CN109825475A - A kind of the induced activation kit and its application method of DC-CTL cell - Google Patents
A kind of the induced activation kit and its application method of DC-CTL cell Download PDFInfo
- Publication number
- CN109825475A CN109825475A CN201910100720.8A CN201910100720A CN109825475A CN 109825475 A CN109825475 A CN 109825475A CN 201910100720 A CN201910100720 A CN 201910100720A CN 109825475 A CN109825475 A CN 109825475A
- Authority
- CN
- China
- Prior art keywords
- cell
- ctl
- pipe
- added
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of induced activation kit of DC-CTL cell and its application methods, the kit includes box cover and box body, the tray interior is equipped with a sponge support, the sponge, which drags, to be set there are six fixation hole, the reagent bottle of built-in modulation CTL cytokine activation liquid IL2 is placed in six fixation holes respectively, the reagent bottle of built-in modulation CTL cytokine activation liquid IFN-γ, the reagent bottle of built-in modulation DC cytokine activation liquid IL-4 and GM-CSF mixed liquor, the reagent bottle of built-in modulation DC cytokine activation liquid TNF-α, the reagent bottle of built-in lymphocyte separation medium, the reagent bottle of built-in serum-free medium.Therefore, the induced activation kit forms of DC-CTL cell of the present invention are simple, practical and convenient, and reagent bottle is split type structure, are convenient for cleaning and sterilizing, reduce the risk of microbiological contamination in incubation, can cultivate the uniform DC-CTL cell of mass in a short time.
Description
Technical field
The present invention relates to kit or reagent box technical fields, and in particular to a kind of induced activation reagent of DC-CTL cell
Box and its application method.
Background technique
Tumor biotherapy is a big hot spot in current tumor research field, and wherein tumour adoptive immunotherapy is in tumour
In occupation of critical role in biological therapy.For promoting the reconstruction of patients immune system, eliminating microresidual disease and marrow
Purification all has good result.A critical issue in tumour adoptive immunotherapy is that the suitable tumor-killing of searching is thin
Born of the same parents.It is prolonged to explore, it was recognized that the basis of tumour immunity is the presence and immune system pair of tumour specific antigen
The effective response of tumour cell, and recognize that cellular immunity plays a key effect in immunotherapy of tumors, especially have anti-
The Dendritic Cells (DC, dendritic cell) of former presentation ability and the CTL cell with killing ability, to be tumour
Immunization therapy is possibly realized.
In recent years deeply to the research of DC-CTL and research and development, the induction and culture of more mature DC-CTL have been formd
Method, but the reagent used in cell cultivation process is inconsistent compared with multi-standard, the heavy workload in incubation, incubation time
Long, microbiological contamination risk is big, and many researchers is made to be difficult to cultivate the uniform DC-CTL cell of mass in a short time.
Therefore, it is necessary to the kit and its application method of a kind of new DC-CTL cell induced activation be designed, to overcome
Drawbacks described above.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of induced activation kit of DC-CTL cell and its application sides
Method can cultivate the uniform DC-CTL cell of mass in a short time.
One of mesh of the invention is to provide a kind of induced activation kit of DC-CTL cell, including band box cover and box
Body, the tray interior are equipped with a sponge support, which, which drags, sets there are six fixation hole, place respectively in six fixation holes
There is the reagent bottle of content modulation CTL cytokine activation liquid IL-2, content modulates the reagent of CTL cytokine activation liquid IFN-γ
Bottle, content modulate the reagent bottle of DC cytokine activation liquid IL-4 and GM-CSF mixed liquor, and content modulates DC cytokine activation
The reagent bottle of liquid TNF-α, the reagent bottle of content lymphocyte separation medium and the reagent bottle of content serum-free medium.
Preferably, the concentration of IL-2 is 300-1200IU/ml, IFN-γ concentration is 400-1600U/ml, IL-4 and GM-
It is 600-1500U/ml that the concentration of IL-4, which is 50-300ng/ml and GM-CSF concentration, in CSF mixed liquor, and the concentration of TNF-α is
300-1200U/ml。
Preferably, it is 1000U/ that the concentration of IL-4, which is 100ng/ml and GM-CSF concentration, in IL-4 and GM-CSF mixed liquor
ml U/ml。
Preferably, the concentration of TNF-α is 500U/ml.
