CN110283785A - A kind of method that gamma delta T-NK cell co-cultures - Google Patents
A kind of method that gamma delta T-NK cell co-cultures Download PDFInfo
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Abstract
The invention discloses a kind of methods that gamma delta T-NK cell co-cultures, the method expanded more particularly to gamma delta T cells and NK cell co-culturing, inducing, first passing through stimulation induction mononuclearcell amplification is gamma delta T cells, simultaneously by acquiring peripheral blood separation screening and induced amplification being stimulated to go out NK cell, pass through gamma delta T cells and the common induced activation culture of NK cell afterwards, a variety of amplification factors combination Co stituation induced amplification gamma delta T cells and NK cell are utilized simultaneously, the cell mixing of acquisition has quantity big, amplification times are high, the features such as cytotoxicity is strong, with extraordinary clinical value.And the co-culturing, inducing amplification method improves cell culture efficiency, shortens incubation time, toxigenic capacity is greatly lowered, also reduce culture technique difficulty, simplify the therapeutic process in clinic, simplify treatment means, also reduce treatment cost.
Description
Technical field:
The present invention relates to field of biotechnology, and in particular to the method for gamma delta T cells and the amplification of NK cell co-culturing, inducing,
And gamma delta T-NK the cell composition obtained using this method.
Background technique:
Gamma delta T cells are that one kind can kill cancer cell, tumor stem cell, and can identify the immunocyte of cancer antigen.
Gamma delta T cells are the T cells for executing inherent immunity function, and TCR is made of γ and δ chain.Such T cell is mainly divided
It is distributed in the mucous membranes such as enteron aisle respiratory tract and urogenital tract and subcutaneous tissue, the 0.5%- of CD3+T cell is only accounted in peripheral blood
1%.Its TCR lack of diversity, can the certain complete polypeptide antigens of Direct Recognition.The antigen type that gamma delta T cells are identified has
Limit: 1. HSP；2. the lipid antigen of infection cell surface C D1 molecule deduction；3. certain virus proteins are expressed in infection cell table
The virus protein in face；4. the phosphorylation antigen in bacterial lysates.
Gamma delta T cells are that one kind can kill cancer cell, tumor stem cell, and can identify the immunocyte of cancer antigen, it
It is lethal relatively strong, but tumor stem cell killing is not so good as NK cell.Therefore, it is mainly used for killing cancer cell and DC cell is assisted to know
Not Fa Xian cancer cell antigen, these antigens are then killed or are passed to other cells.Meanwhile gamma delta T cells mainly divide
It is distributed on skin and mucous membrane tissue, therefore prominent for the treatment of cancer effect in terms of mucous membrane, such as alimentary canal, respiratory tract, life
Grow the cancer significant effect of system aspects.
Gamma delta T cells are mainly reached maturity in thymus gland, generate gamma delta T cells receptor (TCR) by V (D) J genetic recombination.
By the gene rearrangement of specificity, break up from a common lymphoid cell precursors (common lymphoidprecursor, CLP)
For the T cell system of express alpha beta receptor and γ δ receptor.The influence of missing, therefore γ δ are not processed vulnerable to antigen and presented to gamma delta T cells
T cell has very high clinical tumor immunization therapy potential using value.Gamma delta T cells are in cancer immunosurveillance and antitumor exempt from
It plays a significant role in epidemic disease reaction.
Gamma delta T cells effect is also embodied in:
1. gamma delta T cells can have an effect with panimmunity cell, anti-tumor immune response is participated in.
2. gamma delta T cells can cause rapidly effective antitumour immune response in tumorigenic early stage.
3. gamma delta T cells have important protective effect during antineoplastic immune.
4. gamma delta T can utilize cellulotoxic effect killing tumor cell, the occurrence and development of tumour are prevented.
5. gamma delta T cells can secrete correlation factor, these factors can amplify tumor signal.
6. gamma delta T cells can secrete perforin, inducing apoptosis of tumour cell.
