CN106222139B - A method of High Fragmentation activity til cell is largely prepared using concretionary pernicious Pleural effusions - Google Patents
A method of High Fragmentation activity til cell is largely prepared using concretionary pernicious Pleural effusions Download PDFInfo
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Abstract
The present invention relates to a kind of methods for largely preparing High Fragmentation activity til cell using concretionary pernicious Pleural effusions.This method can harvest til cell (190.1 ± 46.7) × 10 when cultivating the 21st~28 day8A (n=12), cell are expanded up to 182.5 ± 46.8 times；Wherein, CD3+Cell accounts for about 98.55% ± 2.24%, CD3+CD8+Cell accounts for about 76.11% ± 6.88%, CD3+CD4+Cell accounts for about 24.09% ± 6.73%, CD3+CD56+Cell accounts for about 53.36% ± 9.47%, and CD4+CD25+FoxP3+Tregs cell only accounts for 2.87% ± 1.65%, to the killing activity of tumour cell up to 60.57% ± 5.34% (4h51Cr method for releasing, effect/target=40:1)；Compared with when not handling grumeleuse, the present invention can make the til cell quantity of harvest have more about 30%.
Description
Technical field
The invention belongs to field of cell culture, and in particular to a kind of concretionary pernicious Pleural effusions of utilization largely prepare height and kill
Hurt the method for active til cell.
Background technique
Tumor infiltrating lymphocyte (tumor-infiltrating lymphocytes, TIL) is in tumor tissues or to swell
A heterogeneous lymphocyte populations present in tumor regional lymph nodes, including by the preactivated CD3 of tumour antigen+CD4+Th cell
(T helper lymphocytes) and CD3+CD8+CTL cell (cytotoxic T lymphocytes), can be restricted with MHC
Mode specific recognition, killing tumor cell；A certain number of nonspecific CD3 of tumour antigen+CD4+Or CD3+CD8+T is thin
Born of the same parents；A small amount of CD3-CD56+NK cell (natural killers), with non-MHC restrictive one killing tumor cell；In addition,
Also contain a certain number of CD4 with immunosuppressive action+CD25+FoxP3+Tregs (regulatory T cells) and
IDCs cell (immature dendritic cells) etc., TGF-β, VEGF, PGE2, IL-6, IL-10 with high level expression
Equal molecules constitute inhibitive ability of immunity microenvironment together, prevent the continuous activation of specific for tumour antigen lymphocyte, make it difficult to
Tumor cytotoxicity is effectively played, tumor immune escape is caused.
[Rosenberg SA, et in the clinical research that National Institutes of Health Cancer Institute carries out
al.Durable complete responses in heavily pretreated patients with metastatic
Melanoma using T-cell transfer immunotherapy.Clinical Cancer Research, 2011,17
(13): 4550-4557 it], is separated simultaneously from the melanoma cancer tissue or its regional lymph nodes that operation excision or biopsy obtain
Massive amplification prepares til cell, and then joint lymph pre cleaning scheme, which is adopted, is transfused Advanced Malignant melanoma patients body
Interior, treated effect (CR+PR) was up to 52%, CR patient's longest paracmasis more than 82 months；In treating effective case, visitor
The tumor regression of sight almost occurs in all metastatic lesions, such as brain, lung, liver, bone, lymph node, subcutaneous tissue.This table
Bright, til cell has huge clinical application potentiality in anti-tumor immunotherapy.
But separation prepares til cell from tumor tissues or regional lymph nodes, need to undergo shearing, the enzyme of operation stripping and slicing
A series of cumbersome, lengthy procedures such as solution and cell separation, culture screening, extensive amplification；Meanwhile complicated in vitro operation makes
Til cell faces very high pollution risk, and culture is easily caused to fail；Moreover, most late tumor patients do not have
Standby surgical condition can not obtain til cell from operation stripping and slicing；Though some patients can implement biopsy of performing the operation or carry out, obtain
Tumor tissues are usually very little, it is difficult to therefrom amplify enough til cells, be unable to satisfy the needs of clinical treatment.So passing through
Tumor tissues obtain til cell adopt be transfused antineoplaston faced during clinical implementation it is many be difficult to overcome it is tired
It is difficult.
Patients' Chang Yin tumour splanchnocoel such as lung cancer, breast cancer, gastric cancer, colorectal cancer or oophoroma shifts and pernicious chest occurs
Ascites, a large amount of Clinical Evidences show that there are tumour antigen reactivity til cells in pernicious Pleural effusions, have potential extract
Utility value.However, there are one by the cell factors such as Tregs, iDCs and IL-10, TGF-β, VEGF in pernicious Pleural effusions
The inhibitive ability of immunity microenvironment of composition；Meanwhile an important mechanisms as immunologic escape, the usual low expression MHC- of tumour cell
I/class Ⅱmolecule, can not effective submission all kinds of tumour antigens, cause insufficient or different swollen of tumor response til cell activation
Tumour-reactive T lymphocyte clonal activation is unbalanced, rare numbers and generally existing functional defect.
On the one hand, extraction prepares til cell and adopt when being transfused antineoplaston from pernicious Pleural effusions, infused cells
The quantity of the ratio of middle tumour-specific til cell, quantity and the Tregs cell mixed is influence clinical efficacy important
Factor.Chinese patent literature CN104946589A is using the antibody coating magnetic bead of PD-1, LAG-3 or TIM-3 to pernicious chest/ascites
Middle isolated mononuclearcell is screened, and is then expanded with OKT3 and IL-2 stimulation screening cell, which not can solve because swollen
The presence of oncocyte I/class Ⅱmolecule of low expression MHC- and inhibitive ability of immunity microenvironment and caused by pernicious chest/ascites tumour it is special
The problem of anisotropic til cell activation is insufficient, rare numbers, it is difficult to filter out the tumour-specific til cell of high abundance；Moreover
Due to also having the developed by molecule such as PD-1, LAG-3 or TIM-3 on Tregs and other tumour antigen non-specificity til cells, they
Also it can be entered with immunomagnetic beads in screening cell and obtain massive amplification, influence whether tomour specific in culture finished product
The purity and anti-tumor effect of property til cell.As for other til cell preparation method, such as Chinese patent literature
CN103468641A and CN1560234A stimulates Pleural effusions using for example anti-OX-40 of non-specific activator or OKT3, PHA, IL-2 etc.
