CN106222139A - A kind of method utilizing concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number - Google Patents
A kind of method utilizing concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number Download PDFInfo
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- CN106222139A CN106222139A CN201610620449.7A CN201610620449A CN106222139A CN 106222139 A CN106222139 A CN 106222139A CN 201610620449 A CN201610620449 A CN 201610620449A CN 106222139 A CN106222139 A CN 106222139A
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Abstract
The present invention relates to a kind of method utilizing concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number.The method, can gather in the crops til cell (190.1 ± 46.7) × 10 when cultivating the 21st～28 day8Individual (n=12), cell amplification reaches 182.5 ± 46.8 times；Wherein, CD3+Cell accounts for 98.55% ± 2.24%, CD3+CD8+Cell accounts for 76.11% ± 6.88%, CD3+CD4+Cell accounts for 24.09% ± 6.73%, CD3+CD56+Cell accounts for 53.36% ± 9.47%, and CD4+CD25+FoxP3+Tregs cell only accounts for 2.87% ± 1.65%, to the killing activity of tumor cell up to 60.57% ± 5.34% (4h51Cr method for releasing, effect/target=40:1)；Compared with when not processing grumeleuse, the present invention can make the til cell quantity of results have more about 30%.
Description
Technical field
The invention belongs to field of cell culture, be specifically related to one and utilize concretionary pernicious ascites pleural fluid to prepare height in a large number to kill
The method of wound activity til cell.
Background technology
Tumor infiltrating lymphocyte (tumor-infiltrating lymphocytes, TIL) is at tumor tissues or swollen
A heterogeneous lymphocyte populations present in tumor regional lymph nodes, including by the preactivated CD3 of tumor 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；The nonspecific CD3 of a number of tumor 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；It addition,
Possibly together with a number of CD4 with immunosuppressive action+CD25+FoxP3+Tregs (regulatory T cells) and
IDCs cells (immature dendritic cells) etc., with TGF-β, VEGF, PGE2, IL-6, IL-10 of high level expression
Equimolecular constitutes inhibitive ability of immunity microenvironment together, stops the continuous activation of specific for tumour antigen lymphocyte so that it is be difficult to
Effectively play Tumor cytotoxicity, cause tumor immune escape to occur.
[Rosenberg SA, et in the clinical research that NIH's ICR is carried out
al.Durable complete responses in heavily pretreated patients with metastatic
Melanoma using T-cell transfer immunotherapy.Clinical Cancer Research, 2011,17
(13) melanoma cancer tissue: 4550-4557], obtained from excision or biopsy or its regional lymph nodes separate also
Til cell is prepared in a large amount of amplifications, and then associating lymph pre cleaning scheme is adopted infusion Advanced Malignant melanoma patients body
In, treated effect (CR+PR) up to 52%, CR patient's the longest catabasis was more than 82 months；In treating effective case, visitor
The tumor regression seen almost occurs in all of metastatic lesion, such as brain, lung, liver, bone, lymph node, subcutaneous tissue etc..This table
Bright, til cell has huge clinical practice potentiality in anti-tumor immunotherapy.
But, from tumor tissues or regional lymph nodes, til cell is prepared in separation, need to experience the operation shearing of stripping and slicing, enzyme
A series of loaded down with trivial details, lengthy procedure such as solution and cell separation, cultivation screening, extensive amplification；Meanwhile, complicated manipulation in vitro makes
Til cell faces the highest pollution risk, easily causes cultivating unsuccessfully；And, most late tumor patients do not have
Standby surgical condition, it is impossible to obtain til cell from operation stripping and slicing；Though some patients can implement operation or carry out biopsy, but obtain
Tumor tissues is the most very little, it is difficult to therefrom amplify enough til cells, it is impossible to meet the needs of clinical treatment.So, pass through
Tumor tissues obtain til cell carry out adopting infusion antineoplaston face during clinical implementation many be difficult to overcome tired
Difficult.
Patient's Chang Yin tumor splanchnocoels such as pulmonary carcinoma, breast carcinoma, gastric cancer, colorectal cancer or ovarian cancer shift and pernicious breast occur
Ascites, a large amount of Clinical Evidences show to there is tumor antigen reactivity til cell in pernicious ascites pleural fluid, have potential extraction
Value.But, pernicious ascites pleural fluid exists one by the cytokine such as Tregs, iDCs and IL-10, TGF-β, VEGF
The inhibitive ability of immunity microenvironment constituted；Meanwhile, as an important mechanisms of immunologic escape, the usual low expression MHC-of tumor cell
I/class Ⅱmolecule, it is impossible to effectively all kinds of tumor antigen of submission, causes tumor response til cell activation deficiency, or different swelling
Tumour-reactive T lymphocyte clone activates unbalanced, rare numbers and generally there is functional defect.
On the one hand, from pernicious ascites pleural fluid extraction prepare til cell carry out adopting infusion antineoplaston time, infused cells
The ratio of middle tumour-specific til cell, quantity and the quantity of Tregs cell mixed are all affect clinical efficacy important
Factor.Chinese patent literature CN104946589A utilizes the antibody of PD-1, LAG-3 or TIM-3 to be coated magnetic bead to pernicious breast/ascites
The mononuclearcell of middle separation screens, and then stimulates screening cell amplification with OKT3 and IL-2, and this method can not solve because of swollen
Oncocyte low expression MHC-I/class Ⅱmolecule and the existence of inhibitive ability of immunity microenvironment and in pernicious breast/ascites of causing tumor special
Opposite sex til cell activation is not enough, the problem of rare numbers, it is difficult to filter out abundant tumour-specific til cell；Moreover,
Owing to also having PD-1, LAG-3 or TIM-3 equimolecular to express on Tregs and the non-specific til cell of other tumor antigens, they
Enter into as well as immunomagnetic beads in screening cell and expanded in a large number, influencing whether to cultivate tomour specific in finished product
The purity of property til cell and anti-tumor effect.As for other til cell preparation method, such as Chinese patent literature
CN103468641A and CN1560234A, utilizes the stimulation of chest ascites such as the most anti-OX-40 or OKT3 of non-specific activator, PHA, IL-2
The propagation of medium-sized lymphocyte, causes in the til cell that amplification obtains, except the CD3 containing specific for tumour antigen+CD4+Th1
Cell and CD3+CD8+CTLs extracellular, possibly together with the nonspecific CD3 of substantial amounts of tumor antigen+CD4+Or CD3+CD8+T cell,
NK cell and there is the Tregs cell of immunosuppressive action, the til cell of the results killing-efficiency to patient tumors cell
Low, it is difficult to constantly, to play antitumor action in the patient efficiently.
