CN209113921U - A kind of memory T cell amplification in vitro kit - Google Patents
A kind of memory T cell amplification in vitro kit Download PDFInfo
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- CN209113921U CN209113921U CN201821331481.4U CN201821331481U CN209113921U CN 209113921 U CN209113921 U CN 209113921U CN 201821331481 U CN201821331481 U CN 201821331481U CN 209113921 U CN209113921 U CN 209113921U
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Abstract
The utility model discloses a kind of memory T cell amplification in vitro kits, including box body and openable and closable box cover, equipped with several the first grooves for placing cell culture bags in the box body, place the second groove of cell-stimulating addition reagent bottle D, place the third groove of the first cell factor reagent bottle E, place the 4th groove of the second cell factor reagent bottle F, it places the 5th groove of several clips and places the 6th groove of several syringes, the cell culture bags include the liquid outlet for being provided with the body bag of the first cell culture fluid A and being connected to the body bag, it is provided with the first auxiliary bag of the second cell culture fluid B, it is provided with the second auxiliary bag and sample tap of cell culture addition of C, the liquid outlet, first auxiliary bag and second auxiliary bag and the body bag connecting pipe are equipped with control switch , the sample tap is externally provided with pipe sleeve.This kit prepares that capacity is big, and closure is strong, and contaminated probability is small and easy to operate.
Description
Technical field
The utility model relates to a kind of memory T cell amplification in vitro kits, belong to kit technical field.
Background technique
Lymphocyte considers from functionality viewpoint, can be divided into initial lymphocyte (lymphocyte) and memory lymphocyte
(memory lymphocyte).Initial lymphocyte is that not yet had CD45RA antigen by antigenic stimulus, cell surface expression
Lymphocyte, it is known that it is activated in during regional nodes etc. meet and are stimulated with antigen.Memory leaching
Bar cell is to be subjected to antigenic stimulus (either differential stimulus or nonspecific stimulation) and expressing has CD45RO antigen
Lymphocyte.T lymphocyte and bone-marrow-derived lymphocyte can also be divided into according to respective state T cells and memory T cell,
Naive B cell and memory B cell.Memory T cell can be further divided into effect type memory (effector memory；EM)T
Cell and central type remember (central memory；CM) T cell.Central type memory T cell, which has, goes back to the nest to lymph node, meets head-on
Invade the function of intracorporal foreign matter and antigen, effect type memory T cell have in should where position, capture foreign matter and anti-
Former effect.Research is found: the proliferative capacity and anti-tumor activity of memory T cell in vivo are superior to effect type memory T cell
And T effector cell, in addition, memory T cell is of great significance to the stable state of environment in maintenance immunity of organism and reconstruction.Memory
T cell when meeting with same antigen again in vivo can prompt activation play immunological effect, while there is self-renewal capacity, can
More long-term immunoprotection is provided, more particularly suitable immune effector cell may be provided for adoptive cell therapy.It is expected to
The antineoplastic immune of longer-term is established after feeding back in vivo.Therefore, it is contemplated that make preparation energy as main component containing memory T cell group
It is enough that high therapeutic effect is obtained in adoptive immunotherapy.But status is not force to lymphocyte supplier
A large amount of easily acquisitions is are supplied in the clinical and method of required memory T cell in the case where very big burden.And existing use
Each reagent of some kits need to individually prepare, there are certain pollution risks when taking.
Summary of the invention
Technical problem to be solved by the utility model is to provide a kind of capacity for preparing is big, closure is strong, contaminated probability
Small and easy to operate memory T cell amplification in vitro kit.
In order to solve the above technical problems, the technical solution adopted by the utility model is:
A kind of memory T cell amplification in vitro kit, including box body and openable and closable box cover, the box body are interior equipped with several
For placing the first groove of cell culture bags, the second groove for placing cell-stimulating addition reagent bottle D, for placing the
If the third groove of a cell factor reagent bottle E, the 4th groove for placing the second cell factor reagent bottle F are used to place
The 5th groove and the 6th groove for placing several syringes of dry seal folder, the cell culture bags include being provided with the
The body bag of one cell culture fluid A and the liquid outlet being connected to the body bag are provided with the first of the second cell culture fluid B
Auxiliary bag, the second auxiliary bag and sample tap for being provided with cell culture addition of C, the liquid outlet, first auxiliary bag with
And second auxiliary bag and the body bag connecting pipe are equipped with control switch, the sample tap is externally provided with pipe sleeve.
The body bag capacity is 100-500mL, and the first auxiliary bag capacity is 10-50mL, second auxiliary bag
Capacity is 1-50mL, and the cell-stimulating addition reagent bottle D capacity is 2-5mL, the capacity of the first cell factor reagent bottle E
For 0.5-2mL, the capacity of the second cell factor reagent bottle F is 0.5-2mL.
The specification of the syringe is 1mL, 10mL, 100mL, and quantity is each 2-5.
The cell culture bags integral outer has sterile plastic packaging film.