The proliferation of medium size lymphocyte causes in the til cell that amplification obtains, in addition to the CD3 containing specific for tumour antigen+CD4+Th1
Cell and CD3+CD8+CTLs is extracellular, also contains the nonspecific CD3 of a large amount of tumour antigen+CD4+Or CD3+CD8+T cell,
NK cell and Tregs cell with immunosuppressive action, the killing-efficiency of the til cell of harvest to patient tumors cell
It is low, it is difficult to constantly, efficiently to play antitumor action in patient's body.
On the other hand, since pernicious Pleural effusions are tissue exudates, contain concentration cellulose proteinogen, cause easily to occur
Solidification, thus need to be added in acquisition enough heparin sodiums or EDTA-K2 carry out it is anticoagulant.However, some pernicious Pleural effusions from
It has been solidified when body, has shown as the visible fibrin clot not of uniform size in fresh drainage-fluid.Anti-coagulants is for inhibiting to dislike
Solidification after property Pleural effusions are in vitro has a certain effect, but can not eliminate the grumeleuse formed.In addition, some pernicious chest and abdomen
Water (especially hemorrhagic pernicious Pleural effusions), though having been subjected to fully anticoagulation, does not find any solidification sign, in its centrifugation
It still will appear the grumeleuse for being not easy to dissociate in the cell precipitation formed afterwards.A large number of studies show that containing in the grumeleuse of pernicious Pleural effusions
A large amount of til cells.When carrying out density gradient centrifugation, these grumeleuses can interfere layering, influence the separation of mononuclearcell；Together
When, since grumeleuse is due to being sunken to centrifuge tube tube bottom with the high specific gravities cell component such as red blood cell, granulocyte, causing to be rejected,
This may cause a large amount of loss of til cell.
High Fragmentation activity til cell is largely prepared using concretionary pernicious Pleural effusions however, having no in the prior art
Report.
Therefore, the method that the concretionary pernicious Pleural effusions of research and utilization largely prepare High Fragmentation activity til cell has weight
Want meaning.
Summary of the invention
For this purpose, the present invention provides and a kind of largely prepares High Fragmentation activity til cell using concretionary pernicious Pleural effusions
Method.
In order to solve the above technical problems, the present invention is achieved through the following technical solutions:
The present invention provides a kind of method for largely preparing High Fragmentation activity til cell using concretionary pernicious Pleural effusions,
The following steps are included:
(1) collection of concretionary pernicious Pleural effusions: under aseptic condition, concretionary pernicious Pleural effusions are collected, and be added
Heparin, so that the concentration in the pernicious Pleural effusions containing the heparin is 11.0~16.5U/mL；
(2) in concretionary pernicious Pleural effusions free cell and grumeleuse separation: by the concretionary pernicious Pleural effusions
It is filtered, separates the grumeleuse in pernicious Pleural effusions with free cell, obtain free cell-A；
(3) release of the decomposition of grumeleuse and free cell: the grumeleuse is placed in AIM-V complete medium-I suitable
Under conditions of cultivate 3~5 days, filter, obtain free cell-B；
(4) separation of mononuclearcell: the free cell-A and the free cell-B are moved into centrifuge tube respectively,
Centrifugation, discards supernatant liquid；Sedimentation cell is resuspended, the Ficoll that specific gravity is 1.076~1.078 is laid in and is layered on liquid, centrifugation；
(5) washing of mononuclearcell: the mononuclearcell on Ficoll layering liquid interface is collected, AIM-V is added without blood
Clear culture medium mixes, and centrifugation discards supernatant liquid；
(6) the directional induction activation of til cell: cell is resuspended with the AIM-V complete medium-I, and it is close to adjust cell
Degree is 1.5 × 106~3.0 × 106A/mL is cultivated 4~6 days under appropriate conditions, and complete with the AIM-V during culture
Half amount of culture medium-I is changed liquid at least 1 time；
(7) advantage pcr of til cell: from culture the 5th~7 day, being resuspended cell with AIM-V complete medium-II, and
Adjusting cell density is 1.0 × 106~2.0 × 106A/mL continues to cultivate under appropriate conditions, carries out cell count daily
And cell density is adjusted to 1.0 × 10 with the AIM-V complete medium-II6~2.0 × 106A/mL；
(8) collection of til cell: at culture the 21st~28 day, cell suspension is collected, centrifugation is washed with 0.9%NaCl solution
It washs, cell is resuspended in 0.9%NaCl mixed solution, it is spare.
Preferably, the above-mentioned side that High Fragmentation activity til cell is largely prepared using concretionary pernicious Pleural effusions of the present invention
Method, comprising the following steps:
(1) collection of concretionary pernicious Pleural effusions: under aseptic condition, concretionary pernicious Pleural effusions are collected, and be added
Heparin, so that the concentration in the pernicious Pleural effusions containing the heparin is 12.5~15.0U/mL；
(2) in concretionary pernicious Pleural effusions free cell and grumeleuse separation: by the concretionary pernicious Pleural effusions
It is filtered, separates the grumeleuse in pernicious Pleural effusions with free cell, obtain free cell-A；
(3) release of the decomposition of grumeleuse and free cell: the grumeleuse is placed in AIM-V complete medium-I suitable
Under conditions of cultivate 3~5 days, filter, obtain free cell-B；
(4) separation of mononuclearcell: the free cell-A and the free cell-B are moved into centrifuge tube respectively,
500 × g is centrifuged 5min, discards supernatant liquid；Sedimentation cell is resuspended, the Ficoll that specific gravity is 1.076~1.078 is laid in and is layered
On liquid, 1600 × g is centrifuged 15min；
(5) washing of mononuclearcell: the mononuclearcell on Ficoll layering liquid interface is collected, AIM-V is added without blood
Clear culture medium mixes, and 300 × g is centrifuged 5min, discards supernatant liquid；
(6) the directional induction activation of til cell: cell is resuspended with AIM-V complete medium-I, and adjusts cell density and is
1.5×106~3.0 × 106A/mL is cultivated 4~6 days under appropriate conditions, is cultivated completely during culture with the AIM-V
Half amount of base-I is changed liquid 1 time；
(7) advantage pcr of til cell: from culture the 5th~7 day, being resuspended cell with AIM-V complete medium-II, and
Adjusting cell density is 1.0 × 106~2.0 × 106A/mL continues to cultivate under appropriate conditions, carries out cell count daily
And cell density is adjusted to 1.0 × 10 with the AIM-V complete medium-II6~2.0 × 106A/mL；
(8) collection of til cell: at culture the 21st~28 day, cell suspension is collected, 300 × g is centrifuged 5min, with 0.9%
NaCl solution washing, cell is resuspended in 0.9%NaCl mixed solution, spare.
