CN104762261A - Tumor infiltrating lymphocytes separation method - Google Patents
Tumor infiltrating lymphocytes separation method Download PDFInfo
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- CN104762261A CN104762261A CN201510124216.3A CN201510124216A CN104762261A CN 104762261 A CN104762261 A CN 104762261A CN 201510124216 A CN201510124216 A CN 201510124216A CN 104762261 A CN104762261 A CN 104762261A
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Abstract
The invention relates to a cells separating technology, and provides a tumor infiltrating lymphocytes separation method. The method is characterized by adding tumor tissue masses in a trypsin-EDTA solution for immersion and incubation, fully digesting and dispersing the tumor tissue masses; separating and collecting single cell and being resuspended by the trypsin-EDTA solution for multitime density gradient centrifugation, incubating and separating by an erythrocyte lysate, using calf serum-containing PBS buffer or resuspending to obtain the dissolved tumor infiltrating lymphocytes. In the invention, an enzymatic digestion method and a machinery method are combined for dual separating the enzymatic digestion with high efficiency, compared with the machinery method or the enzymatic digestion method, the survival rate of the obtained tumor infiltrating lymphocytes is high, and the method is simple and easy to operate. The obtained tumor infiltrating lymphocytes can be massively propagated through in vitro stimulation of interleukin 2 (1L-2), have antineoplastic activity with specialty and high efficiency, and provides powerful basis and new approach for treating tumour.
Description
Technical field
The invention belongs to cell separation technology, relate to a kind of separation method of tumor infiltrating lymphocyte.
Background technology
The people such as Rosenberg in 1986 first reported the lymphocyte be separated to from mouse entity knurl, be referred to as " tumor infiltrating lymphocyte (tumor infiltrating lymphocyte; TIL) ", to be the T cell be mainly present in mesenchyma stroma of tumors be a main heterogeneous lymphocyte population, is gathered in around tumour mostly or its interstitial is that shell-like is around cancer nests.Further research finds, this cell can be bred in a large number in vitro after interleukin II (IL-2) stimulates, and there is the effect of stronger specific killing tumour cell, its antitumor action than Tumor-infiltrating lymphocytes (Lymphokine activated killer cell, LAK) high 50-100 doubly.Such as studies have found that, the TIL of melanoma patients only produces killing activity to Autologous tumor cell, and normal to own cells.
Til cell phenotype has heterogeneity.In general, in the original position TIL of fresh preparation, the overwhelming majority is T cell.In the til cell in different tumour source, there were significant differences for the ratio of CD4+T cell and CD8+T cell.Except melanoma, tumor of head and neck, based on CD8+T cell in other tumours.In the TIL of fresh separated, CD25+ cell percentage is lower, and along with the external prolongation adding IL-2 incubation time, CD25+ cell percentage raises gradually.The mark (CD16, CD56) of NK cell has the trend first increasing and reduce afterwards in the external IL-2 of the adding culturing process of TIL.Therefore, TIL has the specificity of anti-self tumor in kinds of tumors (as kidney and melanoma), and the biotherapy be expected as more effectively carrying out tumour from now on even prevents to provide strong foundation and new approach.
Current separation TIL majority adopts mechanical process or enzyme digestion, not only spends a large amount of manpower and materials, and reduces the cytoactive being separated rear TIL.How being effectively separated TIL is the technical problem being badly in need of solving, for the biotherapy studying tumour immunity microenvironment and tumour further provides a strong technique means.
Summary of the invention
The technical problem to be solved in the present invention is, overcomes deficiency of the prior art, provides a kind of separation method of tumor infiltrating lymphocyte.
