CN105385657B - NKT cell culture method and application - Google Patents
NKT cell culture method and application Download PDFInfo
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Abstract
The invention relates to a culture method and application of NKT cells. The NKT cell culture method comprises a step of co-culturing macrophages and NKT cells. Macrophages have utility in enhancing NKT cell killing activity. The present invention selects the mononuclear cells from the mononuclear cells, and then adopts GM-CSF with a certain concentration to induce the mononuclear cells to become macrophages. Suspension cells were cultured into NKT cells, after which both macrophages and NKT cells were co-cultured. The macrophage can directly phagocytize cells and has a certain antigen presenting function, so that the macrophage and the macrophage are co-cultured, and the killing activity of the NKT cell is directly improved.
Description
Technical field
The present invention relates to field of cell culture more particularly to a kind of cultural methods and purposes of NKT cell.
Background technique
Autoimmune cell therapy is one of important method of tumor biotherapy.Autoimmune cell therapy is will be from certainly
Mononuclearcell is isolated in peripheral body, under the action of panimmunity active factors, by vitro culture, effective activation and
Amplification is immunocompetent cell, including NK cell, CIK cell and NKT (naturalkillerT) cell etc., then inputs patient's body
It is interior, make immunocyte direct killing tumour cell or virus infected cell, or adjust and the treatment of the immune function of enhancing body
Method
NKT cell be human peripheral blood single nucleus cell in vitro through the cytokine profiles such as IL-2 stimulation after, acquisition with
Express CD3+CD6+Immune effector cell based on mark kills tumor activity with T lymphocyte, thin for lethal effect of new generation
Born of the same parents.The constancy for being mainly characterized by T cell receptor (TCR) gene expression, the restricted and cell factor of CD1d of NKT cell
Rapid, the high-level property generated.NKT cell can enhance immune response but also inhibit immune response, thus in antitumor, anti-sense
Dye inhibits to play a significant role in autoimmune disease and transplantation tolerance.Though NKT cell is also with CD3+CD6+The cell of mark
For effector cell, but the difference with CIK cell, NKT is from CD4 in peripheral blood-CD8-The cell of mark.
The preparation method of NKT cell is usually the mononuclearcell (PBMC) being directly separated in peripheral blood at present, then will
PBMC is cultivated 2 weeks in the culture medium containing certain density IL-2, IL-15 and is obtained NKT cell.But it cultivates in the prior art
The NKT cell killing activity arrived is lower.
Summary of the invention
In view of this, it is necessary to for the low problem of NKT cell killing activity in the prior art, provide a kind of NKT cell
Cultural method and purposes.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of cultural method of NKT cell includes the steps that co-culturing macrophage and NKT cell.
Preferably, the cultural method of the NKT cell, comprising the following steps:
The sorting of S1, cell:
Monocyte is sub-elected from PBMC；
The induction of S2, macrophage:
The step S1 monocyte sorted is induced as macrophage；
The induction of S3, NKT cell:
Step S1 is sorted remaining cell to induce as NKT cell；
S4, macrophage and NKT cell co-culture:
The S2 macrophage obtained and S3 the NKT cell obtained are co-cultured 7-8 days.
Preferably, tumor cell lysis liquid supernatant stimulating expression of macrophage is used in the step S2.
Preferably, in the step S2 macrophage induction method particularly includes: use the culture of macrophage induced medium
Monocyte, culture to the 4th day addition tumor cell lysis liquid supernatant, culture to the 6th day collection macrophage；The macrophage is thin
The composition of born of the same parents' induced medium are as follows: 500-1000U/mL GM-CSF (granulocyte and macrophage colony stimulating factor), volume point
Number 3-10% autologous plasma, surplus are 15 serum free medium of X-VIVO.
Preferably, final concentration of protein is 1-10 μ g/mL, preferably 5 μ g/mL in the tumor cell lysis liquid supernatant.Tumour
Cell lysate supernatant can promote the proliferation and maturation of macrophage, enhance Macrophages For Tumor recognition reaction and
Killing activity.The tumor cell lysis liquid is prepared by multigelation method.
