CN108753717A - A kind of method of amplification in vitro tumor infiltrating lymphocyte TIL - Google Patents
A kind of method of amplification in vitro tumor infiltrating lymphocyte TIL Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
- C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
Abstract
The invention discloses a kind of method of amplification in vitro tumor infiltrating lymphocyte TIL, step is：Step S1, til cell detaches：It takes cancer beside organism to shred and immerses collagenase solution digestion, filtering, PBS is rinsed, and collects filtrate, supernatant is abandoned in centrifugation, PBS is added, cell suspension is made, make discontinuous density gradient centrifugation with lymphocyte separation medium, the lymphocyte that lower interface is collected after centrifugation, after PBS is cleaned, by 1 × 106The concentration of/mL is suspended in the culture medium containing 10% people's AB types serum and IL-2 and cultivates, and every 3~5d is changed the liquid once；Step S2, culture and amplification：When the lymphocyte growth in hole to 1 × 107When, it is transferred in the culture bottle containing anti-CD49d McAb, AntiCD28 McAb and drug and is incubated, IL-2 is added afterwards for 24 hours and continuously cultivates, the 22~25d of fresh culture culture for replacing the IL-2 containing comparable sodium is measured every 2~3d half.
Description
Technical field
The invention belongs to biological fields, are related to a kind of method of amplification in vitro tumor infiltrating lymphocyte TIL.
Background technology
Immunization therapy is a kind of emerging oncotherapy mode, has been listed in the 4th kind after operation, radiotherapy, chemotherapy
Therapeutic modality gradually plays a significant role in the complex treatment of tumour.2010, U.S. FDA approval was first thin in the world
The Provenge listings of born of the same parents' immunotherapeutic product, fully show value of the cellular immunotherapy in oncotherapy.
Tumor infiltrating lymphocyte (tumor-infiltrating lymphocyte, TIL) is present in mesenchyma stroma of tumors
The heterogeneous lymphocyte population based on lymphocyte, be gathered in mostly around tumour or its interstitial in shell-like surround cancer
Nest.Its main component is the T lymphocytes being present in mesenchyma stroma of tumors, and fraction is the unrestricted NK cells of MHC, common
Characterized by expression T cell receptor (T cell receptor, TCR).TIL come from tumor tissue sections, can specific recognition swell self
Tumor has the special restricted dissolving tumor promotions of MHC.The Rosenberg etc. of U.S. NCI is confirmed for the first time, from solid tumor mass
The TIL of separation after IL-2 activation and large amplification, can be used for the treatment of advanced melanoma patient, therapeutic effect in vitro
LAK (the lymphokine activated killer cells) cell therapy technology (document developed before significantly larger than：
Expansion of human tumor infiltrating lymphocytes for use in immunotherapy
trials.J Immunol Methods 1987).In recent years the more clinical tests report display delivered, til cell immunization therapy
20%~40% advanced melanoma can be cured, fully shows TIL as cellular immunotherapy means in oncotherapy
Potentiality (document：A new approach to the adoptive immunotherapy of cancer with tumor-
infiltrating lymphocytes.Science 1986)。
But there are 2 points of deficiencies for the method for amplification in vitro TIL at present：
First, amplification efficiency is not high, and a large amount of feedbacks for limiting tumor patient TIL use；
Second, the lethality of tumour cell is need to be improved, after feeding back in tumor patient body not to the lethality of tumour
Foot.
Invention content
The present invention is directed to overcome the shortage of prior art, a kind of method of amplification in vitro tumor infiltrating lymphocyte TIL is provided,
On the one hand the amplification efficiency for improving til cell, on the other hand improves its lethality to tumour cell.
Technical solution of the present invention is as follows：
A kind of method of amplification in vitro tumor infiltrating lymphocyte TIL formally uses containing anti-CD49d McAb before culture, resists
The culture medium of CD28 monoclonal antibodies and furans fish pelargonium ketone A or furans fish pelargonium ketone B are incubated for 24 hours.
