CN107502589A - A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method - Google Patents

A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method Download PDF

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CN107502589A
CN107502589A CN201710662000.1A CN201710662000A CN107502589A CN 107502589 A CN107502589 A CN 107502589A CN 201710662000 A CN201710662000 A CN 201710662000A CN 107502589 A CN107502589 A CN 107502589A
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culture
mononuclearcell
tumor infiltrating
infiltrating lymphocyte
cell
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李霞云
潘新
贺伟
刘世红
卢家堃
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Beijing Century Biotechnology Co Ltd
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Beijing Century Biotechnology Co Ltd
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    • C12N2501/50—Cell markers; Cell surface determinants
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells

Abstract

The invention discloses a kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method, comprise the following steps：S1, tumor infiltrating lymphocyte and mononuclearcell are taken in number ratio 1:1‑1:20 mixing；S2, add and co-culture base, til cell culture medium and MNC cell culture mediums are comprised at least in the co-cultivation base；S3, it is placed in 37 DEG C, CO2 incubator cultures；S4, count daily, fluid infusion, maintain cell density 1 × 105/ml‑1×107/ml；S5, cultivate 10 20 days, cell is harvested by centrifugation.The present invention proposes a kind of quick, co-culture method of high efficiently multiplying tumor infiltrating lymphocyte, and discloses the several influence factors for having synergy to proliferative effect.The proliferating cancer lymphocyte infiltration of the present invention and the co-culture method of mononuclearcell, the proliferation times of tumor infiltrating lymphocyte significantly improve, while have the advantages of CD3 positive, CD4 feminine genders and CD8 positives.

Description

A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method
Technical field
The invention belongs to GVT field of cell culture, and in particular to a kind of tumor infiltrating lymphocyte and single core Cell co-culture method.
Background technology
Immunotherapy of tumors is natural defense mechanism by transferring host or to give some biological agents anti-swollen to obtain Knurl effect, according to three classes of mechanism of action point：Activeimmunotberapy (tumor vaccine), adoptive treatment is immune and non-specificity is exempted from Epidemic disease conditioning agent.Adoptive immunotherapy by Activation In Vitro and feeds back, eliminated swollen by separating autologous or allosome lymphocyte Knurl, the key of adoptive cell therapy are to produce the effector cell that quantity is enough, can identify tumour antigen.
Nineteen twenty-two, Mac Carty propose tumor infiltrating lymphocyte (Tumor Infiltrating Lymphocyte, TIL concept), after this tumor infiltrating lymphocyte just persistently become people be keen to the most concern a kind of anticarcinogenic effect Cell.Research concentrates on the action effect that tumor infiltrating lymphocyte is directed to variety classes tumour.It is thin for tumor-infiltrated lymph The in-vitro multiplication of born of the same parents is rarely reported.
CN102174469A discloses a kind of method of effectively culture tumor infiltrating lymphocyte.From patient's Pleural effusions or swollen Mononuclearcell is extracted in tumor tissue, restructuring human fibrin and the cluster differentiation coated culture of the antibody of group 3 are inoculated into after washing In bottle, after cultivating 24 hours, interleukin II is added, was trained afterwards every 2-3 days with the serum-free containing interleukin II, Pleural effusions supernatant Foster base expansion bottle, Secondary Culture, the 12nd~13 day addition apoptosis regulatory protein survivin and Mucin1 activation killing activity, the 14th Its harvesting.CN103374548A discloses a kind of efficient amplification nutrient solution of tumor infiltrating lymphocyte, described culture Contain IL-2, IL-12, anti-CD28 and anti-CTLA-4 in liquid.Compared with prior art, the present invention, which uses, contains IL-2, IL- The serum free medium of the combination of cytokines such as 12, anti-CD28 and anti-CTLA-4, the amplification efficiency of til cell substantially carry Height is higher than the amplification efficiency of conventional amplification cultivation liquid 3~10 times；And it can effectively suppress Treg lymphocytes to cytotoxicity The suppression function of T lymphocytes, the lymphocyte of amplification, which has, extremely strong kills knurl effect.
