CN105969731A - Method for preparing high-killing-activity TIL cells in batches from malignant pleuroperitoneal fluid - Google Patents
Method for preparing high-killing-activity TIL cells in batches from malignant pleuroperitoneal fluid Download PDFInfo
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- CN105969731A CN105969731A CN201610620430.2A CN201610620430A CN105969731A CN 105969731 A CN105969731 A CN 105969731A CN 201610620430 A CN201610620430 A CN 201610620430A CN 105969731 A CN105969731 A CN 105969731A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2302—Interleukin-2 (IL-2)
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/24—Interferons [IFN]
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/515—CD3, T-cell receptor complex
Abstract
The invention relates to a method for preparing high-killing-activity TIL cells in batches from malignant pleuroperitoneal fluid. The method comprises the steps that (338.6+/-134.2)*10<8>(n=25) TIL cells can be obtained when culture is performed for 21-28 d, the cells are amplified by 179.6+/-24.2 times, wherein, CD3+cells account for 98.61%+/-3.22%, CD3+CD8+cells account for 74.56%+/-5.19%, CD3+CD4+cells account for 27.42%+/-6.35%, CD3+CD56+cells account for 51.88%+/-7.49%, and CD4+CD25+FoxP3+ Tregs cells only account for 3.27%+/-1.75%. The killing activity for tumor cells can reach 66.54%+/-5.18% (according to a 4h51Cr release method, the effect/target ratio is equal to 40:1).
Description
Technical field
The invention belongs to field of cell culture, be specifically related to one and utilize pernicious ascites pleural fluid to prepare height in a large number
The method of killing activity til cell.
Background technology
Tumor infiltrating lymphocyte (tumor-infiltrating lymphocytes, TIL) is in tumor group
Knit or a heterogeneous lymphocyte populations present in tumor region lymph node, including pre-by tumor antigen
The CD3 of activation+CD4+Th cell (T helper lymphocytes) and CD3+CD8+CTL cell
(cytotoxic T lymphocytes), can swell with MHC restrictive one specific recognition, killing
Oncocyte；The nonspecific CD3 of a number of tumor antigen+CD4+Or CD3+CD8+T cell；
A small amount of CD3-CD56+NK cell (natural killers), kills with non-MHC restrictive one
Tumor cell；It addition, there is immunosuppressive action possibly together with a number of
CD4+CD25+FoxP3+Tregs (regulatory T cells) and iDCs cell (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 continuing of specific for tumour antigen lymphocyte
Activation so that it is be difficult to effectively play Tumor cytotoxicity, cause tumor immune escape to occur.
[Rosenberg in the clinical research that NIH's ICR is carried out
SA,et al.Durable complete responses in heavily pretreated patients with
metastatic melanoma using T-cell transfer immunotherapy.Clinical Cancer
Research, 2011,17 (13): 4550-4557], from excision or biopsy obtain pernicious
Melanoma tissue or its regional lymph nodes separate and til cell is prepared in amplification in a large number, then combine
Lymph pre cleaning scheme is adopted in infusion Advanced Malignant melanoma patients body, treated effect
(CR+PR) up to 52%, CR patient's the longest catabasis was more than 82 months；Treating effective case
In, objective tumor regression almost occurs in all of metastatic lesion, as brain, lung, liver, bone,
Lymph node, subcutaneous tissue etc..This shows, til cell has huge in anti-tumor immunotherapy
Clinical practice potentiality.
But, from tumor tissues or regional lymph nodes, til cell is prepared in separation, need to experience operation and cut
The shearing of block, enzymolysis and cell separation, cultivation screening, extensive amplification etc. are a series of loaded down with trivial details, unrestrained
Growth process；Meanwhile, complicated manipulation in vitro makes til cell face the highest pollution risk, easily
Cause cultivating unsuccessfully；And, most late tumor patients do not have surgical condition, it is impossible to from
Operation stripping and slicing obtains til cell；Though some patients can implement operation or carry out biopsy, but acquisition is swollen
Tumor tissue is the most very little, it is difficult to therefrom amplify enough til cells, it is impossible to meet clinical treatment
Need.So, obtain til cell by tumor tissues and carry out adopting infusion antineoplaston in clinic
Implementation process faces many difficulties being difficult to and overcoming.
Patient's Chang Yin tumor splanchnocoel transfers such as pulmonary carcinoma, breast carcinoma, gastric cancer, colorectal cancer or ovarian cancer
And pernicious ascites pleural fluid occurs, it is anti-that a large amount of Clinical Evidences show to there is tumor antigen in pernicious ascites pleural fluid
Answering property til cell, has potential extraction and application and is worth.But, pernicious ascites pleural fluid exists one
The inhibitive ability of immunity being made up of the cytokine such as Tregs, iDCs and IL-10, TGF-β, VEGF
Microenvironment；Meanwhile, as an important mechanisms of immunologic escape, the usual low expression of tumor cell
MHC-I/class Ⅱmolecule, it is impossible to effectively all kinds of tumor antigen of submission, causes tumor response TIL thin
Born of the same parents activate deficiency, or different tumor response T lymphocyte clones activates unbalanced, rare numbers
And generally there is functional defect.
