CN108795858A - The screening technique of high anti-cancer activity T cell and application - Google Patents
The screening technique of high anti-cancer activity T cell and application Download PDFInfo
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Abstract
The present invention provides a kind of screening techniques of high anti-cancer activity T cell, include the following steps：The human peripheral blood single nucleus cell of separation is provided；CD25 antibody is provided and is coated with magnetic bead；Detect the ratio of CD3 and CD25 double positive cells；Remove CD25 positive cells to screen using CD25 antibody coating magnetic bead, obtains targeted cell subsets.The present invention is coated with magnetic bead come negative T cell specific subgroup of the screening with high anti-cancer activity using CD25 antibody for the first time, it is simple and easy to operate, at low cost, convenient for obtaining, isolated growth situation is good, stimulates the high-quality cellular immunity product that amplification property is strong, tumor-killing ability is high and lasting again.The present invention also provides application of the screening technique in immune cell therapy.
Description
Technical field
The present invention relates to biotechnologies, and in particular to a kind of screening technique of high anti-cancer activity T cell and application.
Background technology
Immune cell therapy is a kind of emerging tumor treatment model, have become the whole world research hotspot, especially with
Tumor-infiltrating cells (TIL), CAR-T (Chimeric antigen receptor T cell)/TCR-T (mosaic type T cell) technology are main representative,
As global biomedical the main direction of development.Immune cell therapy is to use biotechnology and biological agent from patient's body
After isolating immune cells carry out in vitro culture, activation and amplification, patient's body is fed back to, direct killing or excitating organism are immune anti-
It should carry out killing tumor cell, reach treatment tumour purpose.
Existing cellular immunotherapy carries out culture amplification using unscreened peripheral blood mononuclear cells (PBMC),
The unscreened PBMC obtained in this way, it includes the required killer T cell of immune cell therapy to contain, and immune thin
The unwanted NK cells of born of the same parents' therapy, macrophage etc..Meanwhile killer T cell can be divided into infantilism, maincenter according to its phenotype again
Multiple phenotypes such as memory-type, effect memory-type, effect type, morphological function, amplification property, cytokine secretion ability etc. have very
Big difference.In other words, infantilism, maincenter memory T cells are just suitble to life in unscreened peripheral blood mononuclear cells
Immunocyte product (such cell can be described as " targeted cell subsets ") is produced, and is not added with being given birth to using mononuclearcell for screening
Production not only results in human and material resources cost increase, and useless cell subsets is it is also possible to influence the work(of targeted cell subsets
Can, decline so as to cause the quality of immunocyte product.
Therefore, it is necessary to provide, a kind of production cost is low, and energy high-efficiency sieve selects the side of the target T cell of high anti-cancer activity
Method.
Invention content
In view of this, the purpose of the present invention is to provide a kind of screening techniques of high anti-cancer activity T cell, it is intended to solve existing
Have in technology in immunocyte that useless cell subsets is more, targeted cell subsets are few and active low, ropy problem.
In a first aspect, the present invention provides a kind of screening technique of high anti-cancer activity T cell, include the following steps：
(1) it provides CD25 antibody and is coated with magnetic bead；The human peripheral blood single nucleus cell of separation is provided；
(2) ratio of CD3 and CD25 double positive cells is detected：
The human peripheral blood single nucleus cell isolated in step (1) is resuspended in 15 serum free mediums of X-VIVO, is obtained
To the first cell re-suspension liquid；Be added into the first cell re-suspension liquid the anti-human CD25 antibody of mouse of the first fluorochrome label with
The mouse anti-CD3antibody of second fluorochrome label is incubated 15-50min, using thin after being incubated described in flow cytomery
The percentage of CD3 and CD25 double positive cells in born of the same parents；
(3) CD25 antibody coating magnetic bead is used to screen CD25 feminine gender targeted cell subsets：
The human peripheral blood single nucleus cell that will be isolated in step (1) is resuspended using 15 serum free mediums of X-VIVO,
Obtain the second cell re-suspension liquid；According to the ratio of the CD3 and CD25 double positive cells, CD25 antibody coating magnetic bead is pressed
According to the number of double positive cells ratio be (2-5):1 ratio is added in the second cell re-suspension liquid, after mixing, is placed in thin
40-60min is cultivated in born of the same parents' incubator；
Gained culture mixed liquor is stood in the case where applying magnetic field, supernatant is sucked out, and remove magnetic field；To on gained
15 serum free mediums of X-VIVO are added in clear liquid to be resuspended, obtain the suspension of the targeted cell subsets of feminine gender containing CD25, that is, contain
The suspension of high anti-cancer activity T cell.
T cell phenotypic classification index general at present is CCR7, CD62L and CD45RA/RO, and there are no make CD25
For negative screening T cell phenotypic classification index.Present inventor has found expression during using cellular immunotherapy
The T cell (such as CD4+CD25+) of CD25+ can through antigen, be activated by the signal path of TCR after, inhibit immune response
Tolerance with body is maintained, can also inhibit the activation of other CD4+ or CD8+ normal T-cells, influence their high anti-cancer activity.