Preferably, IFN-γ concentration is 1000U/ml.
Preferably, the concentration of IL-2 is 500IU/ml.
In order to achieve the above object, being used the present invention also provides a kind of application method of the induced activation kit of DC-CTL cell
In the induced activation kit of DC-CTL cell, which includes modulation CTL cytokine activation liquid IL-2, and modulation CTL is thin
Intracellular cytokine activating fluid IFN-γ modulates DC cytokine activation liquid IL-4 and GM-CSF mixed liquor, modulates DC cytokine activation
Liquid TNF-α, lymphocyte separation medium and serum-free medium, include the following steps:
Single milling machine acquires peripheral blood mononuclear cells 1 × 109-3×109；
Blood pretreatment: acquired 50ml peripheral blood mononuclear cells is placed in the centrifuge tube (A pipe) of 1 50ml, from
Heart A manages (400-1200g, 8-30min), and supernatant is transferred to that 50ml centrifuge tube is to be processed spare, and the cell of A pipe lower part makees ficoll
PBMC (peripheral blood mononuclear cells) is separated to use；
The separation of cell: the A that blood pretreatment obtains manages lower confluent monolayer cells and is diluted with physiological saline 1:1, mixes, 40ml is drenched
Bar cell separating liquid ficoll equivalent, which is divided equally, is put into 2 50ml graduated centrifuge tubes (D pipe and E pipe).By diluted cell in A pipe
Suspension equivalent, which is divided equally, is slowly at the uniform velocity added D pipe and E pipe along tube wall.Attention is firmly uniform during being added dropwise, and avoids damage to ficoll-
The integrality at leucocyte interface, centrifugation D pipe and E pipe (300-1200g, 10-35min, centrifuge lifting speed are transferred to most slow), centrifugation
Afterwards, liquid in pipe is divided into three layers, and pipette is drawn after discarding upper layer about 15ml liquid, middle layer interface white cloud and mist layer is narrow
Band (PBMC, about 5ml needed for i.e.) is put into H pipe, and supernatant is abandoned in physiology salt washing 2 times (600g, 8min) afterwards, and every pipe 40ml serum-free is thin
Born of the same parents' culture solution is resuspended cell and separates PBMC obtained；
Inoculating cell: 200ml-600ml PBMC cell suspension is separately added into Tissue Culture Flask T175cm2, it is wet to set saturation
Degree, 37 DEG C, 3.0%-10.0%CO2Culture, after 2 hours, by the taking-up of suspension in Tissue Culture Flask, magnetic bead sorting obtains CD8+T
Lymphocyte freezes as T cell；50ml/ bottles of culture solutions and 50ul DC cell inducible factors are added in attached cell therein
50-300ng/ml IL-4 and 600-1500U/ml GM-CSF mixed liquor is used for DC cell Fiber differentiation；
Culture to the 4th day, into DC Tissue Culture Flask be added 10ul DC cell inducible factors 50-300ng/ml IL-4 and
600-1500U/ml GM-CSF mixed liquor；
Tumour antigen 5ul was added to the 6th day in culture；
Culture was added 10ul DC cell inducible factors TNF-α (300-1200U/ml) to the 7th day；
8th day, mature DC cell was divided into 3 parts by recovery T cell, and 2 parts are used to freeze, 1 part thin with the T of recovery
Born of the same parents co-culture, and activating solution 300-1200IU/ml IL2 are added, 400-1600U/ml IFN-γ induces specific for tumour antigen DC-
CTL, and sample and carry out DC label detection；
9th day, DC-CTL was added in 1 part of DC cell of recovering, while activating solution 300-1200IU/ml IL2,400- is added
1600U/ml IFN-γ cultivates DC-CTL；
10th day, DC-CTL was added in 1 part of DC cell of recovering, while activating solution 300-1200IU/ml IL2,400- is added
1600U/ml IFN-γ cultivates DC-CTL；
11st day, coated cell culture bottle is added in DC-CTL, DC-CTL is expanded；
13rd day, activating solution 300-1200IU/ml IL2,400-1600U/ml IFN-γ and cell culture fluid pair is added
DC-CTL is cultivated；
14th day, after observing cell growth state in detail, random uniform sampling 5ml, trypan blue staining living cell counting,
Dead cell total quantity, while lymphocyte surface markers detection is done in sampling；And confirm cell suspension bacterium, fungi smear, Zhi Yuan
After body and endotoxin are detected as feminine gender, DC-CTL and DC is fed back, collects cell about 4000ml, is centrifuged (1500rpm, 8min), washes
Liquid (the injection physiological saline containing 0.2% human albumin) washs (1500rpm, 8min) 2 times, and washing lotion is according to 500ml physiology
Salt water, 500,000 IU 20% human serum albumins of IL-2,5ml proportion, cell is resuspended, being loaded into cell, to feed back bag hot
Conjunction machine closes nozzle heat seal, and keeps sample and seal up for safekeeping.
