CN104388386B - In-vitro activation amplification method for V delta 1 gamma delta T cell coming from peripheral blood - Google Patents
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Abstract
The invention discloses an in-vitro activation amplification method for V delta 1 gamma delta T cell coming from peripheral blood. The method comprises: getting peripheral whole blood, employing a density gradient centrifugation and immunomagnetic bead sorting method to collecting total gamma delta T cell; resuspending the total gamma delta T cell by using an improved 1640 medium as a culture solution, performing cell culture under the conditions of 5% CO2 and 37 DEG C, and replacing a fresh culture solution every 2-3 days, so as to obtain in-vitro activated amplified gamma delta T cell mainly containing V delta 1 gamma delta T subset. The raw materials are convenient to obtain, the method does not cause an ethical issue and has extremely strong usage value. The obtained cell has relatively high cytotoxic activity of killing solid tumor cells, tumor stem cells and blood system tumor cells. The method for obtaining the cell is simple, efficient and low in cost.
Description
(1) technical field
The present invention relates to a kind of Activation In Vitro amplification method from peripheral blood V δ 1 gamma delta T cells.
(2) background technology
Gamma delta T cells be a class have in panimmunity response and immunopathogenesis reaction important function intrinsic lymph thin
Born of the same parents.Gamma delta T cells are different from most of α β T cell, and its TCR acceptor is made up of two chains of γ and δ.Under physiological conditions, gamma delta T cells
Quantity in peripheral blood accounts for the 0.5%-16% of T lymphocyte.Gamma delta T cells have various kinds of cell subgroup, in Normal human peripheral
In blood mainly based on V γ 9V δ 2 gamma delta T cells subgroup (more than 70%), V δ 1 gamma delta T cells subgroup only accounts for total gamma delta T cells
1%-10%.Gamma delta T cells have the characteristics that：1st, non-MHC is restricted；2nd, cytokine profiles are secreted；3rd, can be damaged by inflammation
Wound, infection and tumour activation；4th, cancer immunosurveillance effect etc..Since the first time in 2003 such as Martin Wilhelm is by gamma delta T
Since cell is used for treating NHL and the clinical testing acquirement certain effect of Huppert's disease, gamma delta T cells
The technical method of Activation In Vitro amplification becomes new study hotspot, and it represents before wide application to the adoptive immunotherapy of tumour
Scape.
The research being currently based on the gamma delta T cells adoptive immunotherapy of tumour is concentrated mainly on V γ 9V δ 2 gamma delta T cells Asia
Group.Different from V γ 9V δ 2 gamma delta T cells, V δ 1 gamma delta T cells are mainly distributed on the tissue such as enteric epithelium, skin, spleen, liver, in periphery
In blood, ratio is very low.Research report V δ 1 gamma delta T cells also have antitumor action, the especially tumour of epithelial origin.Therefore, V δ 1
Gamma delta T cells are likely to become the candidate cell subgroup of the adoptive cellular immunotherapy of tumour.But because its cell quantity is few,
It is particularly necessary that Activation In Vitro amplification obtains a large amount of V δ 1 gamma delta T cells.
Traditional gamma delta T cells Activation In Vitro amplification method mainly with expand V γ 9V δ 2 gamma delta T cells subgroup based on, but
Activation In Vitro amplification after its cytoactive decline, apoptotic cell quantity more to adoptive immunotherapy especially to solid tumor
Therapeutic effect not good.Studies have reported that at present amplification method PHA+IL-2 can be with selective amplification V δ 1 gamma delta T cells, and energy
Effectively improve its killing ability (Blood.2011Jul 28 to hematological system tumor；118(4):992-1001.).But still deposit
In problems with：1) cell amplification efficiency is low, and V δ 1 gamma delta T cells subset proportions are expanded to 40%-60%, and absolute cell numbers amount increases
Plus 40-80 times about, cell purity and quantity all can not reach clinical requirement；2) cytoactive decline and apoptotic cell quantity relatively
Many, the V δ 1 gamma delta T cells subgroup apoptosis of 10%-20% after expanding two weeks；3) after expanding V δ 1 gamma delta T cells to solid tumor cell and
The killing ability of tumor stem cell is not evaluated.
Therefore, if a kind of amplification efficiency can be adopted high the gamma delta T cells obtaining from peripheral blood, kill the cell of tumour
Cytotoxic activity is strong, and purity is high, and simple and fast, method with low cost, then to amplification V δ 1 gamma delta T cells subgroup general in a large number from now on
It is applied to clinical tumor adoptive immunotherapy and has important actual application value.