Therefore, it is necessary to get rid of this part CD25 positive T cells, this negative effect of part cell to high anti-cancer activity T cell is reduced.
In the application, according to CD3 in the human peripheral blood single nucleus cell re-suspension liquid of separation and the hundred of CD25 double positive cells
Divide ratio, is screened CD25 antibody coating magnetic bead is added, the T cell of the CD25 positives is all screened and got rid of, and
Retain the T cell of CD25 feminine genders as cell population of interest, the T cell signal of obtained CD25 negative T cells can be made in this way
Access will not be suppressed, T cell expand in vitro it is unaffected, can also when antitumor preferably play activation kinetic energy, to
Reach potent lasting antitumous effect.Can cultured in vitro be carried out to the cell population of interest that screening obtains again later.This method
The interference of untargeted cells can be greatly reduced, reduce production cost.
In the step of the application (3), after gained culture mixed liquor is stood in the case where applying magnetic field, gained lower layer
The CD25 positive cells for needing to abandon are precipitated as, contain required CD25 feminine genders target cell in gained supernatant.
In the application, about it may separate out (0.5-2) × 10 in volunteer's blood sample per 100mL8A PBMC cells.It is preferred that
Ground, a concentration of (0.5-3) × 10 of the first cell re-suspension liquid7A/100 μ L.
Preferably, the transmitting light of first fluorescent dye and the second fluorescent dye is misaligned, to distinguish.
It is further preferred that first fluorescent dye is allophycocyanin (APC), phycoerythrin (PE：
Phycoerythrin) or phycoerythrin-cyanine type dye 7 (PE-Cy7), glow.
Second fluorescent dye is fluorescein isothiocynate (FITC), green light.
Preferably, cell proportion >=50% of the CD3 positives is presented in the human peripheral blood single nucleus cell of the separation.
Further, if the cell proportion that the CD3 positives are presented in the human peripheral blood single nucleus cell of separation is less than 50% (example
As 30%), this explanation is containing less T cell (in general, almost all of T cell is all the CD3 positives), then to described
In the human peripheral blood single nucleus cell of separation, CD3 immunomagnetic beads are added and are enriched with to obtain the T cell of the CD3 positives, are walked again later
Suddenly (2) and (3).
Preferably, in step (3), the culture is in 37 DEG C, 5%CO2Under conditions of carry out.
Preferably, the CD25 antibody coating magnetic bead is made using following methods：
A, pH is used to be washed, be resuspended for the first buffer solution of 6.0-10.0 load magnetic bead, to bearing after resuspension
It carries in magnetic bead and anti-human CD25 antibody is added, be protected from light at 18-37 DEG C and be incubated 16-24h；It is protected from light in Incubating Solution to gained and closing is added
Agent is incubated 10-30min again later to close the site for being not associated with antibody on magnetic bead, obtains the coating magnetic bead of antibody containing CD25
Reaction solution；
B, the reaction solution is stood, discards supernatant liquid, the second buffer solution is used to gained CD25 antibody coating magnetic bead
It is cleaned for several times, and is resuspended in second buffer solution, obtain CD25 antibody coating magnetic bead after purification, wherein described
Second buffer solution is the phosphate of the pH=7.0-8.0 containing bovine serum albumin(BSA) (BSA) and EDTA (ethylenediamine tetra-acetic acid)
Buffer solution.
It is understood that in step (a), the washing to the load magnetic bead can be that load magnetic bead, first will be housed
The container (such as test tube, conical flask etc.) of buffer solution is placed in the environment in magnetic field (on such as magnetic frame or magnet) and stands, and abandons
Supernatant is removed, the magnetic bead after being washed.It is that the container equipped with magnetic bead after washing is left into magnetic frame or magnet provides when resuspension
The magnetic field environment, then be added be resuspended solution.To antibody containing CD25 be coated with magnetic bead reaction solution washing, be resuspended with etc
Seemingly.The solution of the reaction solution is placed in magnetic field environment first, discards supernatant liquid, obtains the coating magnetic bead of antibody containing CD25；So
Recession demagnetizing field environment, is added the second buffer solution mixing, realizes the cleaning of coating magnetic bead.
Preferably, when using second buffer solution cleaning, after the second buffer solution is added, first it is incubated at room temperature 3-
5min。
In the application, the load magnetic bead is the common magnetic bead of not connected antibody.Preferably, the load magnetic bead includes grain
The common magnetic bead that diameter is 1nm-500 μm.It it is further preferably 100nm-300 μm at grain size.More preferably 5-100 μm.
In an embodiment of the present invention, the load magnetic bead isM-450Epoxy can support type magnetic
Pearl, grain size are 2-10 μm.Can also be other common magnetic beads, however it is not limited toM-450Epoxy magnetic beads.
