CN108929862A - A kind of the T cell preparation and amplification method and its application of malignant tumour Lung metastases companion malignant pleural effusion - Google Patents
A kind of the T cell preparation and amplification method and its application of malignant tumour Lung metastases companion malignant pleural effusion Download PDFInfo
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- CN108929862A CN108929862A CN201810865936.9A CN201810865936A CN108929862A CN 108929862 A CN108929862 A CN 108929862A CN 201810865936 A CN201810865936 A CN 201810865936A CN 108929862 A CN108929862 A CN 108929862A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2302—Interleukin-2 (IL-2)
Abstract
The present invention relates to a kind of malignant tumour advanced stage Lung metastases with the tumor therapeuticing method of Patients with Malignant Pleural Metastases, the described method includes: pleural effusion is collected in drainage in the cancerous hydrothorax that Lung metastases patient occurs, monocyte (PEMC) in metastatic hydrothorax is isolated after centrifugal enrichment, adjusts concentration 5 × 105/ ml is placed in incubator in each hole of 24 orifice plates, expands PEMC with the peripheral blood mononuclear cells (PBMC) and IL-2 use in conjunction of health adult after irradiation, amplification and cell toxicity test of the PBMC after irradiation as trophocyte stimulation PEMC.The present invention also provides purposes of the PEMC being prepared by the above method in the drug of preparation treatment malignant tumour advanced stage Lung metastases companion's malignant pleural effusion.Its advantage is shown: providing a kind of more efficient, relatively convenient and fast PEMC amplification method of technique, amplification efficiency reaches 1000 times, and has high-strength cell toxicant lethal effect.
Description
Technical field
The present invention relates to immunology, molecular biology and technical field of cell biology, specifically, being a kind of pernicious swollen
T cell preparation and amplification method and its application of the tumor Lung metastases with malignant pleural effusion.
Background technique
Malignant pleural effusion refers to the product of thoracic cavity caused by the Malignant tumor of bonal metastasis to pleura for being primary in pleura or other positions
Liquid is also that malignant tumour is common and be difficult to the late complication controlled, and rapid development is often accompanied by uncomfortable in chest, out of breath, cardio palmus
Shape, treatment can cause patient breathing cycle's dysfunction, Hypoproteinemia, anaemia, serious person's even threat to life, shadow not in time
Ring the life quality of patient.And occur that malignant pleural effusion usually shows tumour spread or to advanced stage, tumour is mostly resistance to
Big by chemicotherapy, Sudden Changing Rate, patient survival shortens.Therefore pleural effusion, control metastatic tumor and primary pernicious are efficiently controlled
Tumour, extending tumor patient life cycle, improving life quality is a great problem that current late malignant tumour is treated.
It is known together according to malignant pleural effusion specialist, malignant pleural effusion should consider palliative treatment once making a definite diagnosis as early as possible.
Therapeutic scheme is formulated after fully assessing to patient symptom, ordinary circumstance and expected life span, main purpose is that mitigation breathing is tired
It is difficult.More typical method includes clinical observation, therapeutic thoracentesis, intercostal catheter drainage, pleurodesis, thoracoscopic operation
Treatment etc..Clinic is more using method, but general curative effect is limited, and clinic needs new therapeutic strategy.
Previous literature report, by large dosage of IL-2 intrathoracic injection, can be such that cancerous hydrothorax reduces, or even disappear, local disease
Shape is alleviated.Theoretically mainly LAK cell is activated to generate adoptive immunity effect by IL-2, it is thin that LAK cell can kill tumour
Born of the same parents, while enhancing NK cell activity, body's immunity is adjusted, tumour growth is inhibited.Although not using this method therapeutic effect
One, and large dosage application is likely to occur cardiopulmonary toxic reaction, patient tolerance is poor, but early stage is by improving human autoimmune
The method of effect killing malignant tumour.At present when treatment malignant pleural effusion, thoracic cavity is carried out for the patient of a large amount of pleural effusions
Closed drainage is punctured, and the hydrothorax drained is then recycled as clinical waste by specialized medical waste processing unit, while this
Kind method is palliative treatment means, mostly reduction of patient symptom uncomfortable in chest, temporarily improves respiratory function.
Adoptive cellular immunotherapy (AdopitveCellTherapy, ACT) is the new method of oncotherapy, is used for cancer
The ACT of disease treatment has three kinds of forms of current mainstream, respectively tumor infiltrating lymphocyte (TIL), T cell receptor (TCR) and embedding
Close antigen receptor (CAR-T) cell therapy.Different from traditional treatment mode, tumour immunotherapy is a kind of for human immunity system
It unites rather than enhances the anti-swollen of tumor microenvironment by exciting or transferring the immune system of body directly against the therapy of tumour
Tumor immunity, thus control and killing tumor cell.
Tumor infiltrating lymphocyte is to enter the leucocyte in tumour after being detached from blood flow, when there are massive tumor infiltrations to drench
When bar cell, surface body is started to antitumor immune response.Tumor infiltrating lymphocyte carries out melanoma patients
Adoptive cellular treatment achieves good effect.Monokaryon lymphocyte by comparison in Malignant Pleural is not traditional
TIL, but it is all transferred property tumor antigen presentation that it is similar to TIL, can identify tumour variant antigens site, targeting is strong.
