CN105886468B - A kind of method and application of efficient induction gamma delta T cells - Google Patents
A kind of method and application of efficient induction gamma delta T cells Download PDFInfo
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- CN105886468B CN105886468B CN201510270659.3A CN201510270659A CN105886468B CN 105886468 B CN105886468 B CN 105886468B CN 201510270659 A CN201510270659 A CN 201510270659A CN 105886468 B CN105886468 B CN 105886468B
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Abstract
The present invention provides a kind of for cultivating the culture medium of gamma delta T cells, the cultural method of gamma delta T cells, purposes of the gamma delta T cells efficiently induced and its preparation and the gamma delta T cells efficiently induced obtained by this method in preparation improves patient base's immune function, improves the drug for resisting tumour and anti-infection ability.
Description
Technical field:
The present invention relates to immunocyte in vitro culture field, in particular to a kind of preparation methods of efficiently induction gamma delta T cells
And cell preparation.
Background technique:
T lymphocyte is different according to the composition of T cell receptor (TCR) double-chain peptide in human body, can be divided into TCR α β T cell
(abbreviation α β T cell) and TCR gamma delta T cells (abbreviation gamma delta T cells), wherein gamma delta T cells are with major histocompatibility complex
(Major histocompatibility complex, MHC) unrestricted mode Direct Recognition simultaneously combines antigen molecule, and leads to
The approach such as perforin-granzyme, Fas/FasL, IFN-γ secretion and TNF-related apoptosis-inducing ligand receptor TRAILR are crossed to kill
Hurt tumour cell and virus infected cell.Gamma delta T cells are except effectively in addition to killing tumor cell, capable of can also passing through secretion of gamma-IFN
Etc. cell factors, promote the activation of other immunocytes, therefore play in the innate immunity and adaptive immune response important
Immunosurveillance and immunoregulation effect, it is considered to be the important bridge of the connection innate immunity and adaptive immunity.Gamma delta T cells tool
There is the double characteristic of NK cell and T cell, the important function of reactivity of high level expression NK cell is gone back in addition to expressing TCR γ δ in surface
Energy receptor NKG2D, both acceptor molecules play a significant role during killing of the gamma delta T cells to tumour cell.Wherein,
Single TCR γ δ can be such that gamma delta T cells activate, and NKG2D then plays the role of costimulation in the process, so that gamma delta T cells
Fast activating and corresponding biological effect is generated after identifying antigen, the fast activating when body resists tumour or infection, from
And play antitumor or anti-infectious important function.In addition, research discovery kills tumor with no MHC is restrictive due to gamma delta T cells
Effect shows significant killing activity, therefore gamma delta T cells conduct to a variety of self, allogeneics or xenograft tumor cell
The candidate cell of a kind of important adoptive immunotherapy causes the concern of more and more home and abroad scholars.But another party
Face, since all blood only accounts for 1-5% to gamma delta T cells out of the human body, it is very tired for obtaining the gamma delta T cells of a large amount of high cytotoxic activities
Difficult, thus limit its clinical application.Although prior art mostly uses anti-tcr γ anti-δ or non-peptide phosphonic acid antigen to obtain
A large amount of gamma delta T cells, but be not used widely because expense is relatively expensive and amplification times are limited.
Therefore, for overcome the deficiencies in the prior art, the present invention is provided outside economical and practical, the efficient rapid amplifying human body of one kind
The method of all blood gamma delta T cells resists tumour and anti-infection ability to improve patient base's immune function, provide sickened body.
It is detected simultaneously for needing to carry out it cell quantity, purity, immunophenotype after the preparation of human peripheral gamma delta T cells, it is ensured that its energy
Effectively play certain clinical therapeutic efficacy.
Summary of the invention:
The object of the present invention is to provide a kind of method of economical and practical, efficient rapid amplifying human peripheral blood gamma delta T cells,
Quantity, purity and the killing activity of gamma delta T cells are improved to ensure clinical application.
On the one hand, the present invention provides a kind of culture mediums dedicated for cultivating gamma delta T cells, by nutrient media components I, γ δ
T cell special media component II is constituted, wherein the nutrient media components I includes the ingredient of following content: RPMI-1640 training
Base is supported, wherein the L-Glutamine for being 1-10mM added with concentration, concentration is the recombination human transferrin of 5-20mg/L, and concentration is
The recombinant human albumin of 1-10g/L, concentration are the rh-insulin of 1-10mg/L, and concentration is the vitamin of 10-100mg/L
PP, concentration are the vitamin C of 10-50mg/L, and concentration is the hydrocortisone of 1-50ng/mL, and concentration is the micro member of 1-10mg/L
Element；The gamma delta T special media component II is made of the component of following concentration: PMA concentration is 10-50ng/ml, ion
Mycin concentration is 0.5-5ug/ml, and rhIL-2 concentration is 100-1000IU/ml, and rhIL-18 concentration is 5-20ng/ml.