M-450Epoxy load magnetic beads are the hydrophily magnetic bead that pH is in neutrality, and are non-porous monodisperse, ultra paramagnetic microballon, table
Face is modified with glycidol ether, which has very strong locomitivity in the solution, makes the antibody for being coupled to magnetic bead can be with cell
Suspension lasts interact.When being placed in magnetic field, microballon can lightly draw target cell to tube wall., can be light
The supernatant comprising non-target cell is rapidly poured out or siphoned away to pine.
In an embodiment of the present invention, first buffer solution is the phosphate buffer of pH=7.0-8.0.Into one
Step is preferably the sodium phosphate buffer of pH=7.4-8.0.
In another embodiment of the present invention, first buffer solution is the sodium borate buffer liquid of pH=7.6-9.5.
The incubation is carried out under the conditions of shaking, such as can be carried out in shaking table or gyroscope, to increase each raw material
Mixing, the extent of reaction.
Preferably, in step (a), the additional proportion of the CD25 antibody and the load magnetic bead is (150-300 μ g):(4
×107It is a).
Preferably, in step (a), the sealer includes bovine serum albumin(BSA) (BSA), glycine, lysine and smart ammonia
It is one or more in acid.
It is further preferred that the sealer is BSA.
Preferably, the sealer is in a concentration of 0.01-0.1% (w/v) being protected from light in Incubating Solution.
In the application, second buffer solution is mainly that (antibody containing CD25 is coated with magnetic to the reaction solution after the completion of incubation
Pearl, free magnetic bead) it cleaned, purified.Wherein, the effect of BSA is the site on further closing load magnetic bead；EDTA is main
It is the most of transition metal element ion (such as iron (III), nickel (II), manganese (II) etc.) excluded in solution, in order to avoid influence magnetic bead
And the combination of magnet.
Preferably, in second buffer solution, a concentration of 0.5-5mM of EDTA；A concentration of 0.02-0.2% of BSA
(w/v).For example, in the second buffer solution, the concentration of EDTA can be for 0.5,1,1.5,2,2.5,3,4 or 4.5mM；BSA's
Mass-volume concentration can be 0.02%, 0.04%, 0.06%, 0.1%, 0.15% or 0.2% (w/v).
It is further preferred that in second buffer solution, a concentration of 2mM of EDTA；A concentration of 0.1% (the w/ of BSA
v)。
Preferably, the human peripheral blood single nucleus cell (PBMC) is that separation obtains with the following method：
Peripheral blood mononuclear cells is acquired from the blood samples of patients of extraction, takes middle layer thin after density gradient centrifugation detaches
Born of the same parents are washed using physiological saline or phosphate buffer solution (PBS), obtain PBMC.
Specifically, it acquires blood samples of patients and dilutes；The centrifuge tube equipped with cell density gradient separations liquid is tilted, and along tube wall
The blood sample of patient being slowly added to after dilution, makes blood sample be covered on cell density gradient separations liquid；The centrifuge tube for filling sample is existed
Centrifugation lithium is to centrifuge 15-30min under 200-500g；
Centrifuge tube is taken out later, sucks top layer's blood plasma, middle layer is peripheral blood mononuclear cells layer, and is transferred them to
It is preserved in new centrifuge tube.
The screening technique for the high anti-cancer activity T cell that first aspect present invention provides, is mainly coated with using CD25 antibody
Magnetic bead gets rid of the contaminating cell of the CD25 positives, obtains the cell population of interest with high anti-cancer activity.On the one hand, by screening
The slow virus for avoiding culture medium, serum, genetic modification that the when of carrying out cultured in vitro together with impurity T cell consumed carries
Body etc. reduces the cost for preparing cellular immunity product；On the other hand, the work(of normal T-cell can be inhibited by expressing the cell of CD25
Energy property and amplification property block non-specific inhibition activation signals access, cause T cell to expand difficulty in vitro, while under functionality
Drop.And after carrying out screening separation, CD25 negative cells can more muchly maintain its phenotype, so as in longer time hair
Wave efficient antitumor efficiency；In addition, it is thus also avoided that the cell factor (such as interleukin-22,7,15) needed for normal T-cell growth
It is sponged by CD25 positive cells and T cell is interfered to expand in vitro indirectly.
The screening technique is simple and easy to operate, at low cost, convenient for obtain isolated growth situation is good, stimulate again amplification property it is strong,
Tumor-killing ability height and lasting high-quality cellular immunity product；Used magnetic bead easy can remove, to T lymphocytes
Damage it is small, do not influence subsequently to prepare cellular immunity product；It can carry out large-scale production.
Second aspect, the present invention provide the high anti-cancer activity T cell that a kind of screening technique as described in relation to the first aspect obtains.
