CN104946589A - Isolated culturing method for tumor-specific TIL cells - Google Patents
Isolated culturing method for tumor-specific TIL cells Download PDFInfo
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Abstract
The invention relates to an isolated culturing method for tumor-specific TIL cells. According to the method, one or more from PD-1, LAC-3 and TIM 3 are utilized for serving as markers for separating tumor specific T-lymphocytes in TIL cells, the immunomagnetic bead technology is combined, the tumor specific T-lymphocytes are separated from a TIL cell mass, then in-vitro efficient amplification is conducted, and the tumor-specific TIL cells with high tumor killing activity are obtained. By means of the isolated culturing method for the tumor-specific TIL cells, the separated culturing operation procedure of the tumor-specific TIL cells is made to be simpler, the separated culturing time is obviously shortened, the cost is low, the application and popularization are facilitated, and moreover through the separation, the obtained tumor-specific TIL cells are higher in tumor cell killing activity.
Description
Technical field
The invention belongs to technical field of cell culture, relate to the isolation cultivation method of tumour-specific til cell (tumor infiltrating lymphocyte).
Background technology
Tumor infiltrating lymphocyte (TIL) is present in the lymphocyte in tumor tissues, tumor-draining lymphode, cancerous thoracoascites, compared to peripheral blood lymphocyte, the tumor specific cytotoxic T lymphocyte of the activation containing higher proportion in til cell, this quasi-lymphocyte is after in-vitro separation, amplification, activation, the killing activity of tumour cell is improved, can be used for the adoptive cellular immunotherapy of tumour.
This technology is used to achieve good clinical effectiveness.Rosenberg etc. adopt body irradiation+chemotherapy+til cell treatment malignant melanoma, total effective rate reaches 72% (Rosenberg SA1, Dudley ME.Adoptive Cell Therapy for the Treatment of Patients with Metastatic Melanoma.Curr Opin Immunol.2009Apr, 21 (2): 233 – 240.), Li J etc. adopt chemoradiotherapy+til cell treatment nasopharyngeal carcinoma, 19 people are had to have objective antitumor reaction (Li J1 in 20 people, Chen QY2, He J1, Li ZL1, Tang XF1, Chen SP1, Xie CM3, Li YQ1, Huang LX1, Ye SB1, Ke M1, Tang LQ2, Liu H2, Zhang L2, Guo SS2, Xia JC1, Zhang XS1, Zheng LM1, Guo X2, Qian CN2, Mai HQ2, Zeng YX4.Phase I trial of adoptively transferred tumor-infiltrating lymphocyte immunotherapy following concurrent chemoradiotherapy in patients with locoregionally advanced nasopharyngeal carcinoma.Oncoimmunology.2015Mar 6, 4 (2): e976507.eCollection 2015.), Khammari A etc. express the Adenoviral Therapy of IFN-r with til cell in conjunction with intratumor injection and to feel sick melanoma, achieve disease control rate (the Khammari A1 of 46%, Nguyen JM, Saint-Jean M, Knol AC, Pandolfino MC, Quereux G, Brocard A, Peuvrel L, Saiagh S, Bataille V, Limacher JM, Dreno B.Adoptive T cell therapy combined with intralesional administrations of TG1042 (adenovirus expressing interferon-γ) in metastatic melanoma patients.Cancer Immunol Immunother.2015Apr 7.).
The source of the til cell of current clinical application mainly contains two aspects: 1. the tumor tissues of excision or lymphoglandula; 2. cancerous thoracoascites.From surgical samples, be separated til cell generally need through mechanical shearing, multiple steps such as the enzymolysis of multiple protein enzyme; The method that the til cell in cancerous thoracoascites source is separated is relatively simple, also easily cultivates in vitro.
Due to the cell mass that til cell is a kind of heterogeneity, comprise T cell, B cell, NK cell etc., wherein play the tumor specific cytotoxic T lymphocyte mainly activated of antitumor action, the gordian technique of therefore til cell treatment be how to filter out from TIL activation tumor specific cytotoxic T lymphocyte and carry out amplification cultivation.
Traditional til cell is separated and culturing step complexity, and nearly 2 months whole operating time, wherein the process of separation andpreconcentration tumour-specific til cell is the most complicated, consuming time the longest.First tumor tissues is needed to be made into little block organization or single cell suspension, be divided into a lot of aliquot then to cultivate with heavy dose of IL-2, after cultivating 4-6 week, every part of take a morsel cell and tumour cell carry out Dual culture, determine that there is tumor specific cytotoxic T lymphocyte which part the inside by the content of IFN-r in the supernatant after test Dual culture.Then the cell got in this some holes carries out rapid amplifying cultivation.This working method step is complicated, and the time is long, and sepn process is subject to the impact of operator's subjective factor, and the result difference of different operator's operations is very large.Long operating process easily causes polluting, and cell viability reduces.Therefore have impact on the effect of til cell clinical application largely.