Preferably, in the step S3 NKT cell induction method particularly includes: use NKT cell culture medium incubation step
Remaining cell after S1 sorting, culture to the 6th day collection NKT cell；The composition of the NKT cell culture medium are as follows: 100-
500U/mL IL-2 (interleukin 2), 10-100 μ g/mL IL-15 (interleukin-15), 10-100 μ g/mL IL-21
(IL-21), surplus are 15 serum free medium of X-VIVO.
Preferably, macrophage and NKT cell co-culture in the step S4 method particularly includes: the macrophage for obtaining S2 is thin
The NKT cell that born of the same parents and S3 are obtained was with NKT cell culture medium co-incubation 7-8 days.
Preferably, the condition of culture in step S2-S4 is 37 DEG C, 5%CO2。
Preferably, the step S2-S4 is during the cultivation process according to 1 × 106The cell density of cells/mL adds culture
Liquid and cell factor.
Preferably, macrophage and NKT cell are co-cultured by quantity ratio 1:10-25.
It is highly preferred that macrophage and NKT cell are co-cultured by quantity ratio 1:15-20.
It is further preferred that macrophage and NKT cell are co-cultured by quantity ratio 1:20.
The present invention also provides the purposes of macrophage, macrophage is used to enhance the purposes of NKT cell killing activity.
Compared with prior art, macrophage cultural method provided by the invention has the advantage that
The present invention sorts out monocyte from mononuclearcell, then using certain density GM-CSF to monokaryon
Cell is induced, and macrophage is made.It is suspension cell that PBMC, which sub-elects the remaining cell after monocyte, will be suspended
Cell culture later co-cultures both macrophage and NKT cell at NKT cell.Macrophage can direct phagocyte,
Also there is certain antigen presentation function simultaneously, therefore macrophage and NKT cell are co-cultured, directly enhance NKT cell
Killing activity.
The present invention stimulates macrophage using the tumor cell lysis liquid supernatant of specificity, obtained macrophage
HLA-II antigen is stronger.When being killed such as NKT cell to K562 cell, when cultivating macrophage, using K562
Cell lysate supernatant stimulates macrophage.When NKT cell kills HL60 cell, in culture macrophage
When, macrophage is stimulated using HL60 cell lysate supernatant.
Detailed description of the invention
Fig. 1 is the flow chart of the cultural method of NKT cell of the present invention.
Fig. 2 is cellular morphology figure in the embodiment of the present invention 1.Wherein, Fig. 2A and Fig. 2 B be respectively NKT cell induce the 1st day,
6th day form, Fig. 2 C and Fig. 2 D are respectively the form that NKT cell and macrophage co-culture the 1st day, the 7th day.
Fig. 3 is the flow cytometer detection result figure of the macrophage obtained in the embodiment of the present invention 1.
Fig. 4 is flow cytometer detection result figure in effect example 1 of the present invention.Wherein Fig. 4 A and Fig. 4 B is using 1 side of embodiment
The NKT cell that method obtains, Fig. 4 C and Fig. 4 D are the NKT cell obtained using 1 method of comparative example.
Fig. 5 is growth curve figure in effect example 3.
Specific embodiment
In order to better illustrate the present invention, it is described further in the following with reference to the drawings and specific embodiments.In the present invention
K562 cell (human leukemia cell), HL60 cell (people's promyelocytic leukemia cell) and HepG2 cell (human liver cancer cell)
It is given by Ji'nan University's biological medicine study base, agents useful for same or instrument are available on the market, the detection method etc. used
All be it is known in the art, details are not described herein.
The cultural method of embodiment 1, NKT cell
A kind of cultural method of NKT cell, comprising the following steps:
The sorting of S1, cell: list is sub-elected from PBMC suspension with CD14 sorting kit (being purchased from STEM CELL company)
Nucleus (CD14+Cell), remaining cell is suspension cell (CD14-Cell).
The induction of S2, macrophage: monocyte is resuspended with macrophage induced medium, according to 1 × 106-2×
106The density of cells/mL is seeded in Tissue Culture Flask, is based on 37 DEG C, 5%CO with macrophage Fiber differentiation2Under the conditions of train
It supports, is periodically observed, every 2-3 days according to 1 × 106The cell density of cells/mL adds culture solution and cell factor.Training
Supporting the K562 cell lysate supernatant that the 4th day is added 5 μ g/mL of final concentration of protein is stimulated, and collection part cell carries out within the 5th day
Streaming identification, the 6th day collection macrophage.The composition of the macrophage induced medium are as follows: 500U/mL GM-CSF, volume
5% autologous plasma of score, surplus are 15 serum free medium of X-VIVO.
