CN106244538A - A kind of isolated culture method of the til cell in malignant ascite source - Google Patents
A kind of isolated culture method of the til cell in malignant ascite source Download PDFInfo
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Abstract
The present invention relates to the isolated culture method of the til cell in a kind of malignant ascite source, utilize at least one in PD 1, LAG 3 and TIM 3 as separating the labelling of tumor specific cytotoxic T lymphocyte in til cell, and combine immunomagnetic bead technique tumor specific cytotoxic T lymphocyte is separated from til cell group, again through external efficient amplification, it is thus achieved that the tumour-specific til cell of high anti-tumor activity.The present invention makes the separation and Culture operating process of tumour-specific til cell simpler, hence it is evident that shortening the separation and Culture time, low cost, it is simple to promote the use of, additionally the anti-tumor activity of the tumour-specific til cell of isolated is higher.
Description
Technical field
The invention belongs to technical field of cell culture, relate to malignant ascite source til cell (tumor-infiltrated lymph is thin
Born of the same parents) isolated culture method.
Background technology
Tumor infiltrating lymphocyte (TIL) is present in the pouring in tumor tissues, tumor-draining lymphode, cancerous ascites pleural fluid
Bar cell, compared to peripheral blood lymphocyte, contains the tumor specific cytotoxic T lymphocyte of the activation of higher proportion in til cell,
This quasi-lymphocyte through in-vitro separation, expand, activate after, the killing activity of tumor cell is improved, can be used for tumor
Adoptive cellular immunotherapy.
This technology is used to have been achieved for good clinical effectiveness.Rosenberg etc. use body irradiation+chemotherapy+TIL
Cell therapy malignant melanoma, total effective rate has reached 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. use chemoradiotherapy+til cell treatment nasopharyngeal carcinoma, have 19 in 20 people
People has objective antitumor to react (Li J1, 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
Express the Adenoviral Therapy of IFN-r feel sick melanoma Deng combining intratumor injection with til cell, achieve the Disease epizootic of 46%
Rate (Khammari A1, 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.)。
Mainly there are two aspects in the source of the til cell of at present clinical practice: 1. the tumor tissues of excision or lymph
Knot；2. cancerous ascites pleural fluid.Til cell is separated it is generally required to through mechanical shearing, the enzymolysis of multiple protein enzyme from surgical samples
Etc. multiple steps；The method that the til cell in cancerous ascites pleural fluid source separates is relatively easy, also easily cultivates in vitro.
Owing to til cell is the cell mass of a kind of heterogeneity, comprise T cell, B cell, NK cell etc., wherein play anti-swollen
The tumor specific cytotoxic T lymphocyte of the mainly activation of tumor effect, therefore how the key technology of til cell treatment is from TIL
In filter out the tumor specific cytotoxic T lymphocyte of activation and carry out amplification cultivation.
Traditional til cell separates and incubation step is complicated, nearly 2 months whole operating time, wherein separates and identifies
The process of tumour-specific til cell is the most complicated, the longest.Firstly the need of tumor tissues being fabricated to little block organization or slender
Born of the same parents' suspension, is divided into a lot of aliquot and then cultivates with heavy dose of IL-2, and after cultivating 4-6 week, every part takes a small amount of cell and tumor
Cell co-cultures, and in the supernatant after being co-cultured by test, the content of IFN-r determines that there is tumour-specific T which part the inside
Lymphocyte.Then take the cell in this some holes and carry out rapid amplifying cultivation.This operational approach step is complicated, and the time is long, and
And separation process is easily affected by operator's subjective factors, the result difference of different operator's operations is the biggest.Behaviour for a long time
It is easily caused pollution as process, and cell viability reduces.Therefore the effect of til cell clinical practice it has been largely affected by.