For technical solution problem, solution of the present invention is:
A kind of separation method of tumor infiltrating lymphocyte is provided, comprises the steps:
(1) with after PBS buffer solution tumor tissue, be placed in culture dish, add 5 ~ 10 volumes trysinization immersion doubly and steep 3 hours;
(2) tumor tissue after immersion treatment is trimmed to 0.5mm
3particulate state after, put culture dish in 37 DEG C of shaking baths, tumor tissues and trysinization liquid hatched 1 ~ 2 hour in the lump, tumor tissue is fully digested and breaks up; Collecting cell suspension also crosses 200 order metal screens, and separated and collected individual cells is in 50ml centrifuge tube; Wash culture dish 2 times with trysinization liquid, all liquid is transferred in this 50ml centrifuge tube;
(3) the 50ml centrifuge tube of previous step is placed on and leaves standstill after 10 minutes on ice, centrifugal treating 2 minutes on 50g whizzer; Abandon bottom settlings, transfer supernatant liquor is in another 50ml centrifuge tube, and on 400g whizzer, centrifugal treating 5 minutes, abandons supernatant, and it is resuspended to add 3ml trysinization liquid;
(4) in resuspended centrifuge tube, add density gradient centrifugation liquid to previous step, carry out density gradient centrifugation; Then collect cloud and mist confluent monolayer cells, be transferred in another Glass tubing, it is resuspended to add 3ml trysinization liquid;
The density gradient centrifugation liquid added and the mass ratio of cell suspension are 1:1 ~ 2, and in density gradient centrifugation process, control condition is 500g × 20 minute, accelerate 9 and slow down 4, temperature 10 DEG C;
(5) in resuspended centrifuge tube, add 1 × erythrocyte cracked liquid 2ml to previous step, add 2ml PBS damping fluid after hatching 5 minutes and stop hatching; On 400g whizzer centrifugal 5 minutes, abandon supernatant; After PBS buffer solution cell 2 times, resuspended with the PBS damping fluid containing 10wt% calf serum; The cell dissolved in this centrifuge tube is tumor infiltrating lymphocyte;
Described trysinization liquid is with Hanks' balance liquid for solvent, and solution comprises the DNase I of collagenasetype IV and 0.001wt% of 0.05wt%, and every 200ml Hanks' balance liquid adds O.111g CaCl2.
The qualification of gained tumor infiltrating lymphocyte in the present invention, be adopt flow cytometry partition method to detect surface of cell membrane antibody, the method has following steps:
(1) utilizing the blue lymphocyte to being separated acquisition of placenta to dye, under microscope, carrying out cell counting and determination of activity.
(2) 1 × 10 is taken out
6individual cell (requires viable count >90-95%) in test tube, adds fluorescently-labeled antibody, and mixing, hatches 30 minutes for 4 DEG C.Wash 2 times with PBS, 1500rpm/min, centrifugal 3 minutes, abandon supernatant.Add 300ulPBS liquid (PH=7.4), flow cytometer carries out Testing and appraisal.
Compared with prior art, beneficial effect of the present invention is:
In the present invention, enzymic digestion is separated tumor infiltrating lymphocyte in conjunction with mechanical process Dual high-efficiency, and compared with mechanical process or enzyme digestion, it is high and simple and easy to do that the present invention obtains tumor infiltrating lymphocyte survival rate.Therefore, strong guarantee can be provided for studying the effect of TIL in tumour further.
The method combined with lymphocyte separation medium by trysinization, mechanical separation isolates tumor infiltrating lymphocyte from tumor tissues.The present invention is that clinical study tumour immunity microenvironment provides lymphocyte that is different classes of, different subtype, for inquiring into the immune adjusting mechanism of tumor infiltrating lymphocyte (TILs) and the techniqueflow of providing convenience in the developing effect of the generation of tumour.Its tumor infiltrating lymphocyte survival rate be separated, up to more than 90%, is convenient to application in the separation of lymphocytes of various solid tumor, simple.The separating obtained tumor infiltrating lymphocyte of the method can be bred in a large number in vitro after interleukin II (IL-2) stimulates, and has the anti-tumor activity of differential high efficient, for clinical treatment tumour provides strong foundation and new approach.
Embodiment
Content of the present invention is illustrated further below in conjunction with specific embodiment:
Tumor tissue of the present invention derives from the tumor tissues sample excised in surgical procedure clinically, and belong to medical treatment and abandon refuse, clinical operation process not the present invention relates to content.
When selecting tumor tissues, the position of tumour, scope should be confirmed, note the discriminating with surrounding tissue and necrotic tissue.In shortest time inscribe sampling this (< 30min) after tumor tissues is in vitro, tumor tissues is cut into the tissue block that polylith diameter is about 0.5cm, puts into liquid nitrogen transfer tank in loading sterilizing cryopreservation tube and preserve for a long time.
All ingredients used in the present invention is commercial products and is configured acquisition, and those skilled in the art can skillfully grasp.Be described as follows:
PBS damping fluid (phosphate buffered saline(PBS)), its mass concentration is 10%, commercial.