Preferably, the above-mentioned side that High Fragmentation activity til cell is largely prepared using concretionary pernicious Pleural effusions of the present invention
Method,
The decomposition of step (3) grumeleuse and the release of free cell and the directional induction of step (6) til cell activate
In, the AIM-V complete medium-I contains IFN-γ, IL-2 and inactivated serum；
In the advantage pcr of step (7) til cell, the AIM-V complete medium-II containing IL-2, IFN-γ,
CD 3-resisting monoclonal antibody and inactivated serum.
It is further preferred that the present invention is above-mentioned, using concretionary pernicious Pleural effusions, largely to prepare High Fragmentation activity TIL thin
The method of born of the same parents, comprising the following steps:
(1) collection of concretionary pernicious Pleural effusions: under aseptic condition, concretionary pernicious Pleural effusions are collected, and be added
Heparin, so that the concentration in the pernicious Pleural effusions containing the heparin is 12.5~15.0U/mL；
(2) in concretionary pernicious Pleural effusions free cell and grumeleuse separation: by the concretionary pernicious Pleural effusions
It is filtered, separates the grumeleuse in pernicious Pleural effusions with free cell, obtain free cell-A；
(3) release of the decomposition of grumeleuse and free cell: the grumeleuse is placed in AIM-V complete medium-I suitable
Under conditions of cultivate 3~5 days, filter, obtain free cell-B；
(4) separation of mononuclearcell: the free cell-A and the free cell-B are moved into centrifuge tube respectively,
Centrifugation, discards supernatant liquid；Sedimentation cell is resuspended, the Ficoll that specific gravity is 1.077 is laid in and is layered on liquid, centrifugation；
(5) washing of mononuclearcell: the mononuclearcell on Ficoll layering liquid interface is collected, AIM-V is added without blood
Clear culture medium mixes, and centrifugation discards supernatant liquid；
(6) the directional induction activation of til cell: cell is resuspended with the AIM-V complete medium-I, and it is close to adjust cell
Degree is 2.2 × 106A/mL is cultivated 5 days under appropriate conditions, is changed during culture with-I half amount of AIM-V complete medium
Liquid 1 time；
(7) advantage pcr of til cell: from culture the 6th day, cell is resuspended with AIM-V complete medium-II, and adjust
Ganglion cell's density is 1.5 × 106A/mL continues to cultivate under appropriate conditions, daily to carry out cell count and with the AIM-
Cell density is adjusted to 1.5 × 10 by V complete medium-II6A/mL；
(8) collection of til cell: at culture the 21st~28 day, cell suspension is collected, centrifugation is washed with 0.9%NaCl solution
It washs, cell is resuspended in 0.9%NaCl mixed solution, it is spare.
It is further preferred that the present invention is above-mentioned, using concretionary pernicious Pleural effusions, largely to prepare High Fragmentation activity TIL thin
The method of born of the same parents,
In the concretionary pernicious Pleural effusions of step (2) in the separation of free cell and grumeleuse, millipore filter is utilized
The concretionary pernicious Pleural effusions are filtered.
It is further preferred that the present invention is above-mentioned, using concretionary pernicious Pleural effusions, largely to prepare High Fragmentation activity TIL thin
The method of born of the same parents, the aperture of the millipore filter are 70 μm, and the millipore filter is nylon micro porous filter.
It is further preferred that the present invention is above-mentioned, using concretionary pernicious Pleural effusions, largely to prepare High Fragmentation activity TIL thin
The method of born of the same parents,
The decomposition of step (3) grumeleuse and the release of free cell and the directional induction of step (6) til cell activate
In, the AIM-V complete medium-I contains 500~1000U/mL IFN-γ, the inactivation blood of 10~100U/mL IL-2 and 3%
Clearly；
In the advantage pcr of step (7) til cell, the AIM-V complete medium-II contains 500~2000U/
ML IL-2,100~300U/mL IFN-γ, 20~50ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum.
It is further preferred that the present invention is above-mentioned, using concretionary pernicious Pleural effusions, largely to prepare High Fragmentation activity TIL thin
The method of born of the same parents,
In the separation of step (4) mononuclearcell, with AIM-V serum free medium, RPMI1640 free serum culture
Sedimentation cell is resuspended for base or PBS buffer solution.
It is further preferred that the present invention is above-mentioned, using concretionary pernicious Pleural effusions, largely to prepare High Fragmentation activity TIL thin
The method of born of the same parents,
In the washing of step (5) mononuclearcell, the mononuclearcell is that lymphocyte, DC cell and tumour are thin
The mixture of born of the same parents.
It is further preferred that the present invention is above-mentioned, using concretionary pernicious Pleural effusions, largely to prepare High Fragmentation activity TIL thin
The method of born of the same parents,
Further include being repeated at least once more following steps in the washing of step (5) mononuclearcell: be added AIM-V without
Cell is resuspended in blood serum medium, mixes, and centrifugation discards supernatant liquid.
It is further preferred that the present invention is above-mentioned, using concretionary pernicious Pleural effusions, largely to prepare High Fragmentation activity TIL thin
The method of born of the same parents,
In the collection of step (8) til cell, the 0.9%NaCl mixed solution contains IL-2, people's blood Alb and Portugal
Grape sugar.
It is further preferred that the present invention is above-mentioned, using concretionary pernicious Pleural effusions, largely to prepare High Fragmentation activity TIL thin
The method of born of the same parents,
In the collection of step (8) til cell, the 0.9%NaCl mixed solution contains 1000U/mL IL-2,3%
People's blood Alb and 6mM glucose.
It is further preferred that the present invention is above-mentioned, using concretionary pernicious Pleural effusions, largely to prepare High Fragmentation activity TIL thin
The release of the method for born of the same parents, the decomposition of step (3) grumeleuse and free cell, the directional induction activation of step (6) til cell and
In the advantage pcr of step (7) til cell, the suitable condition refers to: 37 DEG C, 5%CO2And saturated humidity.