Therefore two of TIL treatment large crucial technology, one is the T lymphocyte that will find specific for tumour antigen from tumor tissues, and it is separated; Two is the T lymphocyte of these preciousnesses will be increased in a large number.The tumor specific cytotoxic T lymphocyte with tumour-specific killing activity to be separated from tumor tissues, need to use the surface markers that can be distinguished tumor specific cytotoxic T lymphocyte.T lymphocyte specific for tumour antigen in tumor tissues is a kind of activated T lymphocyte, and PD-1, LAG-3 and TIM-3 are the Inhibitory receptor of abduction delivering after T cell activation, with activation and the function of suppressor T cell after its ligand binding.The T lymphocyte in til cell with tumour-specific killing activity is just present in (Alena Gros etc.PD-1identifies the patient-specific CD8 in the T lymphocyte that this group activated
+tumor-reactive repertoire infiltrating human tumors.The Journal of Clinical Investigation.Volume 124Number 5May 2014).Therefore PD-1, LAG-3 can as the marks being separated tumor specific cytotoxic T lymphocyte in til cell with TIM-3.
Immunomagnetic beads (Immunomagnetic bead, IMB) technology is a kind of immunological technique, and IMB is the ball-type magnetic particle being coated with antibody, can be combined specifically make it to have magnetic responsiveness with target material.The advantage of IMB technology is to ensure that the morphology and function of separated target cell is complete, also have simultaneously highly sensitive, specificity is high, detection speed is fast, reproducible, simple to operate and do not need the advantages such as expensive plant and instrument.Carry out cellular segregation by two kinds of modes by IMB technology, a kind of is that the method directly isolating target cell from cell mixture is called positive separation; Negative separation is called by the method that the irrelevant cell of IMB removal makes target cell be able to purifying.Therefore immunomagnetic beads is utilized directly to be separated from til cell group by the tumor specific cytotoxic T lymphocyte with tumour-specific killing activity.
Bibliographical information is not had to utilize PD-1, LAG-3 and at least one in TIM-3 as the mark being separated tumor specific cytotoxic T lymphocyte in til cell at present, and tumor specific cytotoxic T lymphocyte is separated by binding immunoassay magnetic bead technology from til cell group, then obtain the method for tumour-specific til cell through external efficient amplification.
Summary of the invention
The object of the invention is to easily cause for prior art separation and Culture til cell complicated operation, consuming time, long operating process the deficiency polluted, cell viability reduces, provide a kind of isolation cultivation method of til cell, the tumor specific cytotoxic T lymphocyte with tumour-specific killing activity is separated and carries out a large amount of amplification cultivation by the method from til cell group, operating process is simpler, spended time obviously shortens, be convenient to promote the use of, be separated the tumour-specific til cell killing activity obtained stronger.
Technical scheme of the present invention is: step one, the preparation of tumor tissues single cell suspension, from tumor tissue or cancerous thoracoascites, namely obtain the single cell suspension containing tumor specific cytotoxic T lymphocyte; Step 2: use immunomagnetic bead technique to be separated by described single cell suspension, separate from the mixtures such as tumour cell, the non-specific T lymphocyte of tumour, suppressor T cell, inoblast by tumor specific cytotoxic T lymphocyte, obtain tumour-specific til cell; Step 3: the rapid amplifying of tumour-specific til cell, carries out efficient amplification cultivation by described tumour-specific til cell in efficient amplification culture system, obtains a large amount of tumour-specific til cells with specific killing activity of tumor cells.
Preferably, in step one, the preparation of described tumor tissues single cell suspension, specifically comprises the steps:
1.1 get tumor tissue, remove healthy tissues and necrotic tissue, to be cut into small pieces tissue with scissors, these little tissue block are immersed in serum-free RPMI 1640 substratum containing 0.2-20mg/ml collagenase, 0.01-10mg/ml Unidasa, 0.01-10mg/ml DNA enzymatic, slowly rock overnight incubation, filter, obtain individual cells suspension.
If the single cell suspension obtained containing tumor specific cytotoxic T lymphocyte does not need above-mentioned steps 1 from cancerous thoracoascites, directly enter following steps:
1.2 individual cells suspensions or cancerous thoracoascites D-Hanks balanced salt solution washing, be separated mononuclearcell with Ficoll density gradient method, obtain tumor tissues single cell suspension.
Preferably, in step 2, described single cell suspension is separated by described use immunomagnetic bead technique, specifically comprises the steps:
2.1 press every 1 × 10 in step one gained tumor tissues single cell suspension
8individual cell adds 0.4ml PBS buffered soln, 10-500ul biotin labeling or fluorochrome label (comprise Cy5/Anti-Alexa Fluor 647, Cy7, FITC, PE, the marks such as APC) or unlabelled PD-1, LAG-3, at least one in the antibody-solutions of TIM-3, 10-500ul Fc receptor blocking agent, the 20-1000ul antibiotin magnetic bead corresponding with antibody labeling or anti-fluorescence dye magnetic bead (comprise Anti-Cy5/Anti-Alexa Fluor647 MicroBeads, Anti-Cy7 MicroBeads, Anti-FITC MicroBeads, Anti-PE MicroBeads, Anti-APC MicroBeads) or anti-Ig magnetic bead, mixing, resuspended cell suspension.