Than unscreened cell there is larger cells ex vivo to expand by high anti-cancer activity T cell obtained by the above method
The long-term effectiveness etc. after number, stronger anti-tumor activity, infusion is doubled, therefore can be used for preparing the production of high-quality cellular immunity
Product can have humidification with direct feedback to human body to patient's body immune system.
The third aspect, the present invention provide the high anticancer described in a kind of screening technique or second aspect as described in relation to the first aspect
Application of the active t cell in immune cell therapy.
The application's has the beneficial effect that：
1, for the first time by CD25 antibody coating magnetic bead for screening removal CD25 positive T cells, the mesh of high anti-cancer activity is obtained
T cell is marked, the screening technique is simple and easy to operate, at low cost, and screening efficiency is high；Used magnetic bead easy can remove, to T
The damage of lymphocyte is small, does not influence subsequently to prepare cellular immunity product；It can carry out large-scale production；
2, for than unscreened PMBC cells or the T cell screened through other CCR7, CD62L immunomagnetic beads,
In the CD25 negative T cells that the present invention screens, contaminating cell is few, and cells ex vivo growing state is good, stimulates amplification property strong again,
Tumor-killing ability with persistent high efficiency after feedback human body.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described.Specific embodiment described herein is only used to explain this
Invention, is not intended to limit the present invention.
Fig. 1 is the ratio variation for accounting for total number of cells in the present invention through CD3+CD25+ in the front and back cell of CD25 magnetic beads screening；
Fig. 2 is the situation of change that the present invention screens front and back regulatory T cells ratio through CD25 magnetic beads；
Fig. 3 is the cell Proliferation times of T cell and unscreened PBMC cells that screening technique provided by the invention obtains
Number variation；
Fig. 4 is the T cell obtained through screening technique provided by the invention and unscreened PBMC cells in mouse tumor
To suffering from the treatment results of mice with tumor in model.
Specific implementation mode
As described below is the optional embodiment of the present invention, it is noted that for those skilled in the art
For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as
Protection scope of the present invention.
It is as follows for the reagent used in the embodiment of the present invention below：
[1]M-450Epoxy can support type magnetic bead, match Mo Feishier companies, article No.：14011；
[2] the anti-human CD25 antibody of high-purity, Biolegend companies, article No.：302802；
[3] Ficoll-Paque PLUS lymphocyte separation mediums, GE companies, article No.：17-1440-02；
[4] 15 serum-free haemocyte special culture medias of X-VIVO, Lonza companies, article No.：04-744Q；
[5] phosphate buffer (PBS), Gibco company, article No.：10010023；
[6] the anti-human CD25 of mouse of APC anti-human CD25Antibody, allophycocyanin (APC) fluorescent marker are anti-
Body, Biolegend companies, article No.：302610；
[7] FITC anti-human CD3Antibody, FITC fluorescent marker mouse anti-CD3antibody, Biolegend are public
Department, article No.：300406；
[8]CellTraceTMCFSE Cell Proliferation Kit, Fluoresceincarboxylic acid acetoacetate succinyl are sub-
Amine ester (CFSE), Invitrogen companies, article No.：C34554.
Embodiment 1
A kind of screening technique of high anti-cancer activity T cell, includes the following steps：
(1) preparation of CD25 antibody coating magnetic bead：
1.1 take the grain size of 1mL for 4.5 μmM-450Epoxy can support type magnetic bead (hereinafter referred to as " magnetic
Pearl ") (4,000 ten thousand/mL), it places it in and stands 1min on magnetic frame, discard supernatant, and test tube is taken out from magnetic frame；
The first buffer solution (sodium phosphate buffer of pH=8.0) repeated washing of 1.2 addition 1mL 2 times；
1.3 are resuspended in magnetic bead in the first buffer solution, and the CD25 antibody of 200 μ g is added, and control total volume is 1mL, is filled
Divide mixing；
Gained mixed solution is placed at 20 DEG C on shaking table or gyroscope and gently shakes by 1.4, is protected from light incubation 20 hours；
Then 1.5 are added bovine serum albumin(BSA) (BSA), control it and be protected from light a concentration of 0.02% in Incubating Solution in gained
(w/v), it is incubated 15min at 20 DEG C；
1,6 waits after the completion of being incubated, and the test tube for filling sample is placed on magnetic frame and stands 1min, is discarded supernatant, and by test tube from
It is taken out on magnetic frame；
1.7 prepare the second buffer solution, are the phosphate-buffered containing 0.1%BSA (w/v) and 2mM EDTA, pH=7.4
Liquid (PBS), wherein；Appropriate second buffer solution is added into above-mentioned test tube, mixing is gently shaken, and 5min is incubated at room temperature, with reality
Now to the cleaning of magnetic bead；
The step of the 1.8 above-mentioned 1.6-1.7 of repetition 2-3 times；
1.9 take out test tube from magnetic frame, are resuspended in the second buffer solution of 1mL, complete CD25 antibody and are coated with magnetic
It is prepared by pearl.
