CN109402057A - A kind of cultural method for the DC-CTL cell loading tumour cell excretion body - Google Patents
A kind of cultural method for the DC-CTL cell loading tumour cell excretion body Download PDFInfo
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Abstract
The present invention relates to DC-CTL field of cell culture, in particular to a kind of cultural method of DC-CTL cell for loading tumour cell excretion body.A kind of cultural method for the DC-CTL cell loading tumour cell excretion body, comprising the following steps: the peripheral blood DC cell co-culture of CTL cell and load tumour cell excretion body obtains the DC-CTL cell of load tumour cell excretion body；Wherein, culture culture medium used is CTL culture medium, the component of the CTL culture medium are as follows: AIM-V culture medium, autologous plasma and IL-2, the content of the autologous plasma are the 5% ± 0.5% of the AIM-V culture volume, the final concentration of 1000 ± 100IU/mL of IL-2.The DC-CTL cell of the component of Optimal Medium of the present invention, obtained load tumour cell excretion body has good lethality.
Description
Technical field
The present invention relates to DC-CTL field of cell culture, in particular to a kind of DC- for loading tumour cell excretion body
The cultural method of CTL cell.
Background technique
Cytotoxic T lymphocyte (cytotoxic lymphocyte, CTL) is a species specificity, has killing ability
T cell, specially secrete various cell factors and participate in immunizations.Have to antigenic substances such as certain viruses, tumour cells and kills
Wound effect constitutes the important defence line of body disease-resistant poison, antineoplastic immune with natural killer cells.
The CTL being activated can generate the anti-tumor activity of specificity, and this cytotoxicity mainly includes (1) perforation
Element-particle enzymatic pathway；(2) activate CTL high express FaSL, by with target cell surface FaS antigen binding, can induce target cell
Apoptosis；(3) cytokine profiles such as TNF secretion direct killing target cell；(4) a variety of chemotactic factor (CF)s are generated, the innate immunity is attracted
Cell kills target cell；(5) a variety of cruel enzymes of serine are discharged, promote lethal effect by activating perforin.
The immune response for depending on cytotoxic T cell (CTL) of the protection of intracellular infection and tumour, CTL is to swollen
Oncocyte implements the restricted identification of MHC and specific killing.Inside and outside induction, activation and amplification tumor response CTL are current
The key subjects of immunotherapy of tumors research, have huge potential significance.
The method multidigit cracking tumour cell or tumor tissues of existing DC cell loading tumour antigen, extract egg therein
It is loaded after white.The protein ingredient of extraction is complicated, contains antigen intracellular in need, and after activating DC, DC is thin to T into offering in vivo
Born of the same parents, but the antigen presentation is in the intracellular of tumour cell, because specific killing action can not be played.
In view of this, the present invention is specifically proposed.
Summary of the invention
Dendritic Cells (Dendritic cells, DCs) is internal most effective antigen presenting cell, surface expression B7,
A variety of costimulatory molecules such as MHC- class Ⅱmolecule, ICAM-1 activate T helper cell, it is promoted to secrete the cell factors such as IL-2, pass
Specific antigen signal is passed, final Activation of cytotoxic T cell, therefore, it is a series of immune anti-that DC cell plays starting in vivo
The key effect answered, it is significant in the immune improvement of the immune function and tumour of body.
Most of DC cell origins enter peripheral blood in marrow, by marrow, redistribute in respectively organizing to whole body, DC divides extensively
Each internal organs of whole body in addition to brain are distributed in, but quantity is few, only accounts for 1% or less peripheral blood mononuclear cells.DC precursor
After being distributed to each non-lymphoid organ such as enteron aisle, the heart, liver with blood, develop for immature DC cell.At this point, DC cell has relatively by force
Antigen uptake working ability, and activate the inferior capabilities of T cell.Immature DC cell in various non-lymphoid tissues is taken the photograph
After taking antigen, lymph node, spleen are reached, the reduced capability of hereafter DC cellular uptake antigen processing is up to disappearing, and sensitization T lymph
The ability enhancing that cell, starting are immunoreacted, becomes mature DC cell.
During cultivating lymphocyte in vitro, for utmostly activating T cell, need to be added mature DC cell
With sensitized T cell.The in vitro culture of DC cell usually induces immature DC using peripheral blood mononuclear cells as precursor
Cell.It is extremely complex that DC cell promotees maturation, is related to the collective effect of a variety of factors, such as GM-CSF, TNF, IL-6,
IL-4, IL-1 β, PGE2, polyI:C, CD40L etc..