The content of the invention
The present inventor is amazing to be found that tumor infiltrating lymphocyte (Tumor by constantly experiment Infiltrating Lymphocyte, TIL) with the biological characteristics of mononuclearcell (mononuclear cell, MNC) symbiosis Property, it generates unexpected effect in terms of the high efficiently multiplying of tumor infiltrating lymphocyte, and the present invention is more than being based on It was found that and complete.
It is specific as follows it is an object of the invention to provide the method for high efficiency proliferating cancer lymphocyte infiltration：
A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method, comprise the following steps：
S1, tumor infiltrating lymphocyte and mononuclearcell are taken in number ratio 1:1-1:20 mixing；
S2, add and co-culture base, til cell culture medium and MNC cell culture mediums are comprised at least in the co-cultivation base；
S3, it is placed in 37 DEG C, CO2 incubator cultures；
S4, count daily, fluid infusion, maintain cell density 1 × 105/ml-1×107/ml；
S5, cultivate 10-20 days, cell is harvested by centrifugation.
Preferably, the mixing number ratio of the tumor infiltrating lymphocyte in the S1 steps and mononuclearcell is 1: 10。
Preferably, the co-cultivation base in the S2 steps includes TIL-b culture mediums and AIM-V (invitrogen) is cultivated Base；The minimal medium of the TIL-b culture mediums is RPMI-1640 culture mediums, and the RPMI-1640 culture mediums are added with following The solute of final concentration：10% people source serum, 25mM HEPES, 6000U/ml IL-2,0.055mM beta -mercaptoethanols.
It is further preferred that the percent by volume of the TIL-b culture mediums and AIM-V (invitrogen) culture medium is distinguished For 10-90% and 90-10%.
Still more preferably, the volume basis score of TIL-b culture mediums and AIM-V (invitrogen) culture medium It is 50%.
Preferably, in addition to mononuclearcell pre-treatment step.
It is further preferred that the mononuclearcell pre-treatment step comprises the following steps：
S01, culture medium dissolves mitomycin based on serum-free RPMI-1640 culture mediums, prepares mitomycin Final concentration of 10-30ug/ml culture medium, filtration sterilization, obtains treatment fluid；
S02, number is not less than 1 × 107The treatment fluid that is obtained with 10-15ml S01 steps of CBMC mix Close, 37 DEG C of standing 10-60min；
S03,1500rpm centrifugation 5mim remove supernatant；
S04, washing：Add 15-30ml PBS and cell is resuspended, 1500rpm centrifugations 5mim removes supernatant, repeated 4-5 times.
It is further preferred that in the treatment fluid of the S01 steps, the final concentration of 20ug/ml of mitomycin.
Still more preferably, in the S02 steps, 37 DEG C of standing 30min.
The fluid infusion of S2 steps in the present invention refers to supplementing culture medium (liquid).
The equipment being related to and reagent chemicals in the present invention, it is commercially available customary commercial unless otherwise indicated.
Ratio and percentage in the present invention, unless otherwise indicated, liquid are volume, and solid is quality.
Bleeding of the umbilicus of the mononuclearcell from xenogenic origin of the present invention.
The invention has the advantages that the present invention proposes a kind of quick, high efficiently multiplying tumor infiltrating lymphocyte be total to Cultural method, and disclose several influence factors that there is synergy to proliferative effect.The proliferating cancer infiltration leaching of the present invention The co-culture method of bar cell and mononuclearcell, the proliferation times of tumor infiltrating lymphocyte significantly improve, while have CD3 The advantages of positive, CD4 feminine genders and the CD8 positives.
Brief description of the drawings
The streaming scatter diagram (Ewing' s tumor source) for the tumor infiltrating lymphocyte that Fig. 1 embodiments 10 harvest
1-1, FITC:CD4；1-2, PerCP:CD3；1-3, PE:CD8
The streaming scatter diagram (lung cancer source) for the tumor infiltrating lymphocyte that Fig. 2 embodiments 10 harvest
1-1, FITC:CD4；1-2, PerCP:CD3；1-3, PE:CD8
Embodiment
To describe technology contents, construction feature, institute's reached purpose and effect of the present invention in detail, embodiment is hereby enumerated below It is explained in detail.