From pernicious ascites pleural fluid extraction prepare til cell carry out adopting infusion antineoplaston time, infusion
In cell, the ratio of tumour-specific til cell, quantity and the quantity of Tregs cell mixed are equal
It it is the key factor affecting clinical efficacy.Chinese patent literature CN104946589A utilize PD-1,
The antibody of LAG-3 or TIM-3 is coated magnetic bead to be carried out the mononuclearcell separated in pernicious breast/ascites
Screening, then stimulates screening cell amplification with OKT3 and IL-2, and this method can not solve because of tumor cell
Low expression MHC-I/class Ⅱmolecule and the existence of inhibitive ability of immunity microenvironment and pernicious breast/abdomen of causing
Tumour-specific til cell activation deficiency, the problem of rare numbers in water, it is difficult to filter out high abundance
Tumour-specific til cell；Moreover, due to Tregs and the non-specific TIL of other tumor antigens
Also having PD-1, LAG-3 or TIM-3 equimolecular to express on cell, they are as well as immunomagnetic beads
And enter in screening cell and expanded in a large number, influence whether to cultivate tumour-specific in finished product
The purity of til cell and anti-tumor effect.As for other til cell preparation method, such as China
Patent documentation CN103468641A and CN1560234A, utilizes the most anti-OX-40 of non-specific activator
Or the propagation of the stimulation of chest ascites medium-sized lymphocyte such as OKT3, PHA, IL-2, cause obtaining in amplification
Til cell in, 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, results
Til cell is low to the killing-efficiency of patient tumors cell, it is difficult to constantly, efficiently in the patient
Play antitumor action.
Summary of the invention
To this end, to be solved by this invention be in prior art from pernicious ascites pleural fluid separation and Culture
The technical problem that til cell is inefficient to tumor cell specific killing, and then provide one to utilize evil
The method that property ascites pleural fluid prepares High Fragmentation activity til cell in a large number.
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 pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number,
Comprise the following steps:
(1) collection of pernicious ascites pleural fluid: under aseptic condition, collects pernicious ascites pleural fluid, and adds liver
Element so that in described pernicious ascites pleural fluid, the concentration containing described heparin is 11.0～16.5U/mL；
(2) separation of mononuclearcell: described pernicious ascites pleural fluid is moved in centrifuge tube, centrifugal,
Abandoning supernatant；Sedimentation cell is resuspended, it is laid on the Ficoll layering liquid that proportion is 1.076～1.078,
Centrifugal；
(3) washing of mononuclearcell: collect the mononuclearcell on Ficoll layering liquid interface, add
Enter AIM-V serum-free medium, mixing, centrifugal, abandoning supernatant；
(4) the directional induction activation of til cell: use AIM-V complete medium-I re-suspended cell, and
Regulation cell density is 1.5 × 106～3.0 × 106Individual/mL, cultivates 4～6 days under appropriate conditions, cultivates
Period changes liquid at least 1 time with by described AIM-V complete medium-I half amount；
(5) advantage pcr of til cell: from cultivating the 5th～7 day, with AIM-V complete medium-
II re-suspended cell, and to regulate cell density be 1.0 × 106～2.0 × 106Individual/mL, under appropriate conditions
Continue to cultivate, carry out cell counting every day and utilize described AIM-V complete medium-II by cell density
It is adjusted to 1.0 × 106～2.0 × 106Individual/mL；
(6) collection of til cell: when cultivating the 21st～28 day, collects cell suspension, centrifugal, uses
0.9%NaCl solution washs, and is resuspended in by cell in 0.9%NaCl mixed solution, standby.
Preferably, the present invention is above-mentioned utilizes pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number
Method, comprises the following steps:
(1) collection of pernicious ascites pleural fluid: under aseptic condition, collects pernicious ascites pleural fluid, and adds liver
Element so that in described pernicious ascites pleural fluid, the concentration containing described heparin is 12.5～15.0U/mL；
(2) separation of mononuclearcell: described pernicious ascites pleural fluid is moved in centrifuge tube, 500 × g from
Heart 5min, abandoning supernatant；Sedimentation cell is resuspended, and being laid in proportion is 1.076～1.078
On Ficoll layering liquid, 1600 × g is centrifuged 15min；
(3) washing of mononuclearcell: collect the mononuclearcell on Ficoll layering liquid interface, add
Entering AIM-V serum-free medium, mixing, 300 × g is centrifuged 5min, abandoning supernatant；
(4) the directional induction activation of til cell: use AIM-V complete medium-I re-suspended cell, and
Regulation cell density is 1.5 × 106～3.0 × 106Individual/mL, cultivates 4～6 days under appropriate conditions, cultivates
Period changes liquid 1 time by described AIM-V complete medium-I half amount；
(5) advantage pcr of til cell: from cultivating the 5th～7 day, with AIM-V complete medium-
II re-suspended cell, and to regulate cell density be 1.0 × 106～2.0 × 106Individual/mL, under appropriate conditions
Continue to cultivate, carry out cell counting every day and with described AIM-V complete medium-II, cell density adjusted
Whole is 1.0 × 106～2.0 × 106Individual/mL；
(6) collection of til cell: when cultivating the 21st～28 day, collect cell suspension, 300 × g from
Heart 5min, washs with 0.9%NaCl solution, is resuspended in by cell in 0.9%NaCl mixed solution,
Standby.
