CN108588021A - The method of separation and purifying TIL, acquisition CD4+CD25+ cell subsets - Google Patents
The method of separation and purifying TIL, acquisition CD4+CD25+ cell subsets Download PDFInfo
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- CN108588021A CN108588021A CN201810407959.5A CN201810407959A CN108588021A CN 108588021 A CN108588021 A CN 108588021A CN 201810407959 A CN201810407959 A CN 201810407959A CN 108588021 A CN108588021 A CN 108588021A
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Abstract
The invention discloses a kind of methods of separation and purifying TIL, acquisition CD4+CD25+ cell subsets.By having carried out complete innovation from technique, the TIL without external long-term disposal is detached directly from cancerous lung tissue sample, the extracorporeal treatment time is short, and processing mode is mild, can more retain the functional status of TIL in vivo.Generally acknowledged CD4+CD25+ cell subsets is further isolated from TIL on this basis, in addition from optimizing in terms of culture medium so that the Treg detached in cancerous lung tissue, and for can survive in vitro for Treg functional study and clinically application provide necessary condition.
Description
Technical field
The present invention relates to biotechnology more particularly to a kind of separation and purifying TIL, obtain CD4+CD25+ cells Asia
The method of group.
Background technology
Lung cancer is that the highest malignant tumour of incidence, the death rate are arranged in mortality of malignant tumors in China's Urban population
In first place.Research for the morbidity and treatment of lung cancer is the task of top priority.Clinically for the treatment of tumour, in addition to being cut in operation
Remove, chemotherapy, on the basis of radiotherapy, emerging immunization therapy basis and field with fastest developing speed in application study at present.But
Since tumor-cell antigen is weak, body cannot generate effective immune clearance, current specific treatment target to tumour cell
Point is serious deficient, and which has limited the development of immunotherapy of tumors and clinical applications.The reason of causing tumor immune escape has very much.
First, the function of immune system of tumour patient is low, the major histocompatibility complex (major carried
Histocompatibility complex, MHC) MHC molecule cannot effective submission tumour antigen, lymphocyte cannot be had
Effect activation.Secondly, invasion transfer is the important feature of malignancy, research shows that tumor invasion and metabasis is soaked with lymphocyte
Moisten close relation, chemotactic factor (CF) and its receptor may take part in the invasion and transfer of tumour by lymphocytic infiltration.Cause
It is to find new tumour to exempt from that this, which illustrates the phenotype of regulatory T cells subgroup (abbreviation Treg) and function in tumor infiltrating lymphocyte,
The basis of epidemic disease therapy target.However due to tumor tissues Finite Samples, tumor infiltrating lymphocyte number is rare, and separation is difficult,
And regulatory T cells subgroup accounting smaller therein is even more to be difficult to purifies and separates to come out, and strongly limits the functional study of Treg
And clinical application.
Foxp3 can be based on flow cytometry in peripheral blood mononuclear cells as relatively more generally acknowledged Treg surface markers
In can recognize that Treg subgroups, but Foxp3, as transcription factor in core, the detection of flow cytometry is needed to cell to be detected
It carries out antigen and fixes and break nuclear membrane processing, thus be only capable of the presence of Treg cells in detection tumor infiltrating lymphocyte TIL, but simultaneously
The Treg for providing functional property cannot be efficiently separated.
Invention content
For disadvantages described above, present invention aims at how being detached from tumor tissues and purify TIL, and nothing is detached in turn
The Treg of damage.