The serum that the above-mentioned dedicated gamma delta T cells culture medium of the present invention can also be further 1-10% containing volume ratio, preferably
Autoserum, further preferably 5% autoserum.In the preferred technical solution of the present invention, the nutrient media components I is no blood
Clear culture medium: the microelement is selected from selenium.
Compared with the existing technology, containing in the of the invention culture medium for being exclusively used in culture gamma delta T cells some can promote γ
Delta T cells proliferated specifically and the component of differentiation, more so as to obtain, purity is higher and the stronger γ δ of killing activity
T cell.
The culture medium for being exclusively used in culture gamma delta T cells of the invention has the advantage that
1) it avoids due to using animal blood serum to patient's bring risk and animal blood in gamma delta T cells incubation
Uncertain ingredient in clear is adversely affected caused by cell culture；2) gamma delta T cells yield is increased, improves purity, and drop
The low production cost of cell culture.
On the other hand, the present invention provides a kind of method of efficiently induction gamma delta T cells, i.e., it is thin that one kind was new prepares gamma delta T
The method of born of the same parents, it is characterised in that peripheral blood mononuclear cells is resuspended with nutrient media components I to 1X106-5X106/ ml, the training
The ingredient that base component I includes following content: RPMI-1640 culture medium is supported, wherein the L- glutamy for being 1-10mM added with concentration
Amine, concentration are the recombination human transferrin of 5-20mg/L, and concentration is the recombinant human albumin of 1-10g/L, concentration 1-10mg/L
Rh-insulin, concentration be 10-100mg/L nicotinic acid, concentration be 10-50mg/L vitamin C, concentration 1-
The hydrocortisone of 50ng/mL, concentration are 1-10mg/L microelement, and every 2-3 days replacement culture mediums, used medium is culture
Base component I, is cultivated in incubator, while gamma delta T special media component II, the gamma delta T special media group is added
Divide II to be made of the component of following concentration: PMA concentration is 10-50ng/ml, and ionomycin concentration is 0.5-5ug/ml, rhIL-2
Concentration is 100-1000IU/ml, and rhIL-18 concentration is 5-20ng/ml.
In a preferred embodiment of the invention, the nutrient media components I is serum free medium；The micro member
Element is selected from selenium.In another preferred embodiment of the present invention, aforementioned present invention is prepared in the method for gamma delta T cells, the training
Support the serum that base component I or gamma delta T special media component II can be further 1-10% containing volume ratio.It is highly preferred that
The autoserum for being 5% containing volume ratio.
In carrying out the present invention, in order to compare, optimizer culture medium culture medium, including AIM-V as a control group is used
Culture medium.Cell is being resuspended to 1X10 with gamma delta T cells culture medium for peripheral blood mononuclear cells6-5X106After/ml, according to
The upgrowth situation of cell, every 2-3 days replacement culture mediums, gamma delta T cells culture medium used are respectively two kinds of different gamma delta T cells cultures
(culture medium, the latter wrap base as a control group for above-mentioned gamma delta T cells special culture media i.e. of the invention or optimizer culture medium
Include AIM-V culture medium), it is cultivated in incubator, while supplementing (the gamma delta T specificity culture i.e. of the present invention of the various cell factors of full dose
Base component II), it is lasting to cultivate, obtain the gamma delta T cells of a large amount of higher concentrations.
The third aspect, the present invention provides the gamma delta T cells of the preparation of culture medium through the invention, contain the method for the present invention system
The application of the cell preparation and said preparation of standby gamma delta T cells.The present invention also provides the gamma delta T of culture medium of the present invention preparation is thin
Purposes of the born of the same parents in preparation improves patient base's immune function, improves the drug for resisting tumour and anti-infection ability.
The brief description of accompanying drawing:
Fig. 1 is the gamma delta T cells proliferation times of culture medium preparation of the present invention compared with control group.
Fig. 2 is the gamma delta T cells culture end density of culture medium preparation of the present invention compared with control group.
Fig. 3 is gamma delta T cells flow cytometer detection result of the present invention.
Fig. 4 is that gamma delta T cells of the present invention kill tumor activity testing result.
Specific embodiment
Below with reference to embodiment, the invention will be further described, but should not be understood is to the scope of the present invention
Limitation.