Preferably, the present invention is above-mentioned utilizes pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number
Method,
In the directional induction activation of described step (4) til cell, described AIM-V complete medium-
I containing IFN-γ, IL-2 and inactivated serum；
In the advantage pcr of described step (5) til cell, described AIM-V complete medium-II contains
There are IL-2, IFN-γ, CD 3-resisting monoclonal antibody and inactivated serum.
Pernicious ascites pleural fluid is utilized to prepare High Fragmentation activity TIL in a large number it is further preferred that the present invention is above-mentioned
The method of cell, comprises the following steps:
(1) collection of pernicious ascites pleural fluid: under aseptic condition, collects pernicious ascites pleural fluid, and adds liver
Element so that in described pernicious ascites pleural fluid, the concentration containing described heparin is 12.5～15.0U/mL；
(2) separation of mononuclearcell: described pernicious ascites pleural fluid is moved in centrifuge tube, centrifugal,
Abandoning supernatant；Sedimentation cell is resuspended, it is laid on the Ficoll layering liquid that proportion is 1.077, from
The heart；
(3) washing of mononuclearcell: collect the mononuclearcell on Ficoll layering liquid interface, add
Enter AIM-V serum-free medium, mixing, centrifugal, abandoning supernatant；
(4) the directional induction activation of til cell: use AIM-V complete medium-I re-suspended cell, and
Regulation cell density is 2.2 × 106Individual/mL, cultivates 5 days, under appropriate conditions with described during cultivation
AIM-V complete medium-I half amount changes liquid 1 time；
(5) advantage pcr of til cell: from cultivating the 6th day, with AIM-V complete medium-II
Re-suspended cell, and to regulate cell density be 1.5 × 106Individual/mL, continues to cultivate under appropriate conditions,
Carry out cell counting every day and with described AIM-V complete medium-II, cell density be adjusted to
1.5×106Individual/mL；
(6) collection of til cell: when cultivating the 25th day, collects cell suspension, centrifugal, uses
0.9%NaCl solution washs, and is resuspended in by cell in 0.9%NaCl mixed solution, standby.
Pernicious ascites pleural fluid is utilized to prepare High Fragmentation activity TIL in a large number it is further preferred that the present invention is above-mentioned
The method of cell,
In the directional induction activation of described step (4) til cell, described 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 (5) til cell, described AIM-V complete medium-II contains
There are 500～2000U/mL IL-2,100～300U/mL IFN-γ, 20～50ng/mL CD 3-resisting monoclonal
Antibody and 3% inactivated serum.
Pernicious ascites pleural fluid is utilized to prepare High Fragmentation activity TIL in a large number it is further preferred that the present invention is above-mentioned
The method of cell,
In the separation of described step (2) mononuclearcell, with AIM-V serum-free medium,
RPMI1640 serum-free medium or PBS are resuspended by sedimentation cell.
Pernicious ascites pleural fluid is utilized to prepare High Fragmentation activity TIL in a large number it is further preferred that the present invention is above-mentioned
The method of cell,
In the washing of described step (3) mononuclearcell, described mononuclearcell be lymphocyte,
DC cell and the mixture of tumor cell.
Pernicious ascites pleural fluid is utilized to prepare High Fragmentation activity TIL in a large number it is further preferred that the present invention is above-mentioned
The method of cell,
In the washing of described step (3) mononuclearcell, also include being repeated at least once more following steps:
Add AIM-V serum-free medium re-suspended cell, mixing, be centrifuged, abandoning supernatant.
Pernicious ascites pleural fluid is utilized to prepare High Fragmentation activity TIL in a large number it is further preferred that the present invention is above-mentioned
The method of cell,
In the washing of described step (3) mononuclearcell, also include being repeated at least once more following steps:
Adding AIM-V serum-free medium re-suspended cell, mixing, 300 × g is centrifuged 5min, supernatant discarded
Liquid.
Pernicious ascites pleural fluid is utilized to prepare High Fragmentation activity TIL in a large number it is further preferred that the present invention is above-mentioned
The method of cell,
The directional induction activation of described step (4) til cell and the advantage of step (5) til cell
In amplification, described suitable condition refers to: 37 DEG C, 5%CO2And saturated humidity.
Pernicious ascites pleural fluid is utilized to prepare High Fragmentation activity TIL in a large number it is further preferred that the present invention is above-mentioned
The method of cell,
In the collection of described step (6) til cell, described 0.9%NaCl mixed solution contain IL-2,
Human blood Alb and glucose.
Pernicious ascites pleural fluid is utilized to prepare High Fragmentation activity TIL in a large number it is further preferred that the present invention is above-mentioned
The method of cell,
In the collection of described (6) til cell, described 0.9%NaCl mixed solution contains
1000U/mL IL-2,3% human blood Alb and 6mM glucose.