The induction of S3, NKT cell: suspension cell is resuspended with NKT cell culture medium, according to 1 × 106-2×106cells/
The density of mL is seeded in Tissue Culture Flask, with NKT cell culture medium in 37 DEG C, 5%CO2Under the conditions of cultivate.Periodically falling
The state for setting microscopically observation cell, every 1-2 days according to 1 × 106The cell density of cells/mL adds culture solution and thin
Intracellular cytokine.6th day collection NKT cell.The composition of the NKT cell culture medium are as follows: 100U/mL IL-2,50 μ g/mL IL-
15,100 μ g/mL IL-21, surplus are 15 serum free medium of X-VIVO.
S4, macrophage and NKT cell co-culture: the macrophage and NKT cell that the 6th day is collected according to 1:10 number
Ratio is measured, with 1 × 106-2×106The density of cells/mL is inoculated with, and is co-cultured with NKT cell with culture medium, and culture is (total to the 14th day
Culture 8 days) collect cell coculture.
The form of two kinds of cells is as shown in Figure 2 when NKT cellular morphology and co-cultivation.Fig. 2A and Fig. 2 B is respectively NKT cell
Induce the form of the 1st day, the 6th day；NKT cell induces the 1st day, and cell is less, small volume, when culture was to the 6th day, cell number
Increase, volume becomes larger, and nucleocytoplasmic ratio is more obvious.Fig. 2 C and Fig. 2 D are respectively that NKT cell and macrophage co-culture the 1st day, the 7th
It form；After co-culturing with macrophage, faster, cell volume is bigger for NKT cell Proliferation.The flow cytometer detection knot of macrophage
Fruit is as shown in figure 3, by Fig. 3 A and Fig. 3 B it is found that the CD14 of macrophage-CD11b+Ratio be 98.7%, illustrate the present embodiment
The purity of the macrophage of middle acquisition is very high.
Autologous plasma, the PBMC used can be prepared by a conventional method.The specific method of PBMC is prepared in the present invention such as
Under:
Peripheral blood in anticoagulant tube is transferred in centrifuge tube, 400-500g is centrifuged 5-10min, by upper layer after centrifugation
Plasma collection carries out hot inactivation in another centrifuge tube in 56 DEG C of water-bath, then with 0.22 μM of disposable filter
Filtering, label is placed on 4 DEG C of refrigerators, spare.
Lower layer's haemocyte is transferred in 50mL centrifuge tube, the physiological saline that two volumes are added is diluted.Separately take one
The sepmate centrifuge tube (being purchased from STEM CELL company) of Zhi Xin, is added Ficoll lymphocyte separation medium and (is purchased from STEM CELL
Company) 12mL, the haemocyte after dilution is transferred to sepmate centrifuge tube, 600-800g is centrifuged 15-20min.It, will after centrifugation
Supernatant liquid is poured out, and physiological saline is added and carries out washing 1 time, obtains PBMC.
The cultural method of embodiment 2, NKT cell
A kind of cultural method of NKT cell, comprising the following steps:
The sorting of S1, cell: monocyte (CD14 is sub-elected from PBMC suspension with CD14 sorting kit+Cell),
Remaining cell is suspension cell (CD14-Cell).
The induction of S2, macrophage: monocyte is resuspended with macrophage induced medium, according to 1 × 106-2×
106The density of cells/mL is seeded in Tissue Culture Flask, is based on 37 DEG C, 5%CO with macrophage Fiber differentiation2Under the conditions of train
It supports, is periodically observed, every 2-3 days according to 1 × 106The cell density of cells/mL adds culture solution and cell factor.Training
Supporting the 4th day addition HL60 cell lysate supernatant is stimulated, and the 5th day collection part cell carries out streaming identification, is collected within the 6th day
Macrophage.The composition of the macrophage induced medium are as follows: 1000U/mL GM-CSF, 3% autologous plasma of volume fraction,
Surplus is 15 serum free medium of X-VIVO.