Preferably, the specific steps are：
Step S1, til cell detaches
It takes the fresh cancer beside organism of cancer patient's operation excision to be soaked in PBS, rejects the clot, fat and necrosis of surrounding
Tissue, PBS are cleaned one time, are cut into 1mm3Size immerses in the IV Collagenase Type solution of 0.1 μ g/mL, and 37 DEG C of water-baths digest 2h.
With the screen filtration in 100 μm of apertures after digestion, tissue block is rinsed with PBS, collects filtrate.Cell filtrate at normal temperatures from
The heart, 1500r/min × 5min.It abandons supernatant and makees discontinuous density with lymphocyte separation medium after cell suspension is made in addition PBS
Gradient centrifugation.The lymphocyte that lower interface is collected after centrifugation, after PBS is cleaned, by 1 × 106The concentration of/mL is suspended in
In X-VIVO-15 culture mediums containing 10% people's AB types serum and IL-2, be inoculated on 12 orifice plates, the holes 3mL/, be placed in 37 DEG C, 5%
CO2Culture, every 3~5d are changed the liquid once in incubator.
Step S2, culture and amplification
When the lymphocyte growth in hole to 1 × 107When, it is transferred to containing anti-CD49d McAb, AntiCD28 McAb and furans
It is incubated in the culture bottle of fish pelargonium ketone A or furans fish pelargonium ketone B, IL-2 is added in incubation afterwards for 24 hours, is placed in 37 DEG C, 5%CO2Culture
It is continuously cultivated in case, measures the fresh X-VIVO-15 culture mediums of replacement IL-2 containing comparable sodium in incubation every 2~3d half,
The TIL of amplification in vitro is harvested after 22~25d of culture.
Preferably, in step S1 IL-2 a concentration of 2000IU/mL.
Preferably, a concentration of 20ng/mL of anti-CD49d McAb.
Preferably, a concentration of 20ng/mL of AntiCD28 McAb.
Preferably, a concentration of 20 μ g/mL of furans fish pelargonium ketone A or furans fish pelargonium ketone B.
Preferably, in step S2 IL-2 a concentration of 4000IU/mL.
A kind of culture medium for amplification in vitro tumor infiltrating lymphocyte TIL, by adding in X-VIVO-15 culture mediums
The furans fish pelargonium ketone A or furans fish pelargonium ketone B of 20 μ g/mL is added to be formulated.
Preferably, the also anti-CD49d McAb containing 20ng/mL.
Preferably, the also AntiCD28 McAb containing 20ng/mL.
A kind of method of amplification in vitro tumor infiltrating lymphocyte TIL formally uses containing anti-CD49d McAb before culture, resists
The culture medium of CD28 monoclonal antibodies and fructus alpiniae oxyphyllae terpinum A or fructus alpiniae oxyphyllae terpinum B are incubated for 24 hours.
Preferably, the specific steps are：
Step S1, til cell detaches
It takes the fresh cancer beside organism of cancer patient's operation excision to be soaked in PBS, rejects the clot, fat and necrosis of surrounding
Tissue, PBS are cleaned one time, are cut into 1mm3Size immerses in the IV Collagenase Type solution of 0.1 μ g/mL, and 37 DEG C of water-baths digest 2h.
With the screen filtration in 100 μm of apertures after digestion, tissue block is rinsed with PBS, collects filtrate.Cell filtrate at normal temperatures from
The heart, 1500r/min × 5min.It abandons supernatant and makees discontinuous density with lymphocyte separation medium after cell suspension is made in addition PBS
Gradient centrifugation.The lymphocyte that lower interface is collected after centrifugation, after PBS is cleaned, by 1 × 106The concentration of/mL is suspended in
In X-VIVO-15 culture mediums containing 10% people's AB types serum and IL-2, be inoculated on 12 orifice plates, the holes 3mL/, be placed in 37 DEG C, 5%
CO2Culture, every 3~5d are changed the liquid once in incubator.