It can verify that PEMC has high tumor-targeting and killing ability by vitro cytotoxicity experiment.And PEMC has compared with TIL
A variety of advantages: (1) patient is mostly advanced tumor, can not be resistant to operation, it is difficult to obtain tumor tissues or lymphnode metastatic, and turn
The collection of shifting property hydrothorax is the drainage that carries out for patients in remission, itself does not increase any wound burden to patient；
(2) patient survival is short, and traditional method for extracting and amplification TIL period are longer, and obtain PEMC at once by Centrifugation method DNA, can
It is quickly expanded, meets clinical treatment requirement in time；(3) PEMC extracted in the hydrothorax of mutation composite transferring tumor is more former
The TIL extracted in the tumour of position may identify more tumour specific antigens.Therefore PEMC turns in treatment malignant tumour advanced stage lung
The more traditional TIL of patient moved with malignant pleural effusion has more advantages, while also providing for late tumor patient immune
Treat new method.
The adoptive cellular immunotherapy of tumour achieves remarkable effect in some malignant tumours, especially in hematological system
In tumour, the treatment of adoptive T cell is because its targeting is high, the advantages such as significant in efficacy have become a kind of effective novel immune and treat
Method.But the therapeutic advance in solid tumor is slow, and curative effect is unstable.With the application and breakthrough of new technology, immunization therapy will
As a part indispensable in treating malignant tumor.
Chinese patent application: CN103501816A discloses a kind of method and composition relevant to T cell, wherein making institute
It states T cell and redirects the immunotherapy for being used for CD70 positive malignancies for CD70.In many aspects of the invention, tool is used
There is the T cell of CD70 specificity.In specific aspect, there is the T cell of expression recruit, the T cell include with T- cell by
The overall length CD70 receptor (CD27) of the ζ signal transduction structural domain fusion of nanocrystal composition.This kind of T cell identification CD70 positive tumor is thin
Born of the same parents and have for CD70 positive cancer cell dissolved cell activity.But about a kind of malignant tumour Lung metastases of the present invention with evil
Property pleural effusion T cell preparation with amplification method and its application yet there are no report.
Summary of the invention
The first purpose of this invention is in view of the deficiencies of the prior art, to provide a kind of malignant tumour Lung metastases with pernicious chest
The T cell of chamber hydrops prepares and amplification method.
Second object of the present invention is in view of the deficiencies of the prior art, to provide a kind of malignant tumour Lung metastases with pernicious chest
The purposes of the T cell of chamber hydrops.
To realize above-mentioned first purpose, the technical solution adopted by the present invention is that:
A kind of malignant tumour Lung metastases with the T cell preparation of malignant pleural effusion and amplification method, the method includes with
Lower step:
(1) preparation of peripheral blood mononuclear cells PBMC；
(2) in metastatic hydrothorax monocyte PEMC preparation；
(3) enrichment of primary tumor cell；
(4) target PEMC is expanded under irradiated allosome PBMC joint high dose IL-2 stimulation.
As a preferred embodiment of the invention, the PBMC is by being made in peripheral blood；The preparation method of PBMC
Are as follows:
(1) anticoagulant tube takes peripheric venous blood, sufficiently rocks；
(2) peripheral blood mixes well in equal volume with PBS；
(3) mixing liquid and lymphocyte separation medium are pressed into mixing liquid volume: lymphocyte separation medium volume=4:3 ratio
Example is placed in lymphocyte separation medium upper layer, takes after centrifugal process separation and is suspended in intermediate tunica albuginea confluent monolayer cells；
(4) isometric PBS is added to mix well, supernatant is removed after centrifugation；
(5) serum-free cell culture medium is resuspended, and removes supernatant after centrifugation, is repeated 3 times, and precipitating is the PBMC.
As a preferred embodiment of the invention, the PEMC is by malignant tumour Lung metastases patient's metastatic hydrothorax
In be made, PEMC the preparation method comprises the following steps:
(1) the clinical chest drainage liquid collected aseptically passes through 40 μm of cell strainer removal chest impurities in water, this
Invent patient row closed drainage of pleural cavity institute of the signified sample from Bone and soft-tissue carcinoma Lung metastases with malignant pleural effusion
The tissue fluid drained collects gained；
(2) it according to the requirement of step (1), from the thoracic tissues liquid of drainage, is sub-packed in 50ml centrifuge tube under aseptic condition,
Supernatant is removed after five minutes through 300G centrifugation, collects lower layer's bloody fluid deposition；Obtained liquid deposition and PBS volume ratio will be collected
=1:1 is mixed well in equal volume, slowly gently moves into Ficoll separating liquid upper layer, and 450G is centrifuged 30 minutes, is drawn intermediate white
Film layer.
(3) after completing step (2), the above-mentioned liquid being prepared and PBS volume ratio=1:1 are mixed well in equal volume,
1200rpm/min is centrifuged 5 minutes, removes supernatant, after being resuspended with the culture medium containing 10%FBS, adjusts concentration to 5 × 105/
Ml, every hole 1ml are placed in 24 orifice plates, and 6000IU/mlIL-2 is added in every hole.
(4) on the basis of step (3), 24 orifice plates are placed in 37 DEG C, 5%CO2Incubator in cultivate.
