CN109504658A - A kind of cultural method of placental blood NK cell - Google Patents
A kind of cultural method of placental blood NK cell Download PDFInfo
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Abstract
The present invention relates to field of cell culture, in particular to a kind of cultural method of placental blood NK cell, the following steps are included: being coated with the PBMC cell that inoculation is isolated by placental blood in the culture vessel of CD16 monoclonal antibody, culture, cultivating system is to add following component based on VIVO-15 basal medium: FBS, IL-2, IL-12, IL-15；It after culture 4-4.5 days, spreads cultivation, the cultivating system used that spreads cultivation is to add following component on the basis of GT-T581 basal medium: FBS, IL-2, IL-12, IL-15.The cultural method of placental blood NK cell provided by the invention, operation is easy, low in cost, obtains the NK cell that more meets criterion for clinical use, provides good basis for the application of placental blood NK cell.
Description
Technical field
The present invention relates to field of cell culture, in particular to a kind of cultural method of placental blood NK cell.
Background technique
NK cell is also known as natural killer cells, belongs to large granular lymphocyte, derive from marrow, be distributed in peripheral blood,
Liver and spleen are distributed on a small quantity in lymph node and its hetero-organization, are accounted for the 5%~15% of peripheral blood lymphocytes, are important
Immunocyte.NK cell is the important mechanism of body initial stage defence, the cytotoxic activity with strength.Immunological regulation is played simultaneously
Effect, fast lifting immunity and Quality of rehabilitation scientist have arrived NK cell in first identified in 1975, and this kind of cell can be
Lack direct killing tumour cell in the case where T, B cell.It is different from having cytotoxic CD8+T cell, NK cell killing
Presensitization is not needed when tumour cell can be with the tumour cell of direct killing MHC feminine gender, this makes NK cell in adoptive cellular
It is widely used in immunization therapy.But NK cell ratio shared in peripheral blood lymphocytes is lower, and it is therefore necessary to establish
A kind of NK cell expansion ex vivo technology, for studying the function of NK cell and laying the foundation for the cellular immunotherapy of tumour.Mesh
Preceding people for Cord blood application than wide, but seldom for the application of placental blood.
In recent years, someone expresses the molecules such as IL-15, CD137L simultaneously on K562 cell using the method for genetic engineering,
And the proliferation of NK cell is stimulated with this cell.Or use the methods of magnetic bead screening CD56- cell culture NK cell.Above-mentioned side
Method is all higher in operating condition and cost, causes certain difficulty for the clinical use of NK cell.
In view of this, the present invention is specifically proposed.
Summary of the invention
It is easy to operate the purpose of the present invention is to provide a kind of cultural method of placental blood NK cell, it is at low cost, it can obtain
To the NK cell for largely meeting criterion for clinical use, good basis is provided for the application of placental blood NK cell.
Placenta is the vitals of mass exchange between fetus and parent, by embryo's embryophoric membrane and parent during being human pregnancy
Endometrium organizes colligator official between combining the mothers and sons grown up to.Fetus develops in uterus, obtains nutrition from parent by placenta,
And both sides keep comparable independence.
The collection capacity of placental blood can at most acquire 250mL generally in 150mL or more, can collection capacity be higher than Cord blood.Tire
Primitive hematopoietic stem cell is rich in disk blood, quality and quantity matches in excellence or beauty with marrow, and quantity is higher than candidate stem cell in Cord blood and contains
Amount.
The present invention is using the monocyte extracted in placental blood, cell needed for cultivating, and is placental blood using provide can
Energy.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of cultural method of placental blood NK cell, comprising the following steps:
It is coated with the PBMC cell that inoculation is isolated by placental blood in the culture vessel of CD16 monoclonal antibody, is cultivated, body is cultivated
System is to add following component based on VIVO-15 basal medium: in terms of the volume of cultivating system, 50 ± 2 μ L/mL of FBS,
20 ± 2ng/mL of IL-2 500 ± 10IU/mL, IL-12 2.5 ± 0.2ng/mL, IL-15；
Culture 4-4.5 days, is inoculated in the culture vessel for being coated with CD16 monoclonal antibody and spreads cultivation, the cultivating system that spreads cultivation is with GT-
Following component is added based on T581 basal medium: in terms of the volume for the cultivating system that spreads cultivation, FBS 50 ± 2 μ L/mL, IL-2
500 ± 10IU/mL, IL-12 2.5 ± 0.2ng/mL, IL-1550 ± 2ng/mL；
It is described to spread cultivation at least once.