Step S2, culture and amplification
When the lymphocyte growth in hole to 1 × 107When, it is transferred to containing anti-CD49d McAb, AntiCD28 McAb and intelligence development
It is incubated in the culture bottle of benevolence terpinum A or fructus alpiniae oxyphyllae terpinum B, IL-2 is added in incubation afterwards for 24 hours, is placed in 37 DEG C, 5%CO2Culture
It is continuously cultivated in case, measures the fresh X-VIVO-15 culture mediums of replacement IL-2 containing comparable sodium in incubation every 2~3d half,
The TIL of amplification in vitro is harvested after 22~25d of culture.
Preferably, in step S1 IL-2 a concentration of 2000IU/mL.
Preferably, a concentration of 20ng/mL of anti-CD49d McAb.
Preferably, a concentration of 20ng/mL of AntiCD28 McAb.
Preferably, a concentration of 20 μ g/mL of fructus alpiniae oxyphyllae terpinum A or fructus alpiniae oxyphyllae terpinum B.
Preferably, in step S2 IL-2 a concentration of 4000IU/mL.
A kind of culture medium for amplification in vitro tumor infiltrating lymphocyte TIL, by adding in X-VIVO-15 culture mediums
The fructus alpiniae oxyphyllae terpinum A or fructus alpiniae oxyphyllae terpinum B of 20 μ g/mL is added to be formulated.
Preferably, the also anti-CD49d McAb containing 20ng/mL.
Preferably, the also AntiCD28 McAb containing 20ng/mL.
A kind of method of amplification in vitro tumor infiltrating lymphocyte TIL formally uses containing anti-CD49d McAb before culture, resists
CD28 monoclonal antibodies and the culture medium of mansonone C or mansonone G are incubated for 24 hours.
Preferably, the specific steps are：
Step S1, til cell detaches
It takes the fresh cancer beside organism of cancer patient's operation excision to be soaked in PBS, rejects the clot, fat and necrosis of surrounding
Tissue, PBS are cleaned one time, are cut into 1mm3Size immerses in the IV Collagenase Type solution of 0.1 μ g/mL, and 37 DEG C of water-baths digest 2h.
With the screen filtration in 100 μm of apertures after digestion, tissue block is rinsed with PBS, collects filtrate.Cell filtrate at normal temperatures from
The heart, 1500r/min × 5min.It abandons supernatant and makees discontinuous density with lymphocyte separation medium after cell suspension is made in addition PBS
Gradient centrifugation.The lymphocyte that lower interface is collected after centrifugation, after PBS is cleaned, by 1 × 106The concentration of/mL is suspended in
In X-VIVO-15 culture mediums containing 10% people's AB types serum and IL-2, be inoculated on 12 orifice plates, the holes 3mL/, be placed in 37 DEG C, 5%
CO2Culture, every 3~5d are changed the liquid once in incubator.
Step S2, culture and amplification
When the lymphocyte growth in hole to 1 × 107When, it is transferred to containing anti-CD49d McAb, AntiCD28 McAb and graceful Song
It is incubated in the culture bottle of ketone C or mansonone G, IL-2 is added in incubation afterwards for 24 hours, is placed in 37 DEG C, 5%CO2It is continuously cultivated in incubator,
The fresh X-VIVO-15 culture mediums for measuring replacement IL-2 containing comparable sodium in incubation every 2~3d half, after cultivating 22~25d
Harvest the TIL of amplification in vitro.
Preferably, in step S1 IL-2 a concentration of 2000IU/mL.
Preferably, a concentration of 20ng/mL of anti-CD49d McAb.
Preferably, a concentration of 20ng/mL of AntiCD28 McAb.
Preferably, a concentration of 20 μ g/mL of mansonone C or mansonone G.
Preferably, in step S2 IL-2 a concentration of 4000IU/mL.
A kind of culture medium for amplification in vitro tumor infiltrating lymphocyte TIL, by adding in X-VIVO-15 culture mediums
The mansonone C or mansonone G of 20 μ g/mL is added to be formulated.
Preferably, the also anti-CD49d McAb containing 20ng/mL.
Preferably, the also AntiCD28 McAb containing 20ng/mL.