Embodiment 1
A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method, comprise the following steps：
S1, tumor infiltrating lymphocyte and mononuclearcell are taken in number ratio 1:1 mixing；
S2, add and co-culture base, til cell culture medium and MNC cell culture mediums are comprised at least in the co-cultivation base；
S3, it is placed in 37 DEG C, CO2 incubator cultures；
S4, count daily, fluid infusion, maintain cell density 1 × 106/ml；
S5, cultivate 15 days, cell is harvested by centrifugation.
Co-cultivation base in the S2 steps includes the TIL-b culture mediums and 90% percent by volume of 10% percent by volume AIM-V (invitrogen) culture medium；The minimal medium of the TIL-b culture mediums is RPMI-1640 culture mediums, described RPMI-1640 culture mediums are added with the solute of following final concentration：10% people source serum, 25mM HEPES, 6000U/ml IL-2, 0.055mM beta -mercaptoethanols.
Embodiment 2
A kind of TIL tumor infiltrating lymphocytes and MNC mononuclearcell co-culture methods, comprise the following steps：
S1, TIL tumor infiltrating lymphocytes and MNC mononuclearcells are taken in number ratio 1:1 mixing；
S2, add and co-culture base, til cell culture medium and MNC cell culture mediums are comprised at least in the co-cultivation base；
S3, it is placed in 37 DEG C, CO2 incubator cultures；
S4, count daily, fluid infusion, maintain cell density 1 × 105/ml；
S5, cultivate 20 days, cell is harvested by centrifugation.
Co-cultivation base in the S2 steps includes the TIL-b culture mediums and 90% percent by volume of 10% percent by volume AIM-V (invitrogen) culture medium；The minimal medium of the TIL-b culture mediums is RPMI-1640 culture mediums, described RPMI-1640 culture mediums are added with the solute of following final concentration：10% people source serum, 25mM HEPES, 6000U/ml IL-2, 0.055mM beta -mercaptoethanols.
Embodiment 3
A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method, comprise the following steps：
S1, tumor infiltrating lymphocyte and mononuclearcell are taken in number ratio 1:1 mixing；
S2, add and co-culture base, til cell culture medium and MNC cell culture mediums are comprised at least in the co-cultivation base；
S3, it is placed in 37 DEG C, CO2 incubator cultures；
S4, count daily, fluid infusion, maintain cell density 1 × 108/ml；
S5, cultivate 10 days, cell is harvested by centrifugation.
Co-cultivation base in the S2 steps includes the TIL-b culture mediums and 90% percent by volume of 10% percent by volume AIM-V (invitrogen) culture medium；The minimal medium of the TIL-b culture mediums is RPMI-1640 culture mediums, described RPMI-1640 culture mediums are added with the solute of following final concentration：10% people source serum, 25mM HEPES, 6000U/ml IL-2, 0.055mM beta -mercaptoethanols.
Embodiment 4
A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method, comprise the following steps：
S1, tumor infiltrating lymphocyte and mononuclearcell are taken in number ratio 1:20 mixing；
S2, add and co-culture base, til cell culture medium and MNC cell culture mediums are comprised at least in the co-cultivation base；
S3, it is placed in 37 DEG C, CO2 incubator cultures；
S4, count daily, fluid infusion, maintain cell density 1 × 106/ml；
S5, cultivate 15 days, cell is harvested by centrifugation.
Co-cultivation base in the S2 steps includes the TIL-b culture mediums and 90% percent by volume of 10% percent by volume AIM-V (invitrogen) culture medium；The minimal medium of the TIL-b culture mediums is RPMI-1640 culture mediums, described RPMI-1640 culture mediums are added with the solute of following final concentration：10% people source serum, 25mM HEPES, 6000U/ml IL-2, 0.055mM beta -mercaptoethanols.