To achieve the goals above, the present invention provides a kind of method of separation and purifying TIL, include the following steps:
Step 1：The tumor tissues of 0.5~3g are aseptically subjected to surface washing with 70% alcoholic solution；
Step 2：The tumor tissues of surface washing are placed in 1640 culture medium；
Step 3：Lung, slough and connective tissue are removed, and tumor tissues are fragmented to 0.5~2mm3Size swells
Tumor fragment；
Step 4：Washing operation is carried out to Tumor fragments, specially Tumor fragments are resuspended in 1640 culture medium, wait swelling
After tumor fragment natural subsidence, muddy supernatant liquid is removed, repetition to Tumor fragments wash supreme using 1640 culture medium
Layer liquid clarification；
Step 5：Tumor fragments after washing are resuspended in cell separating liquid, the cell separating liquid is to be added to
The 1640 culture medium of I digestive juice of 0.05% type Ⅳ collagenase and 0.002%DNase, is placed in 37 DEG C of environment temperatures, is stirred with magnetism
Mix the warm digestion of device 4 hours；
Step 6：The mild mechanical cleavage of suspension tissue processor is at single cell suspension after digestion, on 300g centrifuges from
Cell precipitation is obtained after heart 10min；
Step 7：It adds isotonic PBS to be resuspended, and presses 1:1 is added Percoll PLUS separating liquids, on 300g centrifuges
Low acceleration steadily centrifuges 20min, collects middle layer lymphocyte suspension, and 1640 culture medium is used in combination to wash repeatedly 2 removals
Obtain the tumor infiltrating lymphocyte TIL of purifying after Percoll Plus separating liquids, mode of washing be on 300g centrifuges from
Cell precipitation is obtained after heart 10min.
A method of CD4+ cell subsets is obtained, it, will by the TIL for the purifying that the method for detaching and purifying TIL obtains
The lymphocyte precipitate of above-mentioned separation presses 107A/ml is resuspended in sorting buffer solution, and the coated magnetic bead of CD4 antibody is added, passes through
Magnetic bead adsorbs the CD4+ cell subsets in lymphocyte suspension, acquires CD4+T cell subsets.
A method of CD4+CD25+ cell subsets being obtained, it is thin to obtain CD4+ by the method for obtaining CD4+ cell subsets
Born of the same parents' subgroup is protected from light incubation 20 minutes using the fluorescent labeled antibody in the scheme of colour CD4FITC and CD25APC of stream measuring,
It uses and is cleaned in buffer salt solution PBS again, detected on fluidic cell instrument after cleaning, needed for the mating software delineation of instrument
The cell colony to be collected, then this partial mass captured and be recycled in the container of collection by flow cytometer.
A method of culture CD4+CD25+ cell subsets is obtained by the method for obtaining CD4+CD25+ cell subsets
The cell mass sub-elected is added to and is added in the orifice plate of Treg culture mediums in advance by CD4+CD25+ cell subsets, is 37 in temperature
DEG C, 5%CO2, the environment kind culture of 95% humidity 13 days.
The method of the culture CD4+CD25+ cell subsets, it is characterised in that add cell in the Treg culture mediums
The X-VIVO of the factor, 5% human serum, 40ng/mL IL-2,30ng/mL anti-CD3 and 30ng/mL anti-CD28.
Technical scheme of the present invention has carried out complete innovation from technique, directly from cancerous lung tissue sample separation without
The TIL of external long-term disposal, the extracorporeal treatment time is short, and processing mode is mild, can more retain the functional status of TIL in vivo.Separately
Outside from optimizing in terms of culture medium so that the Treg detached in cancerous lung tissue can survive in vitro for Treg functional study and
Clinically application provides necessary condition.
Description of the drawings
Fig. 1 is the flow chart for obtaining CD4+CD25+T cell subsets；
Fig. 2 is that airflow classification collects CD4+CD25+ cell subsets process schematics；
Fig. 3 is that airflow classification collects CD4+CD25+ cell subsets backtest results schematic diagrames.
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation describes, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other without creative efforts
Embodiment shall fall within the protection scope of the present invention.
The Percoll PLUS density gradient media-GE Healthcare that Percoll PLUS refer to
Life Sciences GE products.
X-VIVOTMChemically Defined, Serum-free Hematopoietic Cell Media are lonza
The product of company is mainly free of exogenous growth factor and indefinite substance.