Advantageous effect：
Method provided by the invention by adding a small amount of furans fish pelargonium ketone A, furans fish pelargonium ketone B, benefit in the medium
Intelligence benevolence terpinum A, fructus alpiniae oxyphyllae terpinum B, mansonone C or mansonone G, can not only remarkably promote the in-vitro multiplication of til cell,
Lethality of the til cell to tumour cell can also be significantly improved.Furans fish pelargonium ketone A, furans fish pelargonium ketone B, fructus alpiniae oxyphyllae terpene
Glycol A, fructus alpiniae oxyphyllae terpinum B, mansonone C or mansonone G can be used for preparing til cell culture solution.
Description of the drawings
Fig. 1 is lethality of the til cell to liver cancer cells of furans fish pelargonium ketone compounds incubation；
Fig. 2 is lethality of the til cell to liver cancer cells of fructus alpiniae oxyphyllae terpinum class compound incubation；
Fig. 3 is lethality of the til cell to liver cancer cells of mansonone class compound incubation.
Specific implementation mode
It is specific with reference to the accompanying drawings and examples to introduce essentiality content of the present invention, but the guarantor of the present invention is not limited with this
Protect range.
Embodiment 1：
One, experiment material
X-VIVO-15 culture mediums are purchased from Lonza companies；Phosphate buffer (PBS), 1640 culture mediums of RPMI, CD3 monoclonal antibodies,
CD28 monoclonal antibodies are purchased from Gibco companies；People's AB types serum is provided by Huai'an Blood Center；IL-2 is purchased from the bio-pharmaceuticals of Beijing Fourth Ring
Company；Clostridiopetidase A IV types are purchased from Sigma companies；Lymphocyte separation medium is purchased from the virtue Pacific Ocean, Tianjin company.
HepG2 cell lines freeze for our company, are cultivated using the RPMI 1640 containing 10% fetal calf serum after recovery
Base is placed in 37 DEG C, 5%CO2Secondary culture in incubator.
Furans fish pelargonium ketone A (No. CAS：1143-45-9), furans fish pelargonium ketone B (No. CAS：1143-46-0), fructus alpiniae oxyphyllae
Terpinum A (No. CAS：363610-30-4), fructus alpiniae oxyphyllae terpinum B (No. CAS：363610-32-6), mansonone C (No. CAS：
5574-34-5), mansonone F (No. CAS：5090-88-0), mansonone G (No. CAS：7715-96-0) purity is not less than 98%, changes
It is as follows to learn structural formula.
Two, experimental method
1, the separation of til cell
Take the fresh HCC cancer beside organism of liver cancer patient operation excision to be soaked in PBS, reject the clot of surrounding, fat and
Slough, PBS are cleaned one time, are cut into 1mm3Size immerses IV Collagenase Types (Sigma-Aldrich) solution of 0.1 μ g/mL
In, 37 DEG C of water-baths digest 2h.With the screen filtration in 100 μm of apertures after digestion, tissue block is rinsed with PBS, collects filtrate.Carefully
Born of the same parents' filtrate centrifuges at normal temperatures, 1500r/min × 5min.Supernatant is abandoned, after cell suspension is made in addition PBS, with lymphocyte point
Chaotropic makees discontinuous density gradient centrifugation.The lymphocyte that lower interface is collected after centrifugation, after PBS is cleaned, by 1 ×
106The concentration of/mL is suspended in the X-VIVO-15 culture mediums containing 10% people's AB types serum and IL-2 (2000IU/mL), is inoculated in
On 12 orifice plates, the holes 3mL/ are placed in 37 DEG C, 5%CO2Culture, every 3~5d are changed the liquid once in incubator.
2, experiment packet
Group includes：Furans fish pelargonium ketone A, furans fish pelargonium ketone B, fructus alpiniae oxyphyllae terpinum A, fructus alpiniae oxyphyllae terpinum B, graceful Song
Ketone C, mansonone F or mansonone G incubation groups；The control group of these drugs is not added.
