CN109370987A - The cultural method of peripheral blood CTL cell improvement - Google Patents

The cultural method of peripheral blood CTL cell improvement Download PDF

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CN109370987A
CN109370987A CN201811409379.6A CN201811409379A CN109370987A CN 109370987 A CN109370987 A CN 109370987A CN 201811409379 A CN201811409379 A CN 201811409379A CN 109370987 A CN109370987 A CN 109370987A
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张权
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See More Vision (beijing) Technology Co Ltd
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See More Vision (beijing) Technology Co Ltd
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex

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Abstract

The present invention relates to technical field of cell culture, in particular to a kind of cultural method of peripheral blood CTL cell improvement.It is cultivated this method comprises: 1) will be inoculated in the culture vessel for being coated with CD3 antibody and CD28 antibody by the isolated PBMC of peripheral blood；Cell factor in cultivating system is IFN-γ 900U/mL~1100U/mL；2) IL-1 α and IL-2 are added into cultivating system, final concentration is 900U/mL~1100U/mL；IL-7 40ng/mL~60ng/mL, IL-12 15ng/mL~25ng/mL is added；3) liquid is changed, the cell factor in new cultivating system is IL-2 900U/mL~1100U/mL.This method CTL expanding effect is good, and the CTL accounting activated is higher, T-reg cell accounting is lower.