(3) content of the invention
It is an object of the present invention to provide a kind of Activation In Vitro amplification method from peripheral blood V δ 1 gamma delta T cells, the method
Amplification efficiency is high, and the cytotoxic activity killing tumour is strong, and purity is high, and simple and fast, with low cost, solves thin in prior art
Born of the same parents' amplification efficiency is low, and after amplification, cytoactive declines, and apoptotic cell quantity is many, kills the problem of scarce capacity.
The technical solution used in the present invention is：
The present invention provides a kind of Activation In Vitro amplification method from peripheral blood V δ 1 gamma delta T cells, and described method is：
Take periphery whole blood, the method using density gradient centrifugation and immunological magnetic bead sorting collects total gamma delta T cells；Total gamma delta T cells are used
1640 culture mediums containing improvement are resuspended as nutrient solution, in 5%CO2, carry out cell culture under the conditions of 37 DEG C, renew within every 2-3 days fresh
Nutrient solution, obtains the gamma delta T cells based on V δ 1 gamma delta T subgroup of Activation In Vitro amplification；Described improvement 1640 culture medium composition
For：Volumetric concentration 10% hyclone, (content of penicillin is 10000U/ml to 50mg/mL mycillin, the content of streptomysin
For 10mg/ml), 0.1-10 μ g/ml PHA (phytolectin) and 1-100ng/ml IL-7 (interleukin-17), solvent is
1640 culture mediums.
Further, the method for described density gradient centrifugation and immunological magnetic bead sorting is specially：
(1) take periphery whole blood, with PBS with volume ratio 1:1 dilution, obtains periphery whole blood dilution；
(2) the Ficoll lymphocyte separation medium that proportion is 1.107 is added in centrifuge tube, then that step (1) is outer
All hemodilution liquid is added slowly to Ficoll lymphocyte separation medium upper strata, horizontal centrifuge 400g/min, 20 DEG C, is centrifuged 25min,
Reactant liquor is layered as upper serum, tunica albuginea confluent monolayer cells, Ficoll separation liquid layer, GCL and bottom red blood cell layer successively；Institute
State peripheral blood dilution and Ficoll lymphocyte separation medium volume ratio 3:1；
(3) detached for step (2) tunica albuginea confluent monolayer cells are suctioned out and are transferred to new centrifuge tube, wash 2 with the PBS of 5 times of volumes
Secondary, 1000r/min, it is centrifuged 5min, abandons supernatant, collect cell a；
(4) with 1 × 107The amount of individual cell/40 μ l magnetic bead sorting buffer solution a is obtained with the resuspended step of magnetic bead sorting liquid a (3)
Cell a, be subsequently adding gamma delta T CR antibody, 4 DEG C of incubation 10min, add magnetic bead sorting buffer solution b and gamma delta T CR magnetic bead resists
Body, 4 DEG C of incubation 15min, centrifugation, precipitation is washed 1 time with PBS, centrifugation, abandons supernatant, collects cell b；Described magnetic bead sorting buffer solution b
It is 3 with magnetic bead sorting buffer solution a volume ratio:4；Described gamma delta T CR antibody and magnetic bead sorting buffer solution a volume ratio are 1:4；Described
Gamma delta T CR magnetic bead antibody and magnetic bead sorting buffer solution a volume ratio are 1:2；Described gamma delta T CR magnetic bead antibody is preferably purchased from German U.S. sky
Ni company；Described magnetic bead sorting buffer solution a and magnetic bead sorting buffer solution b is magnetic bead sorting buffer solution, for the ease of statement not
Name with volume difference used by step, letter does not have implication in itself；
(5) add PBS resuspended in the cell b obtaining to step (4), cross MACS magnetic bead sorting post (positive selection separating gamma δ
T cell：Gamma delta T CR magnetic bead antibody combines gamma delta T cells, crosses the cell of magnetic bead sorting post separation gamma delta T CR+), PBS pillar 3
Secondary, 1000r/min, it is centrifuged 5min, abandons supernatant, collect cell c, that is, obtain gamma delta T cells.
Further, the final concentration of 1 μ g/ml of PHA in preferably described 1640 culture mediums.
Further, in preferably described 1640 culture mediums IL-7 final concentration of 20ng/ml.
The present invention improves on the Research foundation of prior art further, using simple cultural method, selective amplification V
About 100 times of δ 1 gamma delta T cells subgroup quantity, ratio is expanded to 80%, and improve its kill solid tumor (inclusion tumor stem cell) and
The cytotoxic activity of hematological system tumor.This technology will promote the application in clinical tumor adoptive immunotherapy for V δ 1 gamma delta T cells.
Beneficial effects of the present invention are embodied in：(1) raw material of the present invention obtains conveniently, there is not ethics problem, possesses pole
Strong use value；(2) cell that the present invention is obtained has compared with High Fragmentation solid tumor cell, tumor stem cell and blood system
The cytotoxic activity of system tumour cell；(3) method of present invention acquisition cell is simple, efficiently, low cost；(4) cell of the present invention
It is suitable for the thin of solid tumor such as colorectal cancer and hematological system tumor such as chronic myelocytic leukemia, Pancytopenia
Born of the same parents' treatment, drug screening and the seed cell as organizational project.