3, the culture of til cell and amplification method
When the lymphocyte growth in hole to 1 × 107When, it is transferred to the anti-CD49d McAb containing 20ng/mL, 20ng/mL
AntiCD28 McAb and 20 μ g/mL furans fish pelargonium ketone A, furans fish pelargonium ketone B, fructus alpiniae oxyphyllae terpinum A, fructus alpiniae oxyphyllae terpinum
B, it is incubated in the culture bottle of mansonone C, mansonone F or mansonone G, is incubated the IL-2 that 4000IU/mL is added afterwards for 24 hours, is placed in 37
DEG C, 5%CO2It is continuously cultivated in incubator, measures the fresh X- of replacement IL-2 containing comparable sodium in incubation every 2~3d half
VIVO-15 culture mediums harvest the TIL of amplification in vitro after cultivating 22~25d and are counted to each group til cell.
4, tumor cell killing potential is detected
HepG2 cell lines freeze for our company, are cultivated using the RPMI 1640 containing 10% fetal calf serum after recovery
Base is placed in 37 DEG C, 5%CO2Secondary culture in incubator takes logarithmic phase to carry out subsequent experimental.
Take the til cell of each group culture 23d as effector cell, using HepG2 liver cancer cells as target cell, by 20:1 effect target
Than 96 orifice plates are added in effector cell and target cell respectively, independent target cell and individual effect groups of cells are separately set, every group sets 3 again
37 DEG C, 5%CO are placed in hole2After being cultivated for 24 hours in incubator, add 10 μ L of MTT (5mg/mL) per hole, continues to cultivate 4h, centrifugation is abandoned
Clearly, after adding 150 μ L of dimethyl sulfoxide (DMSO), oscillation to dissolve 10min per hole, light absorption value A is surveyed with microplate reader 570nm, is counted as follows
Calculate killing activity of each group til cell to liver cancer cells.
Killing rate (%)=[1- (experimental group A values-individual effect cell A values)/independent target cell A values] × 100%
5, statistical procedures
Data are indicated using mean value ± standard deviation, are examined with SPSS17.0 statistical softwares row t, P<0.05 has statistics
Meaning.
Three, experimental result
1, the amplification in vitro of each group til cell
The results are shown in Table 1.When cultivating 23d, furans fish pelargonium ketone A, furans fish pelargonium ketone B, fructus alpiniae oxyphyllae terpinum A, benefit
Intelligence benevolence terpinum B, mansonone C or the til cell sum of mansonone G incubation groups are significantly higher than control group (P<0.05), mansonone F
The til cell sum of incubation group and the difference unobvious (P > 0.05) of control group.
1 each group til cell sum of table
As it can be seen that furans fish pelargonium ketone A, furans fish pelargonium ketone B, fructus alpiniae oxyphyllae terpinum A, fructus alpiniae oxyphyllae terpinum B, mansonone C
Or mansonone G can remarkably promote the in-vitro multiplication of til cell.
2, killing activity of each group til cell to tumour cell
It is furans fish pelargonium ketone A, furans fish pelargonium ketone B, fructus alpiniae oxyphyllae terpinum A, fructus alpiniae oxyphyllae terpinum B, graceful when cultivating 23d
Song's ketone C or the til cell of mansonone G incubation groups are significantly better than control group (P to the killing activity of liver cancer cells<0.05), graceful Song
The til cell of ketone F incubation groups is to the killing activity of liver cancer cells and the difference unobvious (P > 0.05) of control group.
As a result as shown in table 2 and Fig. 1-3.
Killing activity of the 2 each group til cell of table to tumour cell
As it can be seen that furans fish pelargonium ketone A, furans fish pelargonium ketone B, fructus alpiniae oxyphyllae terpinum A, fructus alpiniae oxyphyllae terpinum B, mansonone C
Or mansonone G can significantly improve lethality of the til cell to liver cancer cells.
Embodiment 2：
A kind of til cell culture medium, by added in X-VIVO-15 culture mediums 20 μ g/mL furans fish pelargonium ketone A,
Furans fish pelargonium ketone B, fructus alpiniae oxyphyllae terpinum A, fructus alpiniae oxyphyllae terpinum B, mansonone C or mansonone G are formulated.For cultivating
When til cell, it is only necessary to appropriate anti-CD49d McAb and AntiCD28 McAb are added again, it is easy to use.