Description

The cultural method of peripheral blood CTL cell improvement
Technical field
The present invention relates to technical field of cell culture, in particular to a kind of culture side of peripheral blood CTL cell improvement Method.
Background technique
Cytotoxic T lymphocyte (cytotoxic lymphocyte, CTL), is a species specificity, has killing ability T cell, specially secrete various cell factors and participate in immunizations.Have to antigenic substances such as certain viruses, tumour cells and kills Wound effect constitutes the important defence line of body disease-resistant poison, antineoplastic immune with natural killer cells.
The CTL being activated can generate the anti-tumor activity of specificity, and this cytotoxicity mainly includes (1) perforation The CTL high of element-particle enzymatic pathway (2) activation expresses FaSL, by with target cell surface FaS antigen binding, can induce target cell The cytokine profiles direct killing target cells (4) such as apoptosis (3) TNF secretion generate a variety of chemotactic factor (CF)s, attract the innate immunity thin Born of the same parents, killing target cell (5) discharge a variety of cruel enzymes of serine, promote lethal effect by activating perforin.
The immune response for depending on cytotoxic T cell (CTL) of the protection of intracellular infection and tumour, CTL is to swollen Oncocyte implements the restricted identification of MHC and specific killing.Inside and outside induction, activation and amplification tumor response CTL are current The key subjects of immunotherapy of tumors research, have huge potential significance.However existing CTL extracorporeal culture method, generally deposit Expanding the problems such as CTL multiplying power is low, cell activity is poor.
In view of this, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of novel peripheral blood cells cytotoxic T cell (cytotoxic Lymphocyte, CTL) cultural method, this method is easy to operate, and culture obtains that CTL mass is preferable, can be used for a large amount of of CTL Preparation, makes this method be more conducive to promotion and application.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
The present invention relates to a kind of cultural methods of peripheral blood CTL cell improvement, comprising:
1) by by the isolated PBMC of peripheral blood be inoculated in the culture vessel for being coated with CD3 antibody and CD28 antibody into Row culture；Cell factor in cultivating system is IFN-γ 900U/mL~1100U/mL；
2) IL-1 α and IL-2 are added into cultivating system, final concentration is 900U/mL~1100U/mL；IL-7 is added 40ng/mL~60ng/mL, IL-12 15ng/mL~25ng/mL；
3) liquid is changed, the cell factor in new cultivating system is IL-2 900U/mL~1100U/mL.
Compared with prior art, the invention has the benefit that
CTL expanding effect is good, and the CTL accounting activated is higher, T-reg cell accounting is lower.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the result detected to the surface of dendritic cells marker that one embodiment of the invention culture obtains.
Specific embodiment
The present invention relates to a kind of cultural methods of peripheral blood CTL cell improvement, comprising:
1) by by the isolated PBMC of peripheral blood be inoculated in the culture vessel for being coated with CD3 antibody and CD28 antibody into Row culture；Cell factor in cultivating system is IFN-γ 900U/mL~1100U/mL；
2) IL-1 α and IL-2 are added into cultivating system, final concentration is 900U/mL~1100U/mL；IL-7 is added 40ng/mL~60ng/mL, IL-12 15ng/mL~25ng/mL；
3) liquid is changed, the cell factor in new cultivating system is IL-2 900U/mL~1100U/mL.
In some embodiments, in step 1), cell factor in cultivating system be IFN-γ 950U/mL~ 1050U/mL。
In some embodiments, in step 1), the cell factor in cultivating system is IFN-γ 1000U/mL.
In some embodiments, in step 2), the final concentration that IL-1 α and IL-2 are added in cultivating system is 950U/mL~1050U/mL.
In some embodiments, in step 2), the final concentration that IL-1 α and IL-2 are added in cultivating system is 1000U/mL。
In some embodiments, in step 2), IL-7 50ng/mL is added in cultivating system.
In some embodiments, in step 2), IL-12 20ng/mL is added in cultivating system.
In some embodiments, in step 3), cell factor in new cultivating system be IL-2950U/mL~ 1050U/mL。
In some embodiments, in step 3), the cell factor in new cultivating system is IL-21000U/mL.
In some embodiments, in step 1), method that the culture vessel is coated with CD3 antibody and CD28 antibody Are as follows:
It is 25cm with the culture area2Meter, be added 2.5~3.5mL contain CD3 antibody 400ng/mL~600ng/mL and The coating buffer of CD28 antibody 400ng/mL~600ng/mL is coated with 1.5h~2.5h under cell culture condition.
If non-specifically emphasized, the cell culture condition that herein described method is related to is between 30 to 45 DEG C and 1 To 10% CO2Between, preferably between 36 to 38 DEG C and 4 to 6% CO2Between.
Add CD3, it is to make T cell living that the antibody of CD28, which is the TCR activation and costimulatory signal in order to simulate T cell, Change the state for entering proliferation.
In some embodiments, the concentration of CD3 antibody is 500ng/mL.
In some embodiments, the concentration of CD28 antibody is 500ng/mL.
In some embodiments, the solvent of the coating buffer is D-PBS.
In some embodiments, in step 1), basal medium in the cultivating system be containing 4%~6% from The VIVO culture medium of body blood plasma.
In some embodiments, in step 1), the basal medium in the cultivating system is containing 5% autologous plasma VIVO culture medium.