The present invention also provides for the til cell that said method prepares.
Compared with prior art, technical scheme has the advantage that
(1) side utilizing pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number of the present invention
Method, resists at inducing tumor cell and DC cell upregulation expression MHC-I/class Ⅱmolecule, enhancing tumor
On the basis of former submission, make the tumor response lymphocyte first warp of fresh separated in pernicious ascites pleural fluid
Going through the process of specific activation, clonal expansion, then recycling specific combination of cytokines stimulates it big
Scale expands, and suppresses activation and the propagation of Tregs cell simultaneously, makes in the cell products finally given
Containing a high proportion of tumour-specific til cell, so that it is guaranteed that patient is swollen by prepared til cell
Oncocyte has special, efficient killing activity；
(2) side utilizing pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number of the present invention
Method, can gather in the crops til cell (338.6 ± 134.2) × 10 when cultivating the 21st～28 day8Individual (n=25),
Cell amplification reaches 179.6 ± 24.2 times；Wherein, CD3+Cell accounts for 98.61% ± 3.22%,
CD3+CD8+Cell accounts for 74.56% ± 5.19%, CD3+CD4+Cell accounts for 27.42% ± 6.35%,
CD3+CD56+Cell accounts for 51.88% ± 7.49%, and CD4+CD25+FoxP3+Tregs cell is only
Account for 3.27% ± 1.75%, to the killing activity of tumor cell up to 66.54% ± 5.18% (4h51Cr releases
Put method, effect/target=40:1)；
(3) with utilize 1000U/mL IL-2,50 μ g/mL PHA and 50ng/mL AntiCD3 McAb Dan Ke
The mononuclearcell separated in pernicious ascites pleural fluid is directly carried out stimulating amplification to compare by grand antibody, the present invention
TIL prepared by the described method utilizing pernicious ascites pleural fluid to prepare High Fragmentation activity til cell in a large number is thin
Born of the same parents contain higher proportion of CD3+CD8+Cell and the CD4 of lower ratio+CD25+FoxP3+Tregs
Cell, and the killing activity higher (p ＜ 0.01, t check) to tumor cell.
Detailed description of the invention
In following example of the present invention and experimental example,
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 pernicious ascites pleural fluid prepares High Fragmentation activity til cell in a large number, including
Following steps:
(1) collection of Malignant Pleural: under aseptic condition, collects the Malignant Pleural that pulmonary carcinoma causes
1000mL, and add 1.5 ten thousand U heparin；
(2) separation of mononuclearcell: being moved into by described Malignant Pleural in centrifuge tube, 500 × g is centrifuged
5min, abandoning supernatant；With 50mL AIM-V serum-free medium, sedimentation cell is resuspended, tiling
On the Ficoll layering liquid that proportion is 1.077,1600 × g is centrifuged 15min；
(3) washing of mononuclearcell: the mononuclearcell collected on Ficoll layering liquid interface (contains
Have lymphocyte, DC cell and tumor cell), add AIM-V serum-free medium, blow and beat, mix
Even, 300 × g is centrifuged 5min, abandoning supernatant；It is repeated 2 times following steps: add AIM-V serum-free
Culture medium re-suspended cell, blows and beats, mixes, and 300 × g is centrifuged 5min, abandoning supernatant；
(4) the directional induction activation of til cell: with containing 750U/mL IFN-γ, 55U/mL IL-2
With AIM-V complete medium-I re-suspended cell of 3% inactivated serum, and regulate cell density and be
2.2×106Individual/mL, at 37 DEG C, 5%CO2Cultivate 5 days with under conditions of saturated humidity, use during cultivation
Described AIM-V complete medium-I half amount changes liquid 1 time；
(5) advantage pcr of til cell: from cultivating the 6th day, with containing 1250U/mL IL-2,
The AIM-V of 200U/mL IFN-γ, 35ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum is complete
Culture medium-II re-suspended cell, and to regulate cell density be 1.5 × 106Individual/mL, at 37 DEG C, 5%CO2With
Continue under conditions of saturated humidity to cultivate, carry out cell counting every day and cultivate completely with described AIM-V
Cell density is adjusted to 1.5 × 10 by base-II6Individual/mL；
(6) collection of til cell: when cultivating the 21st day, collects cell suspension, and 300 × g is centrifuged
5min, washs with 0.9%NaCl solution, is resuspended in by cell containing 1000U/mL IL-2,3% people
In the 0.9%NaCl mixed solution of blood Alb and 6mM glucose, standby.
By detection, til cell total amount prepared by the present embodiment is 2.36 × 1010Individual, expand altogether
Increase 159.2 times；Wherein, CD3+Cell accounts for 98.07%, CD3+CD8+Cell accounts for 79.69%,
CD4+CD25+FoxP3+Tregs cell accounts for 2.44%；Killing activity to tumor cell is 63.75%
(4h 51Cr method for releasing, effect/target=40:1).