(5) after step (4) are completed 48 hours, non-attached cell is gently blown afloat, new 24 orifice plate is moved to together with culture medium
In.
(6) according to the growing state of PEMC in step (5), half amount was carried out every 1-3 days and changes liquid, keeps culture medium IL-2 dense
Spend 6000IU/ml.
(7) to cover with PEMC in orifice plate, monokaryon lymphocyte in each pleural effusion covered in hole is collected.
As a preferred embodiment of the invention, the method for the amplification PEMC are as follows:
(1) prepare the amplification same day, take 1 × 106Culture medium of the 10ml containing 10%FBS is added in the PEMC of the above-mentioned preparation of/ml
In, while the allosome PBMC (feeder) by irradiation is added, irradiation dose 40Gy, PEMC:feeder quantity ratio=1:20,
IL3000IU/ml。
(2) it completes step (1) the 4th day, 65% culture solution is changed to the new culture medium containing 10%FBS in culture bottle, and
Keep IL-2 concentration 3000IU/ml.
(3) it completes step (1) the 5th day, irradiated allosome PBMC (feeder), PEMC:feeder is added in cell count
Quantity ratio=1:20 amounts to culture medium of the 50ml containing 10%FBS, keeps IL-2 concentration 3000IU/ml, and T-75 culture bottle is added,
Erect the culture that suspends.
(4) it completes step (1) the 8th day, 65% culture solution is changed to the new culture medium containing 10%FBS in culture bottle, and
Keep IL-2 concentration 3000IU/ml.
(5) it completes step (1) the 9th day, irradiated allosome PBMC (feeder), PEMC:feeder is added in cell count
Quantity ratio=1:20 amounts to culture medium of the 100ml containing 10%FBS, keeps IL-2 concentration 3000IU/ml.
(6) the 16th day collection cell of step (1) is completed, is counted, and primary tumor cell poison is carried out to the PEMC after amplification
Its lethal and targeting of property experimental verification.
As a preferred embodiment of the invention, the enrichment of the Primary Tumor method particularly includes:
(1) the clinical chest drainage liquid collected aseptically passes through 40 μm of cell strainer removal chest impurities in water, this
Inventing signified sample is that Bone and soft-tissue carcinoma Lung metastases are drawn with patient's row closed drainage of pleural cavity of malignant pleural effusion
The tissue fluid of outflow collects gained；
(2) it according to the requirement of step (1), from the thoracic tissues liquid of drainage, is sub-packed in 50ml centrifuge tube under aseptic condition,
Supernatant is removed after five minutes through 300G centrifugation, collects lower layer's bloody fluid deposition；The liquid deposition being collected into and PBS1:1 is isometric
It mixes well, slowly gently moves into and apply Ficoll separating liquid upper layer, 450G is centrifuged 30 minutes, draws intermediate tunica albuginea layer.
(3) after completing step (2), the above-mentioned liquid being prepared is mixed well in equal volume with PBS1:1,1200rpm/
Min is centrifuged 5 minutes, removes supernatant, after being resuspended with the culture medium containing 10%FBS, adjusts concentration to 5 × 105/ ml, every hole
1ml is placed in 24 orifice plates.
(4) on the basis of step (3), 24 orifice plates are placed in 37 DEG C, 5%CO2Incubator in cultivate；
(5) after step (4) are completed 48 hours, non-attached cell is gently blown afloat, new 24 orifice plate is moved to together with culture medium
In separately cultivate, the high sugar HighGlucoseDMEM culture medium 1ml containing 10%FBS is added in foramen primum.
(6) on the basis of step (5), 24 orifice plates are placed in 37 DEG C, 5%CO2Incubator in cultivate；
(7) according to the growing state of attached cell in step (6), its and amplification rate consistent with primary tumo(u)r form is observed
24-48 hours.
(8) culture medium is removed after covering with according to cell in step (7) hole, 500 μ L trypsin digestion and cells, about 2-5 minutes,
The 1500 μ L of DMEM in high glucose containing 10% is added in microscopic observation cell rounding, after collecting cell suspension, 1200rpm/min, and centrifugation 5
Minute, remove supernatant；PBS is added to be resuspended, goes to upper layer after centrifugation, is repeated 3 times.
(9) step (8) are completed and the DMEM in high glucose containing 10% is added afterwards, count 3 × 106Cell is placed in 6cm culture dish, altogether
The above-mentioned culture medium of 6ml is added, is placed in 37 DEG C, 5%CO2Incubator in cultivate.Remaining cell can record, freeze.
As a preferred embodiment of the invention, the cell toxicity test method particularly includes:
(1) adherent primary tumor cell is dissociated using trypsin digestion, preset fluorescer counts 5 × 103/ hole, sets
In 96 orifice plates；
(2) PEMC and Primary Tumor that preparation amplification finishes are co-cultured, by PEMC and primary tumor cell according to effect target
Than the ratio co-cultivation for 20:1,10:1,5:1,1:1, every group of 3 repetitions；
(3) it cultivates to be placed within 4 hours under spectrophotometer and counts reading, calculate PEMC cytotoxicity efficiency.