Since content of the gamma delta T cells in peripheral blood is extremely low, greatly limits gamma delta T cells and exist as adoptive immunity cell
Application clinically.Gamma delta T cells are expanded from peripheral blood mononuclear cells at present, amplification times are low, and cell purity and cell concentration are not
It is high.The gamma delta T cells amplified are difficult to meet clinical demand, even if being amplified by optimizing various inductive conditions and amplification method
Single gamma delta T cells applied in corresponding immunity disease and tumor disease, but apply after and not up to people manage
Effect in thinking.
Cellular immunotherapy is one of current most promising oncotherapy mode, by returning after amplification in vitro or transformation
It is defeated to achieve the purpose that in patient body killing tumor cell or by activation body immune system, enhancing tumor patient itself exempt from
Epidemic disease function is to resist tumour or other diseases.Currently, NK cellular immunotherapy is more and more paid attention to.NK cell accounts for people
The 5-15% of peripheral blood lymphocytes, generally defining its phenotype is CD3-CD56+, and NK cell can be further subdivided into two again
Main subgroup: the CD56highCD16- cell with immunoloregulation function and the CD56dimCD16+ with cytotoxic activity
Cell.NK cell plays important immune surveillance function in viral infection resisting and antitumor early immune reaction, and NK is thin
Born of the same parents do not need identification tumour specific antigen, can directly, quickly play cytotoxic activity.Particularly importantly NK cell energy
Enough effectively intracorporal Multidrug-resistants of removing machine, inhibit the growth and transfer of tumour.
In existing research, single immunocyte or lethal cell is mostly used to carry out cell therapy or facing greatly
In bed treatment by the way of combination therapy, in the prior art, more costly expense is needed using combination therapy, be sufferer
Bring more economic pressures.
Therefore, invent it is a kind of it is practical and efficient, at low cost, technically simple, can massive amplification high quantity, high cytotoxic activity
Gamma delta T cells and NK cell, massive amplification go out immunocyte, carry out cell therapy for numerous patients and provide raw material sources, become
A kind of hope of sufferer treatment at present.
Summary of the invention
For problem described above, the invention proposes a kind of methods that gamma delta T-NK cell co-cultures, and in particular to gamma delta T
The method of cell and the amplification of NK cell co-culturing, inducing, first passing through stimulation induction mononuclearcell amplification is gamma delta T cells, simultaneously
By acquisition peripheral blood separation screening and induced amplification is stimulated to go out NK cell, work is induced by gamma delta T cells and NK cell jointly afterwards
Change culture, while using a variety of amplification factors combination Co stituation induced amplification gamma delta T cells and NK cell, obtaining gamma delta T cells
Cell mixing is co-cultured with NK cell, has the characteristics that quantity is big, amplification times are high, cytotoxicity is strong, there is extraordinary face
Bed application value.
To achieve the goals above, the following technical solution is employed by the present invention:
Method that the gamma delta T-NK cell co-cultures the following steps are included:
1: the acquisition of gamma delta T cells；
The acquisition of 2:NK cell；
3: gamma delta T cells and NK mixing with cells cell object induced amplification co-culture；
The acquisition of 4:15-20 days gamma delta T cells and NK mixing with cells cell.
Wherein the gamma delta T cells are included the following steps:
(1) preparation of mononuclearcell: separation mononuclearcell contains 10vt% with the 50%-100% containing percent by volume
Cell is resuspended in the mixed culture medium of the RMPI 1640 and 0%-50%X-vivo15 of FBS or autoserum, be seeded in culture bottle or
It is cultivated in culture bag；
(2) zoledronic acid and 500~3000U/mL IL-2 that are 1-100 μM containing concentration the induction of gamma delta T cells: is added
It is added after mixed culture medium stimulated in vitro 4 days and contains 10~500ng/mL anti-human CD3Ab, 10~500ng/
MLanti-human CD28Ab, 10~200ng/mL IL-15,10~200ng/mL IL-21,500~3000U/mL IL-2
Mixed culture medium carry out half amount and change liquid, be placed in 37 DEG C, 5%CO2It is cultivated in incubator；
(3) it cultivates 2~3 days, it is well for later use to gamma delta T cells growth conditions.