(2) separation of PBMC cells (human peripheral blood single nucleus cell)
2.1 obtain blood samples of patients (or provide unit by blood such as hospital or blood stations and complete) by the methods of singly adopting；It will
Single blood sampling of acquisition is 1 according to volume ratio:1 ratio and PBS mixings, tighten pipe lid, light and slow reverse mixing 6 times is positioned over
Pipe support, the blood sample diluted；
2.2 tilt the centrifuge tube equipped with cell density gradient separations liquid (Ficoll-paque PREMIUM), slow along tube wall
Slowly the above-mentioned blood sample diluted is added, blood sample is covered in separating liquid, pays attention to that separation liquid layer is avoided to mix with blood sample layer；
2.3 are put into the pipe for filling sample in centrifuge, and centrifugal force 400g is arranged, and 18 DEG C, centrifugation time 30min of temperature is closed
Brake function starts to centrifuge；
2.4 take out centrifuge tube, suck top layer's blood plasma, carefully retain intermediate mononuclearcell (PBMC) layer, will be intermediate
Mononuclearcell layer be transferred to new centrifuge tube, notice that each 50ml centrifuge tubes are at most transferred to 10ml.
(3) ratio of Flow cytometry CD3 and CD25 double positive cells is used
3.1 take 1,000,000, PBMC cells after above-mentioned separation, are resuspended with 15 serum free mediums of X-VIVO of 100 μ L；
3.2 are added the mouse anti-CD3antibody of the mouse anti-human CD25 antibody and FITC fluorescent markers of APC fluorescent markers, at 4 DEG C
Lower incubation 30min；
Cell after 3.3 pairs of incubations uses flow cytomery, obtains wherein CD3 and is accounted for always carefully with CD25 double positive cells
The percentage of born of the same parents' number.
(4) CD25 antibody coating magnetic bead is used to screen CD25 feminine gender targeted cell subsets
4.1 take the mononuclearcell 100,000,000 after above-mentioned separation, with the 15 special trainings of serum-free haemocyte of X-VIVO of 20mL
Foster base is resuspended, and pours into 50mL test tubes；
4.2 take the CD25 obtained in step 1.9 to be coated with magnetic bead, according to the bis- positive cells of the CD3+CD25+ measured in 3.3 steps
Ratio, according to coating magnetic bead and double positive cells number ratio be 3:1 ratio addition CD25 is coated with magnetic bead, soft mixing；
Test tube is put into 37 DEG C, the cell incubator of 5% carbon dioxide, stationary culture 55-60 minutes by 4.3；
After 4.4 cultures, the test tube for filling sample is put on magnetic separation frame, stands 1 minute；
4.5 are sucked out supernatant liquid in test tube, and are transferred in new test tube, this is CD25 feminine gender cell population of interest, simultaneously
The CD25 positive cells for discarding test tube on magnetic frame and being adsorbed on test tube；
The 15 serum-free haemocyte special culture medias of X-VIVO of 4.6 addition 10mL are thin above-mentioned CD25 feminine genders target is resuspended
Born of the same parents group, the suspension of the targeted cell subsets of feminine gender containing CD25 after being screened, that is, the suspension of the T cell containing high anti-cancer activity.
Embodiment 2
A kind of screening technique of high anti-cancer activity T cell, includes the following steps：
(1) preparation of CD25 antibody coating magnetic bead：
1.1 take the grain size of 1mL for 10 μmM-450Epoxy can support type magnetic bead (hereinafter referred to as " magnetic
Pearl ") (4,000 ten thousand/mL), it places it in and stands 1min on magnetic frame, discard supernatant, and test tube is taken out from magnetic frame；
The first buffer solution (sodium borate buffer liquid of pH=8.4) repeated washing of 1.2 addition 1mL 3 times；
1.3 are resuspended in magnetic bead in the first buffer solution, and the CD25 antibody of 300 μ g is added, and control total volume is 1mL, is filled
Divide mixing；
Gained mixed solution is placed at 18 DEG C on shaking table or gyroscope and gently shakes by 1.4, is protected from light incubation 24 hours；
Then 1.5 are added bovine serum albumin(BSA) (BSA), control it and be protected from light a concentration of 0.02% in Incubating Solution in gained
(w/v), it is incubated 30min at 25 DEG C；
1,6 waits after the completion of being incubated, and the test tube for filling sample is placed on magnetic frame and stands 1min, is discarded supernatant, and by test tube from
It is taken out on magnetic frame；
1.7 prepare the second buffer solution, are the phosphate containing 0.02%BSA (w/v) and 0.5mM EDTA, pH=7.0
Buffer solution (PBS), wherein；Appropriate second buffer solution is added into above-mentioned test tube, mixing is gently shaken, and 5min is incubated at room temperature,
To realize the cleaning to magnetic bead；
The step of the 1.8 above-mentioned 1.6-1.7 of repetition 2-3 times；
1.9 take out test tube from magnetic frame, are resuspended in the second buffer solution of 1mL, complete CD25 antibody and are coated with magnetic
It is prepared by pearl.