Therefore the two of TIL treatment big crucial technology, one is intended to find the T of specific for tumour antigen to drench from malignant ascite
Bar cell, and it is separated；Two are intended to the T lymphocyte of these preciousnesses to expand in a large number.Special having tumor
The tumor specific cytotoxic T lymphocyte of opposite sex killing activity is separated from tumor tissues, needs to use one and can distinguish swollen
The surface markers of tumor T lymphocyte specific.T lymphocyte specific for tumour antigen in tumor tissues is that one is the most activated
T lymphocyte, PD-1, LAG-3 and TIM-3 are the Inhibitory receptor of abduction delivering after T cell activation, tie with its part
Activation and the function of T cell is suppressed after conjunction.The T lymphocyte in til cell with tumour-specific killing activity is present in
(Alena Gros etc.PD-1identifies the patient-in the T lymphocyte that this group has activated
specific CD8+tumor-reactive repertoire infiltrating human tumors.The Journal
of Clinical Investigation.Volume 124Number 5May 2014).Therefore PD-1, LAG-3 and TIM-3 are
Can be as separating the labelling of tumor specific cytotoxic T lymphocyte in til cell.
Immunomagnetic beads (Immunomagnetic bead, IMB) technology is a kind of immunological technique, and IMB is for being coated with antibody
Ball-type magnetic particle, specifically can be combined with target substance and be allowed to that there is magnetic responsiveness.The advantage of IMB technology is to protect
Demonstrate,prove and separated the form of target cell and fully functional, the most also have highly sensitive, specificity is high, detection speed is fast, repeated
Good, simple to operate and need not the advantage such as instrument and equipment of costliness.Cell separation is carried out by two ways, one by IMB technology
It is that the method directly isolating target cell from cell mixture is referred to as positive separation；Remove unrelated cell with IMB and make target cell
The method being able to purification is referred to as feminine gender separation.To there is the tomour specific of tumour-specific killing activity hence with immunomagnetic beads
Property T lymphocyte is directly separated out from til cell group.
Document report is not had to utilize at least one in PD-1, LAG-3 and TIM-3 swollen as separating in til cell at present
The labelling of tumor T lymphocyte specific, and combine immunomagnetic bead technique by tumor specific cytotoxic T lymphocyte from til cell group
Separate, then the method obtaining tumour-specific til cell through external efficient amplification.
Summary of the invention
Present invention aims to the operation of prior art separation and Culture til cell complicated, time-consumingly, operate for a long time
The deficiency that process is easily caused pollution, cell viability reduces, it is provided that the isolated culture method of a kind of til cell, the method will
The tumor specific cytotoxic T lymphocyte with tumour-specific killing activity is separated from til cell group and expands in a large number
Increasing and cultivate, operating process is simpler, and the cost time substantially shortens, it is simple to promote the use of, and the tumour-specific TIL of isolated is thin
Born of the same parents' killing activity is higher.
The technical scheme is that step one, the preparation of single cell suspension, i.e. obtain containing tumor from malignant ascite
The single cell suspension of T lymphocyte specific；Step 2: use immunomagnetic bead technique to be separated by described single cell suspension,
Will tumor specific cytotoxic T lymphocyte from tumor cell, tumor non-specific T lymphocyte, suppressor T cell, fibroblast
The mixtures such as dimension cell are separated, obtains tumour-specific til cell；Step 3: tumour-specific til cell quick
Amplification, will carry out efficient amplification cultivation in efficient amplification cultivating system by described tumour-specific til cell, obtain substantial amounts of
There is the tumour-specific til cell of specific killing activity of tumor cells.
Preferably, in step one, the preparation of described single cell suspension, specifically include following steps:
Malignant ascite D-Hanks balanced salt solution washs, and with Ficoll density gradient method separation mononuclearcell, obtains
Tumor tissues single cell suspension.
Preferably, in step 2, described single cell suspension is separated by described use immunomagnetic bead technique, specifically includes
Following steps:
2.1 press every 1 × 10 in step one gained tumor tissues single cell suspension8Individual cell adds 0.4ml PBS buffering
Solution, 10-500ul biotin labeling or fluorochrome label (include Cy5/Anti-Alexa Fluor 647, Cy7, FITC,
The labellings such as PE, APC) or the antibody-solutions of unlabelled PD-1, LAG-3, TIM-3 at least one, 10-500ul Fc receptor
20-1000ul antibiotin magnetic bead or anti-fluorescent dye magnetic bead that blocker is corresponding with antibody labeling (include Anti-Cy5/
Anti-Alexa Fluor647MicroBeads、Anti-Cy7MicroBeads、Anti-FITC MicroBeads、Anti-PE
MicroBeads, Anti-APC MicroBeads) or anti-Ig magnetic bead, mixing, resuspended cell suspension.