Excretion body (exosomes) is a kind of vesicles of lipid molecular film package.Electric cup-shaped under the microscope, diameter is about
For 40-100nm, density is about 1.13-1.19g/mL.Excretion body contains the cytoplasm and lipid envelope ingredient of derived cell, excretion
Internal portion includes mRNA abundant, microRNA and protein component.
The formation of excretion body is the cellular compartment of more vesica endosomes (MVE) to being formed by way of endogenous budding, intracellular vesicle
It is interior to contain protein, microRNA and mRNA.As MVE and cell membrane fusion, intracellular vesicle is just released to outside extracellular become
Body is secreted, the excretion body released can run to the behavior and change physiologically that cell is influenced apart from farther away tissue.
During tumor development, excretion body participates in intercellular information transmitting, and then promotes tumor vascular
It generates and the transfer of solid tumor, the excretion body in tumour source has become one of the hot spot of tumor cells biological study.It is pernicious swollen
Oncocyte adjusts the generation, development and transfer of itself by the change of its cellular environment, and this adjusting can pass through excretion
Body and the tumor cell secretion factor occur.
Tumour cell source excretion body can both help tumour cell escape immunosurveillance, can also activate tumor-specific immunity
Response.Include tumour specific antigen in the excretion body of tumour source, the restricted T cell clone of mhc class i can be generated in vitro,
And tumour source excretion body can drive the cross-protection for relying on T cell to fight homologous and allogeneic tumor in vivo.Tumour
The excretion body surface face that cell is discharged carries MHC molecule and Antigenic Peptide, and the cytotoxic activity of DC-CTL can be enhanced, and stimulation produces
The anti-tumor T cell of raw specificity, and the DC-CTL cell with specificity and non-specific dual Synergistic action is obtained, to future
The treatment of tumour provides a kind of method that difference is previous.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of cultural method for the DC-CTL cell loading tumour cell excretion body, comprising the following steps:
The peripheral blood DC cell co-culture of CTL cell and load tumour cell excretion body obtains outside load tumour cell
Secrete the DC-CTL cell of body；
Wherein, cultivating culture medium used is CTL culture medium, the component of the CTL culture medium are as follows: AIM-V culture medium, from
Body blood plasma and IL-2, the content of the autologous plasma are the 5% ± 0.5% of the AIM-V culture volume, and the IL-2 is dense eventually
Degree is 1000 ± 100IU/mL.
The component of Optimal Medium of the present invention, the DC-CTL cell of obtained load tumour cell excretion body have good
Lethality.
Further, peripheral blood DC cell co-incubation in culture bottle of CTL cell and load tumour cell excretion body
After 65-75 hours, also expand culture.
Further, culture bottle uses T175 culture bottle, adds 70 ± 10mL of CTL culture medium.
It is specifically, described that expand culture be that the cell in culture bottle is put into culture bag and cultivates, add CTL culture medium 400 ±
50mL。
After expanding culture 20-30 hours, the DC-CTL cell of load tumour cell excretion body is obtained.
Further, the CTL cell is prepared by the following method:
After culture vessel pan coating, access PBMC is cultivated, wherein cultivating culture solution used is containing 4%~6%
IFN-γ, final concentration of 1000 ± 100U/ of the IFN-γ in culture solution is added in the VIVO culture medium of autologous plasma wherein
mL；
After culture 20-24 hours, IL-1 α, IL-2, IL-12 and IL-7 are added in culture solution, wherein in culture solution,
IL-1 α concentration is 1000 ± 100U/mL, IL-2 concentration is 1000 ± 100U/mL, IL-12 concentration is that 20 ± 2ng/mL, IL-7 are dense
Degree is 50 ± 3ng/mL, continues to cultivate；
After culture 65-75 hours, cell is transferred to new culture vessel, cultivates in the CTL culture medium, obtains described
CTL cell.
Further, the component of the culture solution includes AIM-V culture medium, autologous plasma, the content of the autologous plasma
It is the 5% ± 0.5% of the AIM-V culture volume.
Further, the coating is carried out using coating buffer, and the component of the coating buffer includes D-PBS, CD3 and CD28,
Final concentration of 500 ± 50ng/mL of final concentration of 500 ± 50ng/mL of the CD3, the CD28.
When coating buffer is coated with, in terms of T25 culture bottle, 3mL coating buffer is added, is placed in incubator and is coated with 2h；It goes to be coated with
The culture vessel of culture vessel pan coating can be obtained in liquid.
Later, in terms of T25 culture bottle, PBMC number of inoculation is 0.5 × 107-2.0×107；After inoculation, add 15mL above-mentioned
Culture solution, that is, CTL initial medium, while IFN-γ be added make its final concentration of 1000 ± 100U/mL, cultivated.
In the present invention, if not specified to condition of culture, condition of culture is 37 DEG C, 5%CO2, saturated humidity
It is cultivated in incubator.