The induction of S3, NKT cell: suspension cell is resuspended with NKT cell culture medium, according to 1 × 106-2×106cells/
The density of mL is seeded in Tissue Culture Flask, with NKT cell culture medium in 37 DEG C, 5%CO2Under the conditions of cultivate.Periodically falling
The state for setting microscopically observation cell, every 1-2 days according to 1 × 106The cell density of cells/mL adds culture solution and thin
Intracellular cytokine.6th day collection NKT cell.The composition of the NKT cell culture medium are as follows: 100U/mL IL-2,60 μ g/mL IL-
15,20 μ g/mL IL-21, surplus are 15 serum free medium of X-VIVO.
S4, macrophage and NKT cell co-culture: the macrophage and NKT cell that the 6th day is collected according to 1:15 number
Ratio is measured, with 1 × 106-2×106The density of cells/mL is inoculated with, and is co-cultured with NKT cell with culture medium, and culture was received to the 14th day
Collect cell coculture.
The cultural method of embodiment 3, NKT cell
A kind of cultural method of NKT cell, comprising the following steps:
The sorting of S1, cell: monocyte (CD14 is sub-elected from PBMC suspension with CD14 sorting kit+Cell),
Remaining cell is suspension cell (CD14-Cell).
The induction of S2, macrophage: monocyte is resuspended with macrophage induced medium, according to 1 × 106-2×
106The density of cells/mL is seeded in Tissue Culture Flask, is based on 37 DEG C, 5%CO with macrophage Fiber differentiation2Under the conditions of train
It supports, is periodically observed, every 2-3 days according to 1 × 106The cell density of cells/mL adds culture solution and cell factor.Training
Supporting the 4th day addition HepG2 cell lysate supernatant is stimulated, and the 5th day collection part cell carries out streaming identification, is received within the 6th day
Collect macrophage.The composition of the macrophage induced medium are as follows: 750U/mL GM-CSF, the self blood of volume fraction 10%
Slurry, surplus are 15 serum free medium of X-VIVO.
The induction of S3, NKT cell: suspension cell is resuspended with NKT cell culture medium, according to 1 × 106-2×106cells/
The density of mL is seeded in Tissue Culture Flask, with NKT cell culture medium in 37 DEG C, 5%CO2Under the conditions of cultivate.Periodically falling
The state for setting microscopically observation cell, every 1-2 days according to 1 × 106The cell density of cells/mL adds culture solution and thin
Intracellular cytokine.6th day collection NKT cell.The composition of the NKT cell culture medium are as follows: 500U/mL IL-2,20 μ g/mL IL-
15,50 μ g/mL IL-21, surplus are 15 serum free medium of X-VIVO.
S4, macrophage and NKT cell co-culture: the macrophage and NKT cell that the 6th day is collected according to 1:20 number
Ratio is measured, with 1-2 × 106The density of cells/mL is inoculated with, and is co-cultured with NKT cell with culture medium, and culture was collected thin to the 14th day
Born of the same parents' coculture.
Comparative example 1
A kind of cultural method of NKT cell, comprising the following steps:
The sorting of S1, cell
Monocyte (CD14 is sub-elected from PBMC suspension with CD14 sorting kit+Cell), remaining cell is to suspend
Cell (CD14-Cell).
The induction and culture of S2 ', NKT cell:
Suspension cell is resuspended with NKT cell culture medium, according to 1 × 106-2×106The density of cells/mL is seeded to carefully
In born of the same parents' culture bottle, with NKT cell culture medium in 37 DEG C, 5%CO2Under the conditions of cultivate.It is periodically observed under inverted microscope thin
The state of born of the same parents, every 1-2 days according to 1 × 106The cell density of cells/mL adds culture solution and cell factor.It cultivates to the 14th
It collects cell coculture.The composition of the NKT cell culture medium are as follows: 100U/mL IL-2,50 μ g/mL IL-15,100
μ g/mL IL-21, surplus are X-VIVO15 serum free medium.