Embodiment 2
The present embodiment utilizes the method that pernicious ascites pleural fluid prepares High Fragmentation activity til cell in a large number, including
Following steps:
(1) collection of malignant ascite: under aseptic condition, collects the malignant ascite that gastric cancer causes
1200mL, and add 1.8 ten thousand U heparin；
(2) separation of mononuclearcell: being moved into by described malignant ascite in centrifuge tube, 500 × g is centrifuged
5min, abandoning supernatant；With 50mL AIM-V serum-free medium, sedimentation cell is resuspended, tiling
On the Ficoll layering liquid that proportion is 1.076,1600 × g is centrifuged 15min；
(3) washing of mononuclearcell: the mononuclearcell collected on Ficoll layering liquid interface (contains
Have lymphocyte, DC cell and tumor cell), add AIM-V serum-free medium, blow and beat, mix
Even, 300 × g is centrifuged 5min, abandoning supernatant；It is repeated 2 times following steps: add AIM-V serum-free
Culture medium re-suspended cell, blows and beats, mixes, and 300 × g is centrifuged 5min, abandoning supernatant；
(4) the directional induction activation of til cell: with containing 500U/mL IFN-γ, 100U/mL IL-
2 and 3% AIM-V complete medium-I re-suspended cell of inactivated serum, and regulate cell density and be
1.5×106Individual/mL, at 37 DEG C, 5%CO2Cultivate 6 days with under conditions of saturated humidity, use during cultivation
Described AIM-V complete medium-I half amount changes liquid 1 time；
(5) advantage pcr of til cell: from cultivating the 7th day, with containing 500U/mL IL-2,
The AIM-V of 300U/mL IFN-γ, 50ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum is complete
Culture medium-II re-suspended cell, and to regulate cell density be 1.0 × 106Individual/mL, at 37 DEG C, 5%CO2With
Continue under conditions of saturated humidity to cultivate, carry out cell counting every day and cultivate completely with described AIM-V
Cell density is adjusted to 1.0 × 10 by base-II6Individual/mL；
(6) collection of til cell: when cultivating the 28th day, collects cell suspension, and 300 × g is centrifuged
5min, washs with 0.9%NaCl solution, is resuspended in by cell containing 1000U/mL IL-2,3% people
In the 0.9%NaCl mixed solution of blood Alb and 6mM glucose, standby.
By detection, til cell total amount prepared by the present embodiment is 2.18 × 1010Individual, expand altogether
Increase 146.5 times；Wherein, CD3+Cell accounts for 99.46%, CD3+CD8+Cell accounts for 77.37%,
CD4+CD25+FoxP3+Tregs cell accounts for 1.35%；Killing activity to tumor cell is 58.33%
(4h 51Cr method for releasing, effect/target=40:1).
Embodiment 3
The present embodiment utilizes the method that pernicious ascites pleural fluid prepares High Fragmentation activity til cell in a large number, including
Following steps:
(1) collection of Malignant Pleural: under aseptic condition, collects the Malignant Pleural that pulmonary carcinoma is caused
1600mL, and add 20,000 U heparin；
(2) separation of mononuclearcell: being moved into by described Malignant Pleural in centrifuge tube, 500 × g is centrifuged
5min, abandoning supernatant；With 50mL AIM-V serum-free medium, sedimentation cell is resuspended, tiling
On the Ficoll layering liquid that proportion is 1.078,1600 × g is centrifuged 15min；
(3) washing of mononuclearcell: the mononuclearcell collected on Ficoll layering liquid interface (contains
Have lymphocyte, DC cell and tumor cell), add AIM-V serum-free medium, blow and beat, mix
Even, 300 × g is centrifuged 5min, abandoning supernatant；It is repeated 2 times following steps: add AIM-V serum-free
Culture medium re-suspended cell, blows and beats, mixes, and 300 × g is centrifuged 5min, abandoning supernatant；
(4) the directional induction activation of til cell: with containing 1000U/mL IFN-γ, 10U/mL IL-
2 and 3% AIM-V complete medium-I re-suspended cell of inactivated serum, and regulate cell density and be
3.0×106Individual/mL, at 37 DEG C, 5%CO2Cultivate 4 days with under conditions of saturated humidity, use during cultivation
Described AIM-V complete medium-I half amount changes liquid 1 time；
(5) advantage pcr of til cell: from cultivating the 5th day, with containing 2000U/mL IL-2,
The AIM-V of 100U/mL IFN-γ, 20ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum is complete
Culture medium-II re-suspended cell, and to regulate cell density be 2.0 × 106Individual/mL, at 37 DEG C, 5%CO2With
Continue under conditions of saturated humidity to cultivate, carry out cell counting every day and cultivate completely with described AIM-V
Cell density is adjusted to 2.0 × 10 by base-II6Individual/mL；
(6) collection of til cell: when cultivating the 21st day, collects cell suspension, and 300 × g is centrifuged
5min, washs with 0.9%NaCl solution, is resuspended in by cell containing 1000U/mL IL-2,3% people
In the 0.9%NaCl mixed solution of blood Alb and 6mM glucose, standby.
By detection, til cell total amount prepared by the present embodiment is 2.78 × 1010Individual, expand altogether
Increase 239.8 times；Wherein, CD3+Cell accounts for 98.46%, CD3+CD8+Cell accounts for 76.05%,
CD4+CD25+FoxP3+Tregs cell accounts for 2.78%；Killing activity to tumor cell is 62.18%
(4h 51Cr method for releasing, effect/target=40:1).