Further, the peripheral blood DC cell of the load tumour cell excretion body is prepared by the following method:
(a) PBMC cell adhere-wall culture removes culture solution, washs cell；
(b) DC culture medium is then added, cultivates 64-80h；
(c) culture bottle is patted, attached cell is floated, adds DC culture medium and tumour cell excretion body, cultivates 40-50h；
(d) it is collected after cell suspending, obtains cell, cell is transferred to and has used the coated culture vessel of autologous plasma
In, DC maturation medium is added, cultivates 45-55h, obtains the peripheral blood DC cell of load tumour cell excretion body.
The peripheral blood DC cell culture processes of load tumour cell excretion body provided by the invention, tumour cell excretion physical efficiency
Enough promote the maturation of DC cell, DC cell function phenotype CD11c/CD83, CD11c/CD86, HLA-DR/CD40 are better than unsupported
The DC of excretion body.
Further, the DC maturation medium adds following components by following content on the basis of AIM-V culture medium:
Autologous plasma percentage by volume be 1% ± 0.2%, GM-CSF, 50 ± 2ng/mL, 50 ± 2ng/mL of IL-4, TNF-α 10 ±
1 ± 0.2 μ g/mL of 2ng/mL, PGE-2.
DC maturation medium provided by the invention provides good basis for DCs maturation.
Further, in step (a), the PBMC cell adhere-wall culture specific steps are as follows:
In terms of T175 culture bottle, PBMC cell 1 × 10 is accessed8-1.5×108It is a, while AIM-V culture medium 50- is added
60mL and autologous plasma, the volume of the autologous plasma are the 0.1% of the AIM-V culture volume, adhere-wall culture 2-3h.
Further, it in step (a), is washed cell 2-3 times using AIM-V culture medium.
Further, in step (b), in terms of T175 culture bottle, the addition volume of the DC culture medium is 30-40mL.
Further, in step (c), in terms of T175 culture bottle, the volume for the DC culture medium added is 15-20mL, addition
Tumour cell excretion body volume be 80-150 μ L.
Further, in step (d), the step of collection, includes: after cell is suspended
Culture bottle is patted, so that cell is suspended, cell liquid is collected；
At this point, there is a large amount of cells adherent in culture bottle, into culture bottle plus PBS, stand at low temperature are patted, and collect cell liquid,
Repeat the step；
The cell liquid of collection is centrifuged, and is removed supernatant, is obtained the cell.
Further, the parameter of the cell liquid centrifugation of collection are as follows: 1400-1500rpm, 3-5min.
Further, the tumour cell excretion body is prepared by the following method:
It is passed on when culture tumour cell to cell length to 85% ± 10% fusion；
Secondary culture 20-30h siphons away culture solution and not adherent cell, and is cleaned, and it is dedicated that excretion body is then added
Complete culture solution is cultivated；
Culture solution is collected after 45-50h into centrifuge tube, obtains the culture solution rich in tumour cell excretion body；
Wherein, the component of the dedicated complete culture solution of excretion body are as follows: without phenol red RPMI-1640 culture solution and without excretion body
The additive amount of FBS, the no excretion body FBS are 10% ± 2% without phenol red RPMI-1640 nutrient solution volume；
The culture solution rich in tumour cell excretion body is isolated to the tumour cell excretion body.
Tumour cell excretion body culture provided by the invention and separation method, method is easy, obtained tumour cell excretion
Body content is high, and other kinds of excretion body is wherein not detected, purity is high.
In the present invention, no excretion body FBS can be prepared by existing method, can also be prepared by the following method: FBS in
The revolving speed of 100000g-120000g is centrifuged 8-14h；
Draw upper layer clarify serum, and use 0.22 μm of membrane filtration, filtrate as without excretion body FBS.
Further, it is described passage according to spread cultivation 3-5 times ratio progress.That is, one bottle of cell passes on to obtain 3-5
Bottle cell.
Further, the culture of the tumour cell is carried out using the common complete medium of tumour cell, and the tumour is thin
The component of the common complete medium of born of the same parents are as follows: RPMI-1640 culture solution and FBS, the additive amount of the FBS are the RPMI-1640
The 10% ± 2% of nutrient solution volume.
Further, it is cleaned 2-3 times with PBS.Cleaning is to further remove the raffinate not removed and other fragments.
Further, the dedicated complete culture solution of excretion body of 4%-6% volume is added in each culture bottle.
In T75 Tissue Culture Flask (250mL), the dedicated complete culture solution of excretion body of 10-15mL is added, such as T75 cell
It can be 10mL, 12mL, 15mL etc. in culture bottle (250mL).