Described cell suspension joins in cell sorting post by 2.2, utilizes magnetic separator to collect tumour-specific til cell.
Preferably, in step 3, the rapid amplifying of described tumour-specific til cell, specifically comprises the steps:
1. tumour-specific til cell step 2 obtained and following reagent and factor mixed culture: CM and AIM-V mixed culture medium, the PBMC of allosome radiation lethal (50Gy), OKT3 antibody.
2. within the 2nd day, add IL-2 100-10000IU/ml.
3. carry out expansion bottle (bag) according to cell count to cultivate, substratum is CM and the AIM-V mixed culture medium containing IL-2.
4. amplification cultivation is to 7-30 days, obtains final a large amount of tumour-specific til cell.
Preferably, in step 3, described CM and AIM-V mixed culture medium is CM substratum, AIM-V substratum mixes according to the ratio of 1:1.
Preferably, in step 3, the PBMC of described allosome radiation lethal (50Gy) and the ratio of described tumour-specific til cell are 10:1-1000:1.
Preferably, in step 3, described OKT3 antibody, adds the ratio of 0.01-0.1ug OKT3 antibody in every 1.0ml substratum.
Use the tumour-specific til cell of isolation cultivation method separation and Culture that the present invention relates to for the treatment of tumour or prevention.
Compared with prior art, the present invention has following beneficial effect:
1. the isolation cultivation method of til cell of the present invention is simpler, and cell culture period obviously shortens, and greatly reduces toxigenic capacity;
2. the present invention utilizes immunomagnetic bead technique to be separated tumor specific cytotoxic T lymphocyte, make disengaging time shorten to 1 day week by 4-5 compared to existing technology, and operating process is simpler, is convenient to promote the use of.
3. tumor specific cytotoxic T lymphocyte is separated by the Inhibitory receptor that the present invention utilizes the T cell of activation to express from the cell mass of complexity, adds the specific aim of separation, makes the anti-tumor activity finally cultivating the tumour-specific til cell obtained stronger.
Accompanying drawing explanation
Fig. 1 til cell that to be embodiment 1 obtain with comparative example 1 kills the result that knurl is tested;
Fig. 2 til cell that to be embodiment 2 obtain with comparative example 2 kills the result that knurl is tested;
Fig. 3 til cell that to be embodiment 3 obtain with comparative example 3 kills the result that knurl is tested;
Fig. 4 til cell that to be embodiment 4 obtain with comparative example 4 kills the result that knurl is tested;
Fig. 5 is the result that til cell that embodiment 5,6 obtains kills knurl experiment;
Embodiment
The present invention is further illustrated by the following examples, but the present invention is not limited to these embodiments.
Embodiment 1
The tumor tissues now getting a liver cancer patient (female, 48 years old) excision is the material preparing tumour-specific til cell, and tumour-specific til cell separation and Culture process is as follows:
Step 1: the preparation of tumor tissues single cell suspension.
1.1 get tumor tissue, remove healthy tissues and necrotic tissue, the little block organization of diameter 2mm is cut into scissors, these little tissue block are immersed containing 2.0mg/ml collagenase (SIGMA, article No.: C9722), 0.5mg/ml Unidasa (SIGMA, article No.: H3506), in serum-free RPMI 1640 substratum of (10-0.01) 0.1mg/ml DNA enzymatic (SIGMA, article No.: D5319), slowly rock overnight incubation.Cross with 100um nylon leaching net and filter cell mass, obtain individual cells suspension.
1.2 individual cells suspension D-Hanks solution wash 2 times, sedimentation cell adds appropriate PBS buffered soln (containing 0.5%BSA, 2mmol EDTA, PH 7.2) be separated mononuclearcell with Ficoll lymphocyte separation medium, resuspended with PBS buffered soln after counting, obtain final tumor tissues single cell suspension.
Step 2: be separated with immunomagnetic bead technique and obtain tumour-specific til cell.
The cell concn of the final gained of 2.1 set-up procedures 1 is every 1.0ml buffered soln 2.5 × 10
8individual cell.
2.2 add 100ul Fc receptor blocking agent (Miltenyi Biotec, article No. 130-059-901), and mixing, 2-8 degree Celsius of refrigerator places 10 minutes.
2.3 add 10-20ml PBS buffered soln 300xg centrifugal 10 minutes, remove supernatant.
2.4 add 0.4ml PBS buffered soln, 100ul CD279 (PD1)-Biotin solution (Miltenyi Biotec, article No. 130-096-162), and mixing, 2-8 degree Celsius of refrigerator places 10 minutes.
2.5 add 10-20ml PBS buffered soln 300xg centrifugal 10 minutes, remove supernatant.