Described cell suspension is joined in cell sorting post by 2.2, utilizes magnetic separator to collect tumour-specific TIL thin
Born of the same parents.
Preferably, in step 3, the rapid amplifying of described tumour-specific til cell, specifically include following steps:
1. tumour-specific til cell step 2 obtained is with following reagent and factor mixed culture: CM and AIM-V mixes
Closing culture medium, allosome radiates PBMC, the OKT3 antibody of lethal (50Gy).
2. within the 2nd day, add IL-2 100-10000IU/ml.
3. carrying out expanding bottle (bag) according to cell number to cultivate, culture medium is CM and the AIM-V mixed culture medium containing IL-2.
4. amplification cultivation to the 7-30 days, obtains final substantial amounts of tumour-specific til cell.
Preferably, in step 3, described CM and AIM-V mixed culture medium is that CM culture medium, AIM-V culture medium are according to 1:1
Ratio mix.
Preferably, in step 3, described allosome radiates the PBMC of lethal (50Gy) and described tumour-specific til cell
Ratio is 10:1-1000:1.
Preferably, in step 3, described OKT3 antibody, add 0.01-0.1ug OKT3 antibody by every 1.0ml culture medium
Ratio.
Use the tumour-specific til cell treatment for tumor of the isolated culture method separation and Culture that the present invention relates to
Or prevention.
Compared with prior art, the present invention has a following beneficial effect:
The isolated culture method of til cell the most of the present invention is simpler, and cell culture period substantially shortens, and is substantially reduced cultivation
Cost；
2. the present invention utilizes immunomagnetic bead technique separation tumor specific cytotoxic T lymphocyte, when making separation compared to existing technology
Between by shortening in 4-5 week 1 day, and operating process is simpler, it is simple to promote the use of.
3. the present invention utilizes Inhibitory receptor that the T cell of activation expresses by tumor specific cytotoxic T lymphocyte from complicated
Cell mass is separated, adds the specific aim of separation, make the tumor finally cultivating the tumour-specific til cell obtained kill
Wound activity is higher.
Accompanying drawing explanation
The til cell that Fig. 1 is embodiment 1 to be obtained with comparative example 1 kills the result of tumor experiment；
The til cell that Fig. 2 is embodiment 2 to be obtained with comparative example 2 kills the result of tumor experiment；
The til cell that Fig. 3 is embodiment 3 to be obtained with comparative example 3 kills the result of tumor experiment；
The til cell that Fig. 4 is embodiment 4 to be obtained with comparative example 4 kills the result of tumor experiment；
Fig. 5 is the result that the til cell that embodiment 5,6 obtains kills tumor experiment；
Detailed description of the invention
The present invention is further illustrated by the following examples, but the present invention is not limited to these specific embodiment parties
Formula.
Embodiment 1
Now taking a liver cancer patient (female, 48 years old) malignant ascite is the material preparing tumour-specific til cell, tomour specific
Property til cell separation and Culture process is as follows:
Step 1: the preparation of single cell suspension.
1.2 ascites D-Hanks solution are washed 2 times, the sedimentation cell appropriate PBS buffer solution of addition (containing 0.5%BSA,
2mmol EDTA, PH 7.2) with Ficoll lymphocyte separation medium separation mononuclearcell, with PBS buffer solution weight after counting
Outstanding, obtain final tumor tissues single cell suspension.
Step 2: with immunomagnetic bead technique isolated tumour-specific til cell.
The cell concentration of the 2.1 final gained of set-up procedure 1 is every 1.0ml buffer solution 2.5 × 108Individual cell.
2.2 add 100ul Fc receptor blocking agent (Miltenyi Biotec, article No. 130-059-901), and mixing, 2-8 takes the photograph
Family name's degree refrigerator is placed 10 minutes.
2.3 add 10-20ml PBS buffer solution 300xg is centrifuged 10 minutes, removes supernatant.