Preferably, in blood pre-treatment step, centrifugation A pipe (700g, 20min).
Preferably, (800g, 20min, centrifuge lifting speed are transferred to most for centrifugation D pipe and E pipe in the separating step of cell
Slowly).
Preferably, setting saturated humidity, 37 DEG C, 5.0%CO in the step of inoculating cell2Culture.
Compared with prior art, the present invention provides a kind of kit of DC-CTL cell induced activation, by built-in using system
The serum free medium of one standard preparation, cytokine activation liquid, lymphocyte separation medium reagent bottle be put into an examination together
In agent box, when cultivating DC-CTL cell without separately configuring culture medium and inducible factor, the time of culture cell, system are shortened
One configuration reagent, reduces the risk of microbiological contamination in incubation, while easy to use, saves manpower, can be in the shorter time
The uniform CTL cell of interior culture mass.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of the kit of DC-CTL cell induced activation of the invention.
Fig. 2 is the partial structural diagram of the kit of DC-CTL cell induced activation of the invention.
Specific embodiment
To make to have further understanding to the purpose of the present invention, construction, feature and its function, hereby cooperate embodiment detailed
It is described as follows.
Some vocabulary is used in specification and claims to censure specific element.Have in fields
Usual skill is, it is to be appreciated that manufacturer may call the same element with different nouns.This specification and right are wanted
Ask book not in such a way that the difference of title is as element is distinguished, but with the difference of element functionally as differentiation
Criterion.Mentioned " comprising " is open term throughout the specification and claims, therefore should be construed to " including but
It is not limited to ".
As depicted in figs. 1 and 2, the induced activation kit of DC-CTL cell of the present invention is by box body 1, box cover 2, sponge support 3,
Reagent bottle 4 is constituted, and the box body 1 and box cover 2 can relatively move, and to expose the opening of box body 1, the sponge support 3 is equipped with six
A fixation hole 301, be placed with respectively in six fixation holes 301 content modulation CTL cytokine activation liquid IL-2 reagent bottle,
Content modulates the reagent bottle of CTL cytokine activation liquid IFN-γ, content modulates DC cytokine activation liquid IL-4 and GM-CSF
The reagent bottle of mixed liquor, content modulate the reagent of the reagent bottle of DC cytokine activation liquid TNF-α, content lymphocyte separation medium
The reagent bottle of bottle, content serum-free medium.
Further, the concentration of IL-2 is 300-1200IU/ml, and IFN-γ concentration is 400-1600U/ml, IL-4 and GM-
It is 600-1500U/ml that the concentration of IL-4, which is 50-300ng/ml and GM-CSF concentration, in CSF mixed liquor, and the concentration of TNF-α is
300-1200U/ml.In the present embodiment, the concentration of IL-4 is that 100ng/ml and GM-CSF is dense in IL-4 and GM-CSF mixed liquor
Degree is 1000U/ml U/ml.The concentration of TNF-α is 500U/ml.In the present embodiment, IFN-γ concentration is 1000U/ml.This implementation
In example, the concentration of IL-2 is 500IU/ml.