It in some embodiments, is 25cm with the culture area in step 1)2Meter, the additional amount of culture medium are 13ml~17ml, cell inoculation amount are 0.8 × 107~1.2 × 107。
It in some embodiments, is 25cm with the culture area in step 1)2Meter, the additional amount of culture medium are 15ml, cell inoculation amount are 1.0 × 107。
In some embodiments, the incubation time of step 1) is 20h~28h.
In some embodiments, the incubation time of step 1) is for 24 hours.
In some embodiments, the incubation time of step 2) is 2d~4d.
In some embodiments, the incubation time of step 2) is 3d.
In some embodiments, in step 3), basal medium in the cultivating system be containing 4%~6% from The AIM-V culture medium of body blood plasma.
In some embodiments, in step 3), the basal medium in the cultivating system is containing 5% autologous plasma AIM-V culture medium.
In some embodiments, in step 3), cell is expanded 2~3 times.
In some embodiments, the incubation time of step 3) is 7d~9d.
In some embodiments, the incubation time of step 3) is 8d.
In some embodiments, step 3) specifically includes:
It a) is 75cm with culture area215~25ml culture medium culture 3d is added in meter；
B) liquid is changed, is 175cm with culture area260ml~80ml culture medium culture 3d is added in meter；
C) liquid is changed, is cultivated using culture bag, 300ml~400ml culture medium culture 4d is added.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is It can be with conventional products that are commercially available.
Embodiment 1
The present embodiment is related to a kind of cultural method of peripheral blood CTL improvement.
1. peripheral blood CTL cell culture medium
1) coating buffer: D-PBS, CD3 monoclonal antibody concentration 400ng/mL, CD28 antibody concentration 600ng/mL.
2) CTL initial medium: VIVO culture medium (contains 4% autologous plasma), IFN-γ 900U/mL.
3) CTL culture medium: AIM-V culture medium (contains 4% autologous plasma), IL-2 concentration 1100U/mL.
2. peripheral blood CTL cultivates cellular processes
1) it cultivates the 0th day, takes T25 culture bottle, 2.5mL coating buffer is added, is placed in incubator and is coated with 2.5h, it is spare.It takes The T25 culture bottle being coated with out, goes coating buffer, takes the PBMC 0.8 × 10 prepared7, it is inoculated into T25 culture bottle, adds 13mlCTL initial medium is placed in 37 DEG C, 5%CO2In incubator.
2) it cultivates the 1st day, takes out culture bottle, IL-1 α and IL-2 is added, final concentration is 900U/mL, and IL-7 is added Culture bottle is replaced in 37 DEG C, 5%CO by 40ng/mL, IL-12 25ng/mL2In incubator.
3) it cultivates the 4th day, cell is transferred completely into T75, adds CTL culture medium 20mL, is placed in 37 DEG C, 5%CO2Incubator In.
4) it cultivates the 7th day, cell is transferred in T175 culture bottle, adds CTL culture medium 70mL, is placed in 37 DEG C, 5%CO2 In incubator.
5) it cultivates the 10th day, cell is transferred to addition 400mLCTL culture medium in culture bag, is placed in 37 DEG C, 5%CO2Training It supports in case.
6) it cultivates the 12nd day, harvests cell.
Embodiment 2
The present embodiment is related to a kind of cultural method of peripheral blood CTL improvement.
1. peripheral blood CTL cell culture medium
1) coating buffer: D-PBS, CD3 monoclonal antibody concentration 600ng/mL, CD28 antibody concentration 400ng/mL.
2) CTL initial medium: VIVO culture medium (contains 6% autologous plasma), IFN-γ 1100U/mL.
3) CTL culture medium: AIM-V culture medium (contains 6% autologous plasma), IL-2 concentration 900U/mL.
2. peripheral blood CTL cultivates cellular processes
1) it cultivates the 0th day, takes T25 culture bottle, 3.5mL coating buffer is added, is placed in incubator and is coated with 1.5h, it is spare.It takes The T25 culture bottle being coated with out, goes coating buffer, takes the PBMC 1.2 × 10 prepared7, it is inoculated into T25 culture bottle, adds 17ml CTL initial medium, is placed in 37 DEG C, 5%CO2In incubator.
2) it cultivates the 1st day, takes out culture bottle, IL-1 α and IL-2 is added, final concentration is 1100U/mL, and IL-7 is added Culture bottle is replaced in 37 DEG C, 5%CO by 60ng/mL, IL-12 15ng/mL2In incubator.
3) it cultivates the 4th day, cell is transferred completely into T75, adds CTL culture medium 20mL, is placed in 37 DEG C, 5%CO2Incubator In.
4) it cultivates the 7th day, cell is transferred in T175 culture bottle, adds CTL culture medium 70mL, is placed in 37 DEG C, 5%CO2 In incubator.
5) it cultivates the 10th day, cell is transferred to addition 400mLCTL culture medium in culture bag, is placed in 37 DEG C, 5%CO2Training It supports in case.
6) it cultivates the 12nd day, harvests cell.
Embodiment 3
The present embodiment is related to a kind of cultural method of peripheral blood CTL improvement.
1. peripheral blood CTL cell culture medium
1) coating buffer: D-PBS, CD3 monoclonal antibody concentration 450ng/mL, CD28 antibody concentration 550ng/mL.
2) CTL initial medium: VIVO culture medium (contains 5% autologous plasma), IFN-γ 950U/mL.
3) CTL culture medium: AIM-V culture medium (contains 5% autologous plasma), IL-2 concentration 1050U/mL.
2. peripheral blood CTL cultivates cellular processes
1) it cultivates the 0th day, takes T25 culture bottle, 2.8mL coating buffer is added, is placed in incubator and is coated with 2.2h, it is spare.It takes The T25 culture bottle being coated with out, goes coating buffer, takes the PBMC 1.1 × 10 prepared7, it is inoculated into T25 culture bottle, adds 14ml CTL initial medium, is placed in 37 DEG C, 5%CO2In incubator.
2) it cultivates the 1st day, takes out culture bottle, IL-1 α and IL-2 is added, final concentration is 1050U/mL, and IL-7 is added Culture bottle is replaced in 37 DEG C, 5%CO by 45ng/mL, IL-12 18ng/mL2In incubator.