2.4 add 0.4ml PBS buffer solution, 100ul CD279 (PD1)-Biotin solution (Miltenyi Biotec,
Article No. 130-096-162), mixing, 2-8 degree Celsius of refrigerator is placed 10 minutes.
2.5 add 10-20ml PBS buffer solution 300xg is centrifuged 10 minutes, removes supernatant.
2.6 add 0.8ml PBS buffer solution, 200ul antibiotin magnetic bead (Miltenyi Biotec, article No. 130-
090-485), mixing, 2-8 degree Celsius of refrigerator is placed 15 minutes.
2.7 add 10-20ml PBS buffer solution 300xg is centrifuged 10 minutes, removes supernatant, adds 1ml buffer solution weight
Hang and obtain cell suspension.
LD post is placed in MACS magnetic separator by 2.8, with 2ml PBS buffer solution rinse once.
2.9 add step 2.5 gained cell suspension in LD post, collect by the unlabelled cell of LD post to A pipe, then
Washing 2 times (use 1ml PBS buffer solution) with PBS buffer solution, washing liquid is also collected in A pipe every time, obtain unlabelled carefully
Born of the same parents.
LD post is taken out from magnetic separator by 2.10, adds 2ml buffer push rod and is come out by the cell of labelling, uses
B pipe is collected, and obtains tumour-specific til cell.
Step 3: the rapid amplifying of tumour-specific til cell.
The tumour-specific til cell that step 2 is obtained by 3.1 is by every 1 × 106Cell mixes training with following reagent and the factor
Support: CM culture medium (adds 25mmol/L HEPES PH 7.2,100IU/mL penicillin in RPMI1640 culture medium
(penicillin), 100ug/mL streptomycin (streptomycin), 2mmol/L glutamine (L-glutamine), 5.5 ×
10-5Mol/L beta-mercaptoethanol (β-mercaptoethanol), 10% people's AB serum) 75ml, 2 × 108The radiation of individual allosome is lethal
(50Gy) PBMC, 4.5ug OKT3 antibody, 75ml AIM-V culture medium.
Within 3.2 the 2nd days, add IL-2 to 6000IU/ml.
3.3 the 5th days, remove 120ml supernatant, add the 120ml 50%CM and 50%AIM-V containing 6000IU/ml IL-2
Mixed culture medium.
Within 3.4 the 6th days, carrying out expanding bottle (bag) according to cell number to cultivate, cell concentration is adjusted to 1 × 106/ ml, culture medium is
The mixed culture medium of 50%CM and 50%AIM-V of 6000IU/ml IL-2.
3.5 amplification cultivation to the 14th day, obtain final substantial amounts of tumour-specific til cell.
Embodiment 2
Now taking a colorectal cancer patients (man, 53 years old) malignant ascite is the material preparing tumour-specific til cell, tumor
Specificity T IL cell separation incubation 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, for Anti-TIM-3-APC
(Miltenyi Biotec, article No. 130-098-936)；The magnetic bead added in step 2.6 is different, for anti-APC magnetic bead
(Miltenyi Biotec, article No. 130-090-855).
Embodiment 3
Taking a colorectal cancer patients (female, 58 years old) malignant ascite is the material preparing tumour-specific til cell, and tumor is special
Opposite sex 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, for Mus
Anti-human LAG-3 antibody (R&D SYSTEMS, article No.: MAB23193, IgG1)；The magnetic bead added in step 2.6 is different, is anti-
Mus IgG1 magnetic bead (Miltenyi Biotec, article No. 130-047-101)
Embodiment 4
The cancer ascites taking a liver cancer patient (man, 58 years old) is the material preparing tumour-specific til cell, tomour specific
Property 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 for 10 minutes, and it is molten that sedimentation cell adds appropriate PBS buffering
Liquid (containing 0.5%BSA, 2mmol EDTA, PH 7.2) separates mononuclearcell with Ficoll, with PBS buffer solution weight after counting
Outstanding, obtain final tumor tissues single cell suspension.
Step 2 and 3 is with embodiment 1.