Preferably, zoledronic acid concentration is 5 μM, anti-human CD3Ab concentration is 50ng/mL, anti-human
It is 50ng/mL, IL-21 concentration be 50ng/mL, IL-2 concentration is 1000U/mL that CD28Ab concentration, which is 50ng/mL, IL-15 concentration,.
Preferably, mononuclearcell derives from peripheral blood, Cord blood, marrow or inductive pluripotent stem cells (iPSC).
Other phosphoantigens also can be used in one embodiment instead of zoledronic acid, including isopentenyl pyrophosphate
(IPP), (E) -4- hydroxy-3-methyl-but-2-ene base pyrophosphate (HMB-PP), ethyl pyrophosphate (EPP), conversion
Ester (FPP), dimethyl-allyl phosphate (DMAP), dimethylallylpyrophosphate ester (DMAPP), ethyl-adenosine triphosphoric acid
Ester (EPPPA), geranylpyrophosphate ester (GPP), yak geranylpyrophosphate ester (GGPP), isopentenyl-adenosine pyrophosphate
(IPPPA), etherophosphoric acid (MEP), the biphosphonate containing nitrogen such as one ethyl ester of pyrophosphoric acid (MEPP).
Preferably, RMPI 1640 in a case study on implementation containing 10vt%FBS or autoserum in mixed culture medium
Ratio is 50vt%, and the ratio of X-vivo15 culture medium is 50vt%.
Wherein NK cell is included the following steps:
(1) mononuclearcell is separated；
(2) pass through the CD in immune sorting removal mononuclearcell3+T lymphocyte；
(3) NK cell culture serum-free cell culture medium and final concentration of 10-500ng/ml IL-15,10- is added
500ng/ml IL-12,10-500ng/ml IL-21,10-500ng/ml IL-18 and 500-2000U/ml IL-2 are seeded in carefully
Born of the same parents' culture bottle changes liquid after in vitro culture 2-5 days entirely, good to cell growth state.
Mononuclearcell described above is after acquiring peripheral blood by the sterile disposable heparin tube venipuncture of anticoagulant heparin,
The mononuclearcell for being obtained by Ficoll density gradient centrifugation or being acquired by single milling machine；Or from Cord blood, bone
The mononuclearcell that marrow and iPSCs induction differentiation obtain.
CD in the immune sorting removal mononuclearcell3+The immune sorting of immunomagnetic beads, film bubble can be used in T lymphocyte
The methods of sorting is immunized with flow cytometer.
The inoculum density of mononuclearcell after the removal CD3+T lymphocyte is 0.1-2 × 106Cells/ml, it is excellent
It is selected as 1 × 106cells/ml。
The concentration of interleukin-15 used is preferably 120-350ng/ml in the combination of cytokines.
The concentration of IL-12 used is preferably 100-300ng/ml in the combination of cytokines.
The concentration of interleukin-21 used is preferably 100-380ng/ml in the combination of cytokines.
The concentration of IL-18 used is preferably 100-300ng/ml in the combination of cytokines.
The concentration of proleulzin used is preferably 1000-1800U/ml in the combination of cytokines.