(2) separation of PBMC cells (human peripheral blood single nucleus cell)：Operating procedure is the same as embodiment 1.
(3) ratio of Flow cytometry CD3 and CD25 double positive cells is used
3.1 take the mononuclearcell 1,000,000 after above-mentioned separation, with the 15 free serum culture base weights of X-VIVO of 100 μ L
It is outstanding；
3.2 are added the mouse anti-CD3antibody of the mouse anti-human CD25 antibody and FITC fluorescent markers of PE-Cy7 fluorescent markers,
It is incubated 30min at 4 DEG C；
Cell after 3.3 pairs of incubations uses flow cytomery, obtains wherein CD3 and is accounted for always carefully with CD25 double positive cells
The percentage of born of the same parents' number.
(4) CD25 antibody coating magnetic bead is used to screen CD25 feminine gender targeted cell subsets
4.1 take the mononuclearcell 0.5 hundred million after above-mentioned separation, special with the 15 serum-free haemocytes of X-VIVO of 20mL
Culture medium is resuspended, and pours into 50mL test tubes；
4.2 take the CD25 obtained in step 1.9 to be coated with magnetic bead, according to the bis- positive cells of the CD3+CD25+ measured in 3.3 steps
Ratio, be 4 according to coating magnetic bead and double positive number ratios:1 ratio addition CD25 is coated with magnetic bead, soft mixing；
Test tube is put into 37 DEG C, the cell incubator of 5% carbon dioxide, stationary culture 40 minutes by 4.3；
After 4.4 cultures, the test tube for filling sample is put on magnetic separation frame, stands 1 minute；
4.5 are sucked out supernatant liquid in test tube, and are transferred in new test tube, this is CD25 feminine gender cell population of interest, simultaneously
The CD25 positive cells for discarding test tube on magnetic frame and being adsorbed on test tube；
4.6 are added the 15 serum-free haemocyte special culture medias of X-VIVO of 20mL, thin above-mentioned CD25 feminine genders target is resuspended
Born of the same parents group, the suspension of the targeted cell subsets of feminine gender containing CD25 after being screened, that is, the suspension of the T cell containing high anti-cancer activity.
Embodiment 3
A kind of preparation method of CD25 antibody coating magnetic bead, includes the following steps：
1.1 take the grain size of 1mL for 50-100 μmM-450Epoxy can support type magnetic bead (hereinafter referred to as
" magnetic bead ") (4,000 ten thousand/mL), it places it in and stands 1min on magnetic frame, discard supernatant, and test tube is taken out from magnetic frame；
The first buffer solution (the Tris- hydrochloride buffers of pH=9.0) repeated washing of 1.2 addition 1mL 3 times；
1.3 are resuspended in magnetic bead in the first buffer solution, and the CD25 antibody of 150 μ g is added, and control total volume is 1mL, is filled
Divide mixing；
Gained mixed solution is placed at 35 DEG C on shaking table or gyroscope and gently shakes by 1.4, is protected from light incubation 16 hours；
Then 1.5 are added bovine serum albumin(BSA) (BSA), control it and be protected from light a concentration of 0.02% in Incubating Solution in gained
(w/v), it is incubated 10min at 35 DEG C；
1,6 waits after the completion of being incubated, and the test tube for filling sample is placed on magnetic frame and stands 1min, is discarded supernatant, and by test tube from
It is taken out on magnetic frame；
1.7 prepare the second buffer solution, are the phosphate-buffered containing 0.2%BSA (w/v) and 5mM EDTA, pH=8.0
Liquid (PBS), wherein；Appropriate second buffer solution is added into above-mentioned test tube, mixing is gently shaken, and 6min is incubated at room temperature, with reality
Now to the cleaning of magnetic bead；
The step of the 1.8 above-mentioned 1.6-1.7 of repetition 2-3 times；
1.9 take out test tube from magnetic frame, are resuspended in the second buffer solution of 1mL, complete CD25 antibody and are coated with magnetic
It is prepared by pearl.
In order to assess the screening efficiency of T cell after above-mentioned CD25 the moon described in the invention selects, the blood of people source is selected
Peripheral blood mononuclear cells separation, the through the invention screening technique are carried out, after carrying out the screening of CD25 magnetic bead feminine genders, to gained
CD25 negative T-lymphocytes, detect the positive rate of CD3, CD25 using flow cytometer, while being arranged without CD25 antibody
It is coated with the control group of magnetic bead screening, result is as shown in Figure 1, 2.
