CN108823161A - A kind of method of cytotoxic T cell and lymphatic endothelial cells co-cultivation - Google Patents
A kind of method of cytotoxic T cell and lymphatic endothelial cells co-cultivation Download PDFInfo
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Abstract
The present invention relates to cell engineering fields, disclose a kind of cultural method of cytotoxic T cell, it is co-cultured by cytotoxic T cell and lymphatic endothelial cells, avoid traditional technological deficiency existing when being co-cultured using Dendritic Cells (DC) as antigen presenting cell, turn out activity is higher, the speed of growth faster, the stronger T cell of killing ability.Method of the invention is at low cost, cell quality is high, and the commercialization suitable for T cell is mass produced.
Description
Technical field
The present invention relates to cell engineering fields more particularly to a kind of cytotoxic T cell to train altogether with lymphatic endothelial cells
Feeding method.
Background technique
The adoptive immunotherapy of cell is to be transfused trouble self or after alloimmune effector cell by Activated in Vitro and amplification
A kind of intracorporal treatment method of person, the method overcome previous effector cell, to be proliferated quantity few, need to largely be transfused IL-2, be promoted white
The disadvantages of cell titer is lower and side effect is big, for promoting reconstruction, the bone marrow purging, minimal residual disease of patients immune system
Removing and the recurrence and the transfer that prevent tumour etc. all have important meaning.
The adoptive immunotherapy of cell has become important branch of biological therapy, such as CAR-T treatment method etc. in recent years
Good targeting, lethal and persistence are shown in clinical test, illustrate huge development potentiality and application
Prospect.Included in the granted CAR-T cell drug of U.S. FDA at present, for treating children and young acute lymphoblastic
The Kymriah (Tisagenlecleucel, CTL-019) of leukaemia, for treating, other therapies are invalid or the past at least receives
Cross the Yescarta for the certain types of adult large B cell lymphoid tumor patient recurred after two schemes treatment
(AxicabtageneCiloleucel, KTE-C10) etc..
For the Amplification Technologies of immunocyte, more stringent requirements are proposed for the development of adoptive immunity cell therapy.At present
The amplification in vitro of immunocyte usually requires exogenous cytokines, such as the auxiliary of IL-2, IL-15, IFN-γ, these factors
Control the amplification and its biological activity of human immune system internal specific cell.In basic research, cytotoxic T is thin
Born of the same parents (CTL) are limited as a kind of effector T cell by MHC, need the activation of antigen presenting cell, Dendritic Cells (DC) is as thin
The important antigen presenting cell (APCs) and CTL of Cytotoxic T Lymphocytes (CTL) immunocyte in-vitro multiplication induction are common
Co culture system in vitro cell, but loosely survival rate is low after cryopreservation resuscitation there are adherent for DC cell, antigen presentation and to T
The defects of cell-stimulating effect is limited causes the technological difficulties much cultivated, and DC cell needs external use specificity anti-
Former or wide spectrum antigen activation, the activation efficiency of these antigenic activation substances is limited, and its residue is likely to result in centainly
Side effect.
The T cell for being clinically used to assist in the treatment of, in health care and Car-T research both at home and abroad at present is directed in commodity
Change many defects in large-scale production, such as higher cost, the antigen submission dendritic cells speed of growth is slow, it is adherent loosely, freeze
After can not reuse, cell activity problem and immunocyte to the cellkilling capacity problem of tumour cell or senescense and damnification and
The present Research such as immunological rejection, need to develop it is a kind of can turn out the T cell that the speed of growth is fast, killing ability is strong, and
Cost is lower, is suitble to the T cell cultural method of large-scale production.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of low costs, the T cell culture side for being suitble to be mass produced
Method, and by the T cell speed of growth of this method culture faster, killing ability it is stronger, can satisfy clinical adjuvant treatment and
The demand of CAR-T treatment method.
To realize the above goal of the invention, the present invention provides a kind of cytotoxic T cells and lymphatic endothelial cells (LEC)
The method of co-cultivation, is co-cultured by cytotoxic T cell and lymphatic endothelial cells, is avoided traditional with dendron shape
Cell (DC) existing technological deficiency when being co-cultured as antigen presenting cell turns out the active higher, speed of growth more
Fastly, the stronger T cell of ability is killed.