Embodiment 1: the culture of gamma delta T cells
By the peripheral blood 15ml of acquisition, peripheral blood mononuclear cells is obtained by density gradient centrifugation.Use nothing of the present invention
The peripheral blood mononuclear cells of acquisition is resuspended to 1X10 blood serum medium component I6/ml.The serum free medium component I is
It is made of the component of following concentration: RPMI-1640 culture medium (purchased from U.S. Gibco company, article No.: 31800-105), wherein
The L-Glutamine for being 5mM added with concentration, concentration are the recombination human transferrin of 10mg/L, and concentration is that the recombined human of 5g/L is white
Albumen, concentration are the rh-insulin of 5mg/L, and concentration is the nicotinic acid of 50mg/L, and concentration is the vitamin C of 30mg/L,
Concentration is the hydrocortisone of 30ng/mL, and concentration is the selenium of 5mg/L.According to the upgrowth situation of cell, the culture of replacement in every 2-3 days
Base, in 37 DEG C, 5%CO2Incubator in cultivate, while supplementing the various cell factors of full dose, that is, be added and be containing PMA concentration
30ng/ml；Ionomycin concentration is 2ug/ml, and rhIL-2 concentration is 500IU/ml, and rhIL-18 concentration is this hair of 10ng/ml
Bright gamma delta T special media component II.
As control, according to the upgrowth situation of cell, every 2-3 days replacement culture mediums, used medium is AIM-V culture medium
Culture medium as a control group, in 37 DEG C, 5%CO2Incubator in cultivate, while supplementing the various cell factors of full dose, that is, be added
It is 30ng/ml containing PMA concentration；Ionomycin concentration is 2ug/ml, and rhIL-2 concentration is 500IU/ml, and rhIL-18 concentration is
The gamma delta T special media component II of the present invention of 10ng/ml respectively expected gamma delta T cells platform at the 0th, 5,10,15,20 day
It is counted after indigo plant dyeing.
Embodiment 2: the culture of gamma delta T cells
By the peripheral blood 20ml of acquisition, peripheral blood mononuclear cells is obtained by density gradient centrifugation.Using containing volume ratio
The peripheral blood mononuclear cells of acquisition is resuspended to 1X10 the nutrient media components I of 5% autoserum6/ml.The nutrient media components
I is made of the component of following concentration: RPMI-1640 culture medium (is purchased from U.S. Gibco company, article No.: 31800-105),
The L-Glutamine for being wherein 5mM added with concentration, concentration are the recombination human transferrin of 10mg/L, and concentration is the recombination of 5g/L
Human albumin, concentration are the rh-insulin of 5mg/L, and concentration is the nicotinic acid of 50mg/L, and the dimension that concentration is 30mg/L is raw
Plain C, concentration are the hydrocortisone of 30ng/mL, and concentration is the selenium of 5mg/L.According to the upgrowth situation of cell, replace within every 2-3 days
Culture medium, in 37 DEG C, 5%CO2Incubator in cultivate, while supplementing the various cell factors of full dose, that is, be added and contain PMA concentration
For 30ng/ml；Ionomycin concentration is 2ug/ml, and rhIL-2 concentration is 500IU/ml, and rhIL-18 concentration is the sheet of 10ng/ml
Invention gamma delta T special media component II.
As control, according to the upgrowth situation of cell, every 2-3 days replacement culture mediums, used medium is containing 5% self blood
Clear AIM-V culture medium culture medium as a control group, in 37 DEG C, 5%CO2Incubator in cultivate, while it is various to supplement full dose
Cell factor, that is, being added containing PMA concentration is 30ng/ml；Ionomycin concentration is 2ug/ml, and rhIL-2 concentration is 500IU/
Ml, rhIL-18 concentration are the gamma delta T special media component II of the present invention of 10ng/ml, respectively at the 0th, 5,10,15,20 day
To being counted after gamma delta T cells Trypan Blue.
3 cell Proliferation effect of embodiment compares
It is counted after the gamma delta T cells Trypan Blue prepared respectively at the 0th, 5,10,15,20 day to embodiment 1.It will meter
For the total number of cells on the number same day divided by the cell number (i.e. the 0th day) before culture, numerical value is the amplification times of cell.With control group
Culture medium carries out cell Proliferation effect and compares.
Find that, at the 5th, 10,15,20 day of culture, two groups of gamma delta Ts are thin by the amplification situation of two groups of cells of Dynamic comparison
Born of the same parents are expanding, and reach amplification highest point for two groups at the 20th day, but the amplification situation of each time point experimental group is better than
Control group.
The results show that the gamma delta T cells obtained by gamma delta T cells special culture media of the present invention, compared with control group culture medium
Compared with, proliferation times apparent increase (see Fig. 1), while cell culture end density is higher (see Fig. 2).