The first cell culture fluid A is RPMI1640, AIM-V, X-VIVO15 or GT-T551 fluid nutrient medium.
Second cell culture fluid B is human serum albumins.
The cell culture addition of C is L-Glutamine containing 200mM, 200mM Sodium Pyruvate, the nonessential ammonia of 200mM
The physiological saline that the streptomysin concentration of base acid, the penicillin of 100mM Hepes, 10KU/mL and 10mg/mL is 0.85%.
The cell-stimulating additive D is the nanoparticle for being coated with monoclonal antibody OKT3 and monoclonal antibody CD28,
Concentration is 2*108The DPBS solution of a nanoparticle/mL.
The first cell factor reagent E be the IFN-γ of the g/mL of μ containing 5-10,10-20 μ g/mL recombinant human il-2,
The recombined human IL-1 α sterile distilled water solution of 10000-20000U/mL.
The second cell factor reagent F be the recombinant human il-2 of the g/mL of μ containing 5-20,10-30 μ g/mL recombined human IL-7 and
The sterile distilled water solution of 10-30 μ g/mL recombined human IL-15.
The utility model is achieved the utility model has the advantages that using cell culture bags as cell culture container, training in the present apparatus
It is big to support bag gas exchange area, can get greater number of cell, the memory T cell largely activated is made, for preclinical
Research or storage save, and closure is strong, also reduces the link of more external liquid feeding by auxiliary bag, can be significantly reduced
Cell contamination and operator infect probability.
Detailed description of the invention
Fig. 1 is embodiment external structure schematic diagram；
Fig. 2 is embodiment overlooking structure diagram；
Fig. 3 is cell culture bags structural schematic diagram in embodiment；
Fig. 4 is reagent bottle structure schematic diagram in embodiment.
Specific embodiment
The utility model is further described with reference to the accompanying drawing.Following embodiment is only used for clearly illustrating this
The technical solution of utility model, and cannot be used as a limitation the limitation protection scope of the utility model.
A kind of memory T cell amplification in vitro kit, as shown in Figure 1, including box body 2 and openable and closable box cover 1, such as Fig. 2
Shown, the foam mat in box body 2 is equipped with 2 and is used to place the first groove 3,4 of cell culture bags, swashs for placing cell
The second groove 5 of addition reagent bottle D living, is used to place second the third groove 6 for placing the first cell factor reagent bottle E
If the 4th groove 7 of cell factor reagent bottle F, the 5th groove 8 for placing several flat mouth clips are used to place dry injection
The 6th groove 9 and operation instructions of device, flat mouth clips place 1-2 only, specification that there are three types of syringes, 1mL, 10mL,
100mL, quantity are each 2-5, as shown in figure 3, cell culture bags include be provided with the body bag 11 of the first cell culture fluid A with
And be connected to body bag 11 liquid outlet 10, be provided with the second cell culture fluid B the first auxiliary bag 12, be provided with cell training
Support the second auxiliary bag 13 and sample tap 14 of addition of C, liquid outlet 10, the first auxiliary bag 12 and the second auxiliary bag 13 and main body
11 connecting pipes of bag are equipped with control switch, are operated by the knob on switch, for the connection and closing of pipeline two sides,
Sample tap 14 is externally provided with aseptic silica gel pipe sleeve, and logical for sample tap 14 is stopped, and concrete specification is as follows, cell culture bags body bag
11 capacity are 200mL, are provided with AIM-V fluid nutrient medium, and 12 capacity of the first auxiliary bag is 10mL, are provided with human seralbumin egg
White, 13 capacity of the second auxiliary bag is 2mL, is provided with L-Glutamine containing 200mM, 200mM Sodium Pyruvate, the nonessential ammonia of 200mM
The physiological saline that the streptomysin concentration of base acid, the penicillin of 100mM Hepes, 10KU/mL and 10mg/mL is 0.85%, cell
Culture bag is made of ethylene-vinyl acetate copolymer (EVA) material, external also to have sterile plastic packaging film, can be reduced thin
The infected probability of born of the same parents' culture bag is provided with as shown in figure 4, cell-stimulating addition reagent bottle D capacity is 4mL and is coated with monoclonal
The nanoparticle of antibody OKT3 and monoclonal antibody CD28, concentration 2*108The DPBS solution of a nanoparticle/mL, first is thin
The capacity of intracellular cytokine reagent bottle E is 1mL, is provided with recombinant human il-2, the 10000U/ of IFN-γ containing 5 μ g/mL, 20 μ g/mL
The capacity of the recombined human IL-1 α sterile distilled water solution of ml, the second cell factor reagent bottle F is 1mL, is provided with containing 20 μ g/mL
Recombinant human il-2,25 μ g/mL recombined human IL-7 and 25 μ g/mL recombined human IL-15 sterile distilled water solution.
This kit application method is as follows:
One, the preparation of cell culture fluid G is clamped among cell culture bags body bag 11 with flat mouth clips, will be connected
First cell culture fluid A of one side pocket of auxiliary bag and the second cell culture fluid B are mixed, and cell culture addition of C is added.