Embodiment 5
A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method, comprise the following steps：
S1, tumor infiltrating lymphocyte and mononuclearcell are taken in number ratio 1:10 mixing；
S2, add and co-culture base, til cell culture medium and MNC cell culture mediums are comprised at least in the co-cultivation base；
S3, it is placed in 37 DEG C, CO2 incubator cultures；
S4, count daily, fluid infusion, maintain cell density 1 × 106/ml；
S5, cultivate 15 days, cell is harvested by centrifugation.
Co-cultivation base in the S2 steps includes the TIL-b culture mediums and 90% percent by volume of 10% percent by volume AIM-V (invitrogen) culture medium；The minimal medium of the TIL-b culture mediums is RPMI-1640 culture mediums, described RPMI-1640 culture mediums are added with the solute of following final concentration：10% people source serum, 25mM HEPES, 6000U/ml IL-2, 0.055mM beta -mercaptoethanols.
Embodiment 6
A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method, comprise the following steps：
S1, tumor infiltrating lymphocyte and mononuclearcell are taken in number ratio 1:10 mixing；
S2, add and co-culture base, til cell culture medium and MNC cell culture mediums are comprised at least in the co-cultivation base；
S3, it is placed in 37 DEG C, CO2 incubator cultures；
S4, count daily, fluid infusion, maintain cell density 1 × 106/ml；
S5, cultivate 15 days, cell is harvested by centrifugation.
Co-cultivation base in the S2 steps includes the TIL-b culture mediums and 10% percent by volume of 90% percent by volume AIM-V (invitrogen) culture medium；The minimal medium of the TIL-b culture mediums is RPMI-1640 culture mediums, described RPMI-1640 culture mediums are added with the solute of following final concentration：10% people source serum, 25mM HEPES, 6000U/ml IL-2, 0.055mM beta -mercaptoethanols.
Embodiment 7
A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method, comprise the following steps：
S1, tumor infiltrating lymphocyte and mononuclearcell are taken in number ratio 1:10 mixing；
S2, add and co-culture base, til cell culture medium and MNC cell culture mediums are comprised at least in the co-cultivation base；
S3, it is placed in 37 DEG C, CO2 incubator cultures；
S4, count daily, fluid infusion, maintain cell density 1 × 106/ml；
S5, cultivate 15 days, cell is harvested by centrifugation.
Co-cultivation base in the S2 steps includes the TIL-b culture mediums and 50% percent by volume of 50% percent by volume AIM-V (invitrogen) culture medium；The minimal medium of the TIL-b culture mediums is RPMI-1640 culture mediums, described RPMI-1640 culture mediums are added with the solute of following final concentration：10% people source serum, 25mM HEPES, 6000U/ml IL-2, 0.055mM beta -mercaptoethanols.
Embodiment 8
A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method, comprise the following steps：
S1, tumor infiltrating lymphocyte and mononuclearcell are taken in number ratio 1:10 mixing；
S2, add and co-culture base, til cell culture medium and MNC cell culture mediums are comprised at least in the co-cultivation base；
S3, it is placed in 37 DEG C, CO2 incubator cultures；
S4, count daily, fluid infusion, maintain cell density 1 × 106/ml；
S5, cultivate 15 days, cell is harvested by centrifugation.
Co-cultivation base in the S2 steps includes the TIL-b culture mediums and 50% percent by volume of 50% percent by volume AIM-V (invitrogen) culture medium；The minimal medium of the TIL-b culture mediums is RPMI-1640 culture mediums, described RPMI-1640 culture mediums are added with the solute of following final concentration：10% people source serum, 25mM HEPES, 6000U/ml IL-2, 0.055mM beta -mercaptoethanols.
Also include mononuclearcell pre-treatment step.