The cultural method of placental blood NK cell provided by the invention, specific selection add phase on different basal mediums
The component answered carries out initial incubation and spreads cultivation, and easy to operate, at low cost, the NK for effectively largely being met criterion for clinical use is thin
Born of the same parents provide good basis for the application of placental blood NK cell.
Further, the culture vessel for being coated with CD16 monoclonal antibody is all made of following methods preparation:
It is 25cm with culture area2The CD16 monoclonal antibody containing 3 ± 1 μ g and the D-PBS of 1 ± 0.2mL, coating is added in meter
3.5h~4.5h.
This is coated under cell culture condition and carries out.
Further, it is coated with the PBMC cell step that inoculation is isolated by placental blood in the culture vessel of CD16 monoclonal antibody
In, density of the PBMC cell in culture vessel is 0.5 × 106A/mL-2.0 × 106A/mL.
As in various embodiments, density of the PBMC cell in culture vessel can be 0.5 × 106A/mL, 1 ×
106A/mL, 1.5 × 106A/mL, 2.0 × 106A/mL etc..
Further, the placental blood obtains PBMC cell using Ficoll density gradient separation.
Further, the multiple to spread cultivation every time is 2-5 times.
As in various embodiments, the multiple to spread cultivation every time can for 2 times, 3 times, 4 times, 5 times and these numerical value it
Between the multiple of any one numerical value etc..
Further, the multiple to spread cultivation every time is 2-3 times.
Further, the number to spread cultivation is 2-4 times.
As in various embodiments, spread cultivation, number can be 2 times, 3 times, 4 times etc..
Further, the number to spread cultivation is 3-4 times.
It is a discovery of the invention that spreading cultivation 3 times with 3 times of the multiple that spreads cultivation, the NK cell for meeting clinical requirement can be effectively obtained.
Further, cultivating system is 25cm with culture area2Meter, the volume of cultivating system are 5-9mL.
Such as in various embodiments, culture area 25cm2, the volume of cultivating system can for 5mL, 6mL, 7mL,
8mL, 9mL etc..
Such as in various embodiments, culture area 75cm2, the volume of cultivating system can for 15mL, 20mL,
22mL, 25mL, 27mL etc..
Such as in various embodiments, culture area 175cm2, the volume of cultivating system can for 35mL, 40mL,
45mL, 50mL, 55mL, 58mL, 60mL, 63mL etc..
Further, the incubation time that spreads cultivation every time is 3-3.5 days.
In the present invention, condition of culture is 37 DEG C, 5%CO2, saturated humidity.
Compared with prior art, the invention has the benefit that
(1) cultural method of placental blood NK cell provided by the invention, it is special using the culture medium and addition of specific components
Fixed trophic factors can effectively excite effective amplification of NK cell, and obtained NK cell meets criterion for clinical use.
(2) cultural method of placental blood NK cell provided by the invention, general cell proliferation times are more, 80 times with
On, the NK cell for meeting criterion for clinical use of more amount is obtained,
(3) cultural method of placental blood NK cell provided by the invention, it is easy to operate, it is at low cost, it is easy to be extended and applied.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 is the result figure that the embodiment of the present invention 1 cultivates that obtained NK cell surface marker is detected；
Fig. 2 is the result figure that the embodiment of the present invention 1 cultivates that obtained NK cell another surface marker is detected.
Specific embodiment
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
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
A kind of cultural method of placental blood NK cell, steps are as follows:
1.1 placental blood NK cell culture mediums
1) basal medium: VIVO-15, GT-T581；
2) additive: FBS, IL-2, IL-12, IL-15, CD16 monoclonal antibody；
1.2 placental blood NK cellular processes
1) it is coated with: taking T25 culture bottle, add 1mLD-PBS, add 3 μ gCD16 monoclonal antibodies, be put into 37 DEG C of incubator coating 4h.
2) PBMC is separated: Ficoll density gradient method separation and Extraction obtains PBMC cell.
It 3) is about 1 × 10 with VIVO-15 culture medium adjustment cell density6/ mL is inoculated into the T25 culture bottle being coated with,
5mL/ bottles.