3) it cultivates the 4th day, cell is transferred completely into T75, adds CTL culture medium 20mL, is placed in 37 DEG C, 5%CO2Incubator In.
4) it cultivates the 7th day, cell is transferred in T175 culture bottle, adds CTL culture medium 70mL, is placed in 37 DEG C, 5%CO2 In incubator.
5) it cultivates the 10th day, cell is transferred to addition 400mLCTL culture medium in culture bag, is placed in 37 DEG C, 5%CO2Training It supports in case.
6) it cultivates the 12nd day, harvests cell.
Embodiment 4
The present embodiment is related to a kind of cultural method of peripheral blood CTL improvement.
1. peripheral blood CTL cell culture medium
1) coating buffer: D-PBS, CD3 monoclonal antibody concentration 500ng/mL, CD28 antibody concentration 500ng/mL.
2) CTL initial medium: VIVO culture medium (contains 5% autologous plasma), IFN-γ 1000U/mL.
3) CTL culture medium: AIM-V culture medium (contains 5% autologous plasma), IL-2 concentration 1000IUmL.
2. peripheral blood CTL cultivates cellular processes
1) it cultivates the 0th day, takes T25 culture bottle, 3mL coating buffer is added, is placed in incubator and is coated with 2h, it is spare.Take out packet By good T25 culture bottle, coating buffer is gone, takes PBMC1.0 × 10 prepared7, it is inoculated into T25 culture bottle, adds 15mLCTL Initial medium is placed in 37 DEG C, 5%CO2In incubator.
2) it cultivates the 1st day, takes out culture bottle, IL-1 α and IL-2 is added, final concentration is 1000U/mL, and IL-7 is added Culture bottle is replaced in 37 DEG C, 5%CO by 50ng/mL, IL-12 20ng/mL2In incubator.
3) it cultivates the 4th day, cell is transferred completely into T75, adds CTL culture medium 20mL, is placed in 37 DEG C, 5%CO2Incubator In.
4) it cultivates the 7th day, cell is transferred in T175 culture bottle, adds CTL culture medium 70mL, is placed in 37 DEG C, 5%CO2 In incubator.
5) it cultivates the 10th day, cell is transferred to addition 400mLCTL culture medium in culture bag, is placed in 37 DEG C, 5%CO2Training It supports in case.
6) it cultivates the 12nd day, harvests cell, do flow cytometer detection；Its result is as shown in Figure 1.
Comparative example 1
Peripheral blood CTL cultivates cellular processes
1) it cultivates the 0th day, takes the PBMC 1.0 × 10 prepared7, it is inoculated into T25 culture bottle (without coating), adds 10mLCTL initial medium is placed in 37 DEG C, 5%CO2In incubator.
2) it cultivates the 1st day, takes out culture bottle, IL-1 α and IL-2 is added, final concentration is 1000U/mL, and IL-7 is added CD3 monoclonal antibody concentration 500ng/mL, CD28 antibody concentration 500ng/mL is added in 50ng/mL, IL-12 20ng/mL；By culture bottle weight Newly it is placed in 37 DEG C, 5%CO2In incubator.
Remaining step is the same as embodiment 4.
Comparative example 2
The replacement of CTL cell culture medium are as follows:
1) CTL initial medium: RPM1-1640 culture medium (contains 5% autologous plasma), IFN-γ 1000U/mL.
2) CTL culture medium: RPM1-1640 culture medium (contains 5% autologous plasma), IL-2 concentration 1000IUmL.
Remaining condition of culture is the same as embodiment 4.
Comparative example 3
The present embodiment is related to the cultural method of peripheral blood CTL a kind of.
1. peripheral blood CTL cell culture medium
1) coating buffer: D-PBS, CD3 monoclonal antibody concentration 500ng/mL, CD28 antibody concentration 500ng/mL.
2) CTL initial medium: VIVO culture medium (contains 5% autologous plasma), IFN-γ 1000U/mL.
3) CTL culture medium: VIVO culture medium (contains 5% autologous plasma), IL-2 concentration 1000IUmL.
2. peripheral blood CTL cultivates cellular processes
1) it cultivates the 0th day, takes T25 culture bottle, 3mL coating buffer is added, is placed in incubator and is coated with 2h, it is spare.Take out packet By good T25 culture bottle, coating buffer is gone, takes PBMC1.0 × 10 prepared7, it is inoculated into T25 culture bottle, adds 10mLCTL Initial medium is placed in 37 DEG C, 5%CO2In incubator.
2) it cultivates the 1st day, takes out culture bottle, IL-1 α and IL-2 is added, final concentration is 1000U/mL, by culture bottle weight Newly it is placed in 37 DEG C, 5%CO2In incubator.
3) it cultivates the 4th day, cell is transferred completely into T75, adds CTL culture medium 20mL, is placed in 37 DEG C, 5%CO2Incubator In.
4) it cultivates the 7th day, cell is transferred in T175 culture bottle, adds CTL culture medium 70mL, is placed in 37 DEG C, 5%CO2 In incubator.
5) it cultivates the 10th day, cell is transferred to addition 400mLCTL culture medium in culture bag, is placed in 37 DEG C, 5%CO2Training It supports in case.
6) it cultivates the 14th day, harvests cell, do flow cytometer detection.
Experimental example
After the cell for collecting embodiment 4 and comparative example 1,2, cell count is carried out, and by flow cytometry to phenotype It is identified.Every group repetition six times, it is as a result as follows:
Group Cell number
Embodiment 4 9.1×108
Comparative example 1 8.4×108
Comparative example 2 3.3×108*
Comparative example 3 5.3×108*
* p < 0.05, vs embodiment 4.
* p < 0.05, vs embodiment 4.
Even if the quantity of cell harvest is still few it can be seen from the results above that 1~3 incubation time of comparative example is longer In the application；In addition, the culture medium prescription of the application optimization can preferably activate CTL, and by control differentiation opportunity, reduce The ratio of T-reg cell.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but those skilled in the art should understand that: its It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features It is equivalently replaced；And these are modified or replaceed, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution The range of art scheme.