In one aspect of the invention, a kind of T cell cultural method is provided, by T cell and lymph in the cultural method
Endothelial cell is co-cultured.Preferably, the T cell is cytotoxic T cell (CTL).
In the specific embodiment of the present invention, by T cell and lymphatic endothelial cells in serum free medium
It is co-cultured, further, cell factor can be added in the medium, optionally, the cell factor is OKT3, IFN-
One or more of γ, IL-2, IL-15, it is preferred that the cell factor is IL-2, it is furthermore preferred that cultural method of the present invention
Contain OKT3, IFN-γ, IL-2 and IL-15 in the culture medium used.After external extensive amplification, IL-2, IL-15,
The killing activity of the amplified production of the inductions such as OKT3 is substantially better than the amplification situation of the immunocyte of Nostoc commune Vanch.Optionally, originally
KSR can also be contained in the culture medium that invention cultural method uses.
In another aspect of the invention, it provides a kind of for cultivating the culture medium of T cell, leaching is contained in the culture medium
Hand shaft endothelial cell, by the co-cultivation of lymphatic endothelial cells and T cell, can obtain activity is higher, the speed of growth faster,
The stronger T cell of killing ability.Preferably, culture medium of the invention can be used for cultivating cytotoxic T cell.
Further, the culture medium for cultivating cytotoxic T cell of the invention is serum free medium, it is preferred that
Wherein contain one or more of OKT3, IFN-γ, IL-2, IL-15, it is furthermore preferred that containing OKT3, IFN- in culture medium
γ, IL-2 and IL-15.
The present invention is based on the antigen presentations of lymphatic endothelium, with the conventional dendron shape of lymphatic endothelial cells substitution
Cell is co-cultured with cytotoxic T cell.Compared to Dendritic Cells, lymphatic endothelium, which has, to be easy to expand, is adherent
It is firm, use can be frozen repeatedly, the advantages such as high-fidelity in amplification procedure.Comparative experiments shows, and with Dendritic Cells and thin
Cytotoxic T cells co-culture, or individually culture cytotoxic T cell is compared, with lymphatic endothelial cells and cytotoxic T cell
Co-culturing the effect in terms of promoting cell Proliferation more preferably (can reach 200 times of multiplications), can more excite CD3+CD4+ heterogeneity cell
The proliferation (double sun rate mean values 26.3%) of group, can be improved the ratio that cell is killed in immunocyte, while improving cell killing
It is also more preferable in terms of active ability.
It is described further below with reference to technical effect of the attached drawing to design of the invention, specific structure and generation, with
It is fully understood from the purpose of the present invention, feature and effect.
Detailed description of the invention
Fig. 1 show the phenotype flow cytometer showed result of the 1st, 10,30 generation cell of lymphatic endothelial cells;Wherein 1-1 is the 1st
The cell phenotype in generation is as a result, 1-2 is the cell phenotype in the 10th generation as a result, 1-3 is the cell phenotype result in the 30th generation;
Fig. 2 is shown after lymphatic endothelial cells and Dendritic Cells place 30 minutes at 4 DEG C, 10 DEG C and 37 DEG C respectively
The adherent situation map of cell;
Fig. 3 show the growth curve of tri- kinds of cultural method T cells of LEC/DC/Mock;
Fig. 4 show the cellular morphology of tri- kinds of cultural method T cells of LEC/DC/Mock, wherein the agglomerating growth of LEC group cell
And could disperse through piping and druming, DC group and Mock group T cell all disperse to grow, and Mock group cell quantity is minimum, and state is worst;
Fig. 5 show tri- kinds of cultural method T cell phenotype flow cytometer showeds of LEC/DC/Mock as a result, LEC group is double as the result is shown
Positive rate highest, DC group are taken second place, and Mock group is minimum.