It is preferably the 4th day that the NK cell changes liquid number of days entirely, and cell concentration is preferably 1 × 106cells/ml。
It is the NK cell non-serum culture tune that required cell factor is expanded by adding cell containing NK that the NK cell, which changes liquid,
Whole NK cell concentration is to 1 × 106cells/ml。
Gamma delta T cells and the co-cultivation of NK mixing with cells cell object include the following steps:
(1) it takes the good gamma delta T cells of above-mentioned growth conditions appropriate, and is placed in culture bottle；
(2) it takes the good NK cell of above-mentioned growth conditions appropriate, and is placed in step 1 gamma delta T cells culture bottle；
(3) into culture bottle be added autoserum RMPI 1640 and X-vivo15 culture medium 1-2 days；1640 He of RMPI
The ratio of X-vivo15 is 1:0.5-5；
(4) 10-500ng/ml IL-15,10-500ng/ml IL-12,10- are added into Tissue Culture Flask within 2-3 days
The mixed culture medium of 500ng/mlIL-21,10-500ng/ml IL-18 and 500-2000U/ml IL-2 carry out half amount and change liquid, set
In 37 DEG C, 5%CO2It is cultivated in incubator；
(5) it cultivates-the 20 day the 5th day, observes cell growth state daily, add the autoserum of factor-containing
RMPI1640 and X-vivo15 culture medium maintains cell concentration 1 × 106Cells/ml is placed in saturated humidity, 37 DEG C, 5.0%
CO2 incubator continues to cultivate, and harvests cell when until reaching required cell quantity.Period, if cell volume is more than 240ml,
Sub-bottle culture is transferred to secondary culture in cell culture bags, 14-18 days harvest mixed cell cultures.
The gamma delta T cells inoculum concentration is 0.1-2 × 106Cells/ml, NK cell inoculation amount are 0.1-1 × 106cells/
ml。
The cell composition includes: gamma delta T cells and NK cell.
The gamma delta T cells and NK cell proportion are 1:0.5-5.
Application of the above-mentioned cell mixture on antineoplaston.
The raw materials used in the present invention and reagent are commercially available in addition to having specified otherwise.
Due to using above-mentioned technical solution, the beneficial effects of the present invention are: (1) the present invention provides a kind of gamma delta T-NK
The application of cell composition and composition that cell co-culturing, inducing amplification method and this method obtain；Preparation method operation letter
It is single, individual cell can be largely obtained during the preparation process, and later period mixed culture cell compatibility is good, can get the γ of massive amplification
Delta T cells and NK cell；(2) and in the case where stimulating factor lacks, incubation cell can mutually promote stimulating growth, contracting
Short incubation time obtains the medical cell amounts for largely meeting clinical demand, gamma delta T cells and NK cell massive amplification simultaneously, mixes
It is strong to close gamma delta T-NK cytotoxicity/activity；(3) cell composition that mode obtains is co-cultured, simplifies and is used for multiple times in clinic
Individual cell carries out immunization therapy, reduces cost, simplifies therapeutic process；(4) combination cell object is co-cultured to kill cancer cell
Wound property is better than using the lethal effect of single immunocyte.
Detailed description of the invention:
Fig. 1 is amplification cultivation gamma delta T cells growth curve and amplification times；
Fig. 2 is amplification cultivation NK cell growth curve and amplification times；
Fig. 3 is amplification cultivation gamma delta T cells and NK cell growth curve and amplification times；
Fig. 4 is that gamma delta T cells, NK cell, gamma delta T cells and NK cell combination cell object are living to the killing of SK-BR-3 cell
Property detection.
Specific embodiment:
Below with reference to specific embodiment, technical scheme in the embodiment of the invention is clearly and completely described.It answers
Understand, described embodiments are some of the embodiments of the present invention, these embodiments are merely to illustrate the present invention rather than limit
The scope of the present invention processed.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art can be to this
Invention makes various changes or modifications, and these equivalent forms also fall within the scope of the appended claims of the present application.
In order to be easy to understand the technical means, the creative features, the aims and the efficiencies achieved by the present invention, tie below
Specific embodiment is closed, the present invention is further explained.