On the other hand, due to pernicious ascites pleural fluid be tissue exudates, containing concentration cellulose proteinogen, cause easily occurring
Solidification, so needing to add enough heparin sodiums or EDTA-K2 carries out anticoagulant when gathering.But, some pernicious ascites pleural fluid from
Solidify during body, show as visible fibrin clot not of uniform size in fresh drainage-fluid.Anticoagulant is disliked for suppression
Property ascites pleural fluid in vitro after solidification there is certain effect, but the grumeleuse formed cannot be eliminated.It addition, some pernicious chest and abdomen
Water (the most courageous and upright pernicious ascites pleural fluid), though processing through anticoagulant fully, not finding any solidification sign, but being centrifuged at it
The cell precipitation of rear formation still there will be the grumeleuse being difficult to dissociate.Numerous studies show, contain in the grumeleuse of pernicious ascites pleural fluid
A large amount of til cells.When carrying out density gradient centrifugation, these grumeleuses can disturb layering, affects the separation of mononuclearcell；With
Time, owing to grumeleuse is owing to being sunken at the bottom of centrifuge tube pipe with the high specific gravity cell component such as erythrocyte, granulocyte, causing being rejected,
This is likely to result in a large amount of loss of til cell.
But, prior art has no and utilizes concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number
Report.
Therefore, the concretionary pernicious ascites pleural fluid of research and utilization is prepared the method for High Fragmentation activity til cell in a large number and is had weight
Want meaning.
Summary of the invention
Concretionary pernicious ascites pleural fluid is utilized to prepare High Fragmentation activity til cell in a large number to this end, the present invention provides a kind of
Method.
For solving above-mentioned technical problem, the present invention is achieved through the following technical solutions:
The present invention provides a kind of method utilizing concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number,
Comprise the following steps:
(1) collection of concretionary pernicious ascites pleural fluid: under aseptic condition, collects concretionary pernicious ascites pleural fluid, and adds
Heparin so that in described pernicious ascites pleural fluid, the concentration containing described heparin is 11.0～16.5U/mL；
(2) in concretionary pernicious ascites pleural fluid, free cell separates with grumeleuse: by described concretionary pernicious ascites pleural fluid
Filter, make the grumeleuse in pernicious ascites pleural fluid separate with free cell, obtain free cell-A；
(3) decomposition of grumeleuse and the release of free cell: described grumeleuse is placed in AIM-V complete medium-I suitably
Under conditions of cultivate 3～5 days, filter, obtain free cell-B；
(4) separation of mononuclearcell: described free cell-A and described free cell-B is moved in centrifuge tube respectively,
Centrifugal, abandoning supernatant；Sedimentation cell is resuspended, it is laid on the Ficoll layering liquid that proportion is 1.076～1.078, centrifugal；
(5) washing of mononuclearcell: collect the mononuclearcell on Ficoll layering liquid interface, add AIM-V depletion of blood
Clear culture medium, mixing, centrifugal, abandoning supernatant；
(6) the directional induction activation of til cell: with described AIM-V complete medium-I re-suspended cell, and it is close to regulate cell
Degree is 1.5 × 106～3.0 × 106Individual/mL, cultivates 4～6 days under appropriate conditions, and complete with described AIM-V during cultivation
Culture medium-I half amount changes liquid at least 1 time；
(7) advantage pcr of til cell: from cultivating the 5th～7 day, with AIM-V complete medium-II re-suspended cell, and
Regulation cell density is 1.0 × 106～2.0 × 106Individual/mL, continues under appropriate conditions to cultivate, carries out cell counting every day
And with described AIM-V complete medium-II, cell density is adjusted to 1.0 × 106～2.0 × 106Individual/mL；
(8) collection of til cell: when cultivating the 21st～28 day, collects cell suspension, centrifugal, washes with 0.9%NaCl solution
Wash, cell is resuspended in 0.9%NaCl mixed solution, standby.
Preferably, the above-mentioned side utilizing concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number of the present invention
Method, comprises the following steps:
(1) collection of concretionary pernicious ascites pleural fluid: under aseptic condition, collects concretionary pernicious ascites pleural fluid, and adds
Heparin so that in described pernicious ascites pleural fluid, the concentration containing described heparin is 12.5～15.0U/mL；
(2) in concretionary pernicious ascites pleural fluid, free cell separates with grumeleuse: by described concretionary pernicious ascites pleural fluid
Filter, make the grumeleuse in pernicious ascites pleural fluid separate with free cell, obtain free cell-A；
(3) decomposition of grumeleuse and the release of free cell: described grumeleuse is placed in AIM-V complete medium-I suitably
Under conditions of cultivate 3～5 days, filter, obtain free cell-B；
(4) separation of mononuclearcell: described free cell-A and described free cell-B is moved in centrifuge tube respectively,
500 × g is centrifuged 5min, abandoning supernatant；Sedimentation cell is resuspended, it is laid in the Ficoll layering that proportion is 1.076～1.078
On liquid, 1600 × g is centrifuged 15min；
(5) washing of mononuclearcell: collect the mononuclearcell on Ficoll layering liquid interface, add AIM-V depletion of blood
Clear culture medium, mixing, 300 × g is centrifuged 5min, abandoning supernatant；
(6) the directional induction activation of til cell: with AIM-V complete medium-I re-suspended cell, and regulate cell density and be
1.5×106～3.0 × 106Individual/mL, cultivates 4～6 days under appropriate conditions, cultivates completely with described AIM-V during cultivation
Base-I half amount changes liquid 1 time；
(7) advantage pcr of til cell: from cultivating the 5th～7 day, with AIM-V complete medium-II re-suspended cell, and
Regulation cell density is 1.0 × 106～2.0 × 106Individual/mL, continues under appropriate conditions to cultivate, carries out cell counting every day
And with described AIM-V complete medium-II, cell density is adjusted to 1.0 × 106～2.0 × 106Individual/mL；
(8) collection of til cell: when cultivating the 21st～28 day, collects cell suspension, and 300 × g is centrifuged 5min, with 0.9%
NaCl solution is washed, and is resuspended in by cell in 0.9%NaCl mixed solution, standby.
