.13 ± 2.8
Hereafter observation cell growth state is carried out every 1-2 days addition TCM-I culture mediums using Countstar calculating instrument daily Cell count.AB group iuntercellular quantity variance is not statistically significant (F=154.12, P=0.103), and difference has system between other groups Meter learns meaning.(F=199.02, P < 0.001)
Embodiment 6
The preparation of TCM composition and clinical application
(1) the TCM cell obtained in above-described embodiment is resuspended with 0.9% medical saline, is counted, is added in cell re-suspension liquid Add human serum albumin 5%, adjusts total volume 100ml.
(2) prepare clinic in advance and 0.9% normal saline bag or bottle are housed with 100ml, above-mentioned cell composition is packed into it In, stick care label.
(3) after cell composition accordingly detects qualification, the self TCM cell therapy of clinical implementation is sent to feed back.
(4) clinical application method is described as follows:
1) approach of TCM composition administration is preferred: carrying out intravenous drip using No. 7 syringe needle transfusion systems in arm or lower limb.
2) the TCM cell composition clinical effective dose includes: composition containing 3x106-2x107/ ml cell, approach 80ml-100ml/ individual, a course for the treatment of are continuously fed back 3-4 times.
3) period is continuous since for the first time or feeds back again in each interval of one day next time.After one course for the treatment of, curative effect is fitted Situation continues to be administered after being spaced two to three moons.If it is necessary, interval time is that can also continue to control again after 1 year or 2 years It treats.
4) therapeutic evaluation is carried out after the treatment of TCM cell clinical reinfusion
After 1-2 course for the treatment of of TCM cell therapy, patients ' life quality is obviously improved before relatively treating, and the scoring of KPS point system mentions High 20-30 points, QOL point system improves 30-40 points, and patient immune function is improved in varying degrees, and wherein cytokines measurement is aobvious Showing can make Th1 Cytokines Level in Patients Undergoing improve 3-6 times.
Embodiment 7
Identification kit preparation and application after TCM sorting and induction
In culture the 15th day, identification kit identified the source TCM TE different time points after being sorted and induced with TCM of the invention The expression of CD45RO+, CD44+ and CD62L.Cell culture 15d, TCM cell proportion accounts for 99.57%, as a result sees Fig. 4
(1) single cell suspension: cell density is adjusted to 5x105-5x106/ml。
(2) sample 100ul is drawn, IgG-FITC/IgG-PE/IgG-ECD, CD3-FITC/CD8-PE, CD8-PE/ is added Each 5ul of CD62L+FITC/CD45RO-ECD, CD62L+FITC/CD45RO-ECD/CD44PE fluorescent labeled antibody, room temperature are protected from light It is incubated for 15min.
(3) cleaning solution 2ml is added, 1600rpm is centrifuged 5min.
(4) upper machine testing after going supernatant, 500ul PBS buffer solution to be resuspended.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by the limit of embodiment System, other any changes made without departing from the spirit and principles of the present invention, modification, combination, substitution, simplification should be Equivalence replacement mode, is included within the scope of the present invention.

Claims (10)

1. a kind of clinical TCM sorting kit or CD8T+ cell sorting kit, TCM induced activation and product
The kit system of amount control identification, which is characterized in that include human peripheral blood TCM sorting or the examination of CD8T+ cell sorting Agent box, TCM Fiber differentiation kit and qualified products kit, the immunocyte sorted from peripheral blood mononuclear cells are special Sign is to try the whole blood of fresh separated or the peripheral blood mononuclear cells recovered after freezing by CD8T+ cell sorting Agent box or TCM sorting kit obtain CD8+T cell or CD8+CD62L+CD45RO+ cell:
(1) human body TCM sorts kit or CD8T+ cell sorting kit includes: CD8 gravity column, Reagent1:CD8 purifying Reagent is made of CD8Fab-Strep and Buffer CI；Reagent2:CD62L+ purified reagent, by CD62L+Fab-Strep It is constituted with Buffer IS；Reagent3:CD45RA- purified reagent is made of CD45RA-Fab-Strep and Buffer IS； Reagent4:Strep-Tactin Magnetic Microbeads.Reagent5: eluent, by D-biotin and Buffer IS is constituted, and reagent consumptive material is packaged in container in the case where meeting GMP standard conditions；
(2) TCM Fiber differentiation kit expands training completely including container and the clinic being fitted into container TCM cell non-serum Nutrient solution TCM-I and TCM cell expands coating buffer TCM-II and inducible factor composition TCM-III:
Clinic expands complete culture solution TCM-I with serum-free TCM cell, containing the necessary carbohydrate of human body cell growth, Somatomedin and hormone, vitamin, human serum albumin, amino acid, inorganic ion, microelement, water, human interleukin-2 and Human interleukin 15, the present invention are not excluded for that culture can be implemented using the culture medium except above-mentioned Serum-free complete medium；
TCM cell expands coating buffer TCM-II, it contains recombined human fibrin fragment and PBS, recombined human fibrin fragment Working concentration be 12.5ug/ml-50ug/ml；