Embodiment 5,6
Taking a liver cancer patient (female, 68 years old) malignant ascite is the material that embodiment 5,6 prepares tumour-specific til cell,
Tumour-specific til cell separation and Culture process is as follows:
Step 1～3 operating process with embodiment 1, difference is:
(1) the quantity difference of addition collagenase, hyaluronidase, DNA enzymatic in step 1.1:
Embodiment 5:0.2mg/ml collagenase, 0.01mg/ml hyaluronidase, 0.01mg/ml DNA enzymatic；
Embodiment 6:20mg/ml collagenase, 10mg/ml hyaluronidase, 10mg/ml DNA enzymatic.
(2) the quantity difference of addition antibody-solutions, Fc receptor blocking agent, antibiotin magnetic bead 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) quantity of PBMC, OKT3 antibody adding IL-2, allosome radiation lethal (50Gy) in step 3 is different:
Embodiment 5:IL-2 100IU/ml, allosome radiate the PBMC 1.0 × 10 of lethal (50Gy)7Individual, OKT3 antibody
1.5ug；
Embodiment 6:IL-2 10000IU/ml, allosome radiate the PBMC 1.0 × 10 of lethal (50Gy)9Individual, OKT3 antibody
15ug。
(4) natural law that in step 3, cell is cultivated is different:
Embodiment 5: amplification cultivation to the 7th day, obtains final substantial amounts of tumour-specific til cell；
Embodiment 6: amplification cultivation to the 30th day, obtains final substantial amounts 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 culture medium, join on 2 grades of density gradient separation liquid (lower floor is 100%Ficoll,
Intermediate layer is the mixed liquor of 75%Ficoll and 25%CM culture fluid), 2000 leave the heart 20 minutes, collect upper strata and intermediate layer it
Between cell obtain the tumor cell that is enriched.Adjusting cell concentration by RPMI 1640 culture medium containing 10%FBS is 2 × 105
Individual/ml, is inoculated in culture bottle cultivation.
The primary amplification of step 2:TIL cell
It is 5 × 10 that single cell suspension CM culture medium adjusts cell concentration5Individual/ml, adds 6000IU/ml IL-2, inoculation
In 24 orifice plates, every hole inoculation 2ml, puts into 37 DEG C, cultivates in 5%CO2 incubator.
The growth of whether apertures endolymph cell how, to carry out once half amount less than one week and change liquid.When in any one hole
Lymphocyte when covering with, by porose in cell mixing, be divided into 2 parts, add the CM containing 6000IU/ml IL-2 and cultivate
Base continues to cultivate.Hereafter, carry out weekly 2 half amounts and change liquid.Or cell concentration is adjusted to 0.8～1.6 × 106Individual/ml.This
Process needs 4-5 week.
Step 3:IFN-r release experiment
3.1 take the autologous tumor cell that the til cell that step 3 obtains obtains with step 2 joins together with the ratio of 1:1
In one 96 orifice bore, add 200ul culture medium Han 10%FBS RPMI 1640, co-culture 12 hours.
3.2 every holes take supernatant 100ul for ELISA method detection IFN-r concentration (BD, article No.: 550612), tumor-killing
Property til cell is defined as above-mentioned IFN-r concentration > 200pg/ml, thus filter out tumoricidal til cell.
The amplification of step 4:TIL cell.
The tumoricidal til cell that step 4 is filtered out by 4.1 is by every 1 × 106Cell mixes with following reagent and the factor
Cultivate: CM culture medium 75ml, 2 × 108Allosome radiates PBMC, the 4.5ug OKT3 antibody of lethal (50Gy), and 75ml AIM-V trains
Support base.
Within 4.2 the 2nd days, add IL-2 to 6000IU/ml.
4.3 the 5th days, remove 120ml supernatant, add the 120ml 50%CM and 50%AIM-V containing 6000IU/ml IL-2
Mixed culture medium.