Further, the culture solution rich in tumour cell excretion body is isolated to the tumour cell excretion body
Steps are as follows:
The culture solution low-temperature centrifugation rich in tumour cell excretion body removes dead cell and fragment, then filtering removal
Vesica；
Obtained liquid carries out ultracentrifugation, abandons supernatant, obtains tumour cell excretion body.
Tumour cell excretion body separation method provided by the invention handles tumour cell by specific method, obtains richness
The culture solution of the body of excretion containing tumour cell, then separates it, obtains that concentration is higher, the higher tumour cell excretion of purity
Body.
Further, the low-temperature centrifugation is to be centrifuged at 4 ± 2 DEG C with 2000-2200g.It is centrifuged, is gone by the rate
Except dead cell and fragment.
Further, the time of the centrifugation is 5-10min.
Further, the aperture of strainer used in filtering removal vesica is 0.22 μm.By filtering, larger vesica is removed.
Further, the liquid obtained carries out in ultracentrifugation step, and the revolving speed of centrifugation is 100000-120000g, centrifugation
Time is 2-3h.
By ultracentrifugation, excretion body precipitates.
It further, further include removing the liquid on centrifuge container tube wall after the abandoning supernatant, centrifuge container bottom
Substance is tumour cell excretion body.
By removing liquid, purer tumour cell excretion body is obtained.
Centrifuge container is generally centrifuge tube.
Further, the separation method is further comprising the steps of: obtained tumour cell excretion body is resuspended with PBS, is protected
It deposits.
It can such as be saved at -20 DEG C.
The tumour cell excretion body of the container bottom obtained is added PBS and is resuspended, and saves.
The isolated tumour cell excretion body of the present invention is detected, as under Electronic Speculum and the inspection of surface markers albumen
It surveys, meets the characteristic of tumour cell excretion body, illustrate that the isolated tumour cell excretion body of the present invention is reliable and stable.
In addition, calculating, calculated with passing on the tumour cell of inoculation, the yield of excretion body be 155.32 ± 34.3 μ g/1 ×
107cells。
Compared with prior art, the invention has the benefit that
(1) separation method of tumour cell excretion body provided by the invention is calculated, excretion with passing on the tumour cell of inoculation
The yield of body is 155.32 ± 34.3 g/1 × 10 μ7cells。
(2) present invention contains massive tumor related antigen by tumour cell excretion body, being capable of efficient activation DC, promotion DC
Cell maturation, so that T cell plays accurate antitumor action subsequent.
(3) it is provided by the invention load tumour cell excretion body peripheral blood DC cell culture processes, with inoculation 1.5 ×
108A cell calculates, and the mature DC cell number of harvest is 1.0 × 108A cell or so, mature DC cell yield 65% with
On, there is very high yield.
(4) cultural method of the DC-CTL cell of load tumour cell excretion body provided by the invention, outside obtained load
The DC-CTL cell for secreting body has good inhibiting rate to corresponding tumour cell.
Detailed description of the invention
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 technical description to be briefly described.
Fig. 1 is that the cell of the embodiment of the present invention 2 carries out streaming phenotype CD11c/CD14 detection figure；
Fig. 2 is that the cell of the embodiment of the present invention 2 carries out streaming phenotype CD11c/CD83 detection figure；
Fig. 3 is that the cell of the embodiment of the present invention 2 carries out streaming phenotype CD11c/CD86 detection figure；
Fig. 4 is that the cell of the embodiment of the present invention 2 carries out streaming phenotype HLA-DR/CD40 detection figure；
Fig. 5 is that the cell of the embodiment of the present invention 2 carries out streaming phenotype HLA-DR/CD80 detection figure；
Fig. 6 is that the cell of comparative example 1 of the present invention carries out streaming phenotype CD11c/CD14 detection figure；
Fig. 7 is that the cell of comparative example 1 of the present invention carries out streaming phenotype CD11c/CD83 detection figure；
Fig. 8 is that the cell of comparative example 1 of the present invention carries out streaming phenotype CD11c/CD86 detection figure；
Fig. 9 is that the cell of comparative example 1 of the present invention carries out streaming phenotype HLA-DR/CD40 detection figure；
Figure 10 is that the cell of comparative example 1 of the present invention carries out streaming phenotype HLA-DR/CD80 detection figure；
Figure 11 is that the cell of experimental example of the present invention carries out streaming phenotype HLA-DR/CD80 detection figure.
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 is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
The preparation of tumour cell excretion body
1, culture medium
The component of the common complete medium of tumour cell are as follows: the additive amount of RPMI-1640 culture solution and FBS, FBS is
The 10% of RPMI-1640 nutrient solution volume.