300- is added in TCM cell inducible factors composition TCM-III, serum-free TCM cell amplification complete culture solution TCM-I 2000u/mL human interferon gamma, 50-200ng/mLCD3 monoclonal antibody, 50-200ng/mLCD28 monoclonal antibody, 50-200u/mL human interleukin 1 α, 30IU/mL IL-7,30IU/mL human interleukin 15,500-2000u/mL IL-2；
Preferred steps TCM is according to cell density 1 × 106/ mL is resuspended with TCM-III, wherein addition 500u/mL IFN-γ, 120u/mL IL-1 α, 1000u/mL IL-2,30IU/mL IL-7,30IU/mL human interleukin 15,80ng/mL CD3 monoclonal antibody, 80ng/mL CD28, carries out amplification cultivation to TCM cell with TCM-I culture solution after second day, by amplification in 12-14 days Culture, harvest reaches the TCM cell of clinical application standard, and carries out endotoxin, activity and Phenotypic examination, cell to cell preparation Qualification result shows that cell activity reaches 95% or more, simultaneously containing the CD8-PE/CD62L+FITC/CD45RO- of higher proportion ECD, CD62L+FITC/CD45RO-ECD/CD44PE cell；
(3) identification kit after TCM is sorted and induced, clinical TCM cell streaming phenotype and the activity identification kit packet Five groups of double-colored antibody compositions are included, are IgG-FITC/IgG-PE/IgG-ECD, CD3-FITC/CD8-PE, CD8-PE/ respectively CD62L+FITC/CD45RO-ECD, CD62L+FITC/CD45RO-ECD/CD44PE and Annexin-FINKTC/PI, cleaning solution 50ml mono- is managed, and fixer 50ml mono- is managed；Every two kinds of the antibody are loaded in 1 brown EP pipe according to volume 1:1 mixing, every 3- 8ul is detectable primary, amounts to 10-50 set, the cleaning solution is phosphate buffer；The fixer is 1% paraformaldehyde；
(4) the invention also includes above-mentioned TCM is sorted kit, the amplification of serum-free TCM cell complete culture solution TCM-I and TCM Cell amplification coating liquid TCM-II and TCM-III are packed into special cleansing container and are prepared into kit.
2. the method for preparing TCM using kit system described in claim 1, which comprises the following steps:
Peripheral blood CD8+T cell or TCM sorting: acquisition peripheral blood in patients 80-200ml or recovery freeze PBMC cell, according to The application method of TCM sorting kit is sorted, the specific steps are as follows:
1) CD8 gravity column is rinsed with 8ml Buffer CI；
2) plus in the gravity column in 1mlReagent1 to step 1；
3) add the gravity column after 2 loading of 2mlBuffer CI rinsing step；
4) PBMC is according to density 1x106-1x107/ ml upper prop or peripheral blood are according to volume 66ml upper prop；
5) it is rinsed pillar four times with 40ml Buffer CI, each 10ml；
6) cell is eluted with Reagent5,400g is centrifuged 6-10min and collects cell, obtains CD8+T cell；
7) remove supernatant, be resuspended with suitable Buffer IS, carry out the sorting of next step；
8) after taking 50-100ul Reagent2 and 150-300ulReagent4 to mix, shaking table shakes 30min；
9) 1-2x108 PBMC 900-1500ul Buffer IS gravity treatment is taken, is added in above-mentioned steps 8,20min is shaken in shaking table； 10) plus 5mlBuffer IS is into step 9, puts and is horizontally arranged on magnetic pole, after static 3min, removes supernatant, then use 5mlBuffer IS gravity treatment；
11) step 10 is repeated；
12) cell of positive mark is resuspended in Reagent5, and the static 10min of room temperature places static 3min on magnetic pole, will contain the positive The supernatant of cell is transferred in new reaction tube；
13) it is washed again once with 5ml Reagent5, repeats step 12；
14) supernatant of mixing twice, 400g are centrifuged 6-10min；
15) supernatant is removed, cell is resuspended with 5mlBuffer IS, shakes 10min on shaking table；
16) it is placed on static 3min on magnetic pole；
17) supernatant is transferred in a new centrifuge tube, 400g is centrifuged 6-10min；
18) step 8 and 9 is repeated, 400g is centrifuged 6-10min and collects cell；
19) remove supernatant, be resuspended with suitable Buffer IS, carry out the sorting of next step；
By the method for positive selection and the choosing of CD45RA- yin twice in succession, obtain CD8+CD62L+CD45RO+ cell, to its into The detection of row endotoxin and phenotypic evaluation；
(2) TCM induction agent box uses
1) TCM-II was made into 12.5ug/ml working solution in D0 days, be coated with into six orifice plates, lain against and be greater than 2 in 37 DEG C of incubators Hour；
2) TCM is according to cell density 1 × 106/ mL is resuspended with TCM-III, wherein addition 500u/mL IFN-γ, 120u/mL IL- 1 α, 1000u/mL IL-2,30IU/mL IL-7,30IU/mL human interleukin 15,80ng/mL CD3 monoclonal antibody, 80ng/mL CD28, Amplification cultivation is carried out to TCM cell with TCM-I culture solution after second day, by 12-14 days amplification cultivations, harvest reached The TCM cell of clinical application standard, and endotoxin, activity and Phenotypic examination are carried out to cell preparation, cellular identification the result shows that, Cell activity reaches 95% or more, simultaneously containing CD8-PE/CD62L+FITC/CD45RO-ECD, CD62L+ of higher proportion FITC/CD45RO-ECD/CD44PE cell；
(3) the TCM cell of quality testing qualification is fitted into 50-100ml physiological saline, and immunocyte preparation is made, and cooperates tumour Clinical treatment needs venous re-transfusion.
