CN102994448A - Method for in-vitro amplification of gamma-delta-T cells - Google Patents
Method for in-vitro amplification of gamma-delta-T cells Download PDFInfo
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Abstract
The invention relates to a method for culturing gamma-delta-T cells, and in particular relates to a method for in-vitro amplification of gamma-delta-T cells, wherein the method comprises the following operating steps of: pre-coating a T75 culture bottle by a TCR-gamma-delta resisting antibody and CD28McAb for later use use; isolating the peripheral blood mononuclear cell (PBMC) of a patient; regulating the PBMC concentration to 1*10<6> 6/ml by a serum-free culture medium which contains 5% of autologous plasma, and transferring PBMC cell suspension into the T75 culture bottle; adding an initial culture medium which contains proper concentrations of Zoledronat, HSP70, 1L-2, 1L-7 and 1L-15; culturing in a saturated humid environment containing 5% of CO2 at 37 DEG C; depending on growth situation of the cell, changing the culture medium every 2-3days, to control the cell concentration at about 2.5*10<6>/ml; meanwhile, compensating full doses of Zoledronat, HSP70, 1L-2, 1L-7 and 1L-15; and continuously culturing for 12-16days, to obtain a great amount of gamma-delta-T cells which are comparatively high in purity.
Description
[technical field]
The present invention relates to the gamma delta T cells cultural method, be specifically related to a kind of method of amplification in vitro gamma delta T cells.
[background technology]
Difference according to T cell surface receptor (TCR) can be divided into the T lymphocyte two kinds: α β T lymphocyte and gamma delta T lymphocytes.In human body, α β T lymphocyte is the main cell group who participates in immunne response, account for 90 ~ 95% of mature T cells, and γ δ Τ cell only accounts for the 0.5%-5% of periphery blood T lymphocyte, but γ δ Τ cell occurs prior to α β Τ cell, mainly be distributed in skin, in small intestine, esophagus, lung and the sexual organ official rank epithelium.Gamma delta T cells belongs to the First Line defense cell of body, in anti-microbial infection immunity, especially in the immune defense function on skin mucosa surface, and plays a significant role in the anti-mycobacterial infections.In addition, research is also found, gamma delta T cells has the restrictive cytotoxicity without MHC, all show significant killing activity to multiple from body, allogeneic or xenograft tumor cell, therefore, gamma delta T cells is causing the concern of more and more Chinese scholars as the candidate cell of the important adoptive immunotherapy of a class.Because gamma delta T cells contained ratio in human body is lower, separation and purification is comparatively difficult to a large amount of gamma delta T cells, thereby has limited its clinical use but on the other hand.Therefore the external efficient amplification of gamma delta T cells is the main method that addresses this problem.
In this field, people have done a lot of good tries, domesticly usually adopt altogether stimulus method of IPP and IL-2 at cultured and amplified in vitro γ δ Τ cell, but relatively costly and amplification times is not enough is not widely used because of expense.
Therefore, in order to overcome the defective of prior art, the invention provides a kind of method of new gamma delta T cells, the method is easy and simple to handle, low price, and amplification γ δ Τ cell efficient is high.
[summary of the invention]
The method that the purpose of this invention is to provide a kind of external efficient amplification gamma delta T cells, purity and the killing activity of raising gamma delta T cells, it is clinical that gamma delta T cells more is applicable to.
For achieving the above object, design a kind of method of amplification in vitro gamma delta T cells, the method is comprised of following steps:
A. be coated with in advance the T75 culturing bottle with for subsequent use with anti-TCR γ anti-δ and CD28McAb,
B. peripheral blood mononuclear cell is changed in the coated T75 culturing bottle, add the serum free medium that contains Zoledronic acid (Zoledronat), HSP70, IL-7, IL-15 and IL-2, put into CO2gas incubator and cultivate the gamma delta T cells that obtained afterwards a large amount of higher degrees in 12 ~ 16 days.
Aforesaid method also has following prioritization scheme:
Anti-TCR γ anti-δ concentration is 1 μ g/ml.
The concentration in serum free medium of described CD28McAb is 500ng/ml.
The concentration of described Zoledronic acid in serum free medium is 1.00 μ g/ml ~ 3.87 μ g/ml.
The concentration of described Zoledronic acid in serum free medium is 1.29 μ g/ml.
The concentration of described HSP70 in serum free medium is 10 μ g/ml ~ 50 μ g/ml.
The concentration of described HSP70 in serum free medium is 20 μ g/ml.
The concentration of described IL-7 in serum free medium is 10ng/ml.