Fig. 6 show 24 hours of tri- kinds of cultural method T cells of LEC/DC/Mock, 48 hours MTT are as a result, wherein NC is
Blank control, T cell (LEC group) killing that lymphatic endothelial cells and T cell co-culture as the result is shown is 60-70%, and DC is thin
T cell (DC group) killing that born of the same parents and T cell co-culture is 40-50%, the T cell killing of the independent culture group (Mock group) of T cell
Effect is only 15% or so.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Those skilled in the art can be with
Present disclosure is used for reference, realization of process parameters is suitably modified.In particular, it should be pointed out that all similar substitutions and modifications are to ability
It is it will be apparent that they are considered as being included in the invention for field technique personnel.Actual conditions are not specified in embodiment
Person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer, being can be with
The conventional products being commercially available.
Embodiment 1:The separation of peripheral blood mononuclear cells (PBMC)
In the 15ml centrifuge tube for being previously added 3mL lymphocyte separation medium (Ficoll), with 10mL combinations of syringe 1mL
5mL blood is slowly added in above-mentioned centrifuge tube and (is injected along wall, keep layering), is centrifuged 30 minutes with 400g by syringe needle,
Centrifuge raising speed 9, reduction of speed 0;The upper plasma of centrifugate is taken out with 3mL sterile dropper, is transferred in 50mL centrifuge tube (all
Blood plasma is incorporated to same centrifuge tube);With the sterile dropper of 3mL take tunica albuginea layer and its upper layer residual plasma and a small amount of lower layer Ficoll,
(the tunica albuginea layer of every 4 15mL centrifuge tubes is incorporated to a 50mL centrifuge tube) is transferred in 50mL centrifuge tube;It is centrifuged to each 50mL
Physiological saline is added in pipe to 45mL scale, is rocked, is vibrated mixing, with 1800rpm centrifugation 10 minutes, centrifuge raising speed 9, reduction of speed
9;Abandon supernatant, after being vibrated with residual liquid multitube and be a 50mL centrifuge tube, with a small amount of physiology salt wash original 50mL centrifuge tube,
Cleaning liquid is also transferred in the same centrifuge tube, after add physiological saline to 45mL scale, rock, vibrate mixing, with
1800rpm is centrifuged 10 minutes, centrifuge raising speed 9, reduction of speed 9;Supernatant is abandoned, with the resuspension of (former blood volume/10) mL blood plasma, after piping and druming mixes
100 μ L are taken to count, separation obtains PMBC.
Embodiment 2:Isolate and purify T cell
After isolating PMBC, cell is diluted to 5 × 107EasySep is added in separating pipe in cells/mlTMHuman
The cocktail antibody (50 μ l/ml) of CD4+ system, mixing room temperature are incubated for 5 minutes, and RapidSpheres is addedTMNanoscale Iron magnetic bead,
Mixing makes even particle distribution, buffer is added to 2.5ml, separating pipe is put into magnetite by upper and lower gentle inversion 2-3 times mixing
Room temperature is incubated for 3-5 minutes, and separating pipe is overturned together with magnetite, supernatant is poured into collecting pipe;Repetition is added in separating pipe
Buffer repeats to bear and selects process to improve cell yield, to isolate and purify to obtain T cell.
Embodiment 3:The culture and passage of lymphatic endothelial cells (LEC)
Lymphatic endothelial cells suspension is obtained using neutral proteinase and collagenase digestion procedure.1 is no less than to Guan Zhongjia
×106A cell adds I anti-, Podoplanin (1:Or CD34 (1 100):100) mix, 4 DEG C ice bath 20 minutes, add containing 4% tire
Phosphate buffer (PBS) about 30mL of cow's serum (FBS) elutes Excess antibody with 1800r/min centrifugation 5 minutes.Add
PBS of the 200uL containing 4%FBS is resuspended, and adds II anti-, PE (1:Or FITC (1 500):500), 4 DEG C ice bath 20 minutes, add containing 4%FBS
PBS about 30mL, with 1800r/min centrifugation 5 minutes, sufficiently elution Excess antibody.Every pipe adds PBS of the 3mL containing 4%FBS to be resuspended
It is to be measured, set up blank and Isotype control.Parameters are set according to instrument manual, sorter is installed, standard fluorescence ball is used
Best Delaytime and Drive value is debugged by after above-mentioned single cell suspension antibody label, is first carried out with a small amount of label cell
Sort trial test.As can showing good separating effect, then large batch of sorting is carried out.Measurement Podoplanin+ after having collected/
CD34-And CD34+/Podoplanin-The content of cell calculates the rate of recovery and cell purity, measures Cell viability with trypan blue.