As a preferred embodiment of the invention, the malignant tumour Lung metastases include left stock with malignant pleural effusion
The transfer of bone osteosarcoma with lung is with left thoracic cavity hydrops, the transfer of right humerus osteosarcoma with lung with right pleural effusion, pelvis osteosarcoma Lung metastases
With right pleural effusion.
To realize above-mentioned second purpose, the technical solution adopted by the present invention is that:
The PEMC that any described the method as above is prepared is in preparation treatment malignant tumour Lung metastases with malignant pleural
Application in the drug of hydrops.
As a preferred embodiment of the invention, the drug is one kind with PEMC immunization therapy as main component
Property vaccine.
The invention has the advantages that:
1, the present invention is identified and kills Lung metastases struma using as the hydrothorax of clinical waste, therefrom being extracted and had
The PEMC of tumor antigen is further used for immunotherapy of tumors after proliferation, killing Pulmonary artery is not only expected to, because its identification is primary
Tumor mutations generate the novel tumor antigen of transfer, can equally have fragmentation effect to the tumour in the primary and circulatory system, for evening
Phase malignant tumor patient provides a kind of new adoptive cellular immunotherapy.
2, the present invention has verified that PEMC has high tumor-targeting and killing ability by vitro cytotoxicity experiment.
PEMC has a variety of advantages compared with TIL: (1) patient is mostly advanced tumor, can not be resistant to operation, it is difficult to obtain tumor tissues or transfer
Property lymph node, and the collection of metastatic hydrothorax is the drainage that carries out for patients in remission, itself does not increase patient
Any wound burden；(2) patient survival is short, and traditional method for extracting and amplification TIL period are longer, and are by Centrifugation method DNA
Quarter obtains PEMC, can quickly be expanded, and meets clinical treatment requirement in time；(3) in the hydrothorax of mutation composite transferring tumor
The PEMC of extraction may identify more tumour specific antigens compared with the TIL extracted in situ tumor.
3, PEMC amplification method provided by the present invention is more efficient, technique is relatively convenient, and amplification efficiency reaches 1000 times,
And has high-strength cell toxicant lethal effect.
Detailed description of the invention
Attached drawing 1 is PEMC quantity isolated in every liter of hydrothorax of different metastatic hydrothorax patients.
Attached drawing 2 is the amplification quantity of different metastatic hydrothorax patient PEMC.
Attached drawing 3 be PEMC attack Primary Tumor mirror under show (arrow；Left figure PEMC:Tumor=10:1, right figure PEMC:
Tumor=1:1).
Attached drawing 4 is lethal effect of the same patient PEMC to Primary Tumor.
Specific embodiment
The invention will be further elucidated with reference to specific embodiments.It should be understood that these embodiments are merely to illustrate this hair
It is bright rather than limit the scope of the invention.In addition, it should also be understood that, after having read the content of the invention recorded, art technology
Personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Fixed range.
Acquisition, amplification, the acquisition of primary tumor cell of embodiment 1PEMC
One, the acquisition of PEMC
The acquisition of the PEMC refer to by Lung metastases patient's malignant pleural effusion through processing culture after from tissue fluid
It separates.
(1) the clinical chest drainage liquid collected aseptically passes through 40 μm of cell strainer removal chest impurities in water, this
Inventing signified sample is that Bone and soft-tissue carcinoma Lung metastases are drawn with patient's row closed drainage of pleural cavity of malignant pleural effusion
The tissue fluid of outflow collects gained；
(2) it according to the requirement of step (1), from the thoracic tissues liquid of drainage, is sub-packed in 50ml centrifuge tube under aseptic condition,
Supernatant is removed after five minutes through 300G centrifugation, collects lower layer's bloody fluid deposition；Obtained liquid deposition and PBS volume ratio will be collected
=1:1 is mixed well in equal volume, slowly gently moves into Ficoll separating liquid upper layer, and 450G is centrifuged 30 minutes, is drawn intermediate white
Film layer.
(3) after completing step (2), the above-mentioned liquid being prepared and PBS volume ratio=1:1 are mixed well in equal volume,
1200rpm/min is centrifuged 5 minutes, removes supernatant, after being resuspended with the AIM-V culture medium containing 10%FBS, adjusting concentration to 5 ×
105/ ml, every hole 1ml are placed in 24 orifice plates, and 6000IU/mlIL-2 is added in every hole.
(4) on the basis of step (3), 24 orifice plates are placed in 37 DEG C, 5%CO2Incubator in cultivate.
(5) after step (4) are completed 48 hours, non-attached cell is gently blown afloat, new 24 orifice plate is moved to together with culture medium
In.
(6) it according to the growing state of PEMC in step (5), carries out within every diaphragm 1-3 days half amount and changes liquid, keep culture medium IL-2 dense
Spend 6000IU/ml.
(7) to cover with PEMC in orifice plate, monokaryon lymphocyte in each pleural effusion covered in hole is collected.
Two, the amplification of PEMC
(1) prepare the amplification same day, take 1 × 106The PEMC of/ml is added in AIM-V culture medium of the 10ml containing 10%FBS, together
When be added by irradiation allosome PBMC (feeder), irradiation dose 40Gy, PEMC:feeder quantity ratio=1:20,
IL3000IU/ml。
(2) it completes step (1) the 4th day, 65% culture solution is changed to the new AIM-V culture containing 10%FBS in culture bottle
Base, and keep IL-2 concentration 3000IU/ml.