The concentration of described IL-15 in serum free medium is 50ng/ml.
Described IL-2 is 1000IU/ml in serum free medium.
The invention provides a kind of extracorporeal culturing method of new gamma delta T cells, compare with the cellar culture method, the superior part of present method have following some:
(1) adopts anti-TCR γ anti-δ and two kinds of antibody sandwiches of CD28McAb, anti-TCR γ anti-δ provides the first signal of gamma delta T cells activation, CD28McAb then provides costimulatory signal for the activation of gamma delta T cells, and the gamma delta T cells of tranquillization is activated fully;
(2) TCR γ δ has huge sequence polymorphism, and existing two quasi-molecules are proved to be TCR γ 2-delta ligand at present: non-peptide micromolecular and the heat shock protein(HSP) of phosphorous acidic group; Desired concn is higher when stimulating γ δ Τ cell with IPP, IPP's is expensive in addition, if be cultured to clinical needed cell quantity then to cultivate cost higher, and Zoledronic acid (Zoledronat) can activate γ δ Τ cell under lower concentration, so the Zoledrona of choice for use suitable concn stimulates γ δ Τ cell, makes its large scale culturing become possibility; HSP70 can not only carry specific tumour antigen, also can be used as the antigen presentation molecule and directly antigen peptide is and passs γ δ Τ cell, makes its activation and proliferation, the dual-use function of performance cell toxicant and secrete cytokines;
(3) application of IL-2, IL-7 and three kinds of combination of cytokines of IL-15.IL-7 is the important factor that promotes human γ δ Τ precursor cell propagation and promote γ δ Τ differentiation and maturation; IL-15 is a kind of multi-functional cytokine, promotes the increment of T cell, and the IL-15 T cell that can suppress to activate and B cell are because the apoptosis due to the factors such as anti-Fas antibody, cytokine exhaustion, dexamethasone in addition; IL-2 is the somatomedin of T cell; Three kinds of cytokines be used in combination the consumption that has both reduced the single kind of factor, make and cultivate cost without obvious raising, what guaranteed again to play a role is comprehensive, has significantly improved increment multiple and the toxicity of gamma delta T cells.
[description of drawings]
Fig. 1 is the comparison diagram of gamma delta T cells amplification times under two groups of cultural methods;
Fig. 2 is the comparison diagram of gamma delta T cells purity under two groups of cultural methods;
Fig. 3 be under two groups of cultural methods gamma delta T cells to the comparison diagram of SGC-7901 cell killing activity;
[embodiment]
In order to make purpose of the present invention, technical scheme and advantage clearer, the present invention is further elaborated.Production unit among the application all is the common equipment of this area, should be appreciated that specific embodiment described herein only in order to explain the present invention, is not intended to limit the present invention.
Embodiment 1:
One, separation and the cultivation of peripheral blood mononuclear cell (PBMC)
Machine gathers PBMC, and the blood sample that gathers is gone to centrifuge tube; 700g, the 10min centrifugation, for subsequent use when drawing the upper plasma cultivation; Blood sample is reduced to original volume, mixing; Diluted blood slowly is added on the Ficoll, 900g, centrifugal 20min; Draw parting liquid interface oyster white mononuclearcell layer; Centrifuge washing 2 times and counting; With γ δ Τ initial medium that cell is resuspended to 1 * 10
6/ ml; According to the growing state of cell, per 2 ~ 3 days replaced mediums, used medium is the serum free medium that contains 5% autologous plasma.At 37 ℃, 5%CO
2Saturated moist environment in cultivate, according to the growing state of cell, per 2 ~ 3 days replaced mediums are controlled at 2.5 * 10 with cell concn
6About/ml, full dose is replenished the various factors simultaneously: wherein test group is replenished Zoledronic acid (Zoledronat), HSP70, IL-2, IL-7 and IL-15, conventional group is replenished IPP and IL-2, continue to cultivate after 12 ~ 16 days, can obtain the gamma delta T cells of a large amount of higher degrees.
Two, the comparison of the cells expanded of experimental technique and ordinary method
Got culturing cell at the 0th, 4,8,12,14 and 16 day from two groups respectively, with counting behind the Trypan Blue, divided by the cell count (namely the 0th day) before cultivating, numerical value is the amplification times of cell with the total cellular score on counting same day.
With this method can two groups of cells of Dynamic comparison the amplification situation, result such as table 1 and shown in Figure 1, at the 4th, 8,12,14 and 16 day that cultivates, two groups gamma delta T cells is all in amplification, but the gamma delta T cells of each time point experimental group amplification situation all is better than conventional group, two groups all reach the amplification vertex in the time of the 14th day, and cell amplification speed slows down thereafter, and microscopically can be observed the apoptotic cell fragment in the 16th day.