Collagenase type IV, namely collagenase IV, commercial, and Invitrogen company produces.
DNase I, i.e. deoxyribonuclease I, commercial, Sigma-Aldrich company produces.
Hanks' balance liquid, i.e. HBSS, commercial, GENMED company produces.
Density gradient centrifugation liquid, namely human lymphocyte parting liquid, commercial, and Sigma-Aldrich company produces.
Specific embodiment 1
1, the separation of tumor infiltrating lymphocyte
(1) with after PBS buffer solution hepatic carcinoma tissue block, be placed in culture dish, add 5 volumes trysinization immersion doubly and steep 3 hours;
(2) tumor tissue after immersion treatment is trimmed to 0.5mm
3particulate state after, put culture dish in 37 DEG C of shaking baths, tumor tissues and trysinization liquid hatched 1 hour in the lump, tumor tissue is fully digested and breaks up; Collecting cell suspension also crosses 200 order metal screens, and separated and collected individual cells is in 50ml centrifuge tube; Wash culture dish 2 times with trysinization liquid, all liquid is transferred in this 50ml centrifuge tube;
(3) the 50ml centrifuge tube of previous step is placed on and leaves standstill after 10 minutes on ice, centrifugal treating 2 minutes on 50g whizzer; Abandon bottom settlings, transfer supernatant liquor is in another 50ml centrifuge tube, and on 400g whizzer, centrifugal treating 5 minutes, abandons supernatant, and it is resuspended to add 3ml trysinization liquid;
(4) in resuspended centrifuge tube, add density gradient centrifugation liquid to previous step, carry out density gradient centrifugation; Then collect cloud and mist confluent monolayer cells, be transferred in another Glass tubing, it is resuspended to add 3ml trysinization liquid;
The density gradient centrifugation liquid added and the mass ratio of cell suspension are 1:1, and in density gradient centrifugation process, control condition is 500g × 20 minute, accelerate 9 and slow down 4, temperature 10 DEG C;
(5) in resuspended centrifuge tube, add 1 × erythrocyte cracked liquid 2ml to previous step, add 2ml PBS damping fluid after hatching 5 minutes and stop hatching; On 400g whizzer centrifugal 5 minutes, abandon supernatant; After PBS buffer solution cell 2 times, resuspended with the PBS damping fluid containing 10wt% calf serum; The cell dissolved in this centrifuge tube is tumor infiltrating lymphocyte;
Described trysinization liquid is with Hanks' balance liquid for solvent, and solution comprises the DNase I of collagenasetype IV and 0.001wt% of 0.05wt%, and every 200ml Hanks' balance liquid adds O.111g CaCl
2.
2, the qualification of tumor infiltrating lymphocyte
(1) utilizing the blue lymphocyte to being separated acquisition of placenta to dye, under microscope, carrying out cell counting and determination of activity.
(2) 1 × 10 is taken out
6individual cell (requires viable count >90-95%) in test tube, adds fluorescently-labeled antibody, and mixing, hatches 30 minutes for 4 DEG C.Wash 2 times with PBS, 1500rpm/min, centrifugal 3 minutes, abandon supernatant.Add 300ulPBS liquid (PH=7.4), flow cytometer carries out Testing and appraisal.
Specific embodiment 2
Compared with embodiment 1, in the present embodiment: separate object is lung cancer tumor tissue block; In step (1), the add-on of trysinization liquid is 10 volumes times; In step (2), shaking bath incubation time is 2 hours; The density gradient centrifugation liquid added in step (4) and the mass ratio of cell suspension are 1:2; All the other contents are all identical with embodiment 1.
Specific embodiment 3
Compared with embodiment 1, in the present embodiment: separate object is gastric cancer tumor tissue block; In step (1), the add-on of trysinization liquid is 7 volumes times; In step (2), shaking bath incubation time is 1.5 hours; The density gradient centrifugation liquid added in step (4) and the mass ratio of cell suspension are 1:1.5; All the other contents are all identical with embodiment 1.