3. memory immune cell sorting according to claim 2, it is characterised in that PBMC is according to density in step (1) 1x106-1x107/ ml upper prop or acquisition peripheral blood whole blood 80-200ml upper prop, the cell for sorting acquisition includes CD8T+ or TCM, Six orifice plates are coated with using TCM-II, clinical application TCM cell is obtained using T75, T175 culture bottle and the production of 1L culture bag, is trained Supporting the time is 12-28 days.
4. memory immune cell according to claim 2, it is characterised in that use recombination TCR retrovirus after induction (after culture the 3rd day) TCM cell is transfected, TCM-II is made into 50ug/ml working solution before transfection, is coated with into six orifice plates, is put down It is put in 37 DEG C of incubators and is greater than 2 hours.
5. memory immune cell according to claim 2, it is characterised in that resisted with DC load hepatic carcinoma within 5 days after culture Former AFP carries out tumour antigen to be stimulated again.
6. memory immune cell according to claim 2, it is characterised in that the quality control in the kit preparation Identification method is required according to GMP, and correlative is sealed and is packed into kit, carries out sterile and endotoxin according to " Chinese Pharmacopoeia " Detection；The quality of cell preparation controls identification method, " the drug registration pipe announced according to state food pharmaceuticals administration general bureau Reason method (revised draft) " in opinion " biological products registration classification and declaration material require (tentative) " carry out quality control, guarantee Safety when clinical application.
7. memory immune cell according to claim 2, it is characterised in that before obtaining TCM of the present invention, use physiology Salt water washing, which is laid equal stress on, hangs described TCM cell 3-4 times, until meet sterile, the endotoxic Testing index that " Chinese Pharmacopoeia " requires, Guarantee that TCM cell of the invention feeding back to patient is remaining nutrient media components of the present invention and inducible factor composition Patient's discomfort will not be caused to react.
8. the TCM cell composition includes 3x106-2x107The TCM cell of the present invention of/ml, effective quantity 50ml-100ml/ The physiological saline of individual combines object, density 1x105-3x107/ ml, preferably 1x106-2x107/ ml further includes the white egg of people's blood It is white；Wherein, based on the composition, the quality volume content of the human serum albumin is 1-5%, preferably 5%.
9. memory immune cell according to claim 2, it is characterised in that the mode that above-mentioned steps TCM composition uses To instil, specifically, the mode using the TCM cell composition is using No. 6 syringe needles or No. 7 syringe needles, infusion apparatuses, vein note It penetrates or intravenous drip is, it is preferable to use the mode of the TCM cell composition is in arm or lower limb using No. 7 syringe needle transfusion systems Intravenous drip is carried out, TCM cell composition described above is all the dosage of an intravenous drip, three-four veins of a course for the treatment of It instils and treats, period is continuous since for the first time or feeds back again in each interval of one day next time, after a course for the treatment of, fits curative effect feelings Condition continues to be administered after being spaced two to three moons, if it is necessary, interval time is that can also continue to control again after 1 year or 2 years It treats.
10. a kind of memory immune cell of any of claims 1-9 is in clinical cancer therapy, viral infection disease Disease and drug in application, including but not limited to kidney, malignant mela noma, lung cancer, liver cancer, colorectal cancer, gastric cancer, lymthoma and Disease of viral infection includes but unlimited virus hepatitis, tuberculosis and AIDS etc..
CN201811194036.2A 2018-10-15 2018-10-15 A kind of optimization it is clinical with TCM sorting, Fiber differentiation and quality control identification kit and application Pending CN109266607A (en)

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Application publication date: 20190125