Table 1
Annotate: * * represents p for the routine group＜0.01,
Three, the γ δ Τ cell purity of experimental technique and ordinary method relatively
Collect the cell of cultivating 0,4,8,12,14 and 16 day, every tube cell adjusts about 10
6Individual, with the centrifugal 2000rpm of cell suspension to be checked, supernatant discarded behind the 3min clock, cell is resuspended among the 100 μ l PBA buffer, and 4
oC or 20min on ice; Add corresponding fluorescent-labeled antibody: anti-TCR γ δ-PE carries out fluorescent dye, and other establishes colleague's contrast; 4
oC or on ice lucifuge hatch 30min; Add 1ml PBA buffer, the centrifugal 3min of 2000rpm washs 3 times; With the 0.3ml Paraformaldehyde 96 that cell is resuspended, cell suspension is transferred in the streaming pipe, upper machine analysis,
Meaning is organized 01 result such as table 2 and shown in Figure 2, and when cultivating beginning, the γ δ Τ cell purity of two groups of PBMC is similar, and the purity of the γ δ Τ cell of each time point experimental group all is higher than corresponding routine group (p＜0.01) thereafter.
Table 2
Annotate: * * represents p for the routine group＜0.01,
Four, the comparison of the γ δ Τ cells in vitro killing activity of experimental technique and ordinary method cultivation
.013 with the SGC-7901 cell that is in logarithmic phase as target cell, it is adjusted into 1 * 10
5/ mL cultivated 14 days γ δ Τ cell action effect cell with two kinds of methods, by 10: 1,20: 1,50: 1 effect target was than effector cell and target cell being mixed while laying effect cell hole, target cell hole, all establish 3 parallel holes for every group, every hole final volume is 200 μ l, 37 ℃, 5%CO
2Hatch in the incubator, every hole adds 20 μ l CCK-8 solution behind the 12h, continues to hatch 4h, the OD value when detecting 450nm with microplate reader, calculate as follows kill rate: kill rate (%)=[1-(experimental port OD value-effect hole OD value/target cell hole OD value)] * 100%
The result is as described in table 3 and Fig. 3, and experimental group all is higher than conventional group to the kill rate of tumour cell, and difference has statistical significance (P＜0.05).
Annotate: * * represents p for the routine group＜0.01.
Claims (10)
1. the method for an amplification in vitro gamma delta T cells is characterized in that the method is comprised of following steps:
A. be coated with in advance the T75 culturing bottle with for subsequent use with anti-TCR γ anti-δ and CD28McAb,
B. peripheral blood mononuclear cell is changed in the coated T75 culturing bottle, add the serum free medium that contains Zoledronic acid, HSP70, IL-7, IL-15 and IL-2, put into CO2gas incubator and cultivate the gamma delta T cells that obtained afterwards a large amount of higher degrees in 12 ~ 16 days.
2. the method for amplification in vitro gamma delta T cells as claimed in claim 1 is characterized in that anti-TCR γ anti-δ concentration is 1 μ g/ml.
3. the method for amplification in vitro gamma delta T cells as claimed in claim 1, the concentration in serum free medium that it is characterized in that described CD28McAb is 500ng/ml.
4. the method for amplification in vitro gamma delta T cells as claimed in claim 1 is characterized in that the concentration of described Zoledronic acid in serum free medium is 1.00 μ g/ml ~ 3.87 μ g/ml.
5. the method for amplification in vitro gamma delta T cells as claimed in claim 1 is characterized in that the concentration of described Zoledronic acid in serum free medium is 1.29 μ g/ml.
6. the method for amplification in vitro gamma delta T cells as claimed in claim 1 is characterized in that the concentration of described HSP70 in serum free medium is 10 μ g/ml ~ 50 μ g/ml.
7. the method for amplification in vitro gamma delta T cells as claimed in claim 1 is characterized in that the concentration of described HSP70 in serum free medium is 20 μ g/ml.
8. the method for amplification in vitro gamma delta T cells as claimed in claim 1 is characterized in that the concentration of described IL-7 in serum free medium is 10ng/ml.
9. the method for amplification in vitro gamma delta T cells as claimed in claim 1 is characterized in that the concentration of described IL-15 in serum free medium is 50ng/ml.
10. the method for amplification in vitro gamma delta T cells as claimed in claim 1 is characterized in that described IL-2 is 1000IU/ml in serum free medium.
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