The present invention also provides the til cells that the above method is prepared.
Compared with prior art, technical solution of the present invention has the advantages that
(1) method of the present invention for largely preparing High Fragmentation activity til cell using concretionary pernicious Pleural effusions is led to
Concretionary pernicious Pleural effusions will be filtered after first, then train the grumeleuse being obtained by filtration under appropriate conditions
It supports, filtering, so as to be separated to more mononuclearcells, and then significantly improves the quantity of the til cell of preparation, then exist
On the basis of inducing tumour cell and DC cell upregulation expression I/class Ⅱmolecule of MHC-, enhancing tumour antigen submission, make pernicious chest
The tumor response lymphocyte of fresh separated undergoes the process of specific activation, clonal expansion first in ascites, then recycles
Specific combination of cytokines stimulates it to expand on a large scale, while inhibiting the activation and proliferation of Tregs cell, makes finally obtained
Contain a high proportion of tumour-specific til cell in cell products, so that it is guaranteed that prepared til cell is to autologous patient tumour
Cell has special, efficient killing activity；
(2) method of the present invention for largely preparing High Fragmentation activity til cell using concretionary pernicious Pleural effusions,
Til cell (190.1 ± 46.7) × 10 can be harvested when cultivating the 21st~28 day8A (n=12), cell amplification up to 182.5 ±
46.8 times；Wherein, CD3+Cell accounts for about 98.55% ± 2.24%, CD3+CD8+Cell accounts for about 76.11% ± 6.88%, CD3+
CD4+Cell accounts for about 24.09% ± 6.73%, CD3+CD56+Cell accounts for about 53.36% ± 9.47%, and CD4+CD25+FoxP3+
Tregs cell only accounts for 2.87% ± 1.65%, to the killing activity of tumour cell up to 60.57% ± 5.34% (4h51Cr release
Method, effect/target=40:1)；
(3) compared with when not handling grumeleuse, the present invention can make the til cell quantity of harvest have more about 30%.
Specific embodiment
In following embodiment of the present invention,
AIM-V serum free medium: GIBCO, lot number: 1678674；
Ficoll is layered liquid: GE Healthcare, lot number 10024695；
IL-2: Shandong Quan Gang medicine company, lot number: 201501005；
IFN-γ: Shanghai Kai Mao biological medicine, lot number: G20150101；
CD 3-resisting monoclonal antibody: eBioscience, lot number: E06305-1691；
People's blood Alb: the long-range medicine company in Sichuan, lot number: 201504045A.
Embodiment 1
The method that the present embodiment largely prepares High Fragmentation activity til cell using concretionary pernicious Pleural effusions, including with
Lower step:
(1) under aseptic condition, it is concretionary pernicious the collection of concretionary Malignant Pleural: to collect 1000mL caused by lung cancer
Hydrothorax 1000mL, and 1.5 ten thousand U heparin are added；
(2) in concretionary Malignant Pleural free cell and grumeleuse separation: be 70 μm of nylon micro porous mistake using aperture
The concretionary Malignant Pleural is filtered by filter, separates the grumeleuse in Malignant Pleural with free cell, must be dissociated thin
Born of the same parents-A；
(3) release of the decomposition of grumeleuse and free cell: the grumeleuse is placed in containing 750U/mL IFN-γ, 55U/mL
In 37 DEG C, 5%CO in the AIM-V complete medium-I of IL-2 and 3% inactivated serum2It is cultivated 3 days under conditions of saturated humidity,
Filtering, obtains free cell-B；
(4) separation of mononuclearcell: the free cell-A and the free cell-B are moved into centrifuge tube respectively,
500 × g is centrifuged 5min, discards supernatant liquid；Sedimentation cell is resuspended, the Ficoll that specific gravity is 1.076~1.078 is laid in and is layered
On liquid, 1600 × g is centrifuged 15min；
(5) washing of mononuclearcell: collect Ficoll layering liquid interface on mononuclearcell (containing lymphocyte,
DC cell and tumour cell), AIM-V serum free medium is added, piping and druming mixes, and 300 × g is centrifuged 5min, discards supernatant liquid；
It is repeated 2 times following steps: AIM-V serum free medium is added, cell is resuspended, piping and druming mixes, and 300 × g is centrifuged 5min, discards
Clear liquid；
(6) the directional induction activation of til cell: blood is inactivated with containing 750U/mL IFN-γ, 55U/mL IL-2 and 3%
Cell is resuspended in clear AIM-V complete medium-I, and cell count total amount is 85.7 × 106A, adjusting cell density is 2.2 × 106
A/mL, in 37 DEG C, 5%CO2It is cultivated 5 days under conditions of saturated humidity, with the AIM-V complete medium-I during culture
Half amount is changed liquid 1 time；
(7) advantage pcr of til cell: from culture the 6th day, with contain 1250U/mL IL-2,200U/mL IFN-
Cell is resuspended in the AIM-V complete medium-II of γ, 35ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum, and adjusts cell
Density is 1.5 × 106A/mL, in 37 DEG C, 5%CO2With continue to cultivate under conditions of saturated humidity, it is daily to carry out cell count simultaneously
Cell density is adjusted to 1.5 × 10 with the AIM-V complete medium-II6A/mL；
(8) collection of til cell: at culture the 21st day, cell suspension is collected, 300 × g is centrifuged 5min, uses 0.9%NaCl
It is molten to be resuspended in the mixing of the 0.9%NaCl containing 1000U/mL IL-2,3% people's blood Alb and 6mM glucose by solution washing for cell
It is spare in liquid.
By detection it is found that til cell total amount manufactured in the present embodiment is 1.62 × 1010It is a, 189.0 times of coamplification；Its
In, CD3+Cell accounts for 99.32%, CD3+CD8+Cell accounts for 77.56%, CD4+CD25+FoxP3+Tregs cell accounts for 3.42%；It is right
The killing activity of tumour cell is 66.84% (4h51Cr method for releasing, effect/target=40:1).