Preferably, the above-mentioned side utilizing concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number of the present invention
Method,
The decomposition of described step (3) grumeleuse activates with the directional induction of the release of free cell and step (6) til cell
In, described AIM-V complete medium-I is containing IFN-γ, IL-2 and inactivated serum；
In the advantage pcr of described step (7) til cell, described 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, to utilize concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity TIL in a large number thin
The method of born of the same parents, comprises the following steps:
(1) collection of concretionary pernicious ascites pleural fluid: under aseptic condition, collects concretionary pernicious ascites pleural fluid, and adds
Heparin so that in described pernicious ascites pleural fluid, the concentration containing described heparin is 12.5～15.0U/mL；
(2) in concretionary pernicious ascites pleural fluid, free cell separates with grumeleuse: by described concretionary pernicious ascites pleural fluid
Filter, make the grumeleuse in pernicious ascites pleural fluid separate with free cell, obtain free cell-A；
(3) decomposition of grumeleuse and the release of free cell: described grumeleuse is placed in AIM-V complete medium-I suitably
Under conditions of cultivate 3～5 days, filter, obtain free cell-B；
(4) separation of mononuclearcell: described free cell-A and described free cell-B is moved in centrifuge tube respectively,
Centrifugal, abandoning supernatant；Sedimentation cell is resuspended, it is laid on the Ficoll layering liquid that proportion is 1.077, centrifugal；
(5) washing of mononuclearcell: collect the mononuclearcell on Ficoll layering liquid interface, add AIM-V depletion of blood
Clear culture medium, mixing, centrifugal, abandoning supernatant；
(6) the directional induction activation of til cell: with described AIM-V complete medium-I re-suspended cell, and it is close to regulate cell
Degree is 2.2 × 106Individual/mL, cultivates 5 days under appropriate conditions, changes by described AIM-V complete medium-I half amount during cultivation
Liquid 1 time；
(7) advantage pcr of til cell: from cultivating the 6th day, with AIM-V complete medium-II re-suspended cell, and adjusts
Ganglion cell's density is 1.5 × 106Individual/mL, continues under appropriate conditions to cultivate, carries out cell counting every day and use described AIM-
Cell density is adjusted to 1.5 × 10 by V complete medium-II6Individual/mL；
(8) collection of til cell: when cultivating the 21st～28 day, collects cell suspension, centrifugal, washes with 0.9%NaCl solution
Wash, cell is resuspended in 0.9%NaCl mixed solution, standby.
It is further preferred that the present invention is above-mentioned, to utilize concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity TIL in a large number thin
The method of born of the same parents,
During free cell separates with grumeleuse in the concretionary pernicious ascites pleural fluid of described step (2), utilize microfilter
Described concretionary pernicious ascites pleural fluid is filtered.
It is further preferred that the present invention is above-mentioned, to utilize concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity TIL in a large number thin
The method of born of the same parents, the aperture of described microfilter is 70 μm, and described microfilter is nylon micro porous filter.
It is further preferred that the present invention is above-mentioned, to utilize concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity TIL in a large number thin
The method of born of the same parents,
The decomposition of described step (3) grumeleuse activates with the directional induction of the release of free cell and step (6) til cell
In, described AIM-V complete medium-I is containing 500～1000U/mL IFN-γ, 10～100U/mL IL-2 and 3% inactivation blood
Clearly；
In the advantage pcr of described step (7) til cell, described AIM-V complete medium-II is containing 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, to utilize concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity TIL in a large number thin
The method of born of the same parents,
In the separation of described step (4) mononuclearcell, with AIM-V serum-free medium, RPMI1640 serum-free culture
Base or PBS are resuspended by sedimentation cell.
It is further preferred that the present invention is above-mentioned, to utilize concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity TIL in a large number thin
The method of born of the same parents,
In the washing of described step (5) mononuclearcell, described mononuclearcell is that lymphocyte, DC cell and tumor are thin
The mixture of born of the same parents.
It is further preferred that the present invention is above-mentioned, to utilize concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity TIL in a large number thin
The method of born of the same parents,
In the washing of described step (5) mononuclearcell, also include being repeated at least once more following steps: add AIM-V without
Blood serum medium re-suspended cell, mixing, centrifugal, abandoning supernatant.
It is further preferred that the present invention is above-mentioned, to utilize concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity TIL in a large number thin
The method of born of the same parents,
In the collection of described step (8) til cell, described 0.9%NaCl mixed solution contains IL-2, human blood Alb and Portugal
Grape sugar.
It is further preferred that the present invention is above-mentioned, to utilize concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity TIL in a large number thin
The method of born of the same parents,
In the collection of described step (8) til cell, described 0.9%NaCl mixed solution contain 1000U/mL IL-2,3%
Human blood Alb and 6mM glucose.
It is further preferred that the present invention is above-mentioned, to utilize concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity TIL in a large number thin
The method of born of the same parents, the decomposition of described step (3) grumeleuse and the release of free cell, the directional induction activation of step (6) til cell and
In the advantage pcr of step (7) til cell, described suitable condition refers to: 37 DEG C, 5%CO2And saturated humidity.
The present invention also provides for the til cell that said method prepares.