Embodiment 4
The present embodiment utilizes the method that pernicious ascites pleural fluid prepares High Fragmentation activity til cell in a large number, including
Following steps:
(1) collection of malignant ascite: under aseptic condition, collects the malignant ascite that colon cancer causes
1000mL, and add 1.5 ten thousand U heparin；
(2) separation of mononuclearcell: being moved into by described malignant ascite in centrifuge tube, 500 × g is centrifuged
5min, abandoning supernatant；With 50mL AIM-V serum-free medium, sedimentation cell is resuspended, tiling
On the Ficoll layering liquid that proportion is 1.077,1600 × g is centrifuged 15min；
(3) washing of mononuclearcell: the mononuclearcell collected on Ficoll layering liquid interface (contains
Have lymphocyte, DC cell and tumor cell), add AIM-V serum-free medium, blow and beat, mix
Even, 300 × g is centrifuged 5min, abandoning supernatant；It is repeated 2 times following steps: add AIM-V serum-free
Culture medium re-suspended cell, blows and beats, mixes, and 300 × g is centrifuged 5min, abandoning supernatant；
(4) the directional induction activation of til cell: with containing 1000U/mL IFN-γ, 50U/mL IL-
2 and 3% AIM-V complete medium-I re-suspended cell of inactivated serum, and regulate cell density and be
3.0×106Individual/mL, at 37 DEG C, 5%CO2Cultivate 5 days with under conditions of saturated humidity, use during cultivation
Described AIM-V complete medium-I half amount changes liquid 1 time；
(5) advantage pcr of til cell: from cultivating the 6th day, with containing 1000U/mL IL-2,
The AIM-V of 200U/mL IFN-γ, 25ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum is complete
Culture medium-II re-suspended cell, and to regulate cell density be 1.0 × 106Individual/mL, at 37 DEG C, 5%CO2With
Continue under conditions of saturated humidity to cultivate, carry out cell counting every day and cultivate completely with described AIM-V
Cell density is adjusted to 1.0 × 10 by base-II6Individual/mL；
(6) collection of til cell: when cultivating the 21st day, collects cell suspension, and 300 × g is centrifuged
5min, washs with 0.9%NaCl solution, is resuspended in by cell containing 1000U/mL IL-2,3% people
In the 0.9%NaCl mixed solution of blood Alb and 6mM glucose, standby.
By detection, til cell total amount prepared by the present embodiment is 2.93 × 1010Individual, expand altogether
Increase 185.6 times；Wherein, CD3+Cell accounts for 99.12%, CD3+CD8+Cell accounts for 77.46%,
CD4+CD25+FoxP3+Tregs cell accounts for 1.88%；Killing activity to tumor cell is 60.32%
(4h 51Cr method for releasing, effect/target=40:1).
Embodiment 5
The present embodiment utilizes the method that pernicious ascites pleural fluid prepares High Fragmentation activity til cell in a large number, including
Following steps:
(1) collection of Malignant Pleural: under aseptic condition, collects the Malignant Pleural that pulmonary carcinoma causes
1200mL, and add 1.8 ten thousand U heparin；
(2) separation of mononuclearcell: being moved into by described Malignant Pleural in centrifuge tube, 500 × g is centrifuged
5min, abandoning supernatant；With 50mL AIM-V serum-free medium, sedimentation cell is resuspended, tiling
On the Ficoll layering liquid that proportion is 1.077,1600 × g is centrifuged 15min；
(3) washing of mononuclearcell: the mononuclearcell collected on Ficoll layering liquid interface (contains
Have lymphocyte, DC cell and tumor cell), add AIM-V serum-free medium, blow and beat, mix
Even, 300 × g is centrifuged 5min, abandoning supernatant；It is repeated 2 times following steps: add AIM-V serum-free
Culture medium re-suspended cell, blows and beats, mixes, and 300 × g is centrifuged 5min, abandoning supernatant；
(4) the directional induction activation of til cell: with containing 1000U/mL IFN-γ, 100U/mL
AIM-V complete medium-I re-suspended cell of IL-2 and 3% inactivated serum, and regulate cell density and be
3.0×106Individual/mL, at 37 DEG C, 5%CO2Cultivate 5 days with under conditions of saturated humidity, use during cultivation
Described AIM-V complete medium-I half amount changes liquid 1 time；
(5) advantage pcr of til cell: from cultivating the 6th day, with containing 2000U/mL IL-2,
The AIM-V of 300U/mL IFN-γ, 50ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum is complete
Culture medium-II re-suspended cell, and to regulate cell density be 1.0 × 106Individual/mL, at 37 DEG C, 5%CO2With
Continue under conditions of saturated humidity to cultivate, carry out cell counting every day and cultivate completely with described AIM-V
Cell density is adjusted to 1.0 × 10 by base-II6Individual/mL；
(6) collection of til cell: when cultivating the 21st day, collects cell suspension, and 300 × g is centrifuged
5min, washs with 0.9%NaCl solution, is resuspended in by cell containing 1000U/mL IL-2,3% people
In the 0.9%NaCl mixed solution of blood Alb and 6mM glucose, standby.