With complete Endothelial cell culture base (Endothelial cell growth medium -2 for
Microvascular endothelial cell, EGM-2-MV) it is resuspended, it counts, adjustment cell density to 1xl05/ mL is followed by
Kind is in the 75ml bottle with 20ng/mL fibronectin (Fibronectin) shop fixtures.After cell grows into 90% fusion,
0.05% trypsin digestion passage.
To the different algebra of LEC culture, the cell phenotype including 1st generation, 10 generations, 30 generations carries out flow cytometer showed, is marked
Marker include CD34, VEGFR3, Podoplanin and HLA.As a result as shown in table 1 and Fig. 1, wherein the 1-1 in Fig. 1 is the
The cell phenotype in 1 generation is as a result, 1-2 is the cell phenotype in the 10th generation as a result, 1-3 is the cell phenotype result in the 30th generation.
Table 1:The 1st, 10,30 generation phenotypic data of lymphatic endothelial cells
CD34 | VEGFR3 | Podoplanin | HLA | |
1st generation | 98.5 | 98.2 | 95.7 | 0.582 |
10th generation | 99.9 | 99.5 | 99.2 | 3.03 |
30th generation | 99.4 | 99.5 | 99.8 | 3.30 |
As shown in the data of Fig. 1 and table 1, the 1st generation of LEC culture, the 10th generation, the 30th generation cell phenotype all keep relatively steady
It is fixed, illustrate that LEC property in extensive amplification will not change, fidelity with higher.
Embodiment 4:Lymphatic endothelial cells (LEC) are compared with Dendritic Cells (DC) adherent degree
By lymphatic endothelial cells (LEC) and Dendritic Cells (DC) respectively at being placed 30 minutes at 4 DEG C, 10 DEG C and 37 DEG C
Afterwards, culture solution is blown and beaten, detects the adherent situation of LEC and DC, as a result as shown in Figure 2.Shown result is as it can be seen that 4 according to fig. 2
DEG C, at 10 DEG C and 37 DEG C, the adherent situation of LEC is better than DC, wherein gap is the most obvious at 10 DEG C.
Embodiment 5:The co-cultivation of lymphatic endothelial cells and CD3+/CD4+T cell
By 107CD3+/CD4+T cell inoculation in the lymphatic endothelials of inactivations in ultraviolet irradiation 30 minutes of degrees of fusion 90%
Cultivated in Tissue Culture Flask, wherein culture solution be serum free medium (KBM 551), add the factor
OKT3, IFN-γ, IL-2, IL-15 and 5% KSR (Gbio).
Embodiment 6:Immunocyte identification
It takes the T cell liquid piping and druming of appropriate culture to the 13rd day to mix, is cleaned cell 2-3 times, used with sterile D-hanks liquid
Cell is resuspended in RPMI-1640 without serum, and adjustment concentration is 1 × 107-3×107/ mL dispenses cell re-suspension liquid to 1.5mL
It in centrifuge tube (100 μ L/ pipe), is added PE-CD3, FITC-CD4, APC-8a antibody (1 μ L/test), is incubated for 1 hour on ice, use
Cell is resuspended in RPMI-1640 of the 400 μ L without serum, carries out immunocyte identification by flow cytometer detection technology.
Embodiment 7:Immunologic cellular activity detection
Cytotoxic T lymphocyte (CTL) cell liquid for taking appropriate culture to the 13rd day will take after agglomerate piping and druming dispersion
100 μ L are mixed well with the platform phenol indigo plant stain of 100 μ L and are placed on glass slide, observe, take pictures, estimate under microscope (× 100)
Calculate Cell viability.