The acquisition of 1 gamma delta T cells of embodiment
The acquisition of gamma delta T cells separates mononuclearcell (PBMCs) from peripheral blood and expands gamma delta T cells:
(1) first 30 minutes unlatching Biohazard Safety Equipments are used；
(2) D-PBS is taken out from refrigerator using preceding, be placed at room temperature for 30 minutes；
(3) 30ml peripheral blood sample (anticoagulant heparin) is transferred to two sterile 50ml centrifuge tubes, every pipe 15ml, then
The sterile D-PBS of 22.5ml is added to every pipe, overturns centrifuge tube repeatedly, mixes well；
(4) two 50ml sterile centrifugation tubes separately are taken, is separately added into 15ml Ficoll-Paque Plus solution, then distinguishes
The blood (by drawing in two sterile tubes in step 3) after diluting in 24ml step 3 is slowly added, is formed and is layered, 20 DEG C,
400 × g is centrifuged 30 minutes；
(5) two 50ml centrifuge tubes in step 4 are put into Biohazard Safety Equipment, then sop up 15ml blood using 10ml suction pipe
Clear layer is fitted into a new sterile 50ml centrifuge tube, 56 DEG C of inactivations, 30 minutes stand-by (for preparing mixed culture medium)；
(6) leukocytic cream (PBMCs) in each 50ml centrifuge tube is drawn, a new 50ml sterile centrifugation is transferred to
Pipe；
(7) to step 6 equipped with being added the sterile PBS of its 3 times of volumes in the centrifuge tube of PBMCs cell suspension, and with sterile
Pipette mixes, and 20 DEG C, 400 × g, is centrifuged 10 minutes；
(8) supernatant is abandoned, the sterile PBS of 50ml is added, PBMC is slowly resuspended；
It (9) 20 DEG C, 400 × g, is centrifuged 10 minutes；
(10) mixed culture of the RMPI1640+50vt%X-vivo15 of 5ml 50vt% autoserum containing step 5 is added
Base mixes, and takes 10 μ l for counting；
(11) by taking the peripheral blood mononuclear cells (PBMCs) of half amount in step 10, certain volume is added and contains azoles
The mixed culture medium of phosphonic acids adjusts cell concentration to 1 × 106cells/ml；The PBMCs addition of the other half amount contains zoledronic acid
1640 culture medium of RPMI, adjustment cell concentration is to 1 × 106cells/ml；
(12) T-175 culture bottle culture cell is used；
(13) Tissue Culture Flask is placed in 5%CO2, carry out cell culture in 37 DEG C of cell incubators；
(14) on day 4 when, all cells are transferred in 50ml sterile centrifugation tube (20 DEG C, 400 × g, be centrifuged 5 minutes),
Replace amplification culture medium；Take half amount contain required amplification factor (anti-human CD3Ab, anti-human CD28Ab,
IL-15, IL-21 and IL-2) mixed culture medium of composition is added in empty Tissue Culture Flask, the cell culture medium after centrifugation
Supernatant is transferred in 50ml sterile centrifugation tube, takes half amount cell culture medium supernatant that cell is resuspended, respectively by the cell after resuspension
It is put into this step Tissue Culture Flask, 5%CO2, 37 DEG C continue to cultivate；
(15) in the 6th, 8,10,12,14,16 and 18 day replacement amplification culture medium.
The cell number of inoculating cell number and amplification cultivation is counted within the 0th, 4,6,8,10,12,14,16,18 and 20 day in culture,
Cell growth curve and cells expanded are made, as shown in Figure 1,30,000,000 cells of inoculation, expand 20 days, total number of cells are reachable
About 26,000,000,000, fully meet clinical application needs.