The component of the dedicated complete culture solution of excretion body are as follows: without phenol red RPMI-1640 culture solution and without excretion body FBS, without outer
The additive amount of body FBS is secreted for 10% without phenol red RPMI-1640 nutrient solution volume.
2, it is prepared without excretion body FBS
Under aseptic condition by FBS pour into it is dedicated surpass from centrifuge tube, every pipe 38.6mL, 4 pipes are put into super after confirming trim
In fast centrifuge, 120000g is centrifuged overnight.
Second day, careful upper layer clarification serum of drawing into new sterile centrifugation tube, 0.22 μm of membrane filtration to it is another from
In heart pipe, it is ensured that the germ-free condition of serum, sealing are spare.
3, tumour cell excretion body cultural method
T75 Tissue Culture Flask, common complete medium culture tumour cell, when cell length to 80% fusion, according to 1:4
It is passed on.
Second day, common complete medium and not adherent cell are carefully siphoned away, is carefully cleaned with PBS 3 times, every bottle of addition
The dedicated complete medium of 10mL excretion body.
Collection culture medium arrives the culture medium rich in tumour cell excretion body into centrifuge tube after 48h.
4, tumour cell excretion body separation method
General low temperature centrifuge and ultracentrifuge are opened in advance, are cooled to 4 DEG C in advance.
The centrifuge tube of culture medium rich in tumour cell excretion body is centrifuged 10min in 2000g, removes dead cell and fragment.
0.22 μm of membrane filtration removes larger vesica.
Filtrate goes in ultracentrifugation pipe, and with PBS trim, 120000g is centrifuged 2h.
It discards supernatant, tube wall residual liquid is carefully siphoned away with aseptic filter paper, tube bottom is tumour cell excretion body.It weighs outer
The weight of body is secreted, is calculated, is calculated with passing on the tumour cell of inoculation, the yield of excretion body is 167 g/1 × 10 μ7cells。
The tumour cell excretion body of obtained tube bottom is hanged each pipe excretion weight with 1mL PBS, mixing, -80 DEG C of preservations.
Embodiment 2
A kind of peripheral blood DC cell culture processes loading tumour cell excretion body, including the following contents:
1, peripheral blood DC cell culture medium
1) DC culture medium
On the basis of AIM-V culture medium, by following content add following components: autologous plasma percentage by volume be 1% ±
0.2%, GM-CSF concentration 50ng/mL, IL-4 concentration 50ng/mL.
2) DC maturation medium
On the basis of AIM-V culture medium, by following content add following components: autologous plasma percentage by volume be 1% ±
1 μ g/mL of 0.2%, GM-CSF concentration 50ng/mL, IL-4 concentration 50ng/mL, TNF-α concentration 10ng/mL, PGE-2 concentration.
2, peripheral blood DC cell culture processes
It cultivates the 0th day, takes 1 T175 culture bottle, obtained PBMC cell is added in culture bottle, every bottle of cell number 1.5
×108, AIM-V culture medium 50mL is added, adds 500 μ L autologous plasmas, incubator adhere-wall culture 2h.
2) after cultivating 2h, culture bottle is taken out, removes supernatant, 30mL AIM-V culture medium is added and washes culture bottle, washes 2 times.
The DC culture medium 30mL prepared, incubator culture is added.
3) the 3rd day, culture bottle is taken out, culture bottle is patted, floats attached cell.It adds DC culture medium 20mL and tumour is thin
It is extracellular to secrete 100 μ L of body, incubator culture.
4) 1 new T75 culture bottle is taken within the 4th day, 7mL autologous plasma is added, is placed in incubator and is coated with overnight.
5) the 5th day, DC culture bottle is taken out, culture bottle is patted, so that cell is suspended, cell liquid is collected into 50mL centrifuge tube
It is interior.Observation has a large amount of cells adherent at this time, and 15mL PBS is added into culture bottle, is placed in 4 DEG C of 15min.Culture bottle is taken out, is patted,
Most cells floating.PBS is poured out, 15mL PBS is added, is placed in 4 DEG C of 5min.After 5min, culture bottle is taken out, is patted, carefully
Born of the same parents float substantially, and PBS is poured into 50mL centrifuge tube, and centrifugation, 1400rpm, 5min after centrifugation, remove supernatant.It takes out the 4th day and wraps
The T75 culture bottle of quilt, goes coating buffer.Cell is transferred in T75 culture bottle, 20mL maturation medium is added.
6) the 7th day, cell, streaming Phenotypic examination are harvested.
Comparative example 1: as different from Example 2, tumour cell excretion body is not added.
The streaming Phenotypic examination for the cell that embodiment 2 and comparative example 1 obtain is as Figure 1-10 shows.