Embodiment 8:Immunocyte kills ability detection
Immunocyte culture cleans the tumour cell (HEPG-2) in T75 culture bottle after two weeks, with sterile D-hanks liquid
2-3 times, de- wall is digested with 1mL pancreatin, and the DMEM/HIGH GLUCOSE with 2mL containing 10%FBS terminates digestion, piping and druming
15mL centrifuge tube is transferred to after so that it is separated into individual cells, former T75 culture bottle is cleaned with 6mL sterile D-hanks liquid, cleaning
Liquid is incorporated to same 15mL centrifuge tube, and 1000rpm is centrifuged 10 minutes;Abandon supernatant, the DMEM/HIGH with 2mL containing 10%FBS
GLUCOSE is counted after being resuspended, and is 1 × 10 with 10%FBS-DMEM/HIGH GLUCOSE adjustment concentration6Add in/mL, each T25
Enter the 200 above-mentioned cell liquid of μ L (containing 2 × 105/ T25), DMEM/HIGH GLUCOSE of the 5mL containing 10%FBS;At 14 days, previous
It, which overlays, is added corresponding immunocyte 2 × 10 in the T25 of tumour cell6/ T25, and each time point setting blank control (NC, no
Add the tumour cell of immunocyte).Cellkilling capacity is verified by MTT experiment, killing for 24 hours/48h cell microscopy is clapped
According to, microscopy is taken pictures again after being cleaned 2-3 times with sterile D-hanks liquid, remaining tumour cell is digested into de- wall with pancreatin, with
After DMEM/HIGH GLUCOSE containing 10%FBS terminates digestion, a small amount of sterile D-hanks liquid is added to be transferred to 15mL centrifuge tube,
1000rpm is centrifuged 10 minutes;Supernatant is abandoned, (NC is about 2 × 10 with the DMEM/HIGH GLUCOSE resuspension in right amount containing 10%FBS4/
200 μ L) 96 orifice plates are spread afterwards, every kind of cell spreads 6 hole of a column, every 200 μ L cell suspension of hole;It is added after being placed 24 hours in incubator
Solution in hole is sucked out after standing 4 hours in incubator by 20 μ L/ hole MTT, the DMSO of 150 μ L is added, then quiet in the incubator
It sets 30 minutes, is measured at 492nm with microplate reader absorbance (OD value).
Embodiment 9:CTL individually cultivates (Mock), DC and CTL co-cultivation, LEC and CTL co-cultured cell proliferative conditions ratio
Compared with
CTL is individually cultivated to (Mock group) respectively, DC and CTL are co-cultured into (DC group), by LEC and CTL into
Row co-cultures (LEC group), and three kinds of cultures carry out operation repetitive, and initial cell density is 1 × 106/mL.Subsequent each fluid infusion behaviour
Work counts cell, and the cell total amount of each operating point is calculated by count results and corresponding volume, will test twice
Count results summarize after using the operating time as horizontal axis, total number of cells be that the longitudinal axis makees cell Proliferation curve, as a result as shown in figure 3,
From the curve can be seen that with LEC co-culture CTL cell Proliferation it is most fast, DC co-culture base take second place, the two it is final proliferation again
Rate is all higher than 100 times;The cell Proliferation individually cultivated is most slow, and final proliferation multiplying power is also not so good as other two kinds of culture mediums.
The activity of three groups of cells is measured when cultivating to the 13rd day with platform phenol indigo plant, wherein before the piping and druming of LEC group raji cell assay Raji with blow
Latter two state is beaten, as a result as shown in Figure 4.As can be seen from Figure 4 it is grown with the cell of LEC culture in agglomeration, needs to blow and beat
Individual cells can be just dispersed into, DC group and Mock group T cell all disperse to grow, and all >=99%, cell is close for the motility rate of three groups of cells
Spend relatively high, in contrast, Mock group cell quantity is minimum, and state is worst.