By detection, til cell total amount prepared by the present embodiment is 3.28 × 1010Individual, expand altogether
Increase 225.4 times；Wherein, CD3+Cell accounts for 98.47%, CD3+CD8+Cell accounts for 71.33%,
CD4+CD25+FoxP3+Tregs cell accounts for 3.62%；Killing activity to tumor cell is 57.67%
(4h 51Cr method for releasing, effect/target=40:1).
Embodiment 6
The present embodiment utilizes the method that pernicious ascites pleural fluid prepares High Fragmentation activity til cell in a large number, including
Following steps:
(1) collection of Malignant Pleural: under aseptic condition, collects the Malignant Pleural that breast carcinoma causes
900mL, and add 1.2 ten thousand U heparin；
(2) separation of mononuclearcell: being moved into by described Malignant Pleural in centrifuge tube, 500 × g is centrifuged
5min, abandoning supernatant；With 50mL AIM-V serum-free medium, sedimentation cell is resuspended, tiling
On the Ficoll layering liquid that proportion is 1.077,1600 × g is centrifuged 15min；
(3) washing of mononuclearcell: the mononuclearcell collected on Ficoll layering liquid interface (contains
Have lymphocyte, DC cell and tumor cell), add AIM-V serum-free medium, blow and beat, mix
Even, 300 × g is centrifuged 5min, abandoning supernatant；It is repeated 2 times following steps: add AIM-V serum-free
Culture medium re-suspended cell, blows and beats, mixes, and 300 × g is centrifuged 5min, abandoning supernatant；
(4) the directional induction activation of til cell: with containing 1000U/mL IFN-γ, 50U/mL IL-
2 and 3% AIM-V complete medium-I re-suspended cell of inactivated serum, and regulate cell density and be
3.0×106Individual/mL, at 37 DEG C, 5%CO2Cultivate 5 days with under conditions of saturated humidity, use during cultivation
Described AIM-V complete medium-I half amount changes liquid 1 time；
(5) advantage pcr of til cell: from cultivating the 6th day, with containing 1000U/mL IL-2,
The AIM-V of 200U/mL IFN-γ, 25ng/mL CD 3-resisting monoclonal antibody and 3% inactivated serum is complete
Culture medium-II re-suspended cell, and to regulate cell density be 1.0 × 106Individual/mL, at 37 DEG C, 5%CO2With
Continue under conditions of saturated humidity to cultivate, carry out cell counting every day and cultivate completely with described AIM-V
Cell density is adjusted to 1.0 × 10 by base-II6Individual/mL；
(6) collection of til cell: when cultivating the 21st day, collects cell suspension, and 300 × g is centrifuged
5min, washs with 0.9%NaCl solution, is resuspended in by cell containing 1000U/mL IL-2,3% people
In the 0.9%NaCl mixed solution of blood Alb and 6mM glucose, standby.
By detection, til cell total amount prepared by the present embodiment is 1.65 × 1010Individual, expand altogether
Increase 174.1 times；Wherein, CD3+Cell accounts for 98.89%, CD3+CD8+Cell accounts for 75.60%,
CD4+CD25+FoxP3+Tregs cell accounts for 2.83%；Killing activity to tumor cell is 64.25%
(4h 51Cr method for releasing, effect/target=40:1).
To sum up, the present invention utilizes the method that pernicious ascites pleural fluid prepares High Fragmentation activity til cell in a large number,
Til cell (338.6 ± 134.2) × 10 can be gathered in the crops when cultivating the 21st～28 day8Individual (n=25), carefully
Born of the same parents' amplification reaches 179.6 ± 24.2 times；Wherein, CD3+Cell accounts for 98.61% ± 3.22%, CD3+CD8+
Cell accounts for 74.56% ± 5.19%, CD3+CD4+Cell accounts for 27.42% ± 6.35%,
CD3+CD56+Cell accounts for 51.88% ± 7.49%, and CD4+CD25+FoxP3+Tregs cell is only
Account for 3.27% ± 1.75%, to the killing activity of tumor cell up to 66.54% ± 5.18% (4h51Cr releases
Put method, effect/target=40:1).
Obviously, above-described embodiment is only for clearly demonstrating example, and not to embodiment party
The restriction of formula.For those of ordinary skill in the field, the most also may be used
To make other changes in different forms.Here without also all of embodiment being given
With exhaustive.And the obvious change thus extended out or variation are still in the guarantor of the invention
Protect among scope.