(4) brief description
Fig. 1 is layered schematic diagram for density gradient centrifugation separating peripheral blood mononuclear cells；
Fig. 2 is the situation of V δ 1 gamma delta T cells amplification in vitro, and A is V δ 1 gamma delta T cells in fresh separated peripheral blood gamma delta T cells
The ratio of subgroup；B is V δ 1 gamma delta T cells proliferative conditions after amplification in vitro culture；C is V δ in gamma delta T cells after cultured and amplified in vitro
The ratio of 1 gamma delta T cells subgroup；D expands the ratio of V δ 1 gamma delta T cells subgroup for low dosage PHA+IL-7；E is high dose PHA+
IL-7 expands the ratio of V δ 1 gamma delta T cells subgroup；
Fig. 3 is that alone PHA cultivates the amplification of V δ 1 gamma delta T cells subgroup and the ratio of apoptosis；
Fig. 4 is that alone IL-7 cultivates the amplification of V δ 1 gamma delta T cells subgroup and the ratio of apoptosis；
Fig. 5 is V δ 1 gamma delta T cells expression activitiy before and after cultured and amplified in vitro；
Fig. 6 is V δ 1 gamma delta T cells toxicity correlation molecule expression before and after cultured and amplified in vitro, and Granzyme B represents particle
Enzyme, Perforin represents perforin, and Fasl represents Apoptosis associated ligands, and TRAIL represents Apoptosis associated ligands,
NKG2D represents NK surface receptor, and LFA-1 represents adhesion molecule, and NKp30 represents that NK surface is subject to
Body, NKp44 represents NK surface receptor, and NKp46 represents NK surface receptor；
Fig. 7 is different cultivating system amplification V δ 1 gamma delta T cells situation, and wherein A is that PHA+IL-7 and PHA+IL-2 cultivates body
System's amplification V δ 1 gamma delta T cells efficiency comparison (left side expands quantity statistics figure for cell, and right side is amplification times statistical chart)；B is
PHA+IL-7 and PHA+IL-2 cultivating system V δ 1 gamma delta T cells breeding ratio relatively (left side is streaming representative graph, and right side is statistical chart)；
C compares that (left side is streaming representative graph, and right side is statistics for PHA+IL-7 and PHA+IL-2 cultivating system V δ 1 gamma delta T cells apoptosis
Figure)；D is that PHA+IL-7 and PHA+IL-2 cultivating system V δ 1 gamma delta T cells kill to colorectal cancer tumour cell and tumor stem cell
Ability compares；
Fig. 8 is the killing ability to tumour cell for V δ 1 gamma delta T cells after cultured and amplified in vitro, and A is V after cultured and amplified in vitro
δ 1 gamma delta T cells direct killing colorectal cancer tumour cell and tumor stem cell ability；B is V δ 1 gamma delta T cells after cultured and amplified in vitro
Kill hematological system tumor cell ability；
Fig. 9 is the tumor suppression situation to human large intestine cancer transplantable tumor mouse for V δ 1 gamma delta T cells after cultured and amplified in vitro, and A is suppression
Tumor-bearing mice tumour growth, B is the life span extending tumor-bearing mice.
(5) specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
The pH value of PBS described in the embodiment of the present invention is 7.4, purchased from Hangzhou Ji Nuo biological medicine technology Co., Ltd.
Embodiment 1：PMNC gradient centrifugation separates
(1) by Ficoll lymphocyte separation medium (GE company of the U.S.) 10ml that proportion is 1.107 once add 50ml from
Heart bottom of the tube.
(2) take peripheral blood (examination ＆ approval of Yuan Nei Ethics Committee are passed through, and meet international convention, Sample is qualified), use PBS
(pH value is 7.4, Hangzhou Ji Nuo biological medicine technology Co., Ltd) is with volume ratio 1:1 dilution, obtains peripheral blood dilution.Will
30ml peripheral blood dilution is slowly added on Ficoll lymphocyte separation medium in step (1) 50ml centrifuge tube (GE company of the U.S.)
Layer, horizontal centrifuge 400g/min, 20 DEG C, be centrifuged 25min, reactant liquor be layered as successively upper serum, tunica albuginea confluent monolayer cells,
Ficoll separates liquid layer, GCL and bottom red blood cell layer；Tunica albuginea confluent monolayer cells (Fig. 1) by enrichment PMNC
Suction out and be transferred to new 50ml centrifuge tube, washed 2 times with 5 times of volumes PBS, 1000r/min, be centrifuged 5min, abandon supernatant, collect thin
Born of the same parents are precipitated, that is, obtain PMNC.