4 gamma delta T cells flow cytometer detection of embodiment
It after the gamma delta T cells that embodiment 1 obtains are washed with PBS buffer solution, is resuspended with PBS buffer solution, takes two part of 50 μ l thin
Born of the same parents' suspension (about 3 × 105Cell) be separately added into 2 streaming pipes, be added fluorescent marker antibody (respectively AntiCD3 McAb-FITC:
Purchased from BD company, article No. 555274: anti-tcr γ δ-APC: it is purchased from BD company, article No.: 555718) dyed, 4 DEG C are protected from light incubation
30min, after buffer washing, suspension cell is analyzed with flow cytometer, is detected in 500 μ l PBS buffer solution.
The results show that culture medium prepares gamma delta T cells and can detecte CD3+T in the cell preparation of preparation through the invention
High enrichment, purity can achieve 97%, and gamma delta T cells day reach peak value, average out to 89.6% from the 14th day to the 20th
(see Fig. 3).
5 gamma delta T cells of embodiment kill tumor activity detection
The K562 tumour cell of logarithmic growth phase is as target cell respectively, with 5X103Cells/well 96 orifice plates of paving, second
The gamma delta T cells for the experimental group culture 14 days that the embodiment of the present invention 1 obtains are added as effector cell, by experimental group and control in it
Group compares, and is incubated for 72h jointly to imitate concentration of the target than 10: 1,20: 1 and 50: 1, each concentration sets 3 parallel holes；Abandon supernatant
After PBS is washed 2 times, CCK-8 (Cell Counting Kit, CCK-8, purchased from Japanese colleague's CCK8 kit, product is added in liquid
Model: CK04) continue in Cell counting Kit to cultivate 4h using liquid, 96 porocyte culture plates are put into detection in microplate reader and exist
Absorbance OD value at 450nm, is calculated according to the following formula cell killing rate:
Killing rate (%)=[1- (experimental port OD value-effect hole OD value/Target cell wells OD value)] X100%
The detection of cytotoxicity: examining each experimental group using t, is that difference has statistical significance with P < 0.05.
As a result as shown in figure 4, showing: either under the conditions of 10: 1,20: 1 and 50: 1 different effect target ratios, or same
Under one effect target specific concentration, experimental group is above control group to the killing rate of tumour cell, and difference has statistical significance (P
< 0.05).
Claims (2)
1. a kind of for cultivating the culture medium of gamma delta T cells, it is characterised in that trained by nutrient media components I and gamma delta T cells specificity
Base component II is supported to constitute；Wherein the nutrient media components I is made of the ingredient of following content: RPMI-1640 culture medium, wherein adding
The L-Glutamine for being 5mM added with concentration, concentration are the recombination human transferrin of 10mg/L, and concentration is the white egg of recombined human of 5g/L
White, concentration is the rh-insulin of 5mg/L, and concentration is the nicotinic acid of 50mg/L, and concentration is the vitamin C of 30mg/L, dense
Degree is the hydrocortisone of 30ng/mL, and concentration is 5mg/L selenium；The gamma delta T special media component II is by following concentration
Component is constituted: PMA concentration is 30ng/ml, and ionomycin concentration is 2ug/ml, and rhIL-2 concentration is 500IU/ml, and rhIL-18 is dense
Degree is 10ng/ml.
2. a kind of method for preparing gamma delta T cells, it is characterised in that being resuspended peripheral blood mononuclear cells with nutrient media components I to 1
×106- 5 × 106/ml；The nutrient media components I is made of the ingredient of following content: RPMI-1640 culture medium, wherein adding
Having concentration is the L-Glutamine of 5mM, and concentration is the recombination human transferrin of 10mg/L, and concentration is the white egg of recombined human of 5g/L
White, concentration is the rh-insulin of 5mg/L, and concentration is the nicotinic acid of 50mg/L, and concentration is the vitamin C of 30mg/L, dense
Degree is the hydrocortisone of 30ng/mL, and concentration is 5mg/L selenium；Every 2-3 days replacement culture mediums, used medium are culture medium group
Divide I, is cultivated in incubator, while gamma delta T special media component II is added；The gamma delta T special media component II
Be made of the component of following concentration: PMA concentration is 30ng/ml, and ionomycin concentration is 2ug/ml, and rhIL-2 concentration is 500IU/
Ml, rhIL-18 concentration are 10ng/ml.
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| CN108315298B (en) * | 2018-02-07 | 2020-10-20 | 安徽古一生物科技有限公司 | Gamma T cell culture method |
| CN108949685B (en) * | 2018-08-02 | 2022-03-29 | 吉林大学第一医院 | Method for in vitro induction and expansion of gamma delta T cells with high killing activity |
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