Embodiment 2
The method that the present embodiment largely prepares High Fragmentation activity til cell using concretionary pernicious Pleural effusions, including with
Lower step:
(1) collection of concretionary malignant ascite: under aseptic condition, concretionary malignant ascite caused by gastric cancer is collected
1200mL, and 1.8 ten thousand U heparin are added；
(2) in concretionary malignant ascite free cell and grumeleuse separation: be 70 μm of nylon micro porous mistake using aperture
The concretionary malignant ascite is filtered by filter, separates the grumeleuse in malignant ascite with free cell, must be dissociated thin
Born of the same parents-A；
(3) release of the decomposition of grumeleuse and free cell: the grumeleuse is placed in containing 500U/mL IFN-γ, 100U/
In 37 DEG C, 5%CO in the AIM-V complete medium-I of mL IL-2 and 3% inactivated serum2With 4 are cultivated under conditions of saturated humidity
It, filtering obtains free cell-B；
(4) separation of mononuclearcell: the free cell-A and the free cell-B are moved into centrifuge tube respectively,
500 × g is centrifuged 5min, discards supernatant liquid；Sedimentation cell is resuspended, the Ficoll that specific gravity is 1.077 is laid in and is layered on liquid,
1600 × g is centrifuged 15min；
(5) washing of mononuclearcell: collect Ficoll layering liquid interface on mononuclearcell (containing lymphocyte,
DC cell and tumour cell), AIM-V serum free medium is added, piping and druming mixes, and 300 × g is centrifuged 5min, discards supernatant liquid；
It is repeated 2 times following steps: AIM-V serum free medium is added, cell is resuspended, piping and druming mixes, and 300 × g is centrifuged 5min, discards
Clear liquid；
(6) the directional induction activation of til cell: blood is inactivated with containing 500U/mL IFN-γ, 100U/mL IL-2 and 3%
Cell is resuspended in clear AIM-V complete medium-I, and cell count total amount is 112.5 × 106It is a, adjust cell density be 1.5 ×
106A/mL, in 37 DEG C, 5%CO2It is cultivated 6 days under conditions of saturated humidity, with the AIM-V complete medium-during culture
I half amounts are changed liquid 1 time；
(7) advantage pcr of til cell: from culture the 7th day, with containing 500U/mL IL-2,300U/mL IFN-γ,
Cell is resuspended in the AIM-V complete medium-II of 50ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum, and it is close to adjust cell
Degree is 1.0 × 106A/mL, in 37 DEG C, 5%CO2With continue to cultivate under conditions of saturated humidity, it is daily to carry out cell count and be used in combination
Cell density is adjusted to 1.0 × 10 by the AIM-V complete medium-II6A/mL；
(8) collection of til cell: at culture the 28th day, cell suspension is collected, 300 × g is centrifuged 5min, uses 0.9%NaCl
It is molten to be resuspended in the mixing of the 0.9%NaCl containing 1000U/mL IL-2,3% people's blood Alb and 6mM glucose by solution washing for cell
It is spare in liquid.
By detection it is found that til cell total amount manufactured in the present embodiment is 1.95 × 1010It is a, 174.1 times of coamplification；Its
In, CD3+Cell accounts for 97.34%, CD3+CD8+Cell accounts for 79.10%, CD4+CD25+FoxP3+Tregs cell accounts for 3.44%；It is right
The killing activity of tumour cell is 61.18% (4h51Cr method for releasing, effect/target=40:1).
Embodiment 3
The method that the present embodiment largely prepares High Fragmentation activity til cell using concretionary pernicious Pleural effusions, including with
Lower step:
(1) collection of concretionary Malignant Pleural: under aseptic condition, concretionary Malignant Pleural caused by lung cancer is collected
1000mL, and 1.5 ten thousand U heparin are added；
(2) in concretionary Malignant Pleural free cell and grumeleuse separation: be 70 μm of nylon micro porous mistake using aperture
The concretionary Malignant Pleural is filtered by filter, separates the grumeleuse in Malignant Pleural with free cell, must be dissociated thin
Born of the same parents-A；
(3) release of the decomposition of grumeleuse and free cell: the grumeleuse is placed in containing 500U/mL IFN-γ, 100U/
In 37 DEG C, 5%CO in the AIM-V complete medium-I of mL IL-2 and 3% inactivated serum2With 5 are cultivated under conditions of saturated humidity
It, filtering obtains free cell-B；
(4) separation of mononuclearcell: the free cell-A and the free cell-B are moved into centrifuge tube respectively,
500 × g is centrifuged 5min, discards supernatant liquid；Sedimentation cell is resuspended, the Ficoll that specific gravity is 1.077 is laid in and is layered on liquid,
1600 × g is centrifuged 15min；
(5) washing of mononuclearcell: collect Ficoll layering liquid interface on mononuclearcell (containing lymphocyte,
DC cell and tumour cell), AIM-V serum free medium is added, piping and druming mixes, and 300 × g is centrifuged 5min, discards supernatant liquid；
It is repeated 2 times following steps: AIM-V serum free medium is added, cell is resuspended, piping and druming mixes, and 300 × g is centrifuged 5min, discards
Clear liquid；
(6) the directional induction activation of til cell: blood is inactivated with containing 1000U/mL IFN-γ, 10U/mL IL-2 and 3%
Cell is resuspended in clear AIM-V complete medium-I, and cell count total amount is 86.6 × 106A, adjusting cell density is 3.0 × 106
A/mL, in 37 DEG C, 5%CO2It is cultivated 4 days under conditions of saturated humidity, with the AIM-V complete medium-I during culture
Half amount is changed liquid 1 time；
(7) advantage pcr of til cell: from culture the 5th day, with contain 2000U/mL IL-2,100U/mL IFN-
Cell is resuspended in the AIM-V complete medium-II of γ, 20ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum, and adjusts cell
Density is 2.0 × 106A/mL, in 37 DEG C, 5%CO2With continue to cultivate under conditions of saturated humidity, it is daily to carry out cell count simultaneously
Cell density is adjusted to 2.0 × 10 with the AIM-V complete medium-II6A/mL；
(8) collection of til cell: at culture the 21st day, cell suspension is collected, 300 × g is centrifuged 5min, uses 0.9%NaCl
It is molten to be resuspended in the mixing of the 0.9%NaCl containing 1000U/mL IL-2,3% people's blood Alb and 6mM glucose by solution washing for cell
It is spare in liquid.