4) the 0th day plus 250 μ L of FBS, add IL-2:2500IU, IL-12:12.5ng, IL-15:100ng.
5) the 4th day, it is coated with 1 T75 culture bottle, adds 3mLD-PBS, add 9 μ g CD16 monoclonal antibodies, be put into 37 DEG C of incubator coatings
4h.T75 bottles are taken out, coating buffer is gone, cell in T25 bottles is transferred in T75 culture bottle, supplement 15mL GT-T 581 is cultivated
Base makes culture medium total amount 20mL, adds IL-2:10000U, IL-12:50ng, IL-15:1 μ g, FBS by final volume of culture:
1mL。
6) 3 T75 culture bottles of the 7th day coating, add 9 μ gCD16 monoclonal antibodies, are put into 37 DEG C of culture luggages by every bottle plus 3mLD-PBS
By 4h.T75 bottles are taken out, coating buffer is gone, cell is uniformly transferred to respectively in 3 T75 culture bottles in T75 bottles by before, often
Bottle plus 14mL GT-T581 culture medium, by 20ml culture medium, system adds IL-2, IL-12, IL-15 and FBS respectively.
7) 3 T175 culture bottles of the 10th day coating, add 30 μ gCD16 monoclonal antibodies, are put into 37 DEG C of cultures by every bottle plus 10mLD-PBS
Luggage is by 4h.T175 bottles are taken out, coating buffer is gone, cell is uniformly transferred to 3 T175 culture bottles respectively in T75 bottles by before
In, IL-2, IL-12, IL-15 and FBS is added by 60mL final volume in every bottle plus 40mL GT-T581 culture medium.
8) the 14th day harvest cell counts cell quantity.
Obtained cell carries out flow cytometer detection, as a result as depicted in figs. 1 and 2.
The results are shown in Table 1 for statistics.
1 cell of table totally expands information
| 0 day placental blood PBMC sum (× 106) | 5 |
| 14th day harvest total number of cells (× 106) | 405 |
| Total number of cells amplification times | 81 |
Method provided by the invention has the advantage that
1. cell can be proliferated 81 times by the placental blood NK cell of this method culture, initial incubation amount, Ke Yiman are increased
Sufficient clinical use cell quantity.
2. by the placental blood NK cell of this method culture, flow cytometer detection result (Fig. 1 and Fig. 2), CD3-CD56+NK cell
Ratio is 36.42%, CD4+CD25+Treg cell proportion is that 1.23%, NK cell proportion is normal, and immunosuppressant cell ratio is very
It is low, meet criterion for clinical use.
Embodiment 2
A kind of cultural method of placental blood NK cell, steps are as follows:
1.1 placental blood NK cell culture mediums
1) basal medium: VIVO-15, GT-T581；
2) additive: FBS, IL-2, IL-12, IL-15, CD16 monoclonal antibody；
1.2 placental blood NK cellular processes
1) it is coated with: taking T25 culture bottle, add 1mLD-PBS, add 3 μ gCD16 monoclonal antibodies, be put into 37 DEG C of incubator coating 4h.
2) PBMC is separated: extracting PBMC cell with Ficoll density gradient separation.
It 3) is about 0.6 × 10 with VIVO-15 culture medium adjustment cell density6/ mL is inoculated into the T25 culture bottle being coated with
In, 5mL/ bottles.
4) the 0th day plus 260 μ L of FBS, add IL-2:2500IU, IL-12:13.5ng, IL-15:90ng.
5) the 4th day, it is coated with 1 T75 culture bottle, adds 3mLD-PBS, add 9 μ g CD16 monoclonal antibodies, be put into 37 DEG C of incubator coatings
4h.T75 bottles are taken out, coating buffer is gone, cell in T25 bottles is transferred in T75 culture bottle, supplement 15mL GT-T 581 is cultivated
Base makes culture medium total amount 20mL, adds IL-2:10000U, IL-12:50ng, IL-15:1 μ g, FBS by final volume of culture:
1mL。
6) 3 T75 culture bottles of the 7th day coating, add 9 μ gCD16 monoclonal antibodies, are put into 37 DEG C of culture luggages by every bottle plus 3mLD-PBS
By 4h.T75 bottles are taken out