Fig. 1 is the flow chart for obtaining CD4+CD25+ cell subsets, it is necessary first to obtain and purify tumor-infiltrated leaching in lung cancer
Bar cell TIL, CD4+CD25+ cell subsets is generally acknowledged in the industry T cell subgroup.
Collection about sample：Patients with lung cancer confirms through pathological examination, and when sampling avoids necrotic area, preoperative not receive
The immunotherapies such as including antibody drug.
Collect tumor infiltrating lymphocyte (TIL)：The tumor tissues for taking size about 1g patients with lung cancer, are aseptically used
70% alcoholic solution quickly carries out surface washing, not impregnate, and only surface clean sterilizes, and balance reduces pollution probability and cell
Damage, transfers in the culture dish containing 1640 culture medium.With tweezers removal lung, slough and knot in 1640 culture medium
Tissue is formed, tiny fragment (about 1mm is then cut into operating scissors3Size), it is resuspended in 1640 culture medium and is placed in
In 15mL centrifuge tubes.After Tumor fragments natural subsidence, muddy supernatant liquid is siphoned away, tumour is washed repeatedly with 1640 culture medium
Fragment is clarified to supernatant liquid twice.Tumor fragments after washing are resuspended in cell separating liquid, the cell separating liquid is
It is added to the 1640 culture medium of I digestive juice of 0.05% type Ⅳ collagenase and 0.002%DNase, is placed in 37 DEG C of environment temperatures, is used
The warm digestion of magnetic stirrer 4 hours.After digestion suspension with the mild mechanical cleavages of Gentle MACS Dissociator at unicellular
Suspension, low acceleration steadily centrifuges 10min and centrifuges to obtain on 300g centrifuges, and isotonic PBS is added after cell precipitation and is resuspended simultaneously
By 1:1 is added Percoll PLUS separating liquids (density 1.09g/ml)；Low acceleration steadily centrifuges on 300g centrifuges
20min, low acceleration steadily centrifuge, and collect middle layer lymphocyte suspension, and 1640 culture medium is used in combination to wash repeatedly 2 removals
PercollPlus separating liquids, the tumor infiltrating lymphocyte TIL of the purifying can be used for subsequently screening.
Tumor infiltrating lymphocyte TIL, which is further purified, isolates CD4+T cell subsets：The lymphocyte precipitate of separation in
107/ ml is resuspended in sorting buffer solution, and the coated magnetic bead of CD4 antibody is added, and is adsorbed in lymphocyte suspension using magnetic pole
CD4+ cells be further enriched with and purify, and obtain CD4+ cell subsets.
CD4+ cell subsets further collects CD4+CD25+ cell subsets by airflow classification instrument：Booting preheating is gone forward side by side
It is sorted after row aseptic process.Flow cytometry sorts the BD FACSARIA III instruments used.Above-mentioned CD4+ cell subsets
It is protected from light incubation 20 minutes by the fluorescent labeled antibody in following stream measuring scheme of colour, PBS cleans machine testing on twice, circle
Cell mass needed for fixed.It is respectively adopted and is drawn a circle to approve using the scheme of colour of CD4FITC and CD25APC stream measurings.Fig. 2 is streaming
CD4+CD25+ cell subsets process schematics are collected in choosing, are the required cell mass of delineation in last P3 boxes.Fig. 3 is streaming point
CD4+CD25+ cell subsets backtest results schematic diagrames are collected in choosing, as being the required cell mass of delineation in B2 boxes.
The cell mass sub-elected is added to and is added in the orifice plate of Treg culture mediums in advance, is 37 DEG C in temperature, 5%CO2,
The environment kind culture of 95% humidity 13 days.X-VIVO, 5% human serum, the 40ng/mL of cell factor are added in Treg culture mediums
IL-2,30ng/mL anti-CD3 and 30ng/mL anti-CD28.
Above disclosed is only an embodiment of the present invention, cannot limit the right model of the present invention with this certainly
It encloses, those skilled in the art can understand all or part of the processes for realizing the above embodiment, and is wanted according to right of the present invention
Equivalent variations made by asking still fall within the range that the present invention is covered.