By detection it is found that til cell total amount manufactured in the present embodiment is 1.78 × 1010It is a, 205.5 times of coamplification；Its
In, CD3+Cell accounts for 96.54%, CD3+CD8+Cell accounts for 75.22%, CD4+CD25+FoxP3+Tregs cell accounts for 3.28%；It is right
The killing activity of tumour cell is 60.07% (4h51Cr method for releasing, effect/target=40:1).
Embodiment 4
The method that the present embodiment largely prepares High Fragmentation activity til cell using concretionary pernicious Pleural effusions, including with
Lower step:
(1) collection of concretionary malignant ascite: under aseptic condition, concretionary malignant ascite caused by colon cancer is collected
1500mL, and 20,000 U heparin are added；
(2) in concretionary malignant ascite free cell and grumeleuse separation: be 70 μm of nylon micro porous mistake using aperture
The concretionary malignant ascite is filtered by filter, separates the grumeleuse in malignant ascite with free cell, must be dissociated thin
Born of the same parents-A；
(3) release of the decomposition of grumeleuse and free cell: the grumeleuse is placed in containing 1000U/mL IFN-γ, 50U/
In 37 DEG C, 5%CO in the AIM-V complete medium-I of mL IL-2 and 3% inactivated serum2With 5 are cultivated under conditions of saturated humidity
It, filtering obtains free cell-B；
(4) separation of mononuclearcell: the free cell-A and the free cell-B are moved into centrifuge tube respectively,
500 × g is centrifuged 5min, discards supernatant liquid；Sedimentation cell is resuspended, the Ficoll that specific gravity is 1.077 is laid in and is layered on liquid,
1600 × g is centrifuged 15min；
(5) washing of mononuclearcell: collect Ficoll layering liquid interface on mononuclearcell (containing lymphocyte,
DC cell and tumour cell), AIM-V serum free medium is added, piping and druming mixes, and 300 × g is centrifuged 5min, discards supernatant liquid；
It is repeated 2 times following steps: AIM-V serum free medium is added, cell is resuspended, piping and druming mixes, and 300 × g is centrifuged 5min, discards
Clear liquid；
(6) the directional induction activation of til cell: blood is inactivated with containing 1000U/mL IFN-γ, 50U/mL IL-2 and 3%
Cell is resuspended in clear AIM-V complete medium-I, and cell count total amount is 136.8 × 106It is a, adjust cell density be 3.0 ×
106A/mL, in 37 DEG C, 5%CO2It is cultivated 5 days under conditions of saturated humidity, with the AIM-V complete medium-during culture
I half amounts are changed liquid 1 time；
(7) advantage pcr of til cell: from culture the 6th day, with contain 1000U/mL IL-2,200U/mL IFN-
Cell is resuspended in the AIM-V complete medium-II of γ, 25ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum, and adjusts cell
Density is 1.0 × 106A/mL, in 37 DEG C, 5%CO2With continue to cultivate under conditions of saturated humidity, it is daily to carry out cell count simultaneously
Cell density is adjusted to 1.0 × 10 with the AIM-V complete medium-II6A/mL；
(8) collection of til cell: at culture the 21st day, cell suspension is collected, 300 × g is centrifuged 5min, uses 0.9%NaCl
It is molten to be resuspended in the mixing of the 0.9%NaCl containing 1000U/mL IL-2,3% people's blood Alb and 6mM glucose by solution washing for cell
It is spare in liquid.
By detection it is found that til cell total amount manufactured in the present embodiment is 3.27 × 1010It is a, 239.0 times of coamplification；Its
In, CD3+Cell accounts for 99.52%, CD3+CD8+Cell accounts for 81.49%, CD4+CD25+FoxP3+Tregs cell accounts for 2.02%；It is right
The killing activity of tumour cell is 68.83% (4h51Cr method for releasing, effect/target=40:1).
Embodiment 5
The method that the present embodiment largely prepares High Fragmentation activity til cell using concretionary pernicious Pleural effusions, including with
Lower step:
(1) collection of concretionary Malignant Pleural: under aseptic condition, concretionary Malignant Pleural caused by lung cancer is collected
700mL, and 10,000 U heparin are added；
(2) in concretionary Malignant Pleural free cell and grumeleuse separation: be 70 μm of nylon micro porous mistake using aperture
The concretionary Malignant Pleural is filtered by filter, separates the grumeleuse in pernicious Pleural effusions with free cell, is obtained free
Cell-A；
(3) release of the decomposition of grumeleuse and free cell: the grumeleuse is placed in containing 1000U/mL IFN-γ, 50U/
In 37 DEG C, 5%CO in the AIM-V complete medium-I of mL IL-2 and 3% inactivated serum2With 3 are cultivated under conditions of saturated humidity
It, obtains free cell-B；
(4) separation of mononuclearcell: the free cell-A and the free cell-B are moved into centrifuge tube respectively,
500 × g is centrifuged 5min, discards supernatant liquid；Sedimentation cell is resuspended, the Ficoll that specific gravity is 1.077 is laid in and is layered on liquid,
1600 × g is centrifuged 15min；
(5) washing of mononuclearcell: collect Ficoll layering liquid interface on mononuclearcell (containing lymphocyte,
DC cell and tumour cell), AIM-V serum free medium is added, piping and druming mixes, and 300 × g is centrifuged 5min, discards supernatant liquid；
It is repeated 2 times following steps: AIM-V serum free medium is added, cell is resuspended, piping and druming mixes, and 300 × g is centrifuged 5min, discards
Clear liquid；
(6) it the directional induction activation of til cell: is inactivated with containing 1000U/mL IFN-γ, 100U/mL IL-2 and 3%
Cell is resuspended in the AIM-V complete medium-I of serum, and cell count total amount is 75.6 × 106It is a, adjust cell density be 3.0 ×
106A/mL, in 37 DEG C, 5%CO2It is cultivated 5 days under conditions of saturated humidity, with the AIM-V complete medium-during culture
I half amounts are changed liquid 1 time；
(7) advantage pcr of til cell: from culture the 6th day, with contain 2000U/mL IL-2,300U/mL IFN-
Cell is resuspended in the AIM-V complete medium-II of γ, 50ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum, and adjusts cell
Density is 1.0 × 106A/mL, in 37 DEG C, 5%CO2With continue to cultivate under conditions of saturated humidity, it is daily to carry out cell count simultaneously
Cell density is adjusted to 1.0 × 10 with the AIM-V complete medium-II6A/mL；
(8) collection of til cell: at culture the 21st day, cell suspension is collected, 300 × g is centrifuged 5min, uses 0.9%NaCl
It is molten to be resuspended in the mixing of the 0.9%NaCl containing 1000U/mL IL-2,3% people's blood Alb and 6mM glucose by solution washing for cell
It is spare in liquid.