(3) it completes step (1) the 5th day, irradiated allosome PBMC (feeder), PEMC:feeder is added in cell count
Quantity ratio=1:20 amounts to AIM-V culture medium of the 50ml containing 10%FBS, keeps IL-2 concentration 3000IU/ml, and T-75 training is added
Bottle is supported, the culture that suspends is erect.
(4) it completes step (1) the 8th day, 65% culture solution is changed to the new AIM-V culture containing 10%FBS in culture bottle
Base, and keep IL-2 concentration 3000IU/ml.
(5) it completes step (1) the 9th day, irradiated allosome PBMC (feeder), PEMC:feeder is added in cell count
Quantity ratio=1:20 amounts to AIM-V culture medium of the 100ml containing 10%FBS, keeps IL-2 concentration 3000IU/ml.
(6) the 16th day collection cell of step (1) is completed, is counted, and primary tumor cell poison is carried out to the PEMC after amplification
Its lethal and targeting of property experimental verification.
Three, the acquisition of primary tumor cell
(1) the clinical chest drainage liquid collected aseptically passes through 40 μm of cell strainer removal chest impurities in water, this
Inventing signified sample is that Bone and soft-tissue carcinoma Lung metastases are drawn with patient's row closed drainage of pleural cavity of malignant pleural effusion
The tissue fluid of outflow collects gained；
(2) it according to the requirement of step (1), from the thoracic tissues liquid of drainage, is sub-packed in 50ml centrifuge tube under aseptic condition,
Supernatant is removed after five minutes through 300G centrifugation, collects lower layer's bloody fluid deposition；The bodies such as obtained liquid deposition and PBS1:1 will be collected
Product mixes well, and slowly gently moves into and applies Ficoll separating liquid upper layer, and 450G is centrifuged 30 minutes, draws intermediate tunica albuginea layer.
(3) after completing step (2), the above-mentioned liquid being prepared is mixed well in equal volume with PBS1:1,1200rpm/
Min is centrifuged 5 minutes, removes supernatant, after being resuspended with the AIM-V culture medium containing 10%FBS, adjusts concentration to 5 × 105/ ml,
Every hole 1ml is placed in 24 orifice plates.
(4) on the basis of step (3), 24 orifice plates are placed in 37 DEG C, 5%CO2Incubator in cultivate；
(5) after step (4) are completed 48 hours, non-attached cell is gently blown afloat, new 24 orifice plate is moved to together with culture medium
In separately cultivate, the high sugar HighGlucoseDMEM culture medium 1ml containing 10%FBS is added in foramen primum.
(6) on the basis of step (5), 24 orifice plates are placed in 37 DEG C, 5%CO2Incubator in cultivate；
(7) according to the growing state of attached cell in step (6), its and amplification rate consistent with primary tumo(u)r form is observed
24-48 hours.
(8) culture medium is removed after covering with according to cell in step (7) hole, 500 μ L trypsin digestion and cells, about 2-5 minutes,
The 1500 μ L of DMEM in high glucose containing 10% is added in microscopic observation cell rounding, after collecting cell suspension, 1200rpm/min, and centrifugation 5
Minute, remove supernatant；PBS is added to be resuspended, goes to upper layer after centrifugation, is repeated 3 times.
(9) step (8) are completed and the DMEM in high glucose containing 10% is added afterwards, count 3 × 106Cell is placed in 6cm culture dish, altogether
The above-mentioned culture medium of 6ml is added, is placed in 37 DEG C, 5%CO2Incubator in cultivate.Remaining cell can record, freeze.
Four, cell toxicity test
(1) it by the primary tumor cell of above-mentioned acquirement after trypsin digestion, is resuspended, eccentric cleaning, is answered with culture medium
Cell quantity is adjusted with culture medium, reaches 1x106Cell/ml.
(2) according to the cell suspension of step (1), the fluorescence enhancement ligand of 5 μ l is added in the cell suspension of 2-4ml.It sets
In 37 DEG C, 5%CO2Incubator in cultivate 30 minutes.
(3) after completing step (2), it is centrifuged 1200rpm/min, is centrifuged 5 minutes, supernatant is removed, PBS is resuspended, is repeated 3 times.
(4) after completing step (3), culture medium is resuspended, adjustment cell to 5x104/ mL, by the primary tumor cell of 100 μ L
(5000 cells) is packed into a V-shaped bottom plate.
(5) after completing step (4), the homologous PEMC cell of 100 μ L is added, according to different cell concentrations.PEMC and original
For tumour ratio from 1:1 to 20:1.
(6) after completing step (5), 37 DEG C are placed in, 5%CO2Incubator in cultivate 4 hours.Take 5 μ L of upper liquid that europium is added
Solution is incubated for 5 minutes at room temperature, measures fluorescer shading value.
(7) after completing step (6), statistics shading value calculates PEMC to the cytotoxic effect of tumour.