2.6 add 0.8ml PBS buffered soln, 200ul antibiotin magnetic bead (Miltenyi Biotec, article No. 130-090-485), and mixing, 2-8 degree Celsius of refrigerator places 15 minutes.
2.7 add 10-20ml PBS buffered soln 300xg centrifugal 10 minutes, remove supernatant, add that 1ml buffered soln is resuspended obtains cell suspension.
LD post is placed in MACS magnetic separator by 2.8, with the rinse of 2ml PBS buffered soln once.
2.9 add step 2.5 gained cell suspension in LD post, collect by the unlabelled cell of LD post in A pipe, then wash 2 times (each use 1ml PBS buffered soln) with PBS buffered soln, and washing lotion is also collected in A pipe, obtains unlabelled cell.
LD post takes out by 2.10 from magnetic separator, adds 2ml damping fluid push rod and is come out by the cell of mark, collects, obtain tumour-specific til cell with B pipe.
Step 3: the rapid amplifying of tumour-specific til cell.
3.1 tumour-specific til cells step 2 obtained are by every 1 × 10
6cell and following reagent and factor mixed culture: CM substratum (adds 25mmol/L HEPES PH 7.2 in RPMI1640 substratum, 100IU/mL penicillin (penicillin), 100ug/mL Streptomycin sulphate (streptomycin), 2mmol/L glutamine (L-glutamine), 5.5 × 10
-5mol/L beta-mercaptoethanol (β-mercaptoethanol), 10% people AB serum) 75ml, 2 × 10
8the PBMC of individual allosome radiation lethal (50Gy), 4.5ug OKT3 antibody, 75ml AIM-V substratum.
Within 3.2 the 2nd days, add IL-2 to 6000IU/ml.
3.3 the 5th days, remove 120ml supernatant, then add the mixed culture medium of 120ml containing 50%CM and 50%AIM-V of 6000IU/ml IL-2.
Within 3.4 the 6th days, carry out expansion bottle (bag) according to cell count to cultivate, cell concn is adjusted to 1 × 10
6/ ml, substratum is the mixed culture medium of 50%CM and 50%AIM-V of 6000IU/ml IL-2.
3.5 amplification cultivation, by the 14th day, obtain final a large amount of tumour-specific til cell.
Embodiment 2
The tumor tissues now getting a colorectal cancer patients (man, 53 years old) excision is the material preparing tumour-specific til cell, and tumour-specific til cell separation and Culture process is as follows:
Step 1 and step 3 are with embodiment 1;
The operating process of step 2 is with embodiment 1, and the antibody added in step 2.4 is different, is Anti-TIM-3-APC (Miltenyi Biotec, article No. 130-098-936); The magnetic bead added in step 2.6 is different, is anti-APC magnetic bead (Miltenyi Biotec, article No. 130-090-855).
Embodiment 3
The tumor tissues getting a colorectal cancer patients (female, 58 years old) excision is the material preparing tumour-specific til cell, and tumour-specific til cell separation and Culture process is as follows:
Step 1 and step 3 are with embodiment 1;
The operating process of step 2 is with embodiment 1, and difference has: the antibody added in step 2.4 is different, is mouse-anti people LAG-3 antibody (R & D SYSTEMS, article No.: MAB23193, IgG1); The magnetic bead added in step 2.6 is different, is against murine IgG1 magnetic bead (Miltenyi Biotec, article No. 130-047-101)
Embodiment 4
The cancerous hydrothorax getting a liver cancer patient (man, 58 years old) is the material preparing tumour-specific til cell, and tumour-specific til cell separation and Culture process is as follows:
Step 1: the preparation of tumor tissues single cell suspension
Cancerous hydrothorax 1000ml, 1500 leave the heart obtains cell precipitation in 10 minutes, sedimentation cell adds appropriate PBS buffered soln (containing 0.5%BSA, 2mmol EDTA, PH 7.2) be separated mononuclearcell with Ficoll, resuspended with PBS buffered soln after counting, obtain final tumor tissues single cell suspension.
Step 2 and 3 is with embodiment 1.
Embodiment 5,6
The tumor tissues getting a liver cancer patient (female, 68 years old) excision is the material that embodiment 5,6 prepares tumour-specific til cell, and tumour-specific til cell separation and Culture process is as follows:
The operating process of step 1 ~ 3 is with embodiment 1, and difference is:
(1) different amts of collagenase, Unidasa, DNA enzymatic is added in step 1.1:
Embodiment 5:0.2mg/ml collagenase, 0.01mg/ml Unidasa, 0.01mg/ml DNA enzymatic;
Embodiment 6:20mg/ml collagenase, 10mg/ml Unidasa, 10mg/ml DNA enzymatic.
(2) different amts of antibody-solutions, Fc receptor blocking agent, antibiotin magnetic bead is added in step 2:
Embodiment 5:10ul antibody-solutions, 10ul Fc receptor blocking agent, 20ul antibiotin magnetic bead;
Embodiment 6:500ul antibody-solutions, 500ul Fc receptor blocking agent, 1000ul antibiotin magnetic bead.