Embodiment 2：Peripheral blood total gamma delta T cells magnetic bead sorting
By in embodiment 1 through the detached PMNC of gradient centrifugation with 1 × 107Cell concentration adds 40 μ l magnetic beads
Sorting buffer solution (German Mei Tian Ni company) is resuspended, is subsequently adding the gamma delta T CR antibody (German Mei Tian Ni company) of 10 μ l, incubates for 4 DEG C
Educate 10min, add 30 μ l magnetic bead sorting buffer solutions and 20 μ l gamma delta T CR magnetic bead antibody (German Mei Tian Ni company), 4 DEG C of incubations
15min, centrifugation, precipitation is washed 1 time with PBS, centrifugation, abandons supernatant, collects cell precipitation.Add 500 μ in the cell precipitation obtaining
L PBS is resuspended, crosses MACS magnetic bead sorting post (German Mei Tian Ni company) and carries out positive selection separation gamma delta T cells, PBS post
Son 3 times, collects cell efflux (rinsing, on magnetic bead sorting post, the efflux getting off), by cell efflux 1000r/min, from
Heart 5min, abandons supernatant, collects cell precipitation, that is, obtains total gamma delta T cells.
Embodiment 3：PHA+IL-7 in vitro culture peripheral blood gamma delta T cells
Improve 1640 culture mediums：Volumetric concentration 10% hyclone, 50mg/mL mycillin (penicillin concn
100000IU/mL, streptomysin concentration is 10mg/mL), 1 μ g/ml PHA (phytolectin) and 20ng/ml IL-7 (leucocyte
Interleukin 7), solvent is 1640 culture mediums (Gibco company of the U.S.).
With improveing 1640 culture mediums as the obtained cell precipitation of the resuspended embodiment of nutrient solution 2, containing 5%CO237 DEG C of perseverances
Warm incubator carries out cell culture, changes fresh medium within every 2-3 days, continuous culture 14 days, obtains cell suspension.Result：Culture
After 14 days, V δ 1 gamma delta T cells subset proportions from<10% is expanded to 80% about (in Fig. 2 shown in A and C).
Comparison：Volumetric concentration 10% hyclone, 50mg/mL mycillin (penicillin concn 100000IU/mL, strepto-
Plain concentration is 10mg/mL), the obtained cell of the resuspended embodiment of 1640 culture mediums 2 of 0.1 μ g/ml PHA and 1ng/ml IL-7 sinks
Form sediment, containing 5%CO237 DEG C of constant incubators carry out cell culture, change fresh medium within every 2-3 days, continuous culture 14 days, obtain
Obtain cell suspension.Result：After culture 14 days, V δ 1 gamma delta T cells are effectively amplified, but amplification is slightly below above-mentioned improvement 1640 and cultivates
Base (in Fig. 2 shown in D).
Comparison：Volumetric concentration 10% hyclone, 50mg/mL mycillin (penicillin concn 100000IU/mL, strepto-
Plain concentration is 10mg/mL), the obtained cell of the resuspended embodiment of 1640 culture mediums 2 of 10 μ g/ml PHA and 100ng/ml IL-7 sinks
Form sediment, containing 5%CO237 DEG C of constant incubators carry out cell culture, change fresh medium within every 2-3 days, continuous culture 14 days, obtain
Obtain cell suspension.Result：After culture 14 days, V δ 1 gamma delta T cells are effectively amplified, but amplification efficiency is slightly below above-mentioned improvement 1640
Culture medium (in Fig. 2 shown in E).
Embodiment 4：PHA+IL-2 in vitro culture peripheral blood gamma delta T cells
The 20ng/ml IL-7 (interleukin-17) improveing in embodiment 3 in 1640 culture mediums is changed to 100IU/ml
IL-2 (interleukin 2), other with embodiment 3, containing 5%CO237 DEG C of constant incubators carry out cell culture, every 2-3
It changes fresh medium, continuous culture 14 days, obtains cell suspension.Result：After culture 14 days, V δ 1 gamma delta T cells subset proportions
From<10% is expanded to 40%-60% (Fig. 3).
Embodiment 5：PHA in vitro culture peripheral blood gamma delta T cells
By the IL-7 improveing in embodiment 3 in 1640 culture mediums remove, other with embodiment 3, containing 5%CO237 DEG C
Constant incubator carries out cell culture, changes fresh medium within every 2-3 days, continuous culture 14 days, obtains cell suspension.Result：PHA
Single factor test can also effectively expand V δ 1 gamma delta T cells, but V δ 1 gamma delta T cells apoptosis more (Fig. 3).