The mononuclearcell pre-treatment step comprises the following steps：
S01, culture medium dissolves mitomycin based on serum-free RPMI-1640 culture mediums, prepares mitomycin Final concentration of 10ug/ml culture medium, filtration sterilization, obtains treatment fluid；
S02, number is not less than 1 × 107The treatment fluid that is obtained with 10-15ml S01 steps of CBMC mix Close, 37 DEG C of standing 60min；
S03,1500rpm centrifugation 5mim remove supernatant；
S04, washing：Add 15-30ml PBS and cell is resuspended, 1500rpm centrifugations 5mim removes supernatant, repeated 4-5 times.
Embodiment 9
A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method, comprise the following steps：
S1, tumor infiltrating lymphocyte and mononuclearcell are taken in number ratio 1:10 mixing；
S2, add and co-culture base, til cell culture medium and MNC cell culture mediums are comprised at least in the co-cultivation base；
S3, it is placed in 37 DEG C, CO2 incubator cultures；
S4, count daily, fluid infusion, maintain cell density 1 × 106/ml；
S5, cultivate 15 days, cell is harvested by centrifugation.
Co-cultivation base in the S2 steps includes the TIL-b culture mediums and 50% percent by volume of 50% percent by volume AIM-V (invitrogen) culture medium；The minimal medium of the TIL-b culture mediums is RPMI-1640 culture mediums, described RPMI-1640 culture mediums are added with the solute of following final concentration：10% people source serum, 25mM HEPES, 6000U/ml IL-2, 0.055mM beta -mercaptoethanols.
Also include mononuclearcell pre-treatment step.
The mononuclearcell pre-treatment step comprises the following steps：
S01, culture medium dissolves mitomycin based on serum-free RPMI-1640 culture mediums, prepares mitomycin Final concentration of 30ug/ml culture medium, filtration sterilization, obtains treatment fluid；
S02, number is not less than 1 × 107The treatment fluid that is obtained with 10-15ml S01 steps of CBMC mix Close, 37 DEG C of standing 10min；
S03,1500rpm centrifugation 5mim remove supernatant；
S04, washing：Add 15-30ml PBS and cell is resuspended, 1500rpm centrifugations 5mim removes supernatant, repeated 4-5 times.
Embodiment 10
A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method, comprise the following steps：
S1, tumor infiltrating lymphocyte and mononuclearcell are taken in number ratio 1:10 mixing；
S2, add and co-culture base, til cell culture medium and MNC cell culture mediums are comprised at least in the co-cultivation base；
S3, it is placed in 37 DEG C, CO2 incubator cultures；
S4, count daily, fluid infusion, maintain cell density 1 × 106/ml；
S5, cultivate 15 days, cell is harvested by centrifugation.
Co-cultivation base in the S2 steps includes the TIL-b culture mediums and 50% percent by volume of 50% percent by volume AIM-V (invitrogen) culture medium；The minimal medium of the TIL-b culture mediums is RPMI-1640 culture mediums, described RPMI-1640 culture mediums are added with the solute of following final concentration：10% people source serum, 25mM HEPES, 6000U/ml IL-2, 0.055mM beta -mercaptoethanols.
Also include mononuclearcell pre-treatment step.
The mononuclearcell pre-treatment step comprises the following steps：
S01, culture medium dissolves mitomycin based on serum-free RPMI-1640 culture mediums, prepares mitomycin Final concentration of 20ug/ml culture medium, filtration sterilization, obtains treatment fluid；
S02, number is not less than 1 × 107The treatment fluid that is obtained with 10-15ml S01 steps of CBMC mix Close, 37 DEG C of standing 30min；
S03,1500rpm centrifugation 5mim remove supernatant；
S04, washing：Add 15-30ml PBS and cell is resuspended, 1500rpm centrifugations 5mim removes supernatant, repeated 4-5 times.
Reference examples 1
A kind of tumor infiltrating lymphocyte cultural method, comprises the following steps：
S1, take tumor infiltrating lymphocyte；
S2, add and co-culture base, til cell culture medium and MNC cell culture mediums are comprised at least in the co-cultivation base；
S3, it is placed in 37 DEG C, CO2 incubator cultures；
S4, count daily, fluid infusion, maintain cell density 1 × 106/ml；
S5, cultivate 15 days, cell is harvested by centrifugation.