Further, it is respectively equipped with label 5 on the bottle body and bottle cap of each reagent bottle 4, has on each label 5 corresponding
The title of content (content) respectively corresponds on the label of different reagent bottles 4 and indicates text: " CTL cytokine activation
Liquid IL2 ", " modulation CTL cytokine activation liquid IFN-γ ", " modulation DC cytokine activation liquid IL-4 and GM-CSF mixing
Liquid ", " modulation DC cytokine activation liquid TNF-α ", " lymphocyte separation medium " or " serum-free medium ", to show difference
Content in reagent bottle 4 is what, and convenient for taking, prevents mistake from taking or obscuring.Preferably, neighbouring reagent bottle 4 on cavernous body 3
Position also there is label, on the label with corresponding content title, convenient for reagent bottle take out after keep in the center, prevent
Reagent bottle achievees the effect that more to standardize.
Further, box cover 2 is connect with box body 1, overturn box cover 2 can with respect to box body 1, alternatively, box cover 2 be it is split type,
To form the box cover 2 of double-door type.In other embodiments, box cover 2 can also be it is plug-type, specifically by designer according to reality
Depending on the situation of border, details are not described herein.
Further, the shape of six fixation holes 301 and the shape of reagent bottle match, in favor of limitation reagent bottle 4
Sliding,.Red in the present embodiment, six fixation holes 301 are circle.
Further, six fixation holes 301 divide two rows of arrangements.Preferably, the two rows fixation hole 301 is parallel to each other.Certainly,
In other embodiments, six fixation holes 301 can Arbitrary distribution, be limited with not formed to interfere, specifically by designer's root
Depending on actual conditions, details are not described herein.
Further, two fixation holes 301 in six fixation holes 301 line up first row, remaining four fixation hole 301
Second row is lined up, but not limited to this, can also be that three fixation holes 301 line up the first row, 301 row of excess-three fixation hole
The first row is lined up at the second row or a fixation hole 301, remaining five fixation hole 301 lines up the second row.
Preferably, two of the first row sizes for fixing 301 holes are greater than the ruler of four fixation holes 301 of the second row
It is very little.In the present embodiment, the diameter that two of the first row fix 301 holes is greater than the diameter of four fixation holes 301 of the second row.
Further, the size of two fixation holes 301 of the first row is identical, four fixation holes 301 of the second row
Size is identical.
Using the DC-CTL cell induced activation kit culture DC-CTL cell the step of it is as follows:
1. single milling machine acquires peripheral blood mononuclear cells 1 × 109-3×109。
2. blood pre-processes: acquired 50ml peripheral blood mononuclear cells is placed in the centrifuge tube (A pipe) of 1 50ml,
It is centrifuged A pipe (400-1200g (preferably 700g), 8-30min (preferably 20min)), supernatant is transferred to 50ml centrifuge tube and waits locating
Manage it is spare, the cell of A pipe lower part be used as ficoll separation PBMC (peripheral blood mononuclear cells).
3. the separation of cell: the A that blood pretreatment obtains manages lower confluent monolayer cells and is diluted with physiological saline 1:1, mixes.By 40ml
Lymphocyte separation medium ficoll equivalent, which is divided equally, is put into 2 50ml graduated centrifuge tubes (D pipe and E pipe).It will be diluted thin in A pipe
Born of the same parents' suspension equivalent, which is divided equally, is slowly at the uniform velocity added D pipe and E pipe along tube wall.Attention is firmly uniform during being added dropwise, and avoids damage to
The integrality at ficoll- leucocyte interface.Being centrifuged D pipe and E pipe, (300-1200g (preferably 800g), 10-35min is (preferably
20min), centrifuge lifting speed is transferred to most slow).After centrifugation, liquid in pipe is divided into three layers, and pipette discards upper layer about 15ml liquid
Absorption is gone up afterwards, middle layer interface white cloud and mist layer narrow band (PBMC, about 5ml needed for i.e.) is put into H pipe, physiological saline (600g,
Supernatant is abandoned after 8min) washing 2 times, every pipe 40ml serum-free cell culture medium is resuspended cell and separates PBMC obtained.