Fig. 1-5 is the streaming phenotypic map of the raji cell assay Raji of embodiment 2, and Fig. 6-10 is the streaming of the raji cell assay Raji of comparative example 1
Phenotypic map.
The result of Fig. 1-10 is more as shown in table 1.
1 result of table compares
After loading tumour cell excretion body, CD14 phenotype is lower, and more mononuclearcells are divided into DC cell, explanation
Tumour cell excretion body can promote the maturation of DC cell, DC cell function phenotype CD11c/CD83, CD11c/CD86, HLA-
DR/CD40 is better than the DC of unsupported excretion body.
Embodiment 3
The cell that embodiment 2 harvests, centrifugation remove culture medium, will load the peripheral blood DC cell of tumour cell excretion body
Co-incubation obtains the DC-CTL cell of load tumour cell excretion body, the specific steps are as follows:
One, solution
1, coating buffer: D-PBS, CD3 monoclonal antibody concentration 500ng/mL, CD28 concentration 500ng/mL.
2, CTL initial medium: VIVO culture medium (contains 5% autologous plasma).
3, CTL culture medium: AIM-V culture medium (contains 5% autologous plasma), IL-2 concentration 1000IU/mL.
Two, peripheral blood DC-CTL cultivates cellular processes
1) it cultivates the 0th day, takes T25 culture bottle, 3mL coating buffer is added, is placed in incubator and is coated with 2h, it is spare.Take out packet
By good T25 culture bottle, coating buffer is gone, takes PBMC1.0 × 10 prepared7, it is inoculated into T25 culture bottle, adds 15mLCTL
Initial medium is added the final concentration of 1000U/mL of IFN-γ, is placed in 37 DEG C, 5%CO2It is cultivated in incubator.
2) it cultivates the 1st day, takes out culture bottle, sequentially add: IL-1 α makes its final concentration of 1000U/mL；IL-2 makes its end
Concentration is 1000U/mL；IL-12 makes its final concentration of 20ng/mL, IL-7 make its final concentration of 50ng/mL, again by culture bottle
It is placed in 37 DEG C, 5%CO2It is cultivated in incubator.
3) it cultivates the 4th day, cell is transferred completely into T75, adds CTL culture medium 20mL, is placed in 37 DEG C, 5%CO2Incubator
Middle culture.
4) it cultivates the 7th day, obtains CTL cell；
The peripheral blood DC cell for the load tumour cell excretion body that obtained CTL cell is obtained with embodiment 2 shifts simultaneously
Into T175, adds CTL culture medium 70mL, be placed in 37 DEG C, 5%CO2It is cultivated in incubator.
5) it cultivates the 10th day, cell is transferred to addition 400mLCTL culture medium in cell culture bags, is placed in 37 DEG C, 5%
CO2It is cultivated in incubator.
6) it cultivates the 12nd day, harvests cell, obtain the DC-CTL cell of load tumour cell excretion body.
The excretion body for extracting lung tumor cell A549 according to the method for embodiment 1 is loaded according to the method for embodiment 2 and 3
It after DC-CTL, is co-cultured with A549 cell, carries out killing experiments in vitro.The results are shown in Table 2.
2 killing experiments in vitro result of table
| CTL:A549 | Load excretion body | Unsupported excretion body |
| 5:1 | 64.58% | 49.27% |
| 10:1 | 63.36% | 38.25% |
| 20:1 | 67.30% | 49.20% |
It is detected by CCK8, the CTL for loading excretion body is calculated to the inhibiting rate of tumour cell higher than unsupported
CTL。
Comparative example 2
Peripheral blood CTL cultivates cellular processes
1) it cultivates the 0th day, takes the PBMC 1.0 × 10 prepared7, it is inoculated into T25 culture bottle (without coating), adds
10mLCTL initial medium is placed in 37 DEG C, 5%CO2In incubator.
2) it cultivates the 1st day, takes out culture bottle, IL-1 α and IL-2 is added, final concentration is 1000U/mL, and IL-7 is added
CD3 monoclonal antibody concentration 500ng/mL, CD28 antibody concentration 500ng/mL is added in 50ng/mL, IL-12 20ng/mL；By culture bottle weight
Newly it is placed in 37 DEG C, 5%CO2In incubator.
CTL cell culture of remaining step with embodiment 3.
Comparative example 3
The replacement of CTL cell culture medium are as follows:
1) CTL initial medium: RPM1-1640 culture medium (contains 5% autologous plasma), IFN-γ 1000U/mL.
2) CTL culture medium: RPM1-1640 culture medium (contains 5% autologous plasma), IL-2 concentration 1000IUmL.
CTL cell culture of remaining condition of culture with embodiment 3.