Embodiment 6：IL-7 in vitro culture peripheral blood gamma delta T cells
By the PHA improveing in embodiment 3 in 1640 culture mediums remove, other with embodiment 3, containing 5%CO237 DEG C of perseverances
Warm incubator carries out cell culture, changes fresh medium within every 2-3 days, continuous culture 14 days, obtains cell suspension.Result：IL-7
Single factor test can not substantially expand V δ 1 gamma delta T cells (Fig. 4).
Embodiment 7：Peripheral blood gamma delta T cells subset proportions detection before and after amplification
(prepared with improvement 1640 culture mediums in embodiment 3) after (prepared by embodiment 2) before expanding respectively, amplification and to obtain
Cell suspension 1000r/min, is centrifuged 5min, abandons supernatant, collects cell precipitation.By 1 × 105Cell is resuspended in 100 μ l and contains 2%
(v/v) the streaming dye solution (Biolegend company of the U.S.) of BSA (bovine serum albumin(BSA)), is separately added into the anti-human V δ of 5 μ l
1TCR.Antibody (GeneTax company of the U.S.) and 5 μ l anti-human V δ 2TCR antibody (Biolegend company of the U.S.), 4 DEG C of incubations
30min, adds 1ml PBS, with 1000r/min, is centrifuged 5min, then by precipitation PBS 2 times and cell precipitation is resuspended
In 500 μ l PBS, in the upper machine testing of FACSCantoII polychrome flow cytometer detection instrument (U.S. company BD).By detection data in
Cell subsets ratio before and after FlowJo is analysing amplified, as shown in A and C in Fig. 2, illustrates PHA+IL-7 selective amplification V δ 1 gamma delta T
Cell.
Embodiment 8：Peripheral blood gamma delta T cells propagation detection after amplification
Cell precipitation (preparation of embodiment 2 method) after embodiment 2 magnetic bead sorting is contained 2% (v/v) BSA's in 1ml
In 0.5mM CFSE (hydroxyl fluorescein diacetate succinimide fat) (Invitrogen company) dyeing liquor, 37 DEG C of incubations
20min, adds the PBS 9ml of 4 DEG C of precoolings to terminate dyeing, and is centrifuged 5min, precipitation PBS 2 times with 1000r/min, obtains
Cell after must dyeing.Cell after dyeing is placed in improvement 1640 culture mediums (composition is with embodiment 3), containing 5%CO2's
37 DEG C of constant incubators carry out cell culture, change fresh medium within every 2-3 days, continuous culture 14 days, obtain cell suspension.Finally
This cell suspension is centrifuged 5min with 1000r/min, abandons supernatant, by 1 × 105Cell is resuspended in 100 μ l and contains 2% (v/v) BSA
The streaming dye solution (Biolegend company of the U.S.) of (bovine serum albumin(BSA)), is separately added into 5 μ l anti-human V δ 1TCR.Antibody
(GeneTax company of the U.S.) and 5 μ l anti-human V δ 2TCR antibody (Biolegend company of the U.S.), 4 DEG C of incubation 30min, add 1ml
PBS, with 1000r/min, is centrifuged 5min, then by precipitation PBS 2 times, and will obtain cell (i.e. precipitation) and be resuspended in 500 μ l
PBS, goes up machine testing in FACSCanto II polychrome flow cytometer detection instrument (U.S. company BD) and analyzes TCRV δ 1+CFSE-Cell and
TCRVδ2+CFSE-Proliferative conditions (in Fig. 2 shown in B), illustrate V δ 1 gamma delta T cells propagation substantially, V δ 2 gamma delta T cells hardly increase
Grow.
Embodiment 9：Peripheral blood V δ 1 gamma delta T cells Activity determination before and after amplification
Detect with cell subsets in embodiment 7, (with improvement in embodiment 3 after (prepared by embodiment 2) before expanding, amplification
1640 culture mediums preparations) the cell suspension 1000r/min that obtains, it is centrifuged 5min, abandons supernatant, collect cell.Cell is resuspended in
100 μ l contain the streaming dye solution (Biolegend company of the U.S.) of 2% (v/v) BSA, are separately added into 5 μ l anti-human V δ 1TCR and resist
Body and the anti-human CD69 antibody of 5 μ l (Biolegend company of the U.S.), 4 DEG C of incubation 30min, add 1ml PBS, with 1000r/min,
Precipitation is resuspended in 500 μ l PBS, in FACSCanto II polychrome flow cytometer detection instrument after precipitation PBS 2 times by centrifugation 5min
Upper machine testing.By detection data in the analysing amplified cytoactive (Fig. 5) in front and back of FlowJo, after amplification in vitro is described, V δ 1 gamma delta T is thin
Cytoactive significantly improves.