The effect assessment of embodiment 2PEMC
1 data
1.1 general information
Clinical patients 3.1st patient is 19 years old male, is diagnosed as the transfer of left femur osteosarcoma with lung with left thoracic cavity hydrops
(EnnekingIII phase)；2nd patient is 23 years old women, is diagnosed as right humerus osteosarcoma with lung transfer with right pleural effusion
(EnnekingIII phase)；3rd patient is 25 years old male, is diagnosed as pelvis osteosarcoma Lung metastases with right pleural effusion
(EnnekingIII phase).
1.2 diagnostic criteria
Isolatism Global focus, generally less than 2cm are often shown as on x-ray rabat, mostly round, edge is compared with finishing, density
It is thin, without obvious burr and leaflet feature.
1.3 are included in standard
(1) meet above-mentioned metastatic tumor of lung diagnostic criteria；
(2) age, men and women was unlimited between 18-60 years old；
(3) subject signs informed consent form.
1.4 reject standard:
The case for not meeting the standard of being included in and being accidentally included in；Though not controlled with this research approach after meeting the standard of being included in and being included in
The case for the treatment of；Non- curative effect reason and adverse reaction and test midway stop the case that falls off；Add with other drugs person；Data is not complete
It cannot the person of statistics.
1.5 terminate and remove clinical test standard: other diseases occur in test, influence to test into passerby；It can not be pre-
Other situations seen.
2 methods
First case patient row left side closed drainage of thoracic cavity operation during being hospitalized, it is daily to drain the light bloody fluid of 800ml；Second
Row right side closed drainage of thoracic cavity operation, daily to drain about 400ml weak yellow liquid during example patient is hospitalized；Third example patient's hospital stay
Between row right side closed drainage of thoracic cavity operation, it is daily to drain the light bloody fluid of about 800ml.It all collects, passes through on the day of daily drainage-fluid
Cell sieve strainer filtering impurity dispenses 50ml centrifuge tube, 1200rpm/min5 minutes, the limpid tissue fluid in upper layer is removed after centrifugation, is added
Entering isometric PBS to mix well, the lower liquid of collection is slowly positioned on Ficoll lymphocyte separation medium by electrical pipette rifle,
Partition method extracts intermediate tunica albuginea layer monocyte, and supernatant is removed after centrifugation, and PBS is resuspended, is centrifuged 3 times.With the AIM-V containing 10%FBS
Culture medium is resuspended, and to cell count is collected, can extract about 2.4-4.5 × 10 in every liter of hydrothorax6, then adjust cell concentration to 5 ×
106/ ml, every hole 1ml are placed in 24 orifice plates, are ready for expanding.
The PEMC that will be isolated from above-mentioned 3 clinical patients, is placed in 24 orifice plates, and 6000UI/mlIL-2 is added in every hole.
24 orifice plates are placed in 37 DEG C of 5%CO2It is cultivated in cell incubator.Non- attached cell is gently blown afloat after 24-48 hours, it will be upper
Layer culture medium moves in 24 new orifice plates, passes through the growing state of PEMC in each hole of micro- sem observation daily.Increase according to cell
It grows situation 1-3 days and changes liquid, the cell in each hole is collected after PEMC covers with cell hole.The peripheral blood of normal person is taken, is gone after centrifugation
Upper plasma is mixed well with isometric PBS, is slowly added on Ficoll lymphocyte separation medium, in centrifugal process separation and Extraction
Between tunica albuginea layer, after centrifugation, the PBMC of the same race of extraction is frozen it is spare, it is to be amplified before carry out x-irradiation 40Gy.Extract 1 × 106/
The PEMC of ml is added in AIM-V culture medium of the 10ml containing 10%FBS, while the PBMC (feeder) by irradiation is added, and irradiates
Dosage is 40Gy, PEMC:feeder quantity ratio=1:20, while 3000IU/mlIL-2 is added, and is resuspended in T25 culture bottle culture.
It cultivates the 4th day, 65% culture solution is changed to the new AIM-V culture medium containing 10%FBS in culture bottle, and keeps IL-2 concentration
3000IU/ml.5th day, cell count, while being added irradiated PBMC (feeder), PEMC:feeder quantity ratio=1:
20, AIM-V culture medium of the 50ml containing 10%FBS is amounted to, IL-2 concentration 3000IU/ml is kept, is resuspended in T75 culture bottle culture.
8th day, 65% culture medium was changed to the new AIM-V culture medium containing 10%FBS in culture bottle, kept IL-2 concentration 3000IU/
Ml, the 9th day, cell count was added irradiated PBMC (feeder), PEMC:feeder quantity ratio=1:20, amounted to 100ml
AIM-V culture medium containing 10%FBS keeps IL-23000IU/ml, is resuspended in T175 culture bottle.Amplification is collected after about 2 weeks
PEMC, counting statistics meet clinical application quantitative requirement.