By detection it is found that til cell total amount manufactured in the present embodiment is 1.66 × 1010It is a, 219.6 times of coamplification；Its
In, CD3+Cell accounts for 99.33%, CD3+CD8+Cell accounts for 74.64%, CD4+CD25+FoxP3+Tregs cell accounts for 3.38%；It is right
The killing activity of tumour cell is 58.26% (4h51Cr method for releasing, effect/target=40:1).
Embodiment 6
The method that the present embodiment largely prepares High Fragmentation activity til cell using concretionary pernicious Pleural effusions, including with
Lower step:
(1) collection of concretionary Malignant Pleural: under aseptic condition, concretionary Malignant Pleural caused by breast cancer is collected
1000mL, and 1.5U heparin is added；
(2) in concretionary Malignant Pleural free cell and grumeleuse separation: be 70 μm of nylon micro porous mistake using aperture
The concretionary Malignant Pleural will be filtered by filter, be separated the grumeleuse in Malignant Pleural with free cell, be obtained free
Cell-A；
(3) release of the decomposition of grumeleuse and free cell: the grumeleuse is placed in containing 1000U/mL IFN-γ, 50U/
4 are cultivated under conditions of 37 DEG C, 5%CO2 and saturated humidity in the AIM-V complete medium-I of mL IL-2 and 3% inactivated serum
It, filtering obtains free cell-B；
(4) separation of mononuclearcell: the free cell-A and the free cell-B are moved into centrifuge tube respectively,
500 × g is centrifuged 5min, discards supernatant liquid；Sedimentation cell is resuspended, the Ficoll that specific gravity is 1.076~1.078 is laid in and is layered
On liquid, 1600 × g is centrifuged 15min；
(5) washing of mononuclearcell: collect Ficoll layering liquid interface on mononuclearcell (containing lymphocyte,
DC cell and tumour cell), AIM-V serum free medium is added, piping and druming mixes, and 300 × g is centrifuged 5min, discards supernatant liquid；
It is repeated 2 times following steps: AIM-V serum free medium is added, cell is resuspended, piping and druming mixes, and 300 × g is centrifuged 5min, discards
Clear liquid；
(6) the directional induction activation of til cell: blood is inactivated with containing 1000U/mL IFN-γ, 50U/mL IL-2 and 3%
Cell is resuspended in clear AIM-V complete medium-I, and cell count total amount is 65.4 × 106A, adjusting cell density is 3.0 × 106
A/mL, in 37 DEG C, 5%CO2It is cultivated 5 days under conditions of saturated humidity, with the AIM-V complete medium-I during culture
Half amount is changed liquid 1 time；
(7) advantage pcr of til cell: from culture the 6th day, with contain 1000U/mL IL-2,200U/mL IFN-
Cell is resuspended in the AIM-V complete medium-II of γ, 25ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum, and adjusts cell
Density is 1.0 × 106A/mL, in 37 DEG C, 5%CO2With continue to cultivate under conditions of saturated humidity, it is daily to carry out cell count simultaneously
Cell density is adjusted to 1.0 × 10 with the AIM-V complete medium-II6A/mL；
(8) collection of til cell: at culture the 21st day, cell suspension is collected, 300 × g is centrifuged 5min, uses 0.9%NaCl
It is molten to be resuspended in the mixing of the 0.9%NaCl containing 1000U/mL IL-2,3% people's blood Alb and 6mM glucose by solution washing for cell
It is spare in liquid.
By detection it is found that til cell total amount manufactured in the present embodiment is 1.28 × 1010It is a, 195.7 times of coamplification；Its
In, CD3+Cell accounts for 97.12%, CD3+CD8+Cell accounts for 78.87%, CD4+CD25+FoxP3+Tregs cell accounts for 1.64%；It is right
The killing activity of tumour cell is 67.82% (4h51Cr method for releasing, effect/target=40:1).
Comparative example 1
The concretionary pernicious Pleural effusions of 2000mL are collected, are divided into 2 parts, 1000mL/ parts, it is special that portion does not carry out grumeleuse
Processing directly carries out the separation (control group, n=12) of mononuclearcell；Another is operated by the experimental method of embodiment 6
(experimental group, n=12)；Then, isolated mononuclearcell is suspended in containing IL-2, IFN- by experimental group and control group
In the AIM-V culture medium of γ and CD 3-resisting monoclonal antibody, the washing of mononuclearcell, til cell are carried out in identical condition
Directional induction activation, the collection of the advantage pcr of til cell and til cell.
By detection it is found that the til cell quantity that is prepared of experimental group have more about 30% than control group [(190.1 ±
46.7)×108vs(144.4±38.2)×108；P < 0.01；T is examined]；Moreover, compared with the control group, experimental group is prepared
Til cell and the til cell tumor cytotoxicity activity (4h having the same that is prepared of control group51Cr method for releasing, effect/
Target=40:1；Target cell lysis rate: 60.57% ± 5.34%vs 58.49% ± 6.27%；P > 0.05；T is examined).