Claims (1)
1. a separation method for tumor infiltrating lymphocyte, is characterized in that, comprises the steps:
(1) with after PBS buffer solution tumor tissue, be placed in culture dish, add 5 ~ 10 volumes trysinization immersion doubly and steep 3 hours;
(2) tumor tissue after immersion treatment is trimmed to 0.5mm
3particulate state after, put culture dish in 37 DEG C of shaking baths, tumor tissues and trysinization liquid hatched 1 ~ 2 hour in the lump, tumor tissue is fully digested and breaks up; Collecting cell suspension also crosses 200 order metal screens, and separated and collected individual cells is in 50ml centrifuge tube; Wash culture dish 2 times with trysinization liquid, all liquid is transferred in this 50ml centrifuge tube;
(3) the 50ml centrifuge tube of previous step is placed on and leaves standstill after 10 minutes on ice, centrifugal treating 2 minutes on 50g whizzer; Abandon bottom settlings, transfer supernatant liquor is in another 50ml centrifuge tube, and on 400g whizzer, centrifugal treating 5 minutes, abandons supernatant, and it is resuspended to add 3ml trysinization liquid;
(4) in resuspended centrifuge tube, add density gradient centrifugation liquid to previous step, carry out density gradient centrifugation; Then collect cloud and mist confluent monolayer cells, be transferred in another Glass tubing, it is resuspended to add 3ml trysinization liquid;
The density gradient centrifugation liquid added and the mass ratio of cell suspension are 1:1 ~ 2, and in density gradient centrifugation process, control condition is 500g × 20 minute, accelerate 9 and slow down 4, temperature 10 DEG C;
(5) in resuspended centrifuge tube, add 1 × erythrocyte cracked liquid 2ml to previous step, add 2ml PBS damping fluid after hatching 5 minutes and stop hatching; On 400g whizzer centrifugal 5 minutes, abandon supernatant; After PBS buffer solution cell 2 times, resuspended with the PBS damping fluid containing 10wt% calf serum; The cell dissolved in this centrifuge tube is tumor infiltrating lymphocyte;
Described trysinization liquid is with Hanks' balance liquid for solvent, and solution comprises the DNase I of collagenasetype IV and 0.001wt% of 0.05wt%, and every 200ml Hanks' balance liquid adds O.111g CaCl
2.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107502589A (en) * | 2017-08-04 | 2017-12-22 | 北京世纪劲得生物技术有限公司 | A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method |
| CN111849895A (en) * | 2020-07-31 | 2020-10-30 | 南京元荟生物科技有限公司 | Separation method and application of tumor infiltrating lymphocytes from ascites |
| CN111849894A (en) * | 2020-07-31 | 2020-10-30 | 南京元荟生物科技有限公司 | Separation method and application of tumor infiltrating lymphocytes |
| CN112852729A (en) * | 2021-04-16 | 2021-05-28 | 天津市环湖医院 | Method for extracting high-quality B cells from bone marrow, peripheral blood and lymphoma tissues |
| CN111849895B (en) * | 2020-07-31 | 2024-04-26 | 南京元荟生物科技有限公司 | Separation method of ascites-derived tumor infiltration lymphocytes and application thereof |
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| CN1560234A (en) * | 2004-02-18 | 2005-01-05 | 南京大学医学院附属鼓楼医院 | Process of caltivating TIL cell by self hydrothorax supernatant |
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| CN1560234A (en) * | 2004-02-18 | 2005-01-05 | 南京大学医学院附属鼓楼医院 | Process of caltivating TIL cell by self hydrothorax supernatant |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107502589A (en) * | 2017-08-04 | 2017-12-22 | 北京世纪劲得生物技术有限公司 | A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method |
| CN111849895A (en) * | 2020-07-31 | 2020-10-30 | 南京元荟生物科技有限公司 | Separation method and application of tumor infiltrating lymphocytes from ascites |
| CN111849894A (en) * | 2020-07-31 | 2020-10-30 | 南京元荟生物科技有限公司 | Separation method and application of tumor infiltrating lymphocytes |
| CN111849895B (en) * | 2020-07-31 | 2024-04-26 | 南京元荟生物科技有限公司 | Separation method of ascites-derived tumor infiltration lymphocytes and application thereof |
| CN111849894B (en) * | 2020-07-31 | 2024-04-26 | 南京元荟生物科技有限公司 | Separation method of tumor-infiltrating lymphocytes and application thereof |
| CN112852729A (en) * | 2021-04-16 | 2021-05-28 | 天津市环湖医院 | Method for extracting high-quality B cells from bone marrow, peripheral blood and lymphoma tissues |
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Application publication date: 20150708 |