By adherent separation from the Malignant Pleural of 3 clinical patients, primary transfer oncocyte is isolated.It will pass through
The tunica albuginea layer that Ficoll separating liquid is collected by centrifugation, is placed in 24 orifice plates, after 24-48 hours, non-attached cell is gently blown afloat, with training
Feeding base is moved in new 24 orifice plate together and is separately cultivated, and the high sugar HighGlucoseDMEM culture medium containing 10%FBS is added in foramen primum
1ml.24 orifice plates are placed in 37 DEG C, 5%CO2Incubator in cultivate, observe the growing state of attached cell, observe its with it is primary swollen
Tumor form is consistent and amplification rate 24-48 hours.Remove culture medium after cell covers in hole, 500 μ L trypsin digestion and cells, about
2-5 minutes, the 1500 μ L of DMEM in high glucose containing 10% was added in microscopic observation cell rounding, after collecting cell suspension, 1200rpm/
Min is centrifuged 5 minutes, removes supernatant；PBS is added to be resuspended, goes to upper layer after centrifugation, is repeated 3 times.The DMEM in high glucose containing 10% is added,
Count 3 × 106Cell is placed in 6cm culture dish, and the above-mentioned culture medium of 6ml is added altogether, is placed in 37 DEG C, 5%CO2Incubator in train
It supports.It by the primary tumor cell of acquirement after trypsin digestion, is resuspended with culture medium, eccentric cleaning, applied culture keynote is whole
Cell quantity reaches 1x106Cell/ml.The fluorescence enhancement ligand of 5 μ l is added in the cell suspension of 2-4ml.It is placed in 37
DEG C, 5%CO2Incubator in cultivate 30 minutes.It is centrifuged 1200rpm/min, is centrifuged 5 minutes, supernatant is removed, PBS is resuspended, and repeats
3 times.Culture medium is resuspended, adjustment cell to 5x104The primary tumor cell (5000 cells) of 100 μ L is packed into a v by/mL
Type bottom plate.The same patient PEMC cell for adding 100 μ L, according to different cell concentrations.PEMC and Primary Tumor ratio are from 1:1
To 20:1.37 DEG C are placed in, 5%CO2Incubator in cultivate 4 hours.Microscopic observation PEMC takes the lethal effect of Primary Tumor
Europium solution is added in 5 μ L of upper liquid in plate hole, is incubated for 5 minutes at room temperature, measures fluorescer shading value.Photometric angle value calculates PEMC pairs
The cytotoxic effect of tumour.It can be seen that the PEMC in same patient's metastatic hydrothorax has very strong cytotoxicity to Primary Tumor
Effect.
3 results
Isolated PEMC quantity in every liter of hydrothorax of different metastatic hydrothorax patients is as shown in Figure 1, different metastatic chests
The amplification quantity of water patient PEMC as shown in Fig. 2, PEMC to the lethal effect result of Primary Tumor as shown in figure 3, can from figure
To see that PEMC has good lethal effect to Primary Tumor；PEMC to the cytotoxicity testing result of tumour as shown in figure 4,
The result shows that the PEMC in same patient's metastatic hydrothorax has very strong cytotoxic effect to Primary Tumor.
4 conclusions
Above embodiments show that the PEMC being prepared by means of the present invention can be used for immunotherapy of tumors, PEMC
Amplification method is more efficient, technique is relatively convenient, and has high-strength cell toxicant lethal effect.
The present invention is identified and kills pulmonary metastases tumour using as the hydrothorax of clinical waste, therefrom being extracted and had
The PEMC of antigen is further used for immunotherapy of tumors after proliferation, is not only expected to killing Pulmonary artery, because its identification is primary swollen
Tumor mutation generates the novel tumor antigen of transfer, can equally have fragmentation effect to the tumour in the primary and circulatory system, is advanced stage
Malignant tumor patient provides a kind of new adoptive cellular immunotherapy.
The present invention has verified that PEMC has high tumor-targeting and killing ability by vitro cytotoxicity experiment.
PEMC has a variety of advantages compared with TIL: (1) patient is mostly advanced tumor, can not be resistant to operation, it is difficult to obtain tumor tissues or transfer
Property lymph node, and the collection of metastatic hydrothorax is the drainage that carries out for patients in remission, itself does not increase patient
Any wound burden；(2) patient survival is short, and traditional method for extracting and amplification TIL period are longer, and are by Centrifugation method DNA
Quarter obtains PEMC, can quickly be expanded, and meets clinical treatment requirement in time；(3) in the hydrothorax of mutation composite transferring tumor
The PEMC of extraction may identify more tumour specific antigens compared with the TIL extracted in situ tumor.
PEMC amplification method provided by the present invention is more efficient, technique is relatively convenient, and amplification efficiency reaches 1000 times, and
Has high-strength cell toxicant lethal effect.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, without departing from the principle of the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (9)
1. a kind of malignant tumour Lung metastases are with the T cell preparation of malignant pleural effusion and amplification method, which is characterized in that the side
Method the following steps are included:
(1) preparation of peripheral blood mononuclear cells PBMC；
(2) in metastatic hydrothorax monocyte PEMC preparation；
(3) enrichment of primary tumor cell；
(4) target PEMC is expanded under irradiated allosome PBMC joint high dose IL-2 stimulation.
2. a kind of malignant tumour Lung metastases according to claim 1 are with the T cell preparation of malignant pleural effusion and amplification side
Method, which is characterized in that the PBMC is by being made in peripheral blood；PBMC's the preparation method comprises the following steps:
(1) anticoagulant tube takes peripheric venous blood, sufficiently rocks；
(2) peripheral blood mixes well in equal volume with PBS；
(3) by mixing liquid and lymphocyte separation medium in mixing liquid volume: lymphocyte separation medium volume=4:3 ratio is set
In lymphocyte separation medium upper layer, is taken after centrifugal process separation and be suspended in intermediate tunica albuginea confluent monolayer cells；
(4) isometric PBS is added to mix well, supernatant is removed after centrifugation；
(5) serum-free cell culture medium is resuspended, and removes supernatant after centrifugation, is repeated 3 times, and precipitating is the PBMC.