Compared with prior art, technical scheme has the advantage that
(1) method utilizing concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number of the present invention, logical
Cross first the most concretionary pernicious ascites pleural fluid to filter, then the grumeleuse being filtrated to get is trained under appropriate conditions
Support, filter, such that it is able to be separated to more mononuclearcell, and then significantly improve the quantity of the til cell of preparation, then exist
On the basis of inducing tumor cell and DC cell upregulation express MHC-I/class Ⅱmolecule, enhancing tumor antigen submission, make pernicious breast
In ascites, first the tumor response lymphocyte of fresh separated experiences the process of specific activation, clonal expansion, then recycles
Specific combination of cytokines stimulates it to expand on a large scale, the activation of suppression Tregs cell simultaneously and propagation, makes to finally give
Containing a high proportion of tumour-specific til cell in cell products, so that it is guaranteed that prepared til cell is to autologous patient tumor
Cell has special, efficient killing activity；
(2) method utilizing concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number of the present invention,
Til cell (190.1 ± 46.7) × 10 can be gathered in the crops when cultivating the 21st～28 day8Individual (n=12), cell amplification reaches 182.5 ±
46.8 times；Wherein, CD3+Cell accounts for 98.55% ± 2.24%, CD3+CD8+Cell accounts for 76.11% ± 6.88%, CD3+
CD4+Cell accounts for 24.09% ± 6.73%, CD3+CD56+Cell accounts for 53.36% ± 9.47%, and CD4+CD25+FoxP3+
Tregs cell only accounts for 2.87% ± 1.65%, to the killing activity of tumor cell up to 60.57% ± 5.34% (4h51Cr discharges
Method, effect/target=40:1)；
(3) compared with when not processing grumeleuse, the present invention can make the til cell quantity of results have more about 30%.
Detailed description of the invention
In following example 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 Pharmaceutical, lot number: 201501005；
IFN-γ: Shanghai Kai Mao biological medicine, lot number: G20150101；
CD 3-resisting monoclonal antibody: eBioscience, lot number: E06305-1691；
The long-range Pharmaceutical in human blood Alb: Sichuan, lot number: 201504045A.
Embodiment 1
The present embodiment utilizes the method that concretionary pernicious ascites pleural fluid prepares High Fragmentation activity til cell in a large number, including with
Lower step:
(1) collection of concretionary Malignant Pleural: under aseptic condition, the 1000mL that collection pulmonary carcinoma causes is concretionary pernicious
Hydrothorax 1000mL, and add 1.5 ten thousand U heparin；
(2) in concretionary Malignant Pleural, free cell separates with grumeleuse: the nylon micro porous mistake utilizing aperture to be 70 μm
Described concretionary Malignant Pleural is filtered by filter, makes the grumeleuse in Malignant Pleural separate with free cell, must dissociate thin
Born of the same parents-A；
(3) decomposition of grumeleuse and the release of free cell: described grumeleuse is placed in containing 750U/mL IFN-γ, 55U/mL
At 37 DEG C, 5%CO in the AIM-V complete medium-I of IL-2 and 3% inactivated serum2Cultivate 3 days with under conditions of saturated humidity,
Filter, obtain free cell-B；
(4) separation of mononuclearcell: described free cell-A and described free cell-B is moved in centrifuge tube respectively,
500 × g is centrifuged 5min, abandoning supernatant；Sedimentation cell is resuspended, it is laid in the Ficoll layering that proportion is 1.076～1.078
On liquid, 1600 × g is centrifuged 15min；
(5) washing of mononuclearcell: collect Ficoll layering liquid interface on mononuclearcell (containing lymphocyte,
DC cell and tumor cell), add AIM-V serum-free medium, blow and beat, mix, 300 × g is centrifuged 5min, abandoning supernatant；
It is repeated 2 times following steps: adding AIM-V serum-free medium re-suspended cell, blow and beat, mix, 300 × g is centrifuged 5min, discards
Clear liquid；
(6) the directional induction activation of til cell: with containing 750U/mL IFN-γ, 55U/mL IL-2 and 3% inactivation blood
Clear AIM-V complete medium-I re-suspended cell, cell counting total amount is 85.7 × 106Individual, regulation cell density is 2.2 × 106
Individual/mL, at 37 DEG C, 5%CO2Cultivate 5 days with under conditions of saturated humidity, with described AIM-V complete medium-I during cultivation
Half amount changes liquid 1 time；
(7) advantage pcr of til cell: from cultivating the 6th day, with containing 1250U/mL IL-2,200U/mL IFN-
AIM-V complete medium-II re-suspended cell of γ, 35ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum, and regulate cell
Density is 1.5 × 106Individual/mL, at 37 DEG C, 5%CO2Cultivate with continuation under conditions of saturated humidity, carry out cell counting also every day
With described AIM-V complete medium-II, cell density is adjusted to 1.5 × 106Individual/mL；
(8) collection of til cell: when cultivating the 21st day, collects cell suspension, and 300 × g is centrifuged 5min, uses 0.9%NaCl
Solution washs, and cell is resuspended in the 0.9%NaCl mixing containing 1000U/mL IL-2,3% human blood Alb and 6mM glucose molten
In liquid, standby.
By detection, til cell total amount prepared by the present embodiment is 1.62 × 1010Individual, coamplification 189.0 times；Its
In, CD3+Cell accounts for 99.32%, CD3+CD8+Cell accounts for 77.56%, CD4+CD25+FoxP3+Tregs cell accounts for 3.42%；Right
The killing activity of tumor cell is 66.84% (4h51Cr method for releasing, effect/target=40:1).
Embodiment 2
The present embodiment utilizes the method that concretionary pernicious ascites pleural fluid prepares High Fragmentation activity til cell in a large number, including with
Lower step:
(1) collection of concretionary malignant ascite: under aseptic condition, collects the concretionary malignant ascite that gastric cancer causes
1200mL, and add 1.8 ten thousand U heparin；
(2) in concretionary malignant ascite, free cell separates with grumeleuse: the nylon micro porous mistake utilizing aperture to be 70 μm
Described concretionary malignant ascite is filtered by filter, makes the grumeleuse in malignant ascite separate with free cell, must dissociate thin
Born of the same parents-A；
(3) decomposition of grumeleuse and the release of free cell: described grumeleuse is placed in containing 500U/mL IFN-γ, 100U/
At 37 DEG C, 5%CO in the AIM-V complete medium-I of mL IL-2 and 3% inactivated serum2With cultivation 4 under conditions of saturated humidity
My god, filter, obtain free cell-B；
(4) separation of mononuclearcell: described free cell-A and described free cell-B is moved in centrifuge tube respectively,
500 × g is centrifuged 5min, abandoning supernatant；Sedimentation cell is resuspended, it is laid on the Ficoll layering liquid that proportion is 1.077,
1600 × g is centrifuged 15min；
(5) washing of mononuclearcell: collect Ficoll layering liquid interface on mononuclearcell (containing lymphocyte,
DC cell and tumor cell), add AIM-V serum-free medium, blow and beat, mix, 300 × g is centrifuged 5min, abandoning supernatant；
It is repeated 2 times following steps: adding AIM-V serum-free medium re-suspended cell, blow and beat, mix, 300 × g is centrifuged 5min, discards
Clear liquid；
(6) the directional induction activation of til cell: with containing 500U/mL IFN-γ, 100U/mL IL-2 and 3% inactivation blood
Clear AIM-V complete medium-I re-suspended cell, cell counting total amount is 112.5 × 106Individual, regulation cell density is 1.5 ×
106Individual/mL, at 37 DEG C, 5%CO2With under conditions of saturated humidity cultivate 6 days, during cultivation with described AIM-V complete medium-
I half amounts change liquid 1 time；
(7) advantage pcr of til cell: from cultivating the 7th day, with containing 500U/mL IL-2,300U/mL IFN-γ,
AIM-V complete medium-II re-suspended cell of 50ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum, and it is close to regulate cell
Degree is 1.0 × 106Individual/mL, at 37 DEG C, 5%CO2Cultivate with continuation under conditions of saturated humidity, carry out cell counting and use every day
Cell density is adjusted to 1.0 × 10 by described AIM-V complete medium-II6Individual/mL；
(8) collection of til cell: when cultivating the 28th day, collects cell suspension, and 300 × g is centrifuged 5min, uses 0.9%NaCl
Solution washs, and cell is resuspended in the 0.9%NaCl mixing containing 1000U/mL IL-2,3% human blood Alb and 6mM glucose molten
In liquid, standby.