Comparative example 4
The present embodiment is related to the cultural method of peripheral blood CTL a kind of.
1. peripheral blood CTL cell culture medium
1) coating buffer: D-PBS, CD3 monoclonal antibody concentration 500ng/mL, CD28 antibody concentration 500ng/mL.
2) CTL initial medium: VIVO culture medium (contains 5% autologous plasma), IFN-γ 1000U/mL.
3) CTL culture medium: VIVO culture medium (contains 5% autologous plasma), IL-2 concentration 1000IUmL.
2. peripheral blood CTL cultivates cellular processes
1) it cultivates the 0th day, takes T25 culture bottle, 3mL coating buffer is added, is placed in incubator and is coated with 2h, it is spare.Take out packet
By good T25 culture bottle, coating buffer is gone, takes PBMC1.0 × 10 prepared7, it is inoculated into T25 culture bottle, adds 10mLCTL
Initial medium is placed in 37 DEG C, 5%CO2In incubator.
2) it cultivates the 1st day, takes out culture bottle, IL-1 α and IL-2 is added, final concentration is 1000U/mL, by culture bottle weight
Newly it is placed in 37 DEG C, 5%CO2In incubator.
3) it cultivates the 4th day, cell is transferred completely into T75, adds CTL culture medium 20mL, is placed in 37 DEG C, 5%CO2Incubator
In.
4) it cultivates the 7th day, cell is transferred in T175 culture bottle, adds CTL culture medium 70mL, is placed in 37 DEG C, 5%CO2
In incubator.
5) it cultivates the 10th day, cell is transferred to addition 400mLCTL culture medium in culture bag, is placed in 37 DEG C, 5%CO2Training
It supports in case.
6) it cultivates the 14th day, harvests cell, do flow cytometer detection.
Experimental example
After collecting the CTL cell of embodiment 3 and the cell of comparative example 1,2, cell count is carried out, and pass through flow cytometry
Phenotype is identified.Every group repetition six times, as a result as shown in Table 3 and Table 4.
3 cell number of table
| Group | Cell number |
| Embodiment 3 | 9.1×108 |
| Comparative example 2 | 8.4×108 |
| Comparative example 3 | 3.3×108* |
| Comparative example 4 | 5.3×108* |
* p < 0.05, vs embodiment 3.
Qualification result of 4 flow cytometry of table to phenotype
* p < 0.05, vs embodiment 3.
Even if the quantity of cell harvest is still few it can be seen from the results above that 2~4 incubation time of comparative example is longer
In the application；In addition, the culture medium prescription of the application optimization can preferably activate CTL, and by control differentiation opportunity, reduce
The ratio of T-reg cell.
It should be noted that the unmentioned condition of culture of the present invention is 37 DEG C, 5%CO2, saturated humidity.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of cultural method for the DC-CTL cell for loading tumour cell excretion body, which comprises the following steps:
The peripheral blood DC cell co-culture of CTL cell and load tumour cell excretion body obtains load tumour cell excretion body
DC-CTL cell；
Wherein, cultivating culture medium used is CTL culture medium, the component of the CTL culture medium are as follows: AIM-V culture medium, self blood
Slurry and IL-2, the content of the autologous plasma are the 5% ± 0.5% of the AIM-V culture volume, and the IL-2 is final concentration of
1000±100IU/mL。
2. cultural method according to claim 1, which is characterized in that the CTL cell is prepared by the following method:
After culture vessel pan coating, access PBMC is cultivated, wherein it is self containing 4%~6% for cultivating culture solution used
IFN-γ, final concentration of 1000 ± 100U/mL of the IFN-γ in culture solution is added in the VIVO culture medium of blood plasma wherein；
After culture 20-24 hours, IL-1 α, IL-2, IL-12 and IL-7 are added in culture solution, wherein in culture solution, IL-1
α concentration is 1000 ± 100U/mL, IL-2 concentration is 1000 ± 100U/mL, IL-12 concentration is 20 ± 2ng/mL, IL-7 concentration is
50 ± 3ng/mL continues to cultivate；
After culture 65-75 hours, cell is transferred to new culture vessel, cultivates in the CTL culture medium, obtains the CTL
Cell；
Further, the component of the culture solution includes AIM-V culture medium, autologous plasma, and the content of the autologous plasma is institute
State the 5% ± 0.5% of AIM-V culture volume；
Further, the coating is carried out using coating buffer, and the component of the coating buffer includes D-PBS, CD3 and CD28, described
Final concentration of 500 ± 50ng/mL of final concentration of 500 ± 50ng/mL of CD3, the CD28.