(3) different amts of PBMC, OKT3 antibody of IL-2, allosome radiation lethal (50Gy) is added in step 3:
The PBMC 1.0 × 10 of embodiment 5:IL-2 100IU/ml, allosome radiation lethal (50Gy)
7individual, OKT3 antibody 1.5ug;
The PBMC 1.0 × 10 of embodiment 6:IL-2 10000IU/ml, allosome radiation lethal (50Gy)
9individual, OKT3 antibody 15ug.
(4) in step 3, the number of days of cell cultures is different:
Embodiment 5: amplification cultivation, by the 7th day, obtains final a large amount of tumour-specific til cell;
Embodiment 6: amplification cultivation, by the 30th day, obtains final a large amount of tumour-specific til cell.
Comparative example 1 ~ 4
The tumor tissues single cell suspension of comparative example 1 Example 1 step 1 preparation is the original material preparing til cell;
The tumor tissues single cell suspension of comparative example 2 Example 2 step 1 preparation is the original material preparing til cell;
The tumor tissues single cell suspension of comparative example 3 Example 3 step 1 preparation is the original material preparing til cell;
The tumor tissues single cell suspension of comparative example 4 Example 4 step 1 preparation is the original material preparing til cell.
The til cell separation and Culture process of comparative example 1 ~ 4 is identical, as follows:
Step 1: prepare autologous tumor cell strain
By resuspended for cell suspension CM substratum, join on 2 grades of density gradient separation liquid that (lower floor is 100%Ficoll, middle layer is the mixed solution of 75%Ficoll and 25%CM nutrient solution), 2000 leave the heart 20 minutes, and the cell collected between upper strata and middle layer obtains the tumour cell be enriched.Adjusting cell concn with RPMI 1640 substratum containing 10%FBS is 2 × 10
5individual/ml, is inoculated in culturing bottle and cultivates.
The primary amplification of step 2:TIL cell
Single cell suspension CM substratum adjustment cell concn is 5 × 10
5individual/ml, adds 6000IU/ml IL-2, is seeded in 24 orifice plates, every hole inoculation 2ml, puts into 37 DEG C, 5%CO2 incubator cultivates.
Whether apertures endolymph Growth of Cells how, is no more than one week and will carries out once partly measuring changing liquid.When the lymphocyte in any one hole covers with, by porose in cell mixing, be divided into 2 parts, add containing 6000IU/ml IL-2 CM substratum continue cultivate.After this, carry out weekly 2 half amounts and change liquid.Or cell concn is adjusted to 0.8 ~ 1.6 × 10
6individual/ml.4-5 week of this process need.
Step 3:IFN-r release experiment
3.1 get the til cell that step 3 obtains joins in 96 orifice bores together with the ratio of 1:1 with the autologous tumor cell that step 2 obtains, and adds 200ul containing 10%FBS RPMI 1640 substratum, Dual culture 12 hours.
3.2 every holes are got supernatant 100ul and are detected IFN-r concentration (BD for ELISA method, article No.: 550612), tumoricidal til cell is defined as above-mentioned IFN-r concentration >200pg/ml, thus filters out tumoricidal til cell.
The amplification of step 4:TIL cell.
4.1 tumoricidal til cells step 4 filtered out are by every 1 × 10
6cell and following reagent and factor mixed culture: CM substratum 75ml, 2 × 10
8the PBMC of allosome radiation lethal (50Gy), 4.5ug OKT3 antibody, 75ml AIM-V substratum.
Within 4.2 the 2nd days, add IL-2 to 6000IU/ml.
4.3 the 5th days, remove 120ml supernatant, then add the mixed culture medium of 120ml containing 50%CM and 50%AIM-V of 6000IU/ml IL-2.
Within 4.4 the 6th days, carry out expansion bottle (bag) according to cell count to cultivate, cell concn is adjusted to 1 × 10
6/ ml, substratum is the mixed culture medium of 50%CM and 50%AIM-V of 6000IU/ml IL-2.
4.5 amplification cultivation, to the 14th day, obtain final til cell.
Kill knurl experiment
Step one: prepare autologous tumor cell strain
By resuspended for embodiment 5,6 step one gained cell suspension CM substratum, join on 2 grades of density gradient separation liquid that (lower floor is 100%Ficoll, middle layer is the mixed solution of 75%Ficoll and 25%CM nutrient solution), 2000 leave the heart 20 minutes, and the cell collected between upper strata and middle layer obtains the tumour cell be enriched.Adjusting cell concn with RPMI 1640 substratum containing 10%FBS is 2 × 10
5individual/ml, is inoculated in culturing bottle and cultivates.
Step 2:
Respectively by the til cell action effect cell arrived final in the tumour-specific til cell finally obtained in above-described embodiment 1 ~ 6 and comparative example 1 ~ 4, the autologous tumor cell strain of the autologous tumor cell obtained with comparative example 1 ~ 4 step 2 respectively and the preparation of above step one is for target cell.