By detection, til cell total amount prepared by the present embodiment is 1.95 × 1010Individual, coamplification 174.1 times；Its
In, CD3+Cell accounts for 97.34%, CD3+CD8+Cell accounts for 79.10%, CD4+CD25+FoxP3+Tregs cell accounts for 3.44%；Right
The killing activity of tumor cell is 61.18% (4h51Cr method for releasing, effect/target=40:1).
Embodiment 3
The present embodiment utilizes the method that concretionary pernicious ascites pleural fluid prepares High Fragmentation activity til cell in a large number, including with
Lower step:
(1) collection of concretionary Malignant Pleural: under aseptic condition, collects the concretionary Malignant Pleural that pulmonary carcinoma is caused
1000mL, and add 1.5 ten thousand U heparin；
(2) in concretionary Malignant Pleural, free cell separates with grumeleuse: the nylon micro porous mistake utilizing aperture to be 70 μm
Described concretionary Malignant Pleural is filtered by filter, makes the grumeleuse in Malignant Pleural separate with free cell, must dissociate thin
Born of the same parents-A；
(3) decomposition of grumeleuse and the release of free cell: described grumeleuse is placed in containing 500U/mL IFN-γ, 100U/
At 37 DEG C, 5%CO in the AIM-V complete medium-I of mL IL-2 and 3% inactivated serum2With cultivation 5 under conditions of saturated humidity
My god, filter, obtain free cell-B；
(4) separation of mononuclearcell: described free cell-A and described free cell-B is moved in centrifuge tube respectively,
500 × g is centrifuged 5min, abandoning supernatant；Sedimentation cell is resuspended, it is laid on the Ficoll layering liquid that proportion is 1.077,
1600 × g is centrifuged 15min；
(5) washing of mononuclearcell: collect Ficoll layering liquid interface on mononuclearcell (containing lymphocyte,
DC cell and tumor cell), add AIM-V serum-free medium, blow and beat, mix, 300 × g is centrifuged 5min, abandoning supernatant；
It is repeated 2 times following steps: adding AIM-V serum-free medium re-suspended cell, blow and beat, mix, 300 × g is centrifuged 5min, discards
Clear liquid；
(6) the directional induction activation of til cell: with containing 1000U/mL IFN-γ, 10U/mL IL-2 and 3% inactivation blood
Clear AIM-V complete medium-I re-suspended cell, cell counting total amount is 86.6 × 106Individual, regulation cell density is 3.0 × 106
Individual/mL, at 37 DEG C, 5%CO2Cultivate 4 days with under conditions of saturated humidity, with described AIM-V complete medium-I during cultivation
Half amount changes liquid 1 time；
(7) advantage pcr of til cell: from cultivating the 5th day, with containing 2000U/mL IL-2,100U/mL IFN-
AIM-V complete medium-II re-suspended cell of γ, 20ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum, and regulate cell
Density is 2.0 × 106Individual/mL, at 37 DEG C, 5%CO2Cultivate with continuation under conditions of saturated humidity, carry out cell counting also every day
With described AIM-V complete medium-II, cell density is adjusted to 2.0 × 106Individual/mL；
(8) collection of til cell: when cultivating the 21st day, collects cell suspension, and 300 × g is centrifuged 5min, uses 0.9%NaCl
Solution washs, and cell is resuspended in the 0.9%NaCl mixing containing 1000U/mL IL-2,3% human blood Alb and 6mM glucose molten
In liquid, standby.
By detection, til cell total amount prepared by the present embodiment is 1.78 × 1010Individual, coamplification 205.5 times；Its
In, CD3+Cell accounts for 96.54%, CD3+CD8+Cell accounts for 75.22%, CD4+CD25+FoxP3+Tregs cell accounts for 3.28%；Right
The killing activity of tumor cell is 60.07% (4h51Cr method for releasing, effect/target=40:1).