Claims (10)
1. utilizing the method that pernicious ascites pleural fluid prepares High Fragmentation activity til cell in a large number, it is special
Levy and be, comprise the following steps:
(1) collection of pernicious ascites pleural fluid: under aseptic condition, collects pernicious ascites pleural fluid, and adds liver
Element so that in described pernicious ascites pleural fluid, the concentration containing described heparin is 11.0～16.5U/mL；
(2) separation of mononuclearcell: described pernicious ascites pleural fluid is moved in centrifuge tube, centrifugal,
Abandoning supernatant；Sedimentation cell is resuspended, it is laid on the Ficoll layering liquid that proportion is 1.076～1.078,
Centrifugal；
(3) washing of mononuclearcell: collect the mononuclearcell on Ficoll layering liquid interface, add
Enter AIM-V serum-free medium, mixing, centrifugal, abandoning supernatant；
(4) the directional induction activation of til cell: use AIM-V complete medium-I re-suspended cell, and
Regulation cell density is 1.5 × 106～3.0 × 106Individual/mL, cultivates 4～6 days under appropriate conditions, cultivates
Period changes liquid at least 1 time by described AIM-V complete medium-I half amount；
(5) advantage pcr of til cell: from cultivating the 5th～7 day, with AIM-V complete medium-
II re-suspended cell, and to regulate cell density be 1.0 × 106～2.0 × 106Individual/mL, under appropriate conditions
Continue to cultivate, carry out cell counting every day and with described AIM-V complete medium-II, cell density adjusted
Whole is 1.0 × 106～2.0 × 106Individual/mL；
(6) collection of til cell: when cultivating the 21st～28 day, collects cell suspension, centrifugal, uses
0.9%NaCl solution washs, and is resuspended in by cell in 0.9%NaCl mixed solution, standby.
The most according to claim 1 pernicious ascites pleural fluid is utilized to prepare High Fragmentation activity TIL in a large number
The method of cell, it is characterised in that
In the directional induction activation of described step (4) til cell, described AIM-V complete medium-
I containing IFN-γ, IL-2 and inactivated serum；
In the advantage pcr of described step (5) til cell, described AIM-V complete medium-II contains
There are IL-2, IFN-γ, CD 3-resisting monoclonal antibody and inactivated serum.
The most according to claim 1 and 2 utilize pernicious ascites pleural fluid prepare in a large number High Fragmentation activity
The method of til cell, it is characterised in that comprise the following steps:
(1) collection of pernicious ascites pleural fluid: under aseptic condition, collects pernicious ascites pleural fluid, and adds liver
Element so that in described pernicious ascites pleural fluid, the concentration containing described heparin is 12.5～15.0U/mL；
(2) separation of mononuclearcell: described pernicious ascites pleural fluid is moved in centrifuge tube, centrifugal,
Abandoning supernatant；Sedimentation cell is resuspended, it is laid on the Ficoll layering liquid that proportion is 1.077, from
The heart；
(3) washing of mononuclearcell: collect the mononuclearcell on Ficoll layering liquid interface, add
Enter AIM-V serum-free medium, mixing, centrifugal, abandoning supernatant；
(4) the directional induction activation of til cell: use AIM-V complete medium-I re-suspended cell, and
Regulation cell density is 2.2 × 106Individual/mL, cultivates 5 days, under appropriate conditions with described during cultivation
AIM-V complete medium-I half amount changes liquid 1 time；
(5) advantage pcr of til cell: from cultivating the 6th day, with AIM-V complete medium-II
Re-suspended cell, and to regulate cell density be 1.5 × 106Individual/mL, continues to cultivate under appropriate conditions,
Carry out cell counting every day and with described AIM-V complete medium-II, cell density be adjusted to
1.5×106Individual/mL；
(6) collection of til cell: when cultivating the 25th day, collects cell suspension, centrifugal, uses
0.9%NaCl solution washs, and is resuspended in by cell in 0.9%NaCl mixed solution, standby.
4. prepare High Fragmentation work in a large number according to the pernicious ascites pleural fluid that utilizes described in any one of claim 1-3
The method of property til cell, it is characterised in that
In the directional induction activation of described step (4) til cell, described 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 (5) til cell, described AIM-V complete medium-II contains
There are 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 work in a large number according to the pernicious ascites pleural fluid that utilizes described in any one of claim 1-4
The method of property til cell, it is characterised in that
In the separation of described step (2) mononuclearcell, with AIM-V serum-free medium,
RPMI1640 serum-free medium or PBS are resuspended by sedimentation cell.
6. prepare High Fragmentation work in a large number according to the pernicious ascites pleural fluid that utilizes described in any one of claim 1-5
The method of property til cell, it is characterised in that
In the washing of described step (3) mononuclearcell, described mononuclearcell be lymphocyte,
DC cell and the mixture of tumor cell.
7. prepare High Fragmentation work in a large number according to the pernicious ascites pleural fluid that utilizes described in any one of claim 1-6
The method of property til cell, it is characterised in that
In the washing of described step (3) mononuclearcell, also include being repeated at least once more following steps:
Add AIM-V serum-free medium re-suspended cell, mixing, be centrifuged, abandoning supernatant.
8. prepare High Fragmentation work in a large number according to the pernicious ascites pleural fluid that utilizes described in any one of claim 1-7
The method of property til cell, it is characterised in that
In the collection of described step (6) til cell, described 0.9%NaCl mixed solution contain IL-2,
Human blood Alb and glucose.
9. prepare High Fragmentation work in a large number according to the pernicious ascites pleural fluid that utilizes described in any one of claim 1-8
The method of property til cell, it is characterised in that
In the collection of described (6) 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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