4. inoculating cell: 200ml-600ml PBMC cell suspension is separately added into Tissue Culture Flask T175cm2, set saturation
Humidity, 37 DEG C, 3.0%-10.0% (preferably 5.0%) CO2 culture, after 2 hours, by the taking-up of suspension in Tissue Culture Flask,
Magnetic bead sorting obtains CD8+T lymphocyte freezes as T cell；50ml/ bottles of culture solutions and 50ul is added in attached cell therein
DC cell inducible factors 50-300ng/ml (such as 100ng/ml) IL-4 and 600-1500U/ml (such as 1000U/ml) GM-CSF
Mixed liquor is used for DC cell Fiber differentiation.
5. to the 4th day, 10ul DC cell inducible factors 50-300 (such as 100) were added into DC Tissue Culture Flask in culture
Ng/ml IL-4 and 600-1500 (such as 1000) U/ml GM-CSF mixed liquor.
6. to the 6th day, tumour antigen 5ul was added in culture.
7. culture to the 7th day, is added 10ul DC cell inducible factors TNF-α (300-1200U/ml, such as 500U/ml).
8. the 8th day, recovery T cell.Mature DC cell is divided into 3 parts, 2 parts for freezing, 1 part with the T that recovers
Cell co-cultures, addition activating solution 300-1200IU/ml (such as 500IU/ml) IL2,400-1600U/ml (such as 1000U/
Ml) IFN-γ induces specific for tumour antigen DC-CTL, and samples and carry out DC label detection.
9. the 9th day, DC-CTL is added in 1 part of DC cell of recovering, at the same be added activating solution 300-1200IU/ml (such as
500IU/ml) IL2,400-1600U/ml (such as 1000U/ml) IFN-γ cultivates DC-CTL.
10. the 10th day, DC-CTL is added in 1 part of DC cell of recovering, at the same be added activating solution 300-1200IU/ml (such as
500IU/ml) IL2,400-1600U/ml (such as 1000U/ml) IFN-γ cultivates DC-CTL.
11. the 11st day, coated cell culture bottle being added in DC-CTL, is expanded to DC-CTL.
12. the 13rd day, addition activating solution 300-1200IU/ml (such as 500IU/ml) IL2,300-1200IU/ml (such as
500IU/ml) IFN-γ and cell culture fluid cultivate DC-CTL.
13. the 14th day, after observing cell growth state in detail, random uniform sampling 5ml, trypan blue staining counted living thin
Born of the same parents, dead cell total quantity, while lymphocyte surface markers detection is done in sampling；And confirm cell suspension bacterium, fungi smear, branch
After substance and endotoxin are detected as feminine gender, DC-CTL and DC is fed back, collects cell about 4000ml, is centrifuged (1500rpm, 8min),
Washing lotion (the injection physiological saline containing 0.2% human albumin) washs (1500rpm, 8min) 2 times, and washing lotion is raw according to 500ml
Manage salt water, 500,000 IU 20% human serum albumins of IL-2 and 5ml proportion, be resuspended cell, be loaded into cell feed back bag use
Heat-sealing machine closes nozzle heat seal, and keeps sample and seal up for safekeeping.
To sum up, the present invention provides the kit and its application method of a kind of DC-CTL cell induced activation, by built-in use
Seek unity of standard prepare serum free medium, cytokine activation liquid, lymphocyte separation medium reagent bottle be put into one together
In kit, when cultivating DC-CTL cell without separately configuration culture medium and inducible factor, the time of culture cell is shortened,
Unified configuration reagent, reduces the risk of microbiological contamination in incubation, while easy to use, saves manpower, can be when shorter
The uniform CTL cell of interior culture mass.
The present invention is described by above-mentioned related embodiment, however above-described embodiment is only to implement example of the invention.
It must be noted that the embodiment disclosed is not limiting as the scope of the present invention.On the contrary, do not depart from spirit of the invention and
It is changed and retouched made by range, belongs to scope of patent protection of the invention.
Claims (10)
1. a kind of induced activation kit of DC-CTL cell, including band box cover and box body, it is characterised in that: the tray interior
Equipped with a sponge support, which, which drags, is set there are six fixation hole, and it is thin to be placed with content modulation CTL in six fixation holes respectively
The reagent bottle of intracellular cytokine activating fluid IL-2, content modulate the reagent bottle of CTL cytokine activation liquid IFN-γ, and it is thin that content modulates DC
The reagent bottle of intracellular cytokine activating fluid IL-4 and GM-CSF mixed liquor, content modulate the reagent bottle of DC cytokine activation liquid TNF-α,
The reagent bottle of content lymphocyte separation medium and the reagent bottle of content serum-free medium.