Co-cultivation base in the S2 steps includes the TIL-b culture mediums and 90% percent by volume of 10% percent by volume AIM-V (invitrogen) culture medium；The minimal medium of the TIL-b culture mediums is RPMI-1640 culture mediums, described RPMI-1640 culture mediums are added with the solute of following final concentration：10% people source serum, 25mM HEPES, 6000U/ml IL-2, 0.055mM beta -mercaptoethanols.
Reference examples 2
A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method, comprise the following steps：
S1, tumor infiltrating lymphocyte and mononuclearcell are taken in number ratio 1:10 mixing；
S2, add and co-culture base, til cell culture medium and MNC cell culture mediums are comprised at least in the co-cultivation base；
S3, it is placed in 37 DEG C, CO2 incubator cultures；
S4, count daily, fluid infusion, maintain cell density 1 × 106/ml；
S5, cultivate 15 days, cell is harvested by centrifugation.
Co-cultivation base in the S2 steps is TIL-b culture mediums；The minimal medium of the TIL-b culture mediums is RPMI-1640 culture mediums, the RPMI-1640 culture mediums are added with the solute of following final concentration：10% people source serum, 25mM HEPES, 6000U/ml IL-2,0.055mM beta -mercaptoethanols.
Reference examples 3
A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method, comprise the following steps：
S1, tumor infiltrating lymphocyte and mononuclearcell are taken in number ratio 1:10 mixing；
S2, add and co-culture base, til cell culture medium and MNC cell culture mediums are comprised at least in the co-cultivation base；
S3, it is placed in 37 DEG C, CO2 incubator cultures；
S4, count daily, fluid infusion, maintain cell density 1 × 106/ml；
S5, cultivate 15 days, cell is harvested by centrifugation.
Co-cultivation base in the S2 steps is AIM-V (invitrogen) culture medium.
The influence experiment that the co-culture method of experimental example 1 is bred to tumor infiltrating lymphocyte
Using the method for embodiment or reference examples to the tumor infiltrating lymphocyte from Ewing' s tumor and lung cancer, cultivate Beginning number tumor infiltrating lymphocyte is 2 × 106, mononuclearcell is according to the ratio in method.
Amplification times are harvesting data/initial cells number of tumor infiltrating lymphocyte.
Table 1 co-cultures the influence to tumor infiltrating lymphocyte propagation
# with * numbers of symbols are identical to represent no significant difference, and number difference indicates significant difference (P<0.05)
As it can be seen from table 1 the tumor infiltrating lymphocyte cultivation effect in Ewing' s tumor source and lung cancer source, embodiment 1-5 is significantly better than reference examples 1, illustrates to co-culture the biology for promoting tumor infiltrating lymphocyte propagation with mononuclearcell Characteristic.In embodiment 1-5, embodiment 5 is more excellent, and the ratio for illustrating to co-culture can also influence cultivation effect, and this effect is not It is linear.Embodiment 5-7 is better than reference examples 2-3, illustrates that co-cultured cell needs the new culture medium different from existing culture medium, The component of culture medium can influence cultivation effect, and this effect and the ratio of co-cultured cell have synergy.
Influence of the mononuclearcell of experimental example 2 pretreatment to tumor infiltrating lymphocyte propagation is tested
It is that co-culture method uses different embodiments from the difference of experimental example 1.
Influence of the mononuclearcell of table 2 pretreatment to tumor infiltrating lymphocyte propagation
＆ with ξ numbers of symbols are identical to represent no significant difference, and number difference indicates significant difference (P<0.05)
From table 2 it can be seen that tumor-infiltrated lymph of the mononuclearcell pretreatment to Ewing' s tumor source and lung cancer source The influence of cell cultivation effect is very big, and compared with the embodiment 7 of no pretreatment, the cultivation effect of embodiment 9 is suitable, embodiment 10 More excellent, embodiment 8 is poor.
Experimental example 3 co-cultures the mass experiment of the tumor infiltrating lymphocyte of gained
To the cell of harvest, with fluorescence and surface antigen markers, counted with flow cytometry analysis.