Claims (10)

1. a kind of cultural method of peripheral blood CTL cell improvement characterized by comprising
1) it will be inoculated in the culture vessel for being coated with CD3 antibody and CD28 antibody and trained by the isolated PBMC of peripheral blood It supports；Cell factor in cultivating system is IFN-γ 900U/mL~1100U/mL；
2) IL-1 α and IL-2 are added into cultivating system, final concentration is 900U/mL~1100U/mL；IL-7 40ng/ is added ML~60ng/mL, IL-12 15ng/mL~25ng/mL；
3) liquid is changed, the cell factor in new cultivating system is IL-2 900U/mL~1100U/mL.
2. the cultural method of peripheral blood CTL cell improvement according to claim 1, which is characterized in that in step 1), institute State the method that culture vessel is coated with CD3 antibody and CD28 antibody are as follows:
It is 25cm with the culture area2Meter is added 2.5~3.5mL and contains CD3 antibody 400ng/mL~600ng/mL and CD28 The coating buffer of antibody 400ng/mL~600ng/mL is coated with 1.5h~2.5h under cell culture condition.
3. the cultural method of peripheral blood CTL cell improvement according to claim 2, which is characterized in that the coating buffer Solvent is D-PBS.
4. the cultural method of peripheral blood CTL cell improvement according to claim 1, which is characterized in that in step 1), institute Stating the basal medium in cultivating system is the VIVO culture medium containing 4%~6% autologous plasma.
5. the cultural method of peripheral blood CTL cell improvement according to claim 4, which is characterized in that in step 1), with The culture area is 25cm2Meter, the additional amount of culture medium are 13ml~17ml, and cell inoculation amount is 0.8 × 107~1.2 × 107。
6. the cultural method of peripheral blood CTL cell improvement according to claim 5, which is characterized in that the culture of step 1) Time is 20h~28h.
7. the cultural method of peripheral blood CTL cell improvement according to claim 1, which is characterized in that the culture of step 2) Time is 2d~4d.
8. the cultural method of peripheral blood CTL cell improvement according to claim 1, which is characterized in that in step 3), institute Stating the basal medium in cultivating system is the AIM-V culture medium containing 4%~6% autologous plasma.
9. the cultural method of peripheral blood CTL cell improvement according to claim 8, which is characterized in that, will in step 3) Cell expands 2~3 times.
10. the cultural method of peripheral blood CTL cell improvement according to claim 9, which is characterized in that the culture of step 3) Time is 7d~9d.
CN201811409379.6A 2018-11-23 2018-11-23 The cultural method of peripheral blood CTL cell improvement Withdrawn CN109370987A (en)

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CN110305843A (en) * 2019-07-12 2019-10-08 赛德特生物科技开发有限公司 Immunocyte of autoimmune disease and the preparation method and application thereof can be improved

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