To sum up, the method that the present invention largely prepares High Fragmentation activity til cell using concretionary pernicious Pleural effusions, is being trained
Til cell (190.1 ± 46.7) × 10 can be harvested when supporting the 21st~28 day8A (n=12), cell are expanded up to 182.5 ± 46.8
Times；Wherein, CD3+Cell accounts for about 98.55% ± 2.24%, CD3+CD8+Cell accounts for about 76.11% ± 6.88%, CD3+CD4+Carefully
Born of the same parents account for about 24.09% ± 6.73%, CD3+CD56+Cell accounts for about 53.36% ± 9.47%, and CD4+CD25+FoxP3+Tregs is thin
Born of the same parents only account for 2.87% ± 1.65%, to the killing activity of tumour cell up to 60.57% ± 5.34% (4h51Cr method for releasing, effect/
Target=40:1)；Compared with when not handling grumeleuse, the present invention can make the til cell quantity of harvest have more about 30%.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (8)
1. a kind of method for largely preparing High Fragmentation activity til cell using concretionary pernicious Pleural effusions, which is characterized in that packet
Include following steps:
(1) collection of concretionary pernicious Pleural effusions: under aseptic condition, collecting concretionary pernicious Pleural effusions, and heparin is added,
So that the concentration in the pernicious Pleural effusions containing the heparin is 11.0~16.5U/mL；
(2) in concretionary pernicious Pleural effusions free cell and grumeleuse separation: the concretionary pernicious Pleural effusions are carried out
Filtering, separates the grumeleuse in pernicious Pleural effusions with free cell, obtains free cell-A；
(3) release of the decomposition of grumeleuse and free cell: the grumeleuse is placed in AIM-V complete medium-I in suitable item
It is cultivated 3~5 days under part, filters, obtain free cell-B；
(4) separation of mononuclearcell: the free cell-A and the free cell-B are moved into centrifuge tube respectively, from
The heart discards supernatant liquid；Sedimentation cell is resuspended, the Ficoll that specific gravity is 1.076~1.078 is laid in and is layered on liquid, centrifugation；
(5) washing of mononuclearcell: the mononuclearcell on Ficoll layering liquid interface is collected, the training of AIM-V serum-free is added
Base is supported, is mixed, centrifugation discards supernatant liquid；
(6) the directional induction activation of til cell: cell is resuspended with the AIM-V complete medium-I, and adjusts cell density and is
1.5×106~3.0 × 106A/mL is cultivated 4~6 days under appropriate conditions, is cultivated completely during culture with the AIM-V
Half amount of base-I is changed liquid at least 1 time；
(7) advantage pcr of til cell: from culture the 5th~7 day, cell is resuspended with AIM-V complete medium-II, and adjust
Cell density is 1.0 × 106~2.0 × 106A/mL continues to cultivate under appropriate conditions, and daily progress cell count is used in combination
Cell density is adjusted to 1.0 × 10 by the AIM-V complete medium-II6~2.0 × 106A/mL；
(8) collection of til cell: at culture the 21st~28 day, collecting cell suspension, and centrifugation is washed with 0.9%NaCl solution,
Cell is resuspended in 0.9%NaCl mixed solution, it is spare；
The AIM-V complete medium-I contains 500~1000U/mL IFN-γ, the inactivation blood of 10~100U/mL IL-2 and 3%
Clearly, the AIM-V complete medium-II contains 500~2000U/mL IL-2,100~300U/mL IFN-γ, 20~50ng/
ML CD 3-resisting monoclonal antibody and 3% inactivated serum.
2. the side according to claim 1 for largely preparing High Fragmentation activity til cell using concretionary pernicious Pleural effusions
Method, which comprises the following steps:
(1) collection of concretionary pernicious Pleural effusions: under aseptic condition, collecting concretionary pernicious Pleural effusions, and heparin is added,
So that the concentration in the pernicious Pleural effusions containing the heparin is 12.5~15.0U/mL；
(2) in concretionary pernicious Pleural effusions free cell and grumeleuse separation: the concretionary pernicious Pleural effusions are carried out
Filtering, separates the grumeleuse in pernicious Pleural effusions with free cell, obtains free cell-A；
(3) release of the decomposition of grumeleuse and free cell: the grumeleuse is placed in AIM-V complete medium-I in suitable item
It is cultivated 3~5 days under part, filters, obtain free cell-B；
(4) separation of mononuclearcell: the free cell-A and the free cell-B are moved into centrifuge tube respectively, from
The heart discards supernatant liquid；Sedimentation cell is resuspended, the Ficoll that specific gravity is 1.077 is laid in and is layered on liquid, centrifugation；
(5) washing of mononuclearcell: the mononuclearcell on Ficoll layering liquid interface is collected, the training of AIM-V serum-free is added
Base is supported, is mixed, centrifugation discards supernatant liquid；
(6) the directional induction activation of til cell: cell is resuspended with the AIM-V complete medium-I, and adjusts cell density and is
2.2×106A/mL cultivates 5 days under appropriate conditions, changes liquid 1 with-I half amount of AIM-V complete medium during culture
It is secondary；
(7) advantage pcr of til cell: from culture the 6th day, cell is resuspended with AIM-V complete medium-II, and adjust thin
Born of the same parents' density is 1.5 × 106A/mL continues to cultivate under appropriate conditions, and daily progress cell count is simultaneously complete with the AIM-V
Cell density is adjusted to 1.5 × 10 by full culture medium-II6A/mL；
(8) collection of til cell: at culture the 21st~28 day, collecting cell suspension, and centrifugation is washed with 0.9%NaCl solution,
Cell is resuspended in 0.9%NaCl mixed solution, it is spare.
3. according to claim 1 largely prepare High Fragmentation activity til cell using concretionary pernicious Pleural effusions
Method, which is characterized in that
In the separation of step (4) mononuclearcell, with AIM-V serum free medium, RPMI1640 serum free medium or
Sedimentation cell is resuspended PBS buffer solution.
4. the side according to claim 1 for largely preparing High Fragmentation activity til cell using concretionary pernicious Pleural effusions
Method, which is characterized in that
In the washing of step (5) mononuclearcell, the mononuclearcell is lymphocyte, DC cell and tumour cell
Mixture.
5. the side according to claim 1 for largely preparing High Fragmentation activity til cell using concretionary pernicious Pleural effusions
Method, which is characterized in that
Further include being repeated at least once more following steps in the washing of step (5) mononuclearcell: AIM-V serum-free is added
Cell is resuspended in culture medium, mixes, and centrifugation discards supernatant liquid.
6. the side according to claim 1 for largely preparing High Fragmentation activity til cell using concretionary pernicious Pleural effusions
Method, which is characterized in that
In the collection of step (8) til cell, the 0.9%NaCl mixed solution contains IL-2, people's blood Alb and glucose.
7. the side according to claim 1 for largely preparing High Fragmentation activity til cell using concretionary pernicious Pleural effusions
Method, which is characterized in that
In the collection of step (8) til cell, the 0.9%NaCl mixed solution contains 1000U/mL IL-2,3% people's blood
Alb and 6mM glucose.
8. the til cell that the described in any item methods of claim 1-7 are prepared.
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| CN111172111B (en) * | 2019-12-26 | 2021-08-27 | 北京科途医学科技有限公司 | Method for preparing suspension tumor cell organoid by using malignant pleural effusion and ascites |
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