1. get effector cell, wash 2 times with RPMI 1640 substratum, be made into 1 × 10 with RPMI 1640 substratum containing 10%FBS
6/ ml cell suspension.
2. get target cell, wash once with RPMI 1640 substratum, be made into 1 × 10
5/ ml cell suspension.
3. be inoculated in 96 orifice plates by effect target than 10:1, arrange effector cell hole and Target cell wells simultaneously, by the multiple hole of often kind of substratum 3,37 degree of 5%CO2 cultivate 4 hours.
4. survey absorbance with mtt assay, calculate and kill ratio of outflow.
Kill ratio of outflow %=1-(test holes-effector cell hole)/Target cell wells.
Result:
What the til cell that embodiment 1 and comparative example 1 obtain killed that knurl tests the results are shown in Figure 1;
What the til cell that embodiment 2 and comparative example 2 obtain killed that knurl tests the results are shown in Figure 2;
What the til cell that embodiment 3 and comparative example 3 obtain killed that knurl tests the results are shown in Figure 3;
What the til cell that embodiment 4 and comparative example 4 obtain killed that knurl tests the results are shown in Figure 4;
The til cell that embodiment 5,6 obtains kill knurl experiment the results are shown in Figure 5;
Conclusion:
As can be seen from embodiment 1 ~ 4 and comparative example 1 ~ 4, compared to the method that comparative example 1 ~ 4 is used, adopt the method for the present invention separation tumor specific cytotoxic T lymphocyte time to shorten to 1 day by 4-5 week, reduce costs, and operating process is simpler, is convenient to promote the use of.
As can be seen from Figure 1, the til cell that the anti-tumor activity of tumour-specific til cell that embodiment 1 obtains obtains higher than comparative example 1;
As can be seen from Figure 2, the til cell that the anti-tumor activity of tumour-specific til cell that embodiment 2 obtains obtains higher than comparative example 2;
As can be seen from Figure 3, the til cell that the anti-tumor activity of tumour-specific til cell that embodiment 3 obtains obtains higher than comparative example 3;
As can be seen from Figure 4, the til cell that the anti-tumor activity of tumour-specific til cell that embodiment 4 obtains obtains higher than comparative example 4;
Therefore, tumor specific cytotoxic T lymphocyte is separated by the Inhibitory receptor that the present invention utilizes the T cell of activation to express from the cell mass of complexity, add the specific aim of separation, make the anti-tumor activity finally cultivating the tumour-specific til cell obtained stronger.
Be to be understood that, although this specification sheets is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of specification sheets is only for clarity sake, those skilled in the art should by specification sheets integrally, technical scheme in each embodiment also through appropriately combined, can form other embodiments that it will be appreciated by those skilled in the art that.
A series of detailed description listed is above only illustrating for feasibility embodiment of the present invention; they are also not used to limit the scope of the invention, all do not depart from the skill of the present invention equivalent implementations done of spirit or change all should be included within protection scope of the present invention.
Claims (10)
1. the isolation cultivation method of tumour-specific til cell, is characterized in that, comprises the steps:
Step one, the preparation of tumor tissues single cell suspension, namely obtains the single cell suspension containing tumor specific cytotoxic T lymphocyte from tumor tissue or cancerous thoracoascites;
Step 2: use immunomagnetic bead technique to be separated by described single cell suspension, obtain tumour-specific til cell;
Step 3: the rapid amplifying of tumour-specific til cell, carries out efficient amplification cultivation by described tumour-specific til cell in efficient amplification culture system, obtains a large amount of tumour-specific til cells with specific killing activity of tumor cells.
2. the isolation cultivation method of tumour-specific til cell as claimed in claim 1, it is characterized in that, in step one, the preparation of described tumor tissues single cell suspension, specifically comprises the steps:
A () gets tumor tissue, remove healthy tissues and necrotic tissue, to be cut into small pieces tissue with scissors, these little tissue block are immersed in serum-free RPMI 1640 substratum containing collagenase, Unidasa, DNA enzymatic, slowly rock overnight incubation, filter, obtain individual cells suspension.
If the single cell suspension obtained containing tumor specific cytotoxic T lymphocyte does not need above-mentioned steps (a) from cancerous thoracoascites, directly enter following steps:
B () individual cells suspension or cancerous thoracoascites D-Hanks balanced salt solution washing, be separated mononuclearcell with Ficoll density gradient method, obtain tumor tissues single cell suspension.
3. the isolation cultivation method of tumour-specific til cell as claimed in claim 1, it is characterized in that, in step 2, described single cell suspension is separated by described use immunomagnetic bead technique, specifically comprises the steps:
C () adds PBS buffered soln, antibody-solutions, Fc receptor blocking agent, magnetic bead in step one gained tumor tissues single cell suspension, mixing, resuspended cell suspension.
D described cell suspension joins in cell sorting post by (), utilize magnetic separator to collect tumour-specific til cell.