Embodiment 10：The cell toxicant such as the peripheral blood V δ 1 gamma delta T cells emiocytosis factor and cell surface molecule before and after amplification
Property correlation molecule detection
Cell surface molecule detects with embodiment 9.With improveing the cell after 1640 culture mediums expand 14 days in embodiment 3
2 μ l lymphocyte activator agent (U.S. company BD) are added, 37 DEG C are incubated 6 hours, and 1000r/min is centrifuged in culture medium 200 μ l
5min, collects cell precipitation, to 1 × 105Add rupture of membranes perforation liquid (U.S. company BD) 100 μ l in cell, be incubated in 4 DEG C
25min, adds 1ml cleaning buffer solution (U.S. company BD), and 1000r/min is centrifuged 5min, and precipitation PBS will sink twice afterwards
Shallow lake is resuspended in 100 μ l streaming dye solution (Biolegend company of the U.S.), is separately added into the anti-human intracellular factor antibody of 5 μ l (beautiful
Biolegend company of state), 4 DEG C of incubation 30min, add 1ml PBS, with 1000r/min, be centrifuged 5min, precipitation PBS 2
After secondary, cell is resuspended in 500 μ l PBS, and in the upper machine testing of FACSCanto II polychrome flow cytometer detection instrument (U.S. company BD)
Cytotoxicity correlation molecule expression (Fig. 6)：Granzyme B (granzyme), Perforin (perforin), (cell withers Fasl
Die associated ligands), TRAIL (Apoptosis associated ligands), NKG2D (NK surface receptor), LFA-1 (adhesion point
Son), NKp30 (NK surface receptor), NKp44 (NK surface receptor), (NKT is thin for NKp46
Cellular surface acceptor).Result：After activation amplification, the expression of V δ 1 gamma delta T cells toxicity correlation molecule significantly improves.
Embodiment 11：PHA+IL-7 and PHA+IL-2 cultivating system expands peripheral blood V δ 1 gamma delta T cells Efficiency testing
With the detection of embodiment 7 cell subsets ratio, respectively to PHA+IL-2 in PHA+IL-7 in embodiment 3 and embodiment 4
Peripheral blood V δ 1 gamma delta T cells of cultivating system amplification, carry out cell absolute counting when cultivating 0,7,14,21 days, compare amplification
Cell absolute quantity and amplification times (A in Fig. 7) that former and later two cultivating systems expand to V δ 1 gamma delta T cells, illustrate PHA+IL-
The amplification efficiency of 7 pairs of V δ 1 gamma delta T cells is apparently higher than PHA+IL-2.
Embodiment 12：The propagation detection of PHA+IL-7 and PHA+IL-2 cultivating system peripheral blood V δ 1 gamma delta T cells
By the cell preliminary making CFSE (with embodiment 5) after embodiment 2 magnetic bead sorting, the cell after dyeing is respectively placed in
PHA+IL-7 (composition is with embodiment 3) and PHA+IL-2 (composition is with embodiment 4) cultivating system, continuous culture 14 days.Using reality
Apply example 8 methods described to carry out flow cytometer detection and analyze TCRV δ 1+CFSE-Cell proliferative conditions (B in Fig. 7), illustrate that PHA+IL-7 trains
In foster system, V δ 1 gamma delta T cells are bred apparently higher than PHA+IL-2 cultivating system.
Embodiment 13：PHA+IL-7 and PHA+IL-2 cultivating system peripheral blood V δ 1 gamma delta T cells apoptosis detects
Nutrient solution after embodiment 3 (PHA+IL-7) and embodiment 4 (PHA+IL-2) cultivating system expand 14 days
5 μ l propidium iodide (PI) (U.S. company BD) are added, incubated at room 15 minutes, in FACSCanto II polychrome streaming in 100 μ l
The upper machine testing TCRV δ 1 of detector (U.S. company BD)+PI+Cell (C in Fig. 7).V δ 1 gamma delta T in conclusion PHA+IL-7 cultivating system
Apoptosis is considerably less than PHA+IL-2 cultivating system.
Embodiment 14：PHA+IL-7 and PHA+IL-2 cultivating system peripheral blood V δ 1 gamma delta T cells are to colorectal cancer tumour cell
Kill ability detection with tumor stem cell
Cell suspension 1000r/min after embodiment 3 (PHA+IL-7) cultivating system is expanded, is centrifuged 5min, abandons supernatant,
Collect cell precipitation.By 1 × 105Cell is resuspended in the streaming dye solution (U.S. that 100 μ l contain 2% (v/v) BSA
Biolegend company), add 5 μ l anti-human V δ 1TCR antibody, 4 DEG C of incubation 30min, add 1ml PBS with 1000r/min, be centrifuged
Cell is resuspended in 2ml PBS after precipitation PBS 2 times by 5min, and flow sorter (U.S. company BD) sorts V δ 1 gamma delta T
Cell.By V δ 1 gamma delta T cells with volume ratio 10:1 effect target ratio, by 1 × 105V δ 1 gamma delta T cells and final concentration 1mM DDAO-SE
The 1 × 10 of (succinimide ester) pre-dyed4Colorectal cancer cells HT29 (purchased from U.S. ATCC) co-cultures 4 hours.Then to common training
5 μ l PI are added, incubated at room 15 minutes, on FACSCanto II polychrome flow cytometer detection instrument (U.S. company BD) in foster system
Machine testing DDAO-SE+PI+Cell situation (D in Fig. 7).