1, after the choosing of assessment CD25 the moon, the case where CD3+CD25+ positive cell groups：
By taking the peripheral blood of people, the CD25 magnetic beads prepared using the method for the present invention, after being screened by CD25 feminine genders
Cell (suspension of the targeted cell subsets of feminine gender containing CD25 i.e. in step 4.6), detected using flow cytomery method
CD3+CD25+ positive cells are in total number of cells the case where accounting, while with the PMBC without the coating magnetic bead screening of CD25 antibody
Cell compares, and the results are shown in Figure 1.
From figure 1 it appears that CD25+ and CD3+CD25+ cells are in the total cell number after the screening of CD25 magnetic beads
Ratio is substantially reduced, this illustrate the screening technique used in the present invention effectively remove CD25+ cells (n=9, n be acquisition not
With the PBMC cells of people source).
2, before and after the choosing of assessment CD25 antibody magnetic bead the moon, ratio situation of the regulatory T cells in total cell：
By taking the peripheral blood of people, the CD25 magnetic beads prepared using the method for the present invention are made after being screened by CD25 feminine genders
Regulatory T cells are detected in PBMC in total number of cells the case where accounting, as a result such as Fig. 2 institutes with flow cytomery method
Show.
Fig. 2 is the situation of change that the present invention screens front and back regulatory T cells ratio through CD25 magnetic beads.(a) abscissa generation in
Table CD25 antibody, ordinate FOXp3, (b) in abscissa represent Helios, ordinate FOXp3.Wherein, regulatory T cells
(Regulatory cells, abbreviation Tregs) is the T cell subgroup of autoimmune response in a kind of control volume, and Foxp3 is control
One of the key transcription factor of regulatory T cells (Treg cells) development and function processed, it is now recognized that naturally-produced FOXp3+
CD25+ positive cells are the characteristic phenotype of nature regulatory T cells；Transcription factor Helios is Ikaros transcription factor families
A member, the Treg cells that Helios selective expressions originate from thymus gland, it is now recognized that FoxP3+Helios+ positive cells be lure
The adaptability regulatory T-cell of artificial delivery life.
(a) is nature regulatory T cells Treg (FOXp3+CD25+ double positive cells) in Fig. 2, is screened in CD25 feminine genders
Afterwards, positive cell is significantly lowered, and the mono- positive cells of CD25 also substantially reduce, but does not influence Foxp3 positive cells in total cell
In accounting quantity；(b) it is to induce the adaptability regulatory T-cell (Helios+Foxp3+) generated, is screened in CD25 feminine genders
Afterwards, double positive cells are significantly lowered, and do not influence the mono- positive cells of Foxp3 and the mono- positive cells of Helios accounting in total cell
Compare quantity.From figure 2 it can be seen that after being screened through CD25 antibody magnetic bead feminine genders, either nature regulatory T cells (FOXp3
+ CD25+ double positive cells, right upper quadrant in the figure of left and right in a), or (FoxP3+Helios+ is bis- for regulatory T cells after induction
Positive cell, right upper quadrant in the figure of left and right in b), their ratios in total cell number are substantially reduced, and illustrate to use in the present invention
Screening technique effectively remove regulatory T cells (Treg cells), promote body autoimmune response.
3, after the choosing of assessment CD25 the moon, the proliferative conditions of cell：
The peripheral blood for taking people, the CD25 magnetic beads prepared using the method for the present invention, after being screened by CD25 feminine genders, in vitro
T cell is cultivated, uses 1:Target cell (the associated tumor cells of 1 quantity：Such as Nalm-6, Raji cell), it is stimulated, respectively
At the 0th, 3,5,7,9 day of culture, detection cell expanded number, the results show that the T cell after the screening of CD25 feminine genders has more preferably
Proliferative capacity, as shown in Figure 3.
It will be outside the target T lymphocytes that obtained by CD25 magnetic bead positive-selectings and the people screened without CD25 immunomagnetic beads
All blood mononuclear cells (PBMC), are cultivated, in vitro by 106The concentration of/mL is inoculated with, and uses 1:The target cell of 1 quantity
(associated tumor cells：Such as Nalm-6, Raji cell) T cell is stimulated, at 37 DEG C, 5%CO2Lower culture, and respectively
At the 0th, 3,5,7,9 day of culture, cells expanded is detected, the results are shown in Figure 3.
From figure 3, it can be seen that from after incubation the 5th day, the target T cell that is obtained after CD25 feminine genders of the present invention screening
With better proliferative capacity.
4, T cell after the choosing of assessment CD25 the moon, to the killing situation of mouse interior tumor：
Nalm6 mouse tumor models are built first, and the immunodeficient mouse for choosing 27 6-10 week old carries out tail vein note
Penetrate 1 × 106A Nalm6 tumour cells obtain Nalm6 mouse tumor models, and these mouse for suffering from tumour are randomly divided into three
Group, every group 9, screening group is to pass through tail vein injection 2 × 106A negative T-lymphocytes screened by CD25 magnetic beads,
It is tail vein injection 2 × 10 not screen group6A unscreened PBMC cells, negative control group are not injecting immune T cell
Mouse group observes mouse tumor situation using mouse 3-D imaging system, and records the Survival of mouse, as a result such as attached drawing 4
It is shown.