4. the isolation cultivation method of tumour-specific til cell as claimed in claim 1, it is characterized in that, in step 3, the rapid amplifying of described tumour-specific til cell, specifically comprises the steps:
E tumour-specific til cell that step 2 obtains by () and following reagent and factor mixed culture: CM and AIM-V mixed culture medium, the PBMC of allosome radiation lethal (50Gy), OKT3 antibody.
F () adds IL-2 on the 2nd day;
G () is carried out expansion bottle (bag) according to cell count and is cultivated, substratum is CM and the AIM-V mixed culture medium containing IL-2;
H () amplification cultivation, to 7-30 days, obtains final a large amount of tumour-specific til cell.
5. the isolation cultivation method of tumour-specific til cell as claimed in claim 2, it is characterized in that, described serum-free RPMI 1640 substratum containing collagenase, Unidasa, DNA enzymatic, the concentration of collagenase is 0.2-20mg/ml, the concentration of Unidasa is 0.01-10mg/ml, and the concentration of DNA enzymatic is 0.01-10mg/ml.
6. the isolation cultivation method of tumour-specific til cell as claimed in claim 3, it is characterized in that, antibody is at least one in vitamin H (Biotin) mark or fluorochrome label or unlabelled PD-1 antibody, LAG-3 antibody and TIM-3 antibody; Magnetic bead is the antibiotin magnetic bead corresponding with antibody labeling or anti-fluorescence dye magnetic bead or anti-Ig magnetic bead.
7. the isolation cultivation method of tumour-specific til cell as claimed in claim 6, is characterized in that, by every 1.0 × 10
8individual cell adds antibody-solutions 10-500ul, Fc receptor blocking agent 10-500ul, magnetic bead 20-1000ul.
8. the isolation cultivation method of tumour-specific til cell as claimed in claim 4, is characterized in that, described CM and AIM-V mixed culture medium is CM substratum, AIM-V substratum mixes according to the ratio of 1:1.
9. the isolation cultivation method of tumour-specific til cell as claimed in claim 4, it is characterized in that, the PBMC of described allosome radiation lethal (50Gy) and the ratio of described tumour-specific til cell are 10:1-1000:1; Described OKT3 antibody, adds the ratio of 0.01-0.1ug OKT3 antibody in every 1.0ml substratum.
10. the tumour-specific til cell as the isolation cultivation method separation and Culture in claim 1 ~ 9 as described in any one is used for treatment or the prevention of tumour.
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Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105969731A (en) * | 2016-07-29 | 2016-09-28 | 解西河 | Method for preparing high-killing-activity TIL cells in batches from malignant pleuroperitoneal fluid |
| CN106119194A (en) * | 2016-08-04 | 2016-11-16 | 英普乐孚生物技术(上海)有限公司 | A kind of isolated culture method of the til cell in Malignant Pleural source |
| CN106222139A (en) * | 2016-07-29 | 2016-12-14 | 解西河 | A kind of method utilizing concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number |
| CN106244538A (en) * | 2016-08-04 | 2016-12-21 | 英普乐孚生物技术(上海)有限公司 | A kind of isolated culture method of the til cell in malignant ascite source |
| CN106754703A (en) * | 2016-12-26 | 2017-05-31 | 浙江丹晖生物科技有限公司 | A kind of til cell amplification in vitro culture medium combination and cultural method |
| CN107384867A (en) * | 2017-08-04 | 2017-11-24 | 北京世纪劲得生物技术有限公司 | A kind of tumor tissues til cell preparation method and special culture media |
| CN107502589A (en) * | 2017-08-04 | 2017-12-22 | 北京世纪劲得生物技术有限公司 | A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method |
| CN107760754A (en) * | 2016-08-18 | 2018-03-06 | 上海厚超生物科技有限公司 | The method of vitro detection immunocyte killing-efficiency |
| CN108753717A (en) * | 2018-06-20 | 2018-11-06 | 淮安诺康生物科技有限公司 | A kind of method of amplification in vitro tumor infiltrating lymphocyte TIL |
| CN109988748A (en) * | 2017-12-29 | 2019-07-09 | 深圳华大生命科学研究院 | A method of tumor specific T cells are screened from TIL |
| CN110452870A (en) * | 2019-05-20 | 2019-11-15 | 河南省肿瘤医院 | A kind of isolated culture method of tumor specific T cells and the product obtained by it |
| CN110643574A (en) * | 2019-10-30 | 2020-01-03 | 西南医科大学 | Preparation method of feeder cells for rapid culture of tumor infiltrating lymphocytes |