Within 4.4 the 6th days, carrying out expanding bottle (bag) according to cell number to cultivate, cell concentration is adjusted to 1 × 106/ ml, culture medium 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 tumor experiment
Step one: prepare autologous tumor cell strain
By resuspended for embodiment 5,6 step one gained cell suspension CM culture medium, join on 2 grades of density gradient separation liquid
(lower floor is 100%Ficoll, and intermediate layer is the mixed liquor of 75%Ficoll and 25%CM culture fluid), 2000 leave 20 points of the heart
Clock, collects the tumor cell that the cell between upper strata and intermediate layer obtains being enriched.Cultivate with the RPMI 1640 containing 10%FBS
The whole cell concentration of keynote is 2 × 105Individual/ml, is inoculated in culture bottle cultivation.
Step 2:
Respectively by final in the tumour-specific til cell finally given in above-described embodiment 1～6 and comparative example 1～4
The til cell action effect cell arrived, the autologous tumor cell obtained with comparative example 1～4 step 2 respectively and above step one system
Standby autologous tumor cell strain is target cell.
1. take effector lymphocyte, wash 2 times by RPMI 1640 culture medium, be made into 1 by the RPMI1640 culture medium containing 10%FBS
×106/ ml cell suspension.
2. take target cell, wash once by RPMI 1640 culture medium, be made into 1 × 105/ ml cell suspension.
3. it is inoculated in 96 orifice plates than 10:1 by effect target, effector lymphocyte hole and Target cell wells are set, by every kind of cultivation simultaneously
The multiple hole of 3, base, 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 hole-effector lymphocyte hole)/Target cell wells.
Result:
The til cell that embodiment 1 and comparative example 1 obtain kills the result of tumor experiment and sees Fig. 1；
The til cell that embodiment 2 and comparative example 2 obtain kills the result of tumor experiment and sees Fig. 2；
The til cell that embodiment 3 and comparative example 3 obtain kills the result of tumor experiment and sees Fig. 3；
The til cell that embodiment 4 and comparative example 4 obtain kills the result of tumor experiment and sees Fig. 4；
The til cell that embodiment 5,6 obtains kills the result of tumor experiment and sees Fig. 5；
Conclusion:
From embodiment 1～4 and comparative example 1～4 it can be seen that the method used compared to comparative example 1～4, use this
The bright method separation tumor specific cytotoxic T lymphocyte time, by shortening in 4-5 week 1 day, reduces cost, and operating process is simpler
Single, it is simple to promote the use of.
It will be seen from figure 1 that the anti-tumor activity of tumour-specific til cell that embodiment 1 obtains is higher than comparative example 1
The til cell obtained；
Figure it is seen that the anti-tumor activity of tumour-specific til cell that embodiment 2 obtains is higher than comparative example 2
The til cell obtained；
From figure 3, it can be seen that the anti-tumor activity of tumour-specific til cell that embodiment 3 obtains is higher than comparative example 3
The til cell obtained；
From fig. 4, it can be seen that the anti-tumor activity of tumour-specific til cell that embodiment 4 obtains is higher than comparative example 4
The til cell obtained；
Therefore, the present invention utilizes Inhibitory receptor that the T cell of activation expresses by tumor specific cytotoxic T lymphocyte from complexity
Cell mass in separate, add the specific aim of separation, make finally to cultivate the tumor of the tumour-specific til cell obtained
Killing activity is higher.
It is to be understood that, although this specification is been described by according to embodiment, but the most each embodiment only comprises one
Individual independent technical scheme, this narrating mode of description is only that for clarity sake those skilled in the art should will say
Bright book is as an entirety, and the technical scheme in each embodiment can also be through appropriately combined, and forming those skilled in the art can
With other embodiments understood.
The a series of detailed description of those listed above is only for the feasibility embodiment of the present invention specifically
Bright, they also are not used to limit the scope of the invention, all equivalent implementations made without departing from skill of the present invention spirit
Or change should be included within the scope of the present invention.
Claims (10)
1. the isolated culture method of tumour-specific til cell, it is characterised in that comprise the steps:
Step one, the preparation of tumor tissues single cell suspension, i.e. obtain containing tumor specific cytotoxic T lymphocyte from malignant ascite
Single cell suspension；
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, will train in efficient amplification by described tumour-specific til cell
The system of supporting carries out efficient amplification cultivation, obtains the substantial amounts of tumour-specific TIL with specific killing activity of tumor cells
Cell.