The acquisition of embodiment 2NK cell
1, the separation of monocyte
1.1 under Biohazard Safety Equipment normal operating conditions, 50mlIt is sterileCentrifuge tube is separately added into Ficoll-Paque Plus
Lymphocyte separation medium；
1.2 patient peripheral's whole blood and DPBS are uniformly diluted in proportion after be slowly injected into and separated in centrifuge tube medium size lymphocyte
Liquid liquid level upper layer；
It 1.320 DEG C, 400 × g, is centrifuged 30 minutes；
1.4 draw the plasma layer in separating pipe using aseptic straw, blood plasma are put into 50ml sterile centrifugation tube, 56 DEG C of water
It bathes 30 minutes and inactivates, 800g × centrifugation 10 minutes, upper plasma is transferred in new 50ml sterile centrifugation tube；
1.5 draw separating obtained mononuclearcell layer (PBMCs) loaded in 50ml sterile centrifugation tube；
1.6 are added DPBS, mix, 20 DEG C, 400 × g, are centrifuged 10 minutes, wash 1 time；
1.7 abandon supernatant, then all cells of 1.6 steps are resuspended into a 50ml sterile centrifugation tube with DPBS, mix, take
20 μ l cell suspensions, which are placed in 1.5ml EP pipe, to be counted.
2, NK cell expansion ex vivo-CD3+T cell removal
2.1, according to the count results of step 1.7, take out 5 × 105Cell is placed in 1.5ml EP pipe, for sorting preceding table
Type detection 20 DEG C of remaining cell, 400 × g, is centrifuged 10 minutes, then wash 1 time, abandons supernatant, with containing 0.5% human serum albumin
And the DPBS (0.5%HSA, 1mM EDTA, DPBS) of 1mM EDTA adjusts cell concentration to 1 × 108Cells/ml is transferred to
In the sterile streaming pipe of 5ml, EasysepTM Human CD3Positive Selection Kit is added by 150 μ l/ml samples
Human CD3Positive Selection Cocktail II in II mixes, is placed at room temperature for 3min；
2.2 by the RapidSphereTM in EasysepTM Human CD3Positive Selection Kit II
50100 mix, and add in the streaming pipe of step 2.1 by 90 μ l/ml samples, mix, are placed at room temperature for 3min；
2.3 with 0.5%HSA is contained, and 1mM EDTA, DPBS are by the body of the cell suspension in the sterile streaming pipe of 5ml in step 2.2
Product polishing is mixed gently to 2.5ml；
The sterile streaming pipe of above-mentioned 5ml is placed in magnet and is stored at room temperature 3min by 2.4, picks up magnet, and cell is fallen in 15ml
In sterile centrifugation tube, takes 20 μ l cell suspensions to be placed in 1.5ml EP pipe and count.
3, NK cell expansion ex vivo-NK cell culture
3.1, according to 2.4 step count results, take out 5 × 105Cell is placed in 1.5ml EP pipe, is examined for phenotype after sorting
It surveys, remaining cell suspension is mended remaining cell volume to 14ml with L500 culture medium, is mixed, 20 DEG C, 400 × g, is centrifuged 5min；
3.2 abandon supernatant, 20 DEG C, 400 × g, are centrifuged 5min；
3.3 inhale abandoning supernatant, with SuperCultureTML500 (L500) culture medium containing 10% autoserum that cell is dense
Degree is adjusted to 1 × 106Cells/ml, IL-2, which is added, makes its final concentration of 1500IU/ml, and cell factor (IL-15, Il- is separately added
12, IL-21 and IL-18 final concentration is 200ng/ml), it mixes.
Cell is placed in 37 DEG C, saturated humidity, cultivated in 5.0%CO2 incubator by 3.4.
3.5 cultures the 4th day, cell are blown even, take 20 μ l cell suspensions to be placed in 1.5ml EP pipe, for counting.It is remaining
Cell is gone in 50ml sterile centrifugation tube, 20 DEG C, 400 × g, is centrifuged 5min；
3.6 abandon supernatant, cell are resuspended with the L500 culture medium containing 10% autoserum of Fresh, and it is dense to adjust cell
It spends to 1 × 106Cells/ml, while IL-2, which is added, makes its final concentration of 1500U/ml that cell factor (IL-15, Il- be added with another
12, IL-21 and IL-18 final concentration is 200ng/ml), it mixes.
Above-mentioned cell is placed in 37 DEG C, saturated humidity, 5.0%CO by 3.72Continue to cultivate in incubator.