2. the induced activation kit of DC-CTL cell according to claim 1, it is characterised in that: the concentration of IL-2 is
300-1200IU/ml, IFN-γ concentration are 400-1600U/ml, and the concentration of IL-4 is 50- in IL-4 and GM-CSF mixed liquor
300ng/ml and GM-CSF concentration is 600-1500U/ml, and the concentration of TNF-α is 300-1200U/ml.
3. the induced activation kit of DC-CTL cell according to claim 2, it is characterised in that: IL-4 and GM-CSF is mixed
It is 1000U/mlU/ml that the concentration for closing IL-4 in liquid, which is 100ng/ml and GM-CSF concentration,.
4. the induced activation kit of DC-CTL cell according to claim 2, it is characterised in that: the concentration of TNF-α is
500U/ml。
5. the induced activation kit of DC-CTL cell according to claim 2, it is characterised in that: IFN-γ concentration is
1000U/ml。
6. the induced activation kit of DC-CTL cell according to claim 2, it is characterised in that: the concentration of IL-2 is
500IU/ml。
7. a kind of application method of the induced activation kit of DC-CTL cell, the induced activation reagent for DC-CTL cell
Box, the kit include modulation CTL cytokine activation liquid IL-2, modulate CTL cytokine activation liquid IFN-γ, modulation DC is thin
Intracellular cytokine activating fluid IL-4 and GM-CSF mixed liquor, modulation DC cytokine activation liquid TNF-α, lymphocyte separation medium, and
Serum-free medium, which comprises the steps of:
Single milling machine acquires peripheral blood mononuclear cells 1 × 109-3×109；
Blood pretreatment: acquired 50ml peripheral blood mononuclear cells is placed in the centrifuge tube (A pipe) of 1 50ml, is centrifuged A
It manages (400-1200g, 8-30min), supernatant is transferred to that 50ml centrifuge tube is to be processed spare, and the cell of A pipe lower part makees ficoll points
It is used from PBMC (peripheral blood mononuclear cells)；
The separation of cell: the A that blood pretreatment obtains manages lower confluent monolayer cells and is diluted with physiological saline 1:1, mixes, and 40ml lymph is thin
Born of the same parents' separating liquid ficoll equivalent, which is divided equally, is put into 2 50ml graduated centrifuge tubes (D pipe and E pipe).By diluted cell suspension in A pipe
Equivalent, which is divided equally, is slowly at the uniform velocity added D pipe and E pipe along tube wall.Attention is firmly uniform during being added dropwise, and it is white thin to avoid damage to ficoll-
The integrality at born of the same parents interface, centrifugation D pipe and E pipe (300-1200g, 10-35min, centrifuge lifting speed are transferred to most slow), after centrifugation,
Liquid in pipe is divided into three layers, and pipette draws upper, middle layer interface white cloud and mist layer narrow band after discarding upper layer about 15ml liquid
(PBMC, about 5ml needed for i.e.) is put into H pipe, and supernatant, every pipe 40ml serum-free cell are abandoned in physiology salt washing 2 times (600g, 8min) afterwards
Culture solution is resuspended cell and separates PBMC obtained；
Inoculating cell: 200ml-600ml PBMC cell suspension is separately added into Tissue Culture Flask T175cm2, set saturated humidity, 37
DEG C, 3.0%-10.0%CO2Culture, after 2 hours, by the taking-up of suspension in Tissue Culture Flask, magnetic bead sorting obtains CD8+T lymph
Cell freezes as T cell；50ml/ bottles of culture solutions and 50ul DC cell inducible factors 50- is added in attached cell therein
300ng/ml IL-4 and 600-1500U/ml GM-CSF mixed liquor is used for DC cell Fiber differentiation；
10ul DC cell inducible factors 50-300ng/ml IL-4 and 600- were added into DC Tissue Culture Flask to the 4th day for culture
1500U/ml GM-CSF mixed liquor；
Tumour antigen 5ul was added to the 6th day in culture；
Culture was added 10ul DC cell inducible factors TNF-α (300-1200U/ml) to the 7th day；