Embodiment 4
The present embodiment utilizes the method that concretionary pernicious ascites pleural fluid prepares High Fragmentation activity til cell in a large number, including with
Lower step:
(1) collection of concretionary malignant ascite: under aseptic condition, collects the concretionary malignant ascite that colon cancer causes
1500mL, and add 20,000 U heparin；
(2) in concretionary malignant ascite, free cell separates with grumeleuse: the nylon micro porous mistake utilizing aperture to be 70 μm
Described concretionary malignant ascite is filtered by filter, makes the grumeleuse in malignant ascite separate with free cell, must dissociate thin
Born of the same parents-A；
(3) decomposition of grumeleuse and the release of free cell: described grumeleuse is placed in containing 1000U/mL IFN-γ, 50U/
At 37 DEG C, 5%CO in the AIM-V complete medium-I of mL IL-2 and 3% inactivated serum2With cultivation 5 under conditions of saturated humidity
My god, filter, obtain free cell-B；
(4) separation of mononuclearcell: described free cell-A and described free cell-B is moved in centrifuge tube respectively,
500 × g is centrifuged 5min, abandoning supernatant；Sedimentation cell is resuspended, it is laid on the Ficoll layering liquid that proportion is 1.077,
1600 × g is centrifuged 15min；
(5) washing of mononuclearcell: collect Ficoll layering liquid interface on mononuclearcell (containing lymphocyte,
DC cell and tumor cell), add AIM-V serum-free medium, blow and beat, mix, 300 × g is centrifuged 5min, abandoning supernatant；
It is repeated 2 times following steps: adding AIM-V serum-free medium re-suspended cell, blow and beat, mix, 300 × g is centrifuged 5min, discards
Clear liquid；
(6) the directional induction activation of til cell: with containing 1000U/mL IFN-γ, 50U/mL IL-2 and 3% inactivation blood
Clear AIM-V complete medium-I re-suspended cell, cell counting total amount is 136.8 × 106Individual, regulation cell density is 3.0 ×
106Individual/mL, at 37 DEG C, 5%CO2With under conditions of saturated humidity cultivate 5 days, during cultivation with described AIM-V complete medium-
I half amounts change liquid 1 time；
(7) advantage pcr of til cell: from cultivating the 6th day, with containing 1000U/mL IL-2,200U/mL IFN-
AIM-V complete medium-II re-suspended cell of γ, 25ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum, and regulate cell
Density is 1.0 × 106Individual/mL, at 37 DEG C, 5%CO2Cultivate with continuation under conditions of saturated humidity, carry out cell counting also every day
With described AIM-V complete medium-II, cell density is adjusted to 1.0 × 106Individual/mL；
(8) collection of til cell: when cultivating the 21st day, collects cell suspension, and 300 × g is centrifuged 5min, uses 0.9%NaCl
Solution washs, and cell is resuspended in the 0.9%NaCl mixing containing 1000U/mL IL-2,3% human blood Alb and 6mM glucose molten
In liquid, standby.
By detection, til cell total amount prepared by the present embodiment is 3.27 × 1010Individual, coamplification 239.0 times；Its
In, CD3+Cell accounts for 99.52%, CD3+CD8+Cell accounts for 81.49%, CD4+CD25+FoxP3+Tregs cell accounts for 2.02%；Right
The killing activity of tumor cell is 68.83% (4h51Cr method for releasing, effect/target=40:1).
Embodiment 5
The present embodiment utilizes the method that concretionary pernicious ascites pleural fluid prepares High Fragmentation activity til cell in a large number, including with
Lower step:
(1) collection of concretionary Malignant Pleural: under aseptic condition, collects the concretionary Malignant Pleural that pulmonary carcinoma causes
700mL, and add 10,000 U heparin；
(2) in concretionary Malignant Pleural, free cell separates with grumeleuse: the nylon micro porous mistake utilizing aperture to be 70 μm
Described concretionary Malignant Pleural is filtered by filter, makes the grumeleuse in pernicious ascites pleural fluid separate with free cell, obtains free
Cell-A；
(3) decomposition of grumeleuse and the release of free cell: described grumeleuse is placed in containing 1000U/mL IFN-γ, 50U/
At 37 DEG C, 5%CO in the AIM-V complete medium-I of mL IL-2 and 3% inactivated serum2With cultivation 3 under conditions of saturated humidity
My god, obtain free cell-B；
(4) separation of mononuclearcell: described free cell-A and described free cell-B is moved in centrifuge tube respectively,
500 × g is centrifuged 5min, abandoning supernatant；Sedimentation cell is resuspended, it is laid on the Ficoll layering liquid that proportion is 1.077,
1600 × g is centrifuged 15min；
(5) washing of mononuclearcell: collect Ficoll layering liquid interface on mononuclearcell (containing lymphocyte,
DC cell and tumor cell), add AIM-V serum-free medium, blow and beat, mix, 300 × g is centrifuged 5min, abandoning supernatant；
It is repeated 2 times following steps: adding AIM-V serum-free medium re-suspended cell, blow and beat, mix, 300 × g is centrifuged 5min, discards
Clear liquid；
(6) the directional induction activation of til cell: with containing 1000U/mL IFN-γ, 100U/mL IL-2 and 3% inactivation
AIM-V complete medium-I re-suspended cell of serum, cell counting total amount is 75.6 × 106Individual, regulation cell density is 3.0 ×
106Individual/mL, at 37 DEG C, 5%CO2With under conditions of saturated humidity cultivate 5 days, during cultivation with described AIM-V complete medium-
I half amounts change liquid 1 time；
(7) advantage pcr of til cell: from cultivating the 6th day, with containing 2000U/mL IL-2,300U/mL IFN-
AIM-V complete medium-II re-suspended cell of γ, 50ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum, and regulate cell
Density is 1.0 × 106Individual/mL, at 37 DEG C, 5%CO2Cultivate with continuation under conditions of saturated humidity, carry out cell counting also every day
With described AIM-V complete medium-II, cell density is adjusted to 1.0 × 106Individual/mL；
(8) collection of til cell: when cultivating the 21st day, collects cell suspension, and 300 × g is centrifuged 5min, uses 0.9%NaCl
Solution washs, and cell is resuspended in the 0.9%NaCl mixing containing 1000U/mL IL-2,3% human blood Alb and 6mM glucose molten
In liquid, standby.
By detection, til cell total amount prepared by the present embodiment is 1.66 × 1010Individual, coamplification 219.6 times；Its
In, CD3+Cell accounts for 99.33%, CD3+CD8+Cell accounts for 74.64%, CD4+CD25+FoxP3+Tregs cell accounts for 3.38%；Right
The killing activity of tumor cell is 58.26% (4h51Cr method for releasing, effect/target=40:1).