| CN110713978A (en) * | 2019-11-16 | 2020-01-21 | 福建省肿瘤医院（福建省肿瘤研究所、福建省癌症防治中心） | Separation method of tumor antigen specific tumor infiltrating T cells |
| CN111542595A (en) * | 2017-11-10 | 2020-08-14 | 北京卡替医疗技术有限公司 | Modified immune cells and uses thereof |
| CN112501308A (en) * | 2020-11-30 | 2021-03-16 | 中国水产科学研究院珠江水产研究所 | General primer for amplification of whole genome of mitochondria of trionyx sinensis |
| CN112638428A (en) * | 2018-06-12 | 2021-04-09 | H·李·莫菲特癌症中心和研究所公司 | Chimeric antigen receptor tumor infiltrating lymphocytes |
| CN112980785A (en) * | 2020-12-21 | 2021-06-18 | 赜誉(上海)生物科技有限公司 | Preparation method of high-activity tumor infiltrating lymphocytes |
| WO2022111573A1 (en) * | 2020-11-25 | 2022-06-02 | 上海君赛生物科技有限公司 | Pharmaceutical composition for enhancing cell killing, and use thereof |
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| CN105969731B (en) * | 2016-07-29 | 2019-10-22 | 青岛市中心医院 | A method of High Fragmentation activity til cell is largely prepared using pernicious Pleural effusions |
| CN106222139A (en) * | 2016-07-29 | 2016-12-14 | 解西河 | A kind of method utilizing concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number |
| CN106222139B (en) * | 2016-07-29 | 2019-10-22 | 青岛市中心医院 | A method of High Fragmentation activity til cell is largely prepared using concretionary pernicious Pleural effusions |
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| CN106119194A (en) * | 2016-08-04 | 2016-11-16 | 英普乐孚生物技术(上海)有限公司 | A kind of isolated culture method of the til cell in Malignant Pleural source |
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| CN107760754A (en) * | 2016-08-18 | 2018-03-06 | 上海厚超生物科技有限公司 | The method of vitro detection immunocyte killing-efficiency |
| CN106754703A (en) * | 2016-12-26 | 2017-05-31 | 浙江丹晖生物科技有限公司 | A kind of til cell amplification in vitro culture medium combination and cultural method |
| CN106754703B (en) * | 2016-12-26 | 2018-03-16 | 浙江丹晖生物科技有限公司 | A kind of til cell amplification in vitro culture medium combination and cultural method |
| CN107502589A (en) * | 2017-08-04 | 2017-12-22 | 北京世纪劲得生物技术有限公司 | A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method |
| CN107384867B (en) * | 2017-08-04 | 2020-09-11 | 北京世纪劲得生物技术有限公司 | Preparation method of tumor tissue TIL cells and special culture medium |
| CN107384867A (en) * | 2017-08-04 | 2017-11-24 | 北京世纪劲得生物技术有限公司 | A kind of tumor tissues til cell preparation method and special culture media |
| US11866731B2 (en) | 2017-11-10 | 2024-01-09 | Chineo Medical Technology Co., Ltd | Modified immune cells and uses thereof |
| CN111542595A (en) * | 2017-11-10 | 2020-08-14 | 北京卡替医疗技术有限公司 | Modified immune cells and uses thereof |
| CN109988748A (en) * | 2017-12-29 | 2019-07-09 | 深圳华大生命科学研究院 | A method of tumor specific T cells are screened from TIL |
| CN112638428A (en) * | 2018-06-12 | 2021-04-09 | H·李·莫菲特癌症中心和研究所公司 | Chimeric antigen receptor tumor infiltrating lymphocytes |
| CN108753717A (en) * | 2018-06-20 | 2018-11-06 | 淮安诺康生物科技有限公司 | A kind of method of amplification in vitro tumor infiltrating lymphocyte TIL |
| CN110452870A (en) * | 2019-05-20 | 2019-11-15 | 河南省肿瘤医院 | A kind of isolated culture method of tumor specific T cells and the product obtained by it |
| CN111961648A (en) * | 2019-05-20 | 2020-11-20 | 河南省肿瘤医院 | Isolated culture method of tumor specific T cells and product obtained by same |
| CN110643574A (en) * | 2019-10-30 | 2020-01-03 | 西南医科大学 | Preparation method of feeder cells for rapid culture of tumor infiltrating lymphocytes |
| CN110713978A (en) * | 2019-11-16 | 2020-01-21 | 福建省肿瘤医院（福建省肿瘤研究所、福建省癌症防治中心） | Separation method of tumor antigen specific tumor infiltrating T cells |
| CN110713978B (en) * | 2019-11-16 | 2023-08-18 | 福建省肿瘤医院（福建省肿瘤研究所、福建省癌症防治中心） | Separation method of tumor antigen specific tumor invasive T cells |
| WO2022111573A1 (en) * | 2020-11-25 | 2022-06-02 | 上海君赛生物科技有限公司 | Pharmaceutical composition for enhancing cell killing, and use thereof |
| CN112501308A (en) * | 2020-11-30 | 2021-03-16 | 中国水产科学研究院珠江水产研究所 | General primer for amplification of whole genome of mitochondria of trionyx sinensis |
| CN112980785A (en) * | 2020-12-21 | 2021-06-18 | 赜誉(上海)生物科技有限公司 | Preparation method of high-activity tumor infiltrating lymphocytes |
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