3. a kind of malignant tumour Lung metastases according to claim 1 are with the T cell preparation of malignant pleural effusion and amplification side
Method, which is characterized in that the PEMC be by malignant tumour Lung metastases patient's metastatic hydrothorax be made, PEMC the preparation method comprises the following steps:
(1) drainage-fluid is placed in the vial of sterilized and heparin processing by Patients with Malignant Pleural Metastases row closed drainage of thoracic cavity operation
In；
(2) after being enriched with the metastatic pleural effusion of malignant tumour Lung metastases patient by centrifugal process, then pass through lymphocyte point
Chaotropic centrifuge separation, to obtain the monocyte drained in metastatic pleural effusion；
(3) isometric PBS is added, goes to upper layer after centrifugation；
(4) PBS is resuspended, then is centrifuged supernatant, is repeated 3 times, and precipitating is the PEMC.
4. a kind of malignant tumour Lung metastases according to claim 1 are with the T cell preparation of malignant pleural effusion and amplification side
Method, which is characterized in that the method for the amplification PEMC are as follows:
(1) PBMC is resuspended in serum free medium, and concentration is adjusted to 1 × 106/ mL is placed in culture bottle；
(2) PBMC accumulated dose 40Gy is irradiated；
(3) by the PBMC after irradiation and the PEMC Combined culture being free in the monokaryon lymphocyte in malignant hydrothorax, add simultaneously
Enter high dose IL-2, concentration 6000U/mL；
(4) liquid being changed every half a day and carrying out cell count, upper layer supernatant is removed in centrifugation after 14 days, and precipitating is PEMC after expanding.
5. a kind of malignant tumour Lung metastases according to claim 1 are with the T cell preparation of malignant pleural effusion and amplification side
Method, which is characterized in that the enrichment of the Primary Tumor method particularly includes:
(1) drainage-fluid is placed in the vial of sterilized and heparin processing by Patients with Malignant Pleural Metastases row closed drainage of thoracic cavity operation
In；
(2) Centrifugation method DNA obtains the monocyte drained in metastatic pleural effusion；
(3) isometric PBS is added, goes to upper layer after centrifugation；
(4) PBS is resuspended, then is centrifuged supernatant, is repeated 3 times；
After (5) 24 orifice plate cultures 24-48 hours, non-attached cell, remaining attached cell change 10%FBS high sugar are gently blown afloat
DMEM culture medium continues to cultivate；
(6) it cultivates 24-48 hours, it is seen that form meets the as primary tumor cell of primary tumo(u)r and rapid amplifying.
6. a kind of malignant tumour Lung metastases according to claim 1 are with the T cell preparation of malignant pleural effusion and amplification side
Method, which is characterized in that the method also includes cell toxicity test, cell toxicity test method particularly includes:
(1) adherent primary tumor cell is dissociated using trypsin digestion, preset fluorescer counts 5 × 103/ hole, is placed in 96
In orifice plate；
(2) PEMC and Primary Tumor that preparation amplification finishes are co-cultured, is according to effect target ratio by PEMC and primary tumor cell
The ratio of 20:1,10:1,5:1,1:1 co-culture, every group of 3 repetitions；
(3) it cultivates to be placed within 4 hours under spectrophotometer and counts reading, calculate PEMC cytotoxicity efficiency.
7. -6 any the method according to claim 1, which is characterized in that the malignant tumour Lung metastases are with malignant pleural effusion
It shifts with left thoracic cavity hydrops, the transfer of right humerus osteosarcoma with lung including left femur osteosarcoma with lung with right pleural effusion, pelvis bone and flesh
Tumor Lung metastases are with right pleural effusion.
8. the PEMC that any the method for claim 1-6 is prepared is in preparation treatment malignant tumour Lung metastases with malignant pleural
Application in the drug of hydrops.
9. applying according to claim 8, which is characterized in that the drug is that one kind is controlled with as main component be immunized of PEMC
The property treated vaccine.
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| CN105087485A (en) * | 2015-07-10 | 2015-11-25 | 上海鑫宸医疗科技有限公司 | Culture method of tumor specific TIL cells |
| CN106456670A (en) * | 2014-04-25 | 2017-02-22 | 蓝鸟生物公司 | Improved methods for manufacturing adoptive cell therapies |
| CN106591232A (en) * | 2017-01-04 | 2017-04-26 | 安徽安龙基因医学检验所有限公司 | Efficient PD-1-CD8+T cell culture method |
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| CN106456670A (en) * | 2014-04-25 | 2017-02-22 | 蓝鸟生物公司 | Improved methods for manufacturing adoptive cell therapies |
| CN105087485A (en) * | 2015-07-10 | 2015-11-25 | 上海鑫宸医疗科技有限公司 | Culture method of tumor specific TIL cells |
| CN106591232A (en) * | 2017-01-04 | 2017-04-26 | 安徽安龙基因医学检验所有限公司 | Efficient PD-1-CD8+T cell culture method |
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| 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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