2. the isolated culture method of tumour-specific til cell as claimed in claim 1, it is characterised in that in step one, institute
State the preparation of tumor tissues single cell suspension, specifically include following steps:
Malignant ascite D-Hanks balanced salt solution washs, and with Ficoll density gradient method separation mononuclearcell, obtains tumor
Tissue monocytes suspension.
3. the isolated culture method of tumour-specific til cell as claimed in claim 1, it is characterised in that in step 2, institute
State use immunomagnetic bead technique to be separated by described single cell suspension, specifically include following steps:
(c) toward step one gained tumor tissues single cell suspension adds PBS buffer solution, antibody-solutions, Fc receptor blocking agent,
Magnetic bead, mixing, resuspended cell suspension.
D described cell suspension is joined in cell sorting post by (), utilize magnetic separator to collect tumour-specific til cell.
4. the isolated culture method of tumour-specific til cell as claimed in claim 1, it is characterised in that in step 3, institute
State the rapid amplifying of tumour-specific til cell, specifically include following steps:
E tumour-specific til cell that step 2 is obtained by () is mixed with following reagent and the factor: CM and AIM-V mixes
Culture medium, allosome radiates PBMC, the OKT3 antibody of lethal (50Gy).
F () adds IL-2 on the 2nd day；
G () carries out expanding bottle (bag) according to cell number and cultivates, culture medium is CM and the AIM-V mixed culture medium containing IL-2；
H () amplification cultivation to the 7-30 days, obtains final substantial amounts of tumour-specific til cell.
5. the isolated culture method of tumour-specific til cell as claimed in claim 2, it is characterised in that described containing collagen
Enzyme, hyaluronidase, serum-free RPMI 1640 culture medium of DNA enzymatic, the concentration of collagenase is 0.2-20mg/ml, hyaluronic acid
The concentration of enzyme is 0.01-10mg/ml, and the concentration of DNA enzymatic is 0.01-10mg/ml.
6. the isolated culture method of tumour-specific til cell as claimed in claim 3, it is characterised in that antibody is biological
In element (Biotin) labelling or fluorochrome label or unlabelled PD-1 antibody, LAG-3 antibody and TIM-3 antibody at least one
Kind；Magnetic bead is the antibiotin magnetic bead corresponding with antibody labeling or anti-fluorescent dye magnetic bead or anti-Ig magnetic bead.
7. the isolated culture method of tumour-specific til cell as claimed in claim 6, it is characterised in that by every 1.0 × 108
Individual cell adds antibody-solutions 10-500ul, Fc receptor blocking agent 10-500ul, magnetic bead 20-1000ul.
8. the isolated culture method of tumour-specific til cell as claimed in claim 4, it is characterised in that described CM and
AIM-V mixed culture medium is that CM culture medium, AIM-V culture medium mix according to the ratio of 1:1.
9. the isolated culture method of tumour-specific til cell as claimed in claim 4, it is characterised in that described allosome spoke
Penetrating the PBMC of lethal (50Gy) with the ratio of described tumour-specific til cell is 10:1-1000:1；Described OKT3 antibody, presses
Every 1.0ml culture medium adds the ratio of 0.01-0.1ug OKT3 antibody.
10. the tumour-specific til cell of the isolated culture method separation and Culture as described in any one in claim 1～9
Treatment or prevention for tumor.
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| US11357841B2 (en) | 2017-01-06 | 2022-06-14 | Iovance Biotherapeutics, Inc. | Expansion of tumor infiltrating lymphocytes with potassium channel agonists and therapeutic uses thereof |
| US11939596B2 (en) | 2017-03-29 | 2024-03-26 | Iovance Biotherapeutics, Inc. | Processes for production of tumor infiltrating lymphocytes and uses of same in immunotherapy |
| US11998568B2 (en) | 2017-03-29 | 2024-06-04 | Iovance Biotherapeutics, Inc. | Processes for production of tumor infiltrating lymphocytes and uses of same in immunotherapy |
| 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 |
| 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 |
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| CN111286486A (en) * | 2019-12-19 | 2020-06-16 | 清华大学 | Method for amplifying large amount of lymphocyte infiltrated by malignant tumor ascites in vitro |
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