Adding method is the knob unclamped at auxiliary bag 12,13 and 11 connecting pipe of body bag, repeatedly squeezes auxiliary bag.
Two, the separation of PBMC after donor signs informed consent form, acquires peripheral blood 50mL, utilizes Ficoll-Paque ladder
The separation that centrifugal process carries out PBMC is spent, mononuclearcell layer is isolated.
Three, the liquid in mononuclearcell layer is drawn into centrifuge tube, and DPBS is added, is then centrifuged for, washs, go after washing
Except supernatant, magnetic bead mixing 10min is added, removes non-T cell after mixing in splitter, again centrifuge washing；It is centrifuged and washes
It washs and operates successively alternately twice, first time 1200rpm is centrifuged 6min, and second of 1500rpm is centrifuged 10min, successively carries out
The operation of four centrifugations and washing.
Four, the cell after step 3 centrifugation is resuspended in a small amount of cell culture fluid G is drawn from sample tap 14 with syringe, be added
1.00% the first cell factor additive E, cell-stimulating additive D.
Five, above-mentioned mixed liquor is added in cell culture bags with syringe, cell culture bags is placed in 37 after mixing
DEG C, cultivate in 5%CO2 incubator.
Six, flat mouth clips are removed in third day, are mixed well cell culture fluid G in cell culture bags, are injected with 1ml
The 1.00% the first cell factor additive E are added in device.
Seven, when cell density reaches 2*106When, the cell suspension of half is extracted with 100ml syringe and another cell is trained
The cell culture fluid supported in bag exchanges mixed culture；
The 1.00% the second cell factor additive F are added with 1ml syringe.
Eight, cultivate fortnight after, from liquid outlet 10 collect T cell, after T cell is centrifuged, brine three times, i.e.,
The memory T cell activated.
The above is only the preferred embodiment of the utility model, it is noted that for the common skill of the art
For art personnel, without deviating from the technical principle of the utility model, several improvement and deformations can also be made, these change
It also should be regarded as the protection scope of the utility model into deformation.
Claims (6)
1. a kind of memory T cell amplification in vitro kit, including box body and openable and closable box cover, characterized in that in the box body
Equipped with several the first grooves for placing cell culture bags, the second groove for placing cell-stimulating addition reagent bottle D, use
In the third groove, the 4th groove for placing the second cell factor reagent bottle F, use of placing the first cell factor reagent bottle E
In the 5th groove for placing several clips and the 6th groove for placing several syringes, the cell culture bags include
Be provided with the body bag of the first cell culture fluid A and be connected to the body bag liquid outlet, be provided with the second cell culture
The first auxiliary bag of liquid B, the second auxiliary bag and sample tap for being provided with cell culture addition of C, the liquid outlet, described first
Auxiliary bag and second auxiliary bag and the body bag connecting pipe are equipped with control switch, and the sample tap is externally provided with pipe
Set.
2. a kind of memory T cell amplification in vitro kit according to claim 1, characterized in that the body bag capacity
For 100-500mL, the first auxiliary bag capacity is 10-50mL, and the second auxiliary bag capacity is 1-50mL, and the cell swashs
Addition reagent bottle D capacity living is 2-5mL, and the capacity of the first cell factor reagent bottle E is 0.5-2mL, second cell
The capacity of factor agents bottle F is 0.5-2mL.
3. a kind of memory T cell amplification in vitro kit according to claim 2, characterized in that the rule of the syringe
Lattice are 1mL, 10mL, 100mL, and quantity is each 2-5.
4. a kind of memory T cell amplification in vitro kit according to claim 1, characterized in that the cell culture bags
Integral outer has sterile plastic packaging film.
5. a kind of memory T cell amplification in vitro kit according to claim 1, characterized in that the first cell training
Nutrient solution A is RPMI1640, AIM-V, X-VIVO15 or GT-T551 fluid nutrient medium.
6. a kind of memory T cell amplification in vitro kit according to claim 1, characterized in that the second cell training
Nutrient solution B is human serum albumins.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201821331481.4U CN209113921U (en) | 2018-08-17 | 2018-08-17 | A kind of memory T cell amplification in vitro kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201821331481.4U CN209113921U (en) | 2018-08-17 | 2018-08-17 | A kind of memory T cell amplification in vitro kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN209113921U true CN209113921U (en) | 2019-07-16 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201821331481.4U Active CN209113921U (en) | 2018-08-17 | 2018-08-17 | A kind of memory T cell amplification in vitro kit |
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| Country | Link |
|---|---|
| CN (1) | CN209113921U (en) |
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2018
- 2018-08-17 CN CN201821331481.4U patent/CN209113921U/en active Active
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| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of utility model: Memory T cell in-vitro amplification kit Effective date of registration: 20200610 Granted publication date: 20190716 Pledgee: Suzhou Rongfeng Technology Microfinance Co.,Ltd. Pledgor: SUZHOU MAOXING BIOTECHNOLOGY Co.,Ltd. Registration number: Y2020320010043 |