The pre- dyeing method of DDAO-SE is：1 μ l DDAO-SE adds 1ml to contain 1 × 104In the suspension of tumour cell, 37 DEG C of incubations
20 minutes, plus 10ml PBS, 1000r/min, it is centrifuged 5min, precipitation PBS twice, collects cell, and as DDAO-SE is pre-
The colorectal cancer cells HT29 of dye.
Respectively colorectal cancer cells HT29 is replaced with colorectal cancer cells HCT116 (U.S. ATCC), colorectal cancer cells Y (experiment
Human large intestine cancer primary cell line is set up in room) and large intestine cancer stem cell HT29S (laboratory foundation), large intestine cancer stem cell HCT116S
(laboratory foundation), large intestine cancer stem cell YS (laboratory foundation), in result as Fig. 7 shown in D.
Using the cell suspension after the amplification of embodiment 4 (PHA+IL-2) cultivating system as comparison under similarity condition.
Embodiment 15：Peripheral blood V δ 1 gamma delta T cells after amplification kill colorectal cancer cells, tumor stem cell and hematological system
Tumour cell ability detects
By the cell suspension 1000r/min with obtaining after improvement 1640 culture medium amplification in embodiment 3, it is centrifuged 5min, abandons
Supernatant, collects cell.By 1 × 105Cell is resuspended in the streaming dye solution (U.S. that 100 μ l contain 2% (v/v) BSA
Biolegend company), add 5 μ l anti-human V δ 1TCR antibody, 4 DEG C of incubation 30min, add 1ml PBS, with 1000r/min, from
Heart 5min, cell is resuspended in 2ml PBS with after PBS 2 times by cell (precipitate), and airflow classification V δ 1 gamma delta T cells are (with real
Apply example 14).By V δ 1 gamma delta T cells with volume ratio 10:1 effect target is trained altogether than the colorectal cancer cells HT29 with 1mM DDAO-SE pre-dyed
Support 4 hours (with embodiment 14).Then 5 μ l PI are added in co-culture system, incubated at room 15 minutes, in FACSCanto
The upper machine testing DDAO-SE of II polychrome flow cytometer detection instrument (U.S. company BD)+PI+Cell situation, acetonideexample 3 (PHA+IL-7)
V δ 1 gamma delta T cells after amplification can effectively kill colorectal cancer cells, large intestine cancer stem cell and chronic myelocytic leukemia and urgency
Property T lymphocytic leukemia cell.Colorectal cancer cells HT29 is replaced with colorectal cancer cells HCT116, colorectal cancer cells Y respectively
With large intestine cancer stem cell HT29S, large intestine cancer stem cell HCT116S, large intestine cancer stem cell YS (A in Fig. 8) and chronic myeloid
Leukaemia (K562) (U.S. ATCC) and Pancytopenia cell (Jurkat) (U.S. ATCC) are (in Fig. 8
B).
Embodiment 16：Tumor suppression ability detection in peripheral blood V δ 1 gamma delta T cells body after amplification
By Human Large Intestine Carcinoma Cells system HT29 with 5 × 104Cell concentration subcutaneous vaccination sets up lotus knurl model to NOD/SCID mouse.
The V δ 1 gamma delta T cells airflow classification after three days, embodiment 3 being expanded acquisition out (with embodiment 14), 1000r/min, centrifugation
5min, abandons supernatant, collects cell precipitation.With 100 μ l PBS re-suspended cells precipitations, (concentration is 1 × 107/ ml), mouse tail vein
Injection 1 × 106V δ 1 gamma delta T cells, 1 time/3 days, totally 5 times.Vernier caliper measurement tumour knurl footpath and tumor-bearing mice Survival.
When the V δ 1 gamma delta T cells treatment group of PHA+IL-7 amplification can suppress the growth (A in Fig. 9) of tumour and extend tumor-bearing mice existence
Between (B in Fig. 9).Under similarity condition, with tail vein injection 100 μ l PBS for comparison.