Method provided by the invention by adding a small amount of furans fish pelargonium ketone A, furans fish pelargonium ketone B, benefit in the medium
Intelligence benevolence terpinum A, fructus alpiniae oxyphyllae terpinum B, mansonone C or mansonone G, can not only remarkably promote the in-vitro multiplication of til cell,
Lethality of the til cell to tumour cell can also be significantly improved.Furans fish pelargonium ketone A, furans fish pelargonium ketone B, fructus alpiniae oxyphyllae terpene
Glycol A, fructus alpiniae oxyphyllae terpinum B, mansonone C or mansonone G can be used for preparing til cell culture solution.
Above-described embodiment is intended to specifically introduce essentiality content of the present invention, but protection scope of the present invention is confined to this specifically
Embodiment.
Claims (10)
1. a kind of method of amplification in vitro tumor infiltrating lymphocyte TIL, it is characterised in that：Using containing anti-before formal culture
The culture medium of CD3 monoclonal antibodies, AntiCD28 McAb and furans fish pelargonium ketone A or furans fish pelargonium ketone B are incubated for 24 hours.
2. according to the method described in claim 1, it is characterized in that, the specific steps are：
Step S1, til cell detaches
It takes the fresh cancer beside organism of cancer patient's operation excision to be soaked in PBS, rejects the clot, fat and downright bad group of surrounding
It knits, PBS is cleaned one time, is cut into 1mm3Size immerses in the IV Collagenase Type solution of 0.1 μ g/mL, and 37 DEG C of water-baths digest 2h.Disappear
With the screen filtration in 100 μm of apertures after change, tissue block is rinsed with PBS, collects filtrate.Cell filtrate centrifuges at normal temperatures,
1500r/min×5min.It abandons supernatant and makees discontinuous density gradient with lymphocyte separation medium after cell suspension is made in addition PBS
Centrifugation.The lymphocyte that lower interface is collected after centrifugation, after PBS is cleaned, by 1 × 106The concentration of/mL, which is suspended in, to be contained
In the X-VIVO-15 culture mediums of 10% people's AB types serum and IL-2, it is inoculated on 12 orifice plates, the holes 3mL/, is placed in 37 DEG C, 5%CO2
Culture, every 3~5d are changed the liquid once in incubator.
Step S2, culture and amplification
When the lymphocyte growth in hole to 1 × 107When, it is transferred to containing anti-CD49d McAb, AntiCD28 McAb and furans India
It is incubated in the culture bottle of certain herbaceous plants with big flowers ketone A or furans fish pelargonium ketone B, IL-2 is added in incubation afterwards for 24 hours, is placed in 37 DEG C, 5%CO2In incubator
It is continuous to cultivate, the fresh X-VIVO-15 culture mediums of replacement IL-2 containing comparable sodium, culture are measured in incubation every 2~3d half
The TIL of amplification in vitro is harvested after 22~25d.
3. according to the method described in claim 2, it is characterized in that：A concentration of 2000IU/mL of IL-2 in step S1.
4. according to the method described in claim 2, it is characterized in that：A concentration of 20ng/mL of anti-CD49d McAb.
5. according to the method described in claim 2, it is characterized in that：A concentration of 20ng/mL of AntiCD28 McAb.
6. according to the method described in claim 2, it is characterized in that：Furans fish pelargonium ketone A's or furans fish pelargonium ketone B is a concentration of
20μg/mL。
7. according to the method described in claim 2, it is characterized in that：A concentration of 4000IU/mL of IL-2 in step S2.
8. a kind of culture medium for amplification in vitro tumor infiltrating lymphocyte TIL, it is characterised in that：By in X-VIVO-15
The furans fish pelargonium ketone A or furans fish pelargonium ketone B that 20 μ g/mL are added in culture medium are formulated.
9. culture medium according to claim 8, it is characterised in that：The also anti-CD49d McAb containing 20ng/mL.
10. culture medium according to claim 8, it is characterised in that：The also AntiCD28 McAb containing 20ng/mL.
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| CN110878282A (en) * | 2019-05-09 | 2020-03-13 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Preparation method of tumor antigen specific T lymphocyte |
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