Claims (5)
1. a kind of method of separation and purifying TIL, includes the following steps:
Step 1：The tumor tissues of 0.5~3g are aseptically subjected to surface washing with 70% alcoholic solution；
Step 2：The tumor tissues of surface washing are placed in 1640 culture medium；
Step 3：Lung, slough and connective tissue are removed, and tumor tissues are fragmented to 0.5~2mm3The tumour of size is broken
Block；
Step 4：Washing operation is carried out to Tumor fragments, specially Tumor fragments are resuspended in 1640 culture medium, wait for that tumour is broken
After block natural subsidence, muddy supernatant liquid is removed, repeats to wash to upper liquid Tumor fragments using 1640 culture medium
Body is clarified；
Step 5：Tumor fragments after washing are resuspended in cell separating liquid, the cell separating liquid is to be added to 0.05% IV
The 1640 culture medium of I digestive juice of Collagenase Type and 0.002%DNase, is placed in 37 DEG C of environment temperatures, is disappeared with magnetic stirrer is warm
Change 4 hours；
Step 6：The mild mechanical cleavage of suspension tissue processor is centrifuged at single cell suspension on 300g centrifuges after digestion
Cell precipitation is obtained after 10min；
Step 7：It adds isotonic PBS to be resuspended, and presses 1:1 is added Percoll PLUS separating liquids, low on 300g centrifuges to add
Speed steadily centrifuges 20min, collects middle layer lymphocyte suspension, and 1640 culture medium is used in combination to wash repeatedly 2 removal Percoll
The tumor infiltrating lymphocyte TIL of purifying is obtained after Plus separating liquids, mode of washing is after centrifuging 10min on 300g centrifuges
Obtain cell precipitation.
2. a kind of method obtaining CD4+ cell subsets, the method for detaching and purifying according to claim 1 TIL obtains pure
The lymphocyte precipitate of above-mentioned separation is pressed 10 by the TIL of change7A/ml is resuspended in sorting buffer solution, and CD4 antibody coating is added
Magnetic bead, by magnetic bead adsorb lymphocyte suspension in CD4+ cell subsets, acquire CD4+ cell subsets.
3. a kind of method obtaining CD4+CD25+ cell subsets, the side according to claim 2 for obtaining CD4+ cell subsets
The CD4+T cell subsets that method obtains, it is anti-using the fluorescent marker in scheme of colour CD4 FITC and the CD25 APC of stream measuring
Body is protected from light incubation 20 minutes, then using being cleaned in buffer salt solution PBS, is detected on fluidic cell instrument after cleaning, use instrument
Mating software draws a circle to approve the required cell colony collected, and then this partial mass is captured by flow cytometer and is recycled to receipts
In the container of collection.
4. it is a kind of culture CD4+CD25+ cell subsets method, according to claim 3 method obtain CD4+CD25+ cells
The cell mass sub-elected is added to and is added in the orifice plate of Treg culture mediums in advance by subgroup, is 37 DEG C in temperature, 5%CO2,
The environment kind culture of 95% humidity 13 days.
5. the method for culture CD4+CD25+ cell subsets according to claim 4, it is characterised in that the Treg culture mediums
X-VIVO, 5% human serum, 40ng/mL IL-2,30ng/mL anti-CD3 and the 30ng/mL anti-of middle addition cell factor
CD28。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2022111573A1 (en) * | 2020-11-25 | 2022-06-02 | 上海君赛生物科技有限公司 | Pharmaceutical composition for enhancing cell killing, and use thereof |
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| CN105969731A (en) * | 2016-07-29 | 2016-09-28 | 解西河 | Method for preparing high-killing-activity TIL cells in batches from malignant pleuroperitoneal fluid |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022111573A1 (en) * | 2020-11-25 | 2022-06-02 | 上海君赛生物科技有限公司 | Pharmaceutical composition for enhancing cell killing, and use thereof |
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Application publication date: 20180928 |