3.8 cultures-the 21 day the 5th day, observe cell growth state daily, add the L500 culture medium of factor-containing
(if any enough blood plasma, can continue to 10% autologous plasma content) maintains cell concentration 1 × 106Cells/ml is placed in saturation
Humidity, 37 DEG C, 5.0%CO2Incubator continues to cultivate, and harvests cell when until reaching required cell quantity.Period, if cell body
Product then sub-bottle culture or is transferred to secondary culture in cell culture bags more than 240ml.
In the cell of the 0th, 4,6,8,10,12,14,16,18,20 and 22 day statistics inoculating cell number and amplification cultivation of culture
Number makes cell growth curve and cells expanded, as shown in Fig. 2, 15,600,000 cells of inoculation, expand 21 days, total number of cells
Clinical application needs can be fully met of about 42,000,000,000, it is seen that a large amount of NK cell can be obtained using the method for the present invention.
3 gamma delta T cells of embodiment and NK mixing with cells cell object co-culture
(1) the good gamma delta T cells 1-3ml of 3-10 days growth conditions is cultivated in Example 1, and is placed in culture bottle；
(2) the good NK cell 1-3ml of 3-10 days growth conditions is cultivated in Example 2, and it is thin to be placed in step 1 gamma delta T
In born of the same parents' culture bottle；
(3) into culture bottle be added autoserum RMPI 1640 and X-vivo15 culture medium 1-2 days；1640 He of RMPI
The ratio of X-vivo15 is 1:1；
(4) 1100ng/ml IL-15,100ng/ml IL-12,100ng/ml are added into Tissue Culture Flask in the 2-3 days
The mixed culture medium of IL-21,80ng/ml IL-18 and 500U/ml IL-2 carry out half amount and change liquid, are placed in 37 DEG C, 5%CO2Culture
It is cultivated in case；
(5) it cultivates-the 16 day the 5th day, observes cell growth state daily, add the autoserum of factor-containing
RMPI1640 and X-vivo15 culture medium maintains cell concentration 1 × 106Cells/ml is placed in saturated humidity, 37 DEG C, 5.0%CO2
Incubator continues to cultivate, and harvests cell when until reaching required cell quantity.Period, if cell volume is more than 240ml, sub-bottle
Cultivate or be transferred to secondary culture in cell culture bags, 17-18 days harvest mixed cell cultures.
In the cell number of the 0th, 4,6,8,10,12,14 and 16 day statistics inoculating cell number and amplification cultivation of culture, production is thin
Intracellular growth curve and amplification times, as shown in figure 3,15,000,000 cell of NK cell inoculation, gamma delta T is inoculated with 20,000,000 cells, amplification 16
It, total number of cells can be of about 110,000,000,000；Total cell number fully meets clinical application needs.
The killing activity of 4 gamma delta T cells of embodiment and NK mixing with cells cell object to human breast cancer cell SK-BR-3
Using 5 μM of CFSE to SK-BR-3 cell dyeing, NK cell or gamma delta T cells or gamma delta T cells and NK cell are mixed
Cell object or PBS and SK-BR-3 cell are closed according to the ratio of 20:1, after 37 DEG C of incubation 4h, the PI dyestuff of 1 μ g/ml of addition, CFSE
+ PI double positive cells are dead cell, as shown in figure 4, gamma delta T cells and NK mixing with cells cell object can be significant enhance cancer
The killing activity of cell, almost 100% killing SK-BR-3 cell.
And have above-described embodiment it can be concluded that, gamma delta T cells are being mixed after separating with NK cell, and two kinds of cells can be with
Proliferation of mutually promoting growth, and cell growth cycle is shortened, and in the case where stimulating factor lacks, gamma delta T cells and NK
Cell massive amplification, and cell mixing toxicity/activity is strong.