8th day, mature DC cell was divided into 3 parts by recovery T cell, 2 parts for freezing, the T cell of 1 part and recovery is total to
Activating solution 300-1200IU/ml IL2,400-1600U/ml IFN-γ induction specific for tumour antigen DC-CTL is added in culture,
And it samples and carries out DC label detection；
9th day, DC-CTL was added in 1 part of DC cell of recovering, while activating solution 300-1200IU/ml IL2,400-1600U/ is added
Ml IFN-γ cultivates DC-CTL；
10th day, DC-CTL was added in 1 part of DC cell of recovering, while activating solution 300-1200IU/ml IL2,400-1600U/ is added
Ml IFN-γ cultivates DC-CTL；
11st day, coated cell culture bottle is added in DC-CTL, DC-CTL is expanded；
13rd day, activating solution 300-1200IU/ml IL2,400-1600U/ml IFN-γ and cell culture fluid is added to DC-
CTL is cultivated；
14th day, after observing cell growth state in detail, random uniform sampling 5ml was trypan blue staining living cell counting, dead thin
Born of the same parents' total quantity, while lymphocyte surface markers detection is done in sampling；And confirm cell suspension bacterium, fungi smear, mycoplasma and
After endotoxin is detected as feminine gender, DC-CTL and DC is fed back, collects cell about 4000ml, is centrifuged (1500rpm, 8min), washing lotion (contains
Have the injection physiological saline of 0.2% human albumin) washing (1500rpm, 8min) 2 times, washing lotion according to 500ml physiological saline,
Cell is resuspended in the proportion of 20% human serum albumins of IL-2,5ml of 500000 IU, and being loaded into cell feedback bag heat-sealing machine will
Nozzle heat seal closing, and keep sample and seal up for safekeeping.
8. the application method of the induced activation kit of DC-CTL cell according to claim 7, it is characterised in that: blood is located in advance
It manages in step, centrifugation A pipe (700g, 20min).
9. the application method of the induced activation kit of DC-CTL cell according to claim 7, it is characterised in that: point of cell
From in step, centrifugation D pipe and E manage (800g, 20min, centrifuge lifting speed are transferred to most slow).
10. the application method of the induced activation kit of DC-CTL cell according to claim 7, it is characterised in that: inoculation is thin
In the step of born of the same parents, saturated humidity, 37 DEG C, 5.0%CO are set2Culture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910100720.8A CN109825475A (en) | 2019-01-31 | 2019-01-31 | A kind of the induced activation kit and its application method of DC-CTL cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910100720.8A CN109825475A (en) | 2019-01-31 | 2019-01-31 | A kind of the induced activation kit and its application method of DC-CTL cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109825475A true CN109825475A (en) | 2019-05-31 |
Family
ID=66863146
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910100720.8A Withdrawn CN109825475A (en) | 2019-01-31 | 2019-01-31 | A kind of the induced activation kit and its application method of DC-CTL cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109825475A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005245430A (en) * | 2003-08-25 | 2005-09-15 | Medeinetto:Kk | Method for inducing ctl |
| CN202107707U (en) * | 2011-06-30 | 2012-01-11 | 苏州市立医院（东区） | CIK (cytokine induced killer) cell induction activating reagent kit |
| CN103255105A (en) * | 2013-05-31 | 2013-08-21 | 陈晚华 | Method for stimulating rapid proliferation of CIK (Cytokine-induced Killer) cells by using DC (Dendritic Cells) |
| CN204137485U (en) * | 2014-09-26 | 2015-02-04 | 深圳市茵冠生物科技有限公司 | For the preparation of the kit of DC-CTL |
| CN104651311A (en) * | 2014-09-03 | 2015-05-27 | 深圳市茵冠生物科技有限公司 | Kit for preparing DC-CTL and application of kit |
-
2019
- 2019-01-31 CN CN201910100720.8A patent/CN109825475A/en not_active Withdrawn