Embodiment 10:Influence of the different cultural methods to immunocyte immunophenotype
CTL is individually cultivated to (Mock group) respectively, DC and CTL are co-cultured into (DC group), by LEC and CTL into
Row co-cultures (LEC group), and three kinds of cultures carry out operation repetitives, takes about 15mL cell liquid for every group when culture was to the 13rd day, separation is carefully
After born of the same parents' cleaning treatment, corresponding antibody is added and carries out flow cytometry, detects the CD3+CD4+ cell proportion of cell, compares LEC/
The streamings of tri- groups of culture cells of DC/MOCK as a result, result as shown in table 2 and Fig. 5.
Table 2:Tri- kinds of cultural method T cell phenotypic ratios (%) of LEC/DC/Mock
CD3+ | CD4+ | CD3+CD4+ | |
LEC | 84.7±0.8 | 88.4±0.64 | 76.3±0.57 |
DC | 66.6±0.44 | 66.0±0.78 | 45.9±0.39 |
Mock | 36.5±0.42 | 33.3±0.75 | 21.1±0.33 |
As shown in the result of table 2 and Fig. 5, the cell of LEC group is double positive and three positive mean values are all higher than 80%, and another two groups
The double positive and three positive mean values of cell are below 50%, should be the result shows that carrying out total training compared to independent culture, and with DC
It supports, LEC and CTL is co-cultured more preferably in the proliferation for promoting CD3+CD4+ cell mass, helps to improve in immunocyte and kills
Hurt the ratio of cell.
Embodiment 11:Influence of the different cultural methods to immunocyte killing activity
CTL is individually cultivated to (Mock group) respectively, DC and CTL are co-cultured into (DC group), by LEC and CTL into
Row co-cultures (LEC group), and three kinds of cultures carry out operation repetitive.When culture was to the 14th day, in the tumour cell T25 completed in advance point
Not Jia Ru 10 times of tumour cell quantity the immunocyte obtained by different cultural methods, wash away and exempt from after killing 24 hours
Epidemic disease cell spreads residual tumor cells in 96 orifice plates, and after placing 8 hours plus MTT and DMSO is operated, and stands after the dissolution of first a ceremonial jade-ladle, used in libation
Measure the OD value of 96 orifice plates.Killing 24 hours will be tested three times, 48 hours MTT results obtain histogram after data processing
(Fig. 6), the cell of LEC group can reach the killing activity greater than 60% when killing 24 hours as seen from Figure 6, and the cell of DC group kills
Wound activity is slightly lower, but can also reach 50%, and the cell killing activity of independent culture group is only capable of reaching 15%;When killing 48 hours,
The killing activity of LEC group and DC group cell is all relatively preferable in 50% or so, LEC group fragmentation effect, and independent culture group killing is lived
Property is then lower than 40%.This reflects as the result is shown in terms of the ability for improving cell killing activity, the side that CTL and LEC are co-cultured
Method is also superior to other two training method.
Claims (9)
1. a kind of T cell cultural method, which is characterized in that carry out T cell and lymphatic endothelial cells in the cultural method
It co-cultures.
2. the method as described in claim 1, which is characterized in that the T cell is cytotoxic T cell.
3. the method as described in claim 1, which is characterized in that by T cell and lymphatic endothelial cells in the cultural method
It is co-cultured in serum free medium.
4. the method as described in claim 1, which is characterized in that in the culture medium that the cultural method uses containing cell because
Son.
5. method as claimed in claim 4, which is characterized in that the cell factor is OKT3, IFN-γ, in IL-2, IL-15
One or more.
6. method as claimed in claim 3, which is characterized in that contain KSR in the culture medium that the cultural method uses.
7. a kind of for cultivating the culture medium of T cell, which is characterized in that contain lymphatic endothelial cells in the culture medium.
8. culture medium as claimed in claim 7, which is characterized in that the culture medium is serum free medium.
9. culture medium as claimed in claim 7 or 8, which is characterized in that in the culture medium containing OKT3, IFN-γ, IL-2,
One or more of IL-15.
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SACHIKO HIROSUE等: "Steady-State Antigen Scavenging, Cross-Presentation, and CD8+ T Cell Priming: A New Role for Lymphatic Endothelial Cells", 《THE JOURNAL OF IMMUNOLOGY》 * |
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