The flow cytometer detection of effect example 1, cell coculture
Example 1, comparative example 1 collect NKT cell coculture and carry out flow cytometer detection.Flow cytometer detection method is as follows: taking 1
×106A LAK cell；250g centrifugation 5min removes supernatant；It is cleaned 2 times with the PBS solution containing 10%FBS；Be protected from light be added CD3 and
2.5 μ L of CD56 antibody is incubated at room temperature 30min；It is cleaned 2 times with the PBS solution containing 10%FBS；It is cultivated with 500mL RPMI 1640
Base weight is hanged cell and is filtered；Flow cytometer is detected.
Flow cytometer detection result is as shown in Figure 4.As shown in Figure 4, after co-culturing with macrophage, the surface of NKT cell is anti-
Former CD3+And CD56+Expression quantity to be apparently higher than and do not co-culture group, and difference has statistical significance.
Effect example 2, the detection of co-cultured cell killing activity
The macrophage and NKT cell that above-described embodiment 1, comparative example 1 are collected using lactic dehydrogenase (LDH) method for releasing
Coculture carries out K562 cell (human leukemia cell), HL60 cell (people's promyelocytic leukemia cell) and HepG2 respectively
The killing activity of cell (human liver cancer cell) detects.
1 killing activity testing result of table
As shown in Table 1, when killing K562, co-cultivation group is more preferable than the fragmentation effect for not co-culturing group, and difference is more apparent；
When killing HL60, co-cultivation group is got well than not co-culturing the fragmentation effect of group, significant difference；When killing HepG2, co-culture
Group is more preferable than the fragmentation effect for not co-culturing group, and difference has statistical significance.
Effect example 3, the influence for co-culturing cell proliferation
By taking embodiment 1 and comparative example 1 as an example, NKT cell Fiber differentiation is investigated and its with macrophage co-cultivation or individually
Cell proliferative conditions during culture.Every 3d extracts 20 μ L cell suspensions and carries out cell count, amounts to number 5 times, it is thin to draw NKT
Born of the same parents' growth curve figure, as a result as shown in Figure 5.As seen from Figure 5, after co-culturing with macrophage, cell Proliferation adds NKT cell
Fastly, hence it is evident that be faster than and not cultivate group, and difference has statistical significance.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (6)
1. a kind of cultural method of NKT cell, it is characterised in that: the following steps are included:
The sorting of S1, cell: monocyte is sub-elected from PBMC；
The induction of S2, macrophage: the step S1 monocyte sorted is induced as macrophage；
The induction of S3, NKT cell: step S1 is sorted into remaining cell and is induced as NKT cell；
S4, macrophage and NKT cell co-culture: the S2 macrophage obtained and S3 the NKT cell obtained are co-cultured 7-8
It；
The induction of macrophage in the step S2 method particularly includes: use macrophage induced medium culture monocyte, training
It supports to the 4th day addition tumor cell lysis liquid supernatant, culture to the 6th day collection macrophage；The macrophage Fiber differentiation
The composition of base are as follows: 500-1000U/mL GM-CSF, volume fraction 3-10% autologous plasma, surplus are the training of 15 serum-free of X-VIVO
Support base.
2. the cultural method of NKT cell according to claim 1, it is characterised in that: the tumor cell lysis liquid supernatant
Middle final concentration of protein is 1-10 μ g/mL.
3. the cultural method of NKT cell according to claim 1, it is characterised in that: NKT cell lures in the step S3
It leads method particularly includes: with remaining cell after NKT cell culture medium incubation step S1 sorting, cultivate thin to the 6th day collection NKT
Born of the same parents；The composition of the NKT cell culture medium are as follows: 100-500U/mL IL-2,10-100 μ g/mL IL-15,10-100 μ g/mL
IL-21, surplus are 15 serum free medium of X-VIVO.
4. the cultural method of NKT cell according to claim 3, it is characterised in that: in the step S4 macrophage and
NKT cell co-cultures method particularly includes: trains the NKT cell NKT cell culture that the S2 macrophage obtained and S3 are obtained
It supports base co-incubation and collects cell coculture after 7-8 days.
5. the cultural method of NKT cell according to claim 1, it is characterised in that: macrophage and NKT cell press quantity
It is co-cultured than 1:10-25.
6. the cultural method of NKT cell according to claim 4, it is characterised in that: macrophage and NKT cell press quantity
It is co-cultured than 1:15-20.
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