Embodiment 6
The present embodiment utilizes the method that concretionary pernicious ascites pleural fluid prepares High Fragmentation activity til cell in a large number, including with
Lower step:
(1) collection of concretionary Malignant Pleural: under aseptic condition, collects the concretionary Malignant Pleural that breast carcinoma causes
1000mL, and add 1.5U heparin；
(2) in concretionary Malignant Pleural, free cell separates with grumeleuse: the nylon micro porous mistake utilizing aperture to be 70 μm
The most described concretionary Malignant Pleural of filter filters, and makes the grumeleuse in Malignant Pleural separate with free cell, obtains free
Cell-A；
(3) decomposition of grumeleuse and the release of free cell: described grumeleuse is placed in containing 1000U/mL IFN-γ, 50U/
The AIM-V complete medium-I of mL IL-2 and 3% inactivated serum is cultivated under conditions of 37 DEG C, 5%CO2 and saturated humidity 4
My god, filter, obtain free cell-B；
(4) separation of mononuclearcell: described free cell-A and described free cell-B is moved in centrifuge tube respectively,
500 × g is centrifuged 5min, abandoning supernatant；Sedimentation cell is resuspended, it is laid in the Ficoll layering that proportion is 1.076～1.078
On liquid, 1600 × g is centrifuged 15min；
(5) washing of mononuclearcell: collect Ficoll layering liquid interface on mononuclearcell (containing lymphocyte,
DC cell and tumor cell), add AIM-V serum-free medium, blow and beat, mix, 300 × g is centrifuged 5min, abandoning supernatant；
It is repeated 2 times following steps: adding AIM-V serum-free medium re-suspended cell, blow and beat, mix, 300 × g is centrifuged 5min, discards
Clear liquid；
(6) the directional induction activation of til cell: with containing 1000U/mL IFN-γ, 50U/mL IL-2 and 3% inactivation blood
Clear AIM-V complete medium-I re-suspended cell, cell counting total amount is 65.4 × 106Individual, regulation cell density is 3.0 × 106
Individual/mL, at 37 DEG C, 5%CO2Cultivate 5 days with under conditions of saturated humidity, with described AIM-V complete medium-I during cultivation
Half amount changes liquid 1 time；
(7) advantage pcr of til cell: from cultivating the 6th day, with containing 1000U/mL IL-2,200U/mL IFN-
AIM-V complete medium-II re-suspended cell of γ, 25ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum, and regulate cell
Density is 1.0 × 106Individual/mL, at 37 DEG C, 5%CO2Cultivate with continuation under conditions of saturated humidity, carry out cell counting also every day
With described AIM-V complete medium-II, cell density is adjusted to 1.0 × 106Individual/mL；
(8) collection of til cell: when cultivating the 21st day, collects cell suspension, and 300 × g is centrifuged 5min, uses 0.9%NaCl
Solution washs, and cell is resuspended in the 0.9%NaCl mixing containing 1000U/mL IL-2,3% human blood Alb and 6mM glucose molten
In liquid, standby.
By detection, til cell total amount prepared by the present embodiment is 1.28 × 1010Individual, coamplification 195.7 times；Its
In, CD3+Cell accounts for 97.12%, CD3+CD8+Cell accounts for 78.87%, CD4+CD25+FoxP3+Tregs cell accounts for 1.64%；Right
The killing activity of tumor cell is 67.82% (4h51Cr method for releasing, effect/target=40:1).
Comparative example 1
Collecting the concretionary pernicious ascites pleural fluid of 2000mL, be divided into 2 parts, 1000mL/ part, it is special that grumeleuse is not carried out by portion
Process, directly carry out the separation (matched group, n=12) of mononuclearcell；Another part is operated by the experimental technique of embodiment 6
(experimental group, n=12)；Then, the mononuclearcell of isolated is all suspended in containing IL-2, IFN-by experimental group and matched group
In the AIM-V culture medium of γ and CD 3-resisting monoclonal antibody, carry out the washing of mononuclearcell, til cell in identical condition
Directional induction activation, the advantage pcr of til cell and the collection of til cell.
By detection, the til cell quantity that experimental group prepares than matched group have more about 30% [(190.1 ±
46.7)×108vs(144.4±38.2)×108；P ＜ 0.01；T checks]；And, compared with matched group, experimental group prepares
Til cell and the til cell for preparing of matched group there is identical tumor cytotoxicity activity (4h51Cr method for releasing, effect/
Target=40:1；Target cell lysis rate: 60.57% ± 5.34%vs 58.49% ± 6.27%；P ＞ 0.05；T checks).
To sum up, the present invention utilizes the method that concretionary pernicious ascites pleural fluid prepares High Fragmentation activity til cell in a large number, in training
Til cell (190.1 ± 46.7) × 10 can be gathered in the crops when supporting the 21st～28 day8Individual (n=12), cell amplification reaches 182.5 ± 46.8
Times；Wherein, CD3+Cell accounts for 98.55% ± 2.24%, CD3+CD8+Cell accounts for 76.11% ± 6.88%, CD3+CD4+Carefully
Born of the same parents account for 24.09% ± 6.73%, CD3+CD56+Cell accounts for 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 tumor cell up to 60.57% ± 5.34% (4h51Cr method for releasing, effect/
Target=40:1)；Compared with when not processing grumeleuse, the present invention can make the til cell quantity of results have more about 30%.
Obviously, above-described embodiment is only for clearly demonstrating example, and not restriction to embodiment.Right
For those of ordinary skill in the field, can also make on the basis of the above description other multi-form change or
Variation.Here without also cannot all of embodiment be given exhaustive.And the obvious change thus extended out or
Change among still in the protection domain of the invention.
Claims (10)
1. one kind utilizes the method that concretionary pernicious ascites pleural fluid prepares High Fragmentation activity til cell in a large number, it is characterised in that bag
Include following steps:
(1) collection of concretionary pernicious ascites pleural fluid: under aseptic condition, collects concretionary pernicious ascites pleural fluid, and adds heparin,
Making the concentration containing described heparin in described pernicious ascites pleural fluid is 11.0～16.5U/mL；
(2) in concretionary pernicious ascites pleural fluid, free cell separates with grumeleuse: carried out by described concretionary pernicious ascites pleural fluid
Filter, make the grumeleuse in pernicious ascites pleural fluid separate with free cell, obtain free cell-A；
(3) decomposition of grumeleuse and the release of free cell: described grumeleuse is placed in AIM-V complete medium-I at suitable bar
Cultivate 3～5 days under part, filter, obtain free cell-B；
(4) separation of mononuclearcell: described free cell-A and described free cell-B is moved in centrifuge tube respectively, from
The heart, abandoning supernatant；Sedimentation cell is resuspended, it is laid on the Ficoll layering liquid that proportion is 1.076～1.078, centrifugal；
(5) washing of mononuclearcell: collect the mononuclearcell on Ficoll layering liquid interface, add the training of AIM-V serum-free
Support base, mixing, be centrifuged, abandoning supernatant；
(6) the directional induction activation of til cell: with described AIM-V complete medium-I re-suspended cell, and regulate cell density and be
1.5×106～3.0 × 106Individual/mL, cultivates 4～6 days under appropriate conditions, cultivates completely with described AIM-V during cultivation
Base-I half amount changes liquid at least 1 time；
(7) advantage pcr of til cell: from cultivating the 5th～7 day, with AIM-V complete medium-II re-suspended cell, and regulates
Cell density is 1.0 × 106～2.0 × 106Individual/mL, continues under appropriate conditions to cultivate, carries out cell counting and use every day
Cell density is adjusted to 1.0 × 10 by described AIM-V complete medium-II6～2.0 × 106Individual/mL；
(8) collection of til cell: when cultivating the 21st～28 day, collects cell suspension, centrifugal, washs with 0.9%NaCl solution,
Cell is resuspended in 0.9%NaCl mixed solution, standby.