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (7)
1. a kind of method that gamma delta T-NK cell co-cultures, it is characterised in that: the method that gamma delta T-NK cell co-cultures includes following
Step:
(1) acquisition of gamma delta T cells；
(2) acquisition of NK cell；
(3) gamma delta T cells and NK mixing with cells cell object induced amplification co-culture；
(4) acquisition of 15-20 days gamma delta T cells and NK mixing with cells cell.
2. the method that gamma delta T-NK cell co-cultures according to claim 1, it is characterised in that: the preparation method of gamma delta T cells
Are as follows:
(1) preparation of mononuclearcell: separation mononuclearcell contains 10vt%FBS with the 50%-100% containing percent by volume
Or cell is resuspended in the mixed culture medium of the RMPI 1640 and 0%-50%X-vivo15 of autoserum, is seeded in culture bottle or training
It supports and is cultivated in bag；
(2) mixing of the zoledronic acid and 500~3000U/mL IL-2 that are 1-100 μM containing concentration the induction of gamma delta T cells: is added
It is added after medium body external stimulus 4 days and contains 10~500ng/mL anti-human CD3Ab, 10~500ng/mL anti-
The mixing training of human CD28Ab, 10~200ng/mL IL-15,10~200ng/mL IL-21,500~3000U/mL IL-2
It supports half amount of base progress and changes liquid, be placed in 37 DEG C, 5%CO2It is cultivated in incubator；
(3) it cultivates 2~3 days, it is well for later use to gamma delta T cells growth conditions.
3. the method that gamma delta T-NK cell co-cultures according to claim 1, it is characterised in that: the acquisition of NK cell includes such as
Lower step: (1) mononuclearcell is separated；
(2) pass through the CD in immune sorting removal mononuclearcell3+T lymphocyte；
(3) NK cell culture serum-free cell culture medium and final concentration of 10-500ng/ml IL-15,10-500ng/ is added
Ml IL-12,10-500ng/ml IL-21,10-500ng/ml IL-18 and 500-2000U/ml IL-2 are seeded in cell culture
Bottle changes liquid after in vitro culture 2-5 days entirely, good to cell growth state.
4. the method that gamma delta T-NK cell co-cultures according to claim 1, it is characterised in that: gamma delta T cells and NK cell are mixed
The co-cultivation of cell object is closed to include the following steps:
(1) it takes the good gamma delta T cells of growth conditions in claim 2 appropriate, and is placed in culture bottle；
(2) it takes the good NK cell of claim 3 growth conditions appropriate, and is placed in above-mentioned steps (1) gamma delta T cells culture bottle
In；
(3) into culture bottle be added autoserum RMPI 1640 and X-vivo15 culture medium 1-2 days；
(4) 10-500ng/ml IL-15,10-500ng/ml IL-12,10-500ng/ml are added into Tissue Culture Flask within 2-3 days
The mixed culture medium of IL-21,10-500ng/ml IL-18 and 500-2000U/ml IL-2 carry out half amount and change liquid, be placed in 37 DEG C,
5%CO2It is cultivated in incubator；
(5) it cultivates-the 18 day the 5th day, observes cell growth state daily, add the RMPI of the autoserum of factor-containing
1640 and X-vivo15 culture medium maintains cell concentration 1 × 106Cells/ml is placed in saturated humidity, 37 DEG C, 5.0%CO2 training
Feeding case continues to cultivate, and harvests cell when until reaching required cell quantity；If cell volume is more than 240ml, sub-bottle culture or
It is transferred to secondary culture in cell culture bags, 14-18 days harvest mixed cell cultures.
5. the method that gamma delta T-NK cell co-cultures according to claim 4, it is characterised in that: RMPI 1640 and X-vivo15
Ratio be 1:0.5-5.
6. a kind of cell composition, it is characterised in that: cell composition includes gamma delta T cells and NK cell.
7. a kind of method for preparing cell composition described in claim 6, it is characterised in that: by any one of claim 1-5 institute
The method preparation stated.
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