3. cultural method according to claim 2, which is characterized in that the peripheral blood DC of the load tumour cell excretion body
Cell is prepared by the following method:
(a) PBMC cell adhere-wall culture removes culture solution, washs cell；
(b) DC culture medium is then added, cultivates 64-80h；
(c) culture bottle is patted, attached cell is floated, adds DC culture medium and tumour cell excretion body, cultivates 40-50h；
(d) it is collected after cell suspending, obtains cell, cell is transferred to and has been used in the coated culture vessel of autologous plasma,
DC maturation medium is added, cultivates 45-55h, obtains the peripheral blood DC cell of load tumour cell excretion body；
Further, the DC maturation medium adds following components by following content: self on the basis of AIM-V culture medium
Plasma volumes percentage be 1% ± 0.2%, GM-CSF, 50 ± 2ng/mL, 50 ± 2ng/mL of IL-4,10 ± 2ng/mL of TNF-α,
PGE-2 1±0.2μg/mL。
4. cultural method according to claim 3, which is characterized in that in step (a), the PBMC cell adhere-wall culture tool
Body step are as follows:
In terms of T175 culture bottle, PBMC cell 1 × 10 is accessed8-1.5×108It is a, at the same be added AIM-V culture medium 50-60mL and
Autologous plasma, the volume of the autologous plasma are the 0.1% of the AIM-V culture volume, adhere-wall culture 2-3h；
Further, it in step (a), is washed cell 2-3 times using AIM-V culture medium；
Further, in step (b), in terms of T175 culture bottle, the addition volume of the DC culture medium is 30-40mL；
Further, in step (c), in terms of T175 culture bottle, the volume for the DC culture medium added is 15-20mL, and addition is swollen
The volume of oncocyte excretion body is 80-150 μ L；
Further, in step (d), the step of collection, includes: after cell is suspended
Culture bottle is patted, so that cell is suspended, cell liquid is collected；
At this point, there is a large amount of cells adherent in culture bottle, into culture bottle plus PBS, stand at low temperature are patted, and are collected cell liquid, are repeated
The step；
The cell liquid of collection is centrifuged, and is removed supernatant, is obtained the cell；
Further, the parameter of the cell liquid centrifugation of collection are as follows: 1400-1500rpm, 3-5min.
5. cultural method according to claim 4, which is characterized in that the tumour cell excretion body is made by the following method
It is standby:
It is passed on when culture tumour cell to cell length to 85% ± 10% fusion；
Secondary culture 20-30h siphons away culture solution and not adherent cell, and is cleaned, and it is dedicated completely that excretion body is then added
Culture solution is cultivated；
Culture solution is collected after 45-50h into centrifuge tube, obtains the culture solution rich in tumour cell excretion body；
Wherein, the component of the dedicated complete culture solution of excretion body are as follows: without phenol red RPMI-1640 culture solution and without excretion body FBS,
The additive amount of the no excretion body FBS is 10% ± 2% without phenol red RPMI-1640 nutrient solution volume；
The culture solution rich in tumour cell excretion body is isolated to the tumour cell excretion body.
6. cultural method according to claim 5, which is characterized in that it is described passage according to spread cultivation 3-5 times ratio progress；
Further, the culture of tumour cell is carried out using the common complete medium of tumour cell, and the tumour cell is commonly complete
The component of full culture medium are as follows: RPMI-1640 culture solution and FBS, the additive amount of the FBS are that the RPMI-1640 cultivates liquid
Long-pending 10% ± 2%.
7. cultural method according to claim 5, which is characterized in that cleaned 2-3 times with PBS；
Further, the dedicated complete culture solution of excretion body of 4%-6% volume is added in each culture bottle.
8. cultural method according to claim 5, which is characterized in that the culture solution warp rich in tumour cell excretion body
The step of isolated tumour cell excretion body, is as follows:
The culture solution low-temperature centrifugation rich in tumour cell excretion body removes dead cell and fragment, then filtering removal vesica；
Obtained liquid carries out ultracentrifugation, abandons supernatant, obtains tumour cell excretion body.
9. tumour cell excretion body separation method according to claim 8, which is characterized in that the low-temperature centrifugation is 4 ± 2
It is centrifuged at DEG C with 2000-2200g；
Further, the time of the centrifugation is 5-10min；
Further, the aperture of strainer used in filtering removal vesica is 0.22 μm.
10. cultural method according to claim 8, which is characterized in that obtained liquid carries out in ultracentrifugation step, from
The revolving speed of the heart is 100000-120000g, centrifugation time 2-3h；
It further, further include by the liquid removal on centrifuge container tube wall, the substance of centrifuge container bottom after the abandoning supernatant
As tumour cell excretion body；
Further, the separation method is further comprising the steps of: obtained tumour cell excretion body is resuspended with PBS, is saved.
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