The side utilizing concretionary pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number the most according to claim 1
Method, it is characterised in that
In the directional induction activation of the decomposition of described step (3) grumeleuse and the release of free cell and step (6) til cell, institute
State AIM-V complete medium-I containing IFN-γ, IL-2 and inactivated serum；
In the advantage pcr of described step (7) til cell, described AIM-V complete medium-II contains IL-2, IFN-γ, resists
CD3 monoclonal antibody and inactivated serum.
The most according to claim 1 and 2 concretionary pernicious ascites pleural fluid is utilized to prepare High Fragmentation activity til cell in a large number
Method, it is characterised in that comprise the following steps:
(1) collection of concretionary pernicious ascites pleural fluid: under aseptic condition, collects concretionary pernicious ascites pleural fluid, and adds heparin,
Making the concentration containing described heparin in described pernicious ascites pleural fluid is 12.5～15.0U/mL；
(2) in concretionary pernicious ascites pleural fluid, free cell separates with grumeleuse: carried out by described concretionary pernicious ascites pleural fluid
Filter, make the grumeleuse in pernicious ascites pleural fluid separate with free cell, obtain free cell-A；
(3) decomposition of grumeleuse and the release of free cell: described grumeleuse is placed in AIM-V complete medium-I at suitable bar
Cultivate 3～5 days under part, filter, obtain free cell-B；
(4) separation of mononuclearcell: described free cell-A and described free cell-B is moved in centrifuge tube respectively, from
The heart, abandoning supernatant；Sedimentation cell is resuspended, it is laid on the Ficoll layering liquid that proportion is 1.077, centrifugal；
(5) washing of mononuclearcell: collect the mononuclearcell on Ficoll layering liquid interface, add the training of AIM-V serum-free
Support base, mixing, be centrifuged, abandoning supernatant；
(6) the directional induction activation of til cell: with described AIM-V complete medium-I re-suspended cell, and regulate cell density and be
2.2×106Individual/mL, cultivates 5 days under appropriate conditions, changes liquid 1 by described AIM-V complete medium-I half amount during cultivation
Secondary；
(7) advantage pcr of til cell: from cultivating the 6th day, with AIM-V complete medium-II re-suspended cell, and regulates thin
Born of the same parents' density is 1.5 × 106Individual/mL, continues under appropriate conditions to cultivate, carries out cell counting complete with described AIM-V every day
Cell density is adjusted to 1.5 × 10 by full culture medium-II6Individual/mL；
(8) collection of til cell: when cultivating the 21st～28 day, collects cell suspension, centrifugal, washs with 0.9%NaCl solution,
Cell is resuspended in 0.9%NaCl mixed solution, standby.
4. prepare High Fragmentation activity TIL in a large number according to the concretionary pernicious ascites pleural fluid that utilizes described in any one of claim 1-3
The method of cell, it is characterised in that
In the directional induction activation of the decomposition of described step (3) grumeleuse and the release of free cell and step (6) til cell, institute
State AIM-V complete medium-I containing 500～1000U/mL IFN-γ, 10～100U/mL IL-2 and 3% inactivated serum；
In the advantage pcr of described step (7) til cell, described AIM-V complete medium-II is containing 500～2000U/mL
IL-2,100～300U/mL IFN-γ, 20～50ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum.
5. prepare High Fragmentation activity TIL in a large number according to the concretionary pernicious ascites pleural fluid that utilizes described in any one of claim 1-4
The method of cell, it is characterised in that
In the separation of described step (4) mononuclearcell, with AIM-V serum-free medium, RPMI1640 serum-free medium or
PBS is resuspended by sedimentation cell.
6. prepare High Fragmentation activity TIL in a large number according to the concretionary pernicious ascites pleural fluid that utilizes described in any one of claim 1-5
The method of cell, it is characterised in that
In the washing of described step (5) mononuclearcell, described mononuclearcell is lymphocyte, DC cell and tumor cell
Mixture.
7. prepare High Fragmentation activity TIL in a large number according to the concretionary pernicious ascites pleural fluid that utilizes described in any one of claim 1-6
The method of cell, it is characterised in that
In the washing of described step (5) mononuclearcell, also include being repeated at least once more following steps: add AIM-V serum-free
Culture medium re-suspended cell, mixing, centrifugal, abandoning supernatant.
8. prepare High Fragmentation activity TIL in a large number according to the concretionary pernicious ascites pleural fluid that utilizes described in any one of claim 1-7
The method of cell, it is characterised in that
In the collection of described step (8) til cell, described 0.9%NaCl mixed solution contains IL-2, human blood Alb and glucose.
9. prepare High Fragmentation activity TIL in a large number according to the concretionary pernicious ascites pleural fluid that utilizes described in any one of claim 1-8
The method of cell, it is characterised in that
In the collection of described step (8) til cell, described 0.9%NaCl mixed solution contains 1000U/mL IL-2,3% human blood
Alb and 6mM glucose.
10. the til cell that the method described in any one of claim 1-9 prepares.
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| CN108588021A (en) * | 2018-05-02 | 2018-09-28 | 深圳市因诺转化医学研究院 | The method of separation and purifying TIL, acquisition CD4+CD25+ cell subsets |
| CN111172111A (en) * | 2019-12-26 | 2020-05-19 | 浙江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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