X-axis represents feeding time (day) in Fig. 4, and y-axis represents mouse survival rate (%), and curve, which moves down representative, has mouse dead
It dies.It can be seen that compared to the PBMC cells screened without CD25 magnetic beads by the survivorship curve of Fig. 4, screened by this method
Obtained immune t-cell, can more protect mice against leads to death because of tumour, especially after injection 30 days.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
Cannot the limitation to the scope of the claims of the present invention therefore be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. a kind of screening technique of high anti-cancer activity T cell, which is characterized in that include the following steps：
(1) it provides CD25 antibody and is coated with magnetic bead；The human peripheral blood single nucleus cell of separation is provided；
(2) ratio of CD3 and CD25 double positive cells is detected：
The human peripheral blood single nucleus cell isolated in step (1) is resuspended in 15 serum free mediums of X-VIVO, obtains
One cell re-suspension liquid；The anti-human CD25 antibody of mouse and second of the first fluorochrome label is added into the first cell re-suspension liquid
The mouse anti-CD3antibody of fluorochrome label is incubated 15-50min, using in the cell after being incubated described in flow cytomery
The percentage of CD3 and CD25 double positive cells；
(3) CD25 antibody coating magnetic bead is used to screen CD25 feminine gender targeted cell subsets：
The human peripheral blood single nucleus cell that will be isolated in step (1) is resuspended using 15 serum free mediums of X-VIVO, is obtained
Second cell re-suspension liquid；According to the ratio of the CD3 and CD25 double positive cells, by CD25 antibody coating magnetic bead according to
Double positive cells number ratio is (2-5):1 ratio is added in the second cell re-suspension liquid, after mixing, is placed in cell culture
40-60min is cultivated in case；
Gained culture mixed liquor is stood in the case where applying magnetic field, supernatant is sucked out, and remove magnetic field；To gained supernatant
Middle 15 serum free mediums of addition X-VIVO are resuspended, and obtain the suspension of the targeted cell subsets of feminine gender containing CD25, that is, contain highly resistance
The suspension of cancer active t cell.
2. screening technique as described in claim 1, which is characterized in that in step (1), the human peripheral blood single nucleus cell of separation
Cell proportion >=50% of the middle CD3 positives.
3. screening technique as described in claim 1, which is characterized in that if CD3 sun in the human peripheral blood single nucleus cell of separation
Property cell proportion be less than 50%, then：
Into the human peripheral blood single nucleus cell of the separation, CD3 immunomagnetic beads are added and are enriched with to obtain the T cell of the CD3 positives, it
Carry out step (2) and (3) again afterwards.
4. screening technique as described in claim 1, which is characterized in that the CD25 antibody coating magnetic bead is to use following methods
It is made：
A, pH is used to be washed, be resuspended for the first buffer solution of 6.0-10.0 load magnetic bead, to the load magnetic after resuspension
Anti-human CD25 antibody is added in pearl, is protected from light at 18-37 DEG C and is incubated 16-24h；It is protected from light in Incubating Solution to gained and sealer is added,
To close the site for being not associated with antibody on magnetic bead, it is incubated 10-30min again later, obtains the anti-of the coating magnetic bead of antibody containing CD25
Answer liquid；
B, the reaction solution is stood, discards supernatant liquid, gained CD25 antibody coating magnetic bead is carried out using the second buffer solution
Cleaning for several times, and is resuspended in second buffer solution, obtains CD25 antibody coating magnetic bead after purification, wherein described second
Buffer solution is the phosphate buffer of the pH=7.0-8.0 containing bovine serum albumin(BSA) and EDTA.
5. screening technique as claimed in claim 4, which is characterized in that in step (a), the CD25 antibody and the load magnetic
The additional proportion of pearl is (150-300 μ g):(4×107It is a).
6. screening technique as claimed in claim 4, which is characterized in that in step (a), the sealer includes bovine serum albumin
In vain, one or more in glycine, lysine and arginine；The sealer is in the quality volume being protected from light in Incubating Solution
A concentration of 0.01-0.1%.
7. screening technique as claimed in claim 4, which is characterized in that in second buffer solution, EDTA's is a concentration of
0.5-5mM；The mass-volume concentration of bovine serum albumin(BSA) is 0.02-0.2%.
8. screening technique as claimed in claim 4, which is characterized in that the load magnetic bead includes the general of 1nm-500 μm of grain size
Logical magnetic bead.
9. screening technique as claimed in claim 8, which is characterized in that the load magnetic bead isM-
450Epoxy magnetic beads, grain size are 2-10 μm.
10. such as application of the claim 1-9 any one of them screening techniques in immune cell therapy.
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