Summarize：
The raw material that the present invention uses：Peripheral blood easily obtains, there is not ethics problem；
The method that the present invention obtains cell is simple, efficient, low cost；
The cell being obtained has thin compared with High Fragmentation solid tumor cell, tumor stem cell and hematological system tumor cell
Born of the same parents' cytotoxic activity；
The present invention obtained cell wide expression cytotoxicity correlation molecule；
The obtained cell of the present invention is suitable for solid tumor such as colorectal cancer and hematological system tumor such as chronic myelocytic leukemia, urgency
The cell therapy of property T lymphocytic leukemia, drug screening and the seed cell as organizational project.
In sum, this method utilizes peripheral blood through the purification process of density gradient centrifugation and magnetic bead sorting, obtains relatively
The gamma delta T cells that high-purity, amplification ability are strong, cytoactive is high, in defined medium, cultured and amplified in vitro, after 14 days, obtains
Sub- compared with V δ 1 gamma delta T cells of the cytotoxic activity of High Fragmentation solid tumor cell, tumor stem cell and hematological system tumor cell
Group, presents wide application prospect in adoptive immunotherapy field, it will to the clinical practice of V δ 1 gamma delta T cells subgroup
Bring tremendous expansion.
Claims (4)
1. a kind of Activation In Vitro amplification method from peripheral blood V δ 1 gamma delta T cells is it is characterised in that described method is：Take
Periphery whole blood, the method using density gradient centrifugation and immunological magnetic bead sorting collects total gamma delta T cells；By total gamma delta T cells with changing
Good 1640 culture mediums carry out resuspended, in 5%CO as nutrient solution2, carry out cell culture under the conditions of 37 DEG C, renew within every 2-3 days fresh
Nutrient solution, obtains the gamma delta T cells based on V δ 1 gamma delta T subgroup of Activation In Vitro amplification；Described improvement 1640 culture medium composition
For：Volumetric concentration 10% hyclone, 50mg/mL mycillin, 0.1-10 μ g/ml PHA and 1-100ng/ml IL-7, solvent
For 1640 culture mediums.
2. the method for claim 1 is it is characterised in that the method for described density gradient centrifugation and immunological magnetic bead sorting has
Body is：
(1) take periphery whole blood, with PBS with volume ratio 1:1 dilution, obtains periphery whole blood dilution；
(2) the Ficoll lymphocyte separation medium that proportion is 1.107 is added in centrifuge tube, then by the peripheral blood of step (1)
Dilution is added slowly to Ficoll lymphocyte separation medium upper strata, horizontal centrifuge 400g/min, 20 DEG C, is centrifuged 25min, reaction
Liquid is layered as upper serum, tunica albuginea confluent monolayer cells, Ficoll separation liquid layer, GCL and bottom red blood cell layer successively；Described outer
All hemodilution liquid and Ficoll lymphocyte separation medium volume ratio 3:1；
(3) detached for step (2) tunica albuginea confluent monolayer cells are suctioned out, are transferred to new centrifuge tube, are washed 2 times with the PBS of 5 times of volumes,
1000r/min, is centrifuged 5min, abandons supernatant, collects cell a；
(4) with 1 × 107It is thin that the resuspended step (3) of amount magnetic bead sorting liquid a of individual cell/40 μ l magnetic bead sorting buffer solution a obtains
Born of the same parents a, is subsequently adding gamma delta T CR antibody, 4 DEG C of incubation 10min, adds magnetic bead sorting buffer solution b and gamma delta T CR magnetic bead antibody, 4 DEG C
Incubation 15min, centrifugation, precipitation is washed 1 time with PBS, centrifugation, abandons supernatant, collects cell b；Described magnetic bead sorting buffer solution b and magnetic bead
Sorting buffer solution a volume ratio is 3:4；Described gamma delta T CR antibody and magnetic bead sorting buffer solution a volume ratio are 1:4；Described gamma delta T CR
Magnetic bead antibody and magnetic bead sorting buffer solution a volume ratio are 1:2；
(5) add PBS resuspended in the cell b obtaining to step (4), MACS magnetic bead sorting post excessively, PBS pillar 3 times,
1000r/min, is centrifuged 5min, abandons supernatant, collects cell c, that is, obtain gamma delta T cells.
3. the method for claim 1 is it is characterised in that the final concentration of 1 μ g/ml of PHA in described improvement 1640 culture medium.
4. the method for claim 1 it is characterised in that in described improvement 1640 culture medium IL-7 final concentration of 20ng/
ml.
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| CN102994448A (en) * | 2012-12-13 | 2013-03-27 | 上海柯莱逊生物技术有限公司 | Method for in-vitro amplification of gamma-delta-T cells |
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| CN102994448A (en) * | 2012-12-13 | 2013-03-27 | 上海柯莱逊生物技术有限公司 | Method for in-vitro amplification of gamma-delta-T cells |
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