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005245430A (en) * | 2003-08-25 | 2005-09-15 | Medeinetto:Kk | Method for inducing ctl |
| CN202107707U (en) * | 2011-06-30 | 2012-01-11 | 苏州市立医院（东区） | CIK (cytokine induced killer) cell induction activating reagent kit |
| CN103255105A (en) * | 2013-05-31 | 2013-08-21 | 陈晚华 | Method for stimulating rapid proliferation of CIK (Cytokine-induced Killer) cells by using DC (Dendritic Cells) |
| CN104651311A (en) * | 2014-09-03 | 2015-05-27 | 深圳市茵冠生物科技有限公司 | Kit for preparing DC-CTL and application of kit |
| WO2016034094A1 (en) * | 2014-09-03 | 2016-03-10 | 深圳市茵冠生物科技有限公司 | Kit for preparing dc-ctl and application of kit |
| CN204137485U (en) * | 2014-09-26 | 2015-02-04 | 深圳市茵冠生物科技有限公司 | For the preparation of the kit of DC-CTL |
Non-Patent Citations (3)
| Title |
|---|
| T.BACHLEITNER-HOFMANN ET AL.: "Stimulation of Autologous Antitumor T-Cell Responses Against Medullary Thyroid Carcinoma Using Tumor Lysate-Pulsed Dendritic Cells", 《THE JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM》 * |
| 刘泽杰 等: "化疗联合DC-CTL治疗不同分期非小细胞肺癌的疗效分析", 《现代肿瘤医学》 * |
| 贾硕 等: "抗原致敏的DC-CTL细胞对肝癌干细胞杀伤作用的实验研究", 《中国实验诊断学》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105713871A (en) | Human chorion mesenchymal stem cell isolated culture method | |
| CN108004211B (en) | A kind of method of Activated in Vitro amplifying natural killer cell | |
| CN104560870A (en) | Method for preparing decidua mesenchymal stem cell | |
| JP4047391B2 (en) | Micropathological patient replica based on whole blood without impurities | |
| CN106801032A (en) | The construction method of people's amnioic epithelium stem cell bank | |
| CN113302276A (en) | Dendritic cell generation apparatus and method | |
| CN106367393B (en) | Prostate Carcinoma of Mice circulating tumor cell system and the separation of prostate cancer circulating tumor cell and cultural method | |
| CN107630002A (en) | A kind of amplification method of umbilical cord blood hematopoietic stem cell | |
| CN106434547A (en) | Method for preparing umbilical cord mesenchymal stem cells | |
| CN110079501A (en) | Mouse breast cancer circulating tumor cell system and its method for building up | |
| CN107354130A (en) | A kind of intermembranous mesenchymal stem cells separation method of human placenia | |
| CN109628402A (en) | A kind of primary organoid cultural method of gastric cancer tumor | |
| CN111548994B (en) | Cell culture medium and method for culturing NK cells by using same | |
| CN111321120B (en) | Separation and culture method of circulating tumor cell line of mouse renal clear cell carcinoma and circulating tumor cell of human-derived renal clear cell carcinoma | |
| CN107722121A (en) | Bee larva bacillus PLMP resists more and its application in immunochromatography paper | |
| CN109825475A (en) | A kind of the induced activation kit and its application method of DC-CTL cell | |
| Schenck et al. | Method for isolating single cells and preparation of clones from human bone marrow cultures. | |
| CN109722386A (en) | A kind of the induced activation kit and its application method of DC-CIK cell | |
| CN104288179B (en) | Dendritic Cells and its preparation method and application | |
| CN108070560A (en) | A kind of isolation and culture method of the primary stomach cancer cell of people | |
| JP7016371B2 (en) | Adhesive cell culture substrate sampling device | |
| CN106834232A (en) | Pituitary adenoma cell system and application thereof | |
| CN102260647A (en) | Method for isolating and purifying breast cancer stem cells | |
| Peters | Preparation of large quantities of pure bovine lymphocytes and a monolayer technique for lymphocyte cultivation | |
| US3726795A (en) | Filtering unit for medical liquids |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20190531 |
|
| WW01 | Invention patent application withdrawn after publication |