From Fig. 1 and Fig. 2, it can be seen that using the method for embodiment 10, to Ewing' s tumor source tumor infiltrating lymphocyte Bred, the cell of harvest, CD3, CD8 overwhelming majority are the positive, and CD4 is feminine gender, is of high quality, and are answered with clinical well With value.
In summary, only the preferred embodiments of the invention, does not limit protection scope of the present invention with this, all according to the present invention The equivalent changes and modifications that the scope of the claims and description are made, it is all within the scope of patent of the present invention covers.

Claims (9)

1. a kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method, it is characterised in that comprise the following steps：
S1, tumor infiltrating lymphocyte and mononuclearcell are taken in number ratio 1:1-1:20 mixing；
S2, add and co-culture base, til cell culture medium and MNC cell culture mediums are comprised at least in the co-cultivation base；
S3, it is placed in 37 DEG C, CO2Incubator culture；
S4, count daily, fluid infusion, maintain cell density 1 × 105/ml-1×107/ml；
S5, cultivate 10-20 days, cell is harvested by centrifugation.
2. tumor infiltrating lymphocyte as claimed in claim 1 and mononuclearcell co-culture method, it is characterised in that described The mixing number ratio of tumor infiltrating lymphocyte and mononuclearcell in S1 steps is 1:10.
3. tumor infiltrating lymphocyte as claimed in claim 1 and mononuclearcell co-culture method, it is characterised in that described Co-cultivation base in S2 steps includes TIL-b culture mediums and AIM-V (invitrogen) culture medium；The TIL-b culture mediums Minimal medium is RPMI-1640 culture mediums, and the RPMI-1640 culture mediums are added with the solute of following final concentration：10% people Source serum, 25mM HEPES, 6000U/ml IL-2,0.055mM beta -mercaptoethanols.
4. tumor infiltrating lymphocyte as claimed in claim 3 and mononuclearcell co-culture method, it is characterised in that described The percent by volume of TIL-b culture mediums and AIM-V (invitrogen) culture medium is respectively 10-90% and 90-10%.
5. tumor infiltrating lymphocyte as claimed in claim 4 and mononuclearcell co-culture method, it is characterised in that described The volume basis score of TIL-b culture mediums and AIM-V (invitrogen) culture medium is 50%.
6. the tumor infiltrating lymphocyte and mononuclearcell co-culture method, its feature as described in claim 1-5 is any exist In, in addition to mononuclearcell pre-treatment step.
7. tumor infiltrating lymphocyte as claimed in claim 6 and mononuclearcell co-culture method, it is characterised in that described Mononuclearcell pre-treatment step comprises the following steps：
S01, culture medium dissolves mitomycin based on serum-free RPMI-1640 culture mediums, and the end for preparing mitomycin is dense The culture medium for 10-30ug/ml is spent, filtration sterilization, obtains treatment fluid；
S02, number is not less than 1 × 107CBMC mixed with the treatment fluid that 10-15ml S01 steps obtain, 37 DEG C stand 10-60min；
S03,1500rpm centrifugation 5mim remove supernatant；
S04, washing：Add 15-30ml PBS and cell is resuspended, 1500rpm centrifugations 5mim removes supernatant, repeated 4-5 times.
8. tumor infiltrating lymphocyte as claimed in claim 7 and mononuclearcell co-culture method, it is characterised in that described In the treatment fluid of S01 steps, the final concentration of 20ug/ml of mitomycin.
9. tumor infiltrating lymphocyte as claimed in claim 8 and mononuclearcell co-culture method, it is characterised in that described In S02 steps, 37 DEG C of standing 30min.
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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
CN115141805A (en) * 2022-08-22 2022-10-04 中山大学孙逸仙纪念医院 CIC cell for simulating human tumor microenvironment, preparation method and sorting method
CN115141805B (en) * 2022-08-22 2023-10-27 中山大学孙逸仙纪念医院 CIC cell simulating human tumor microenvironment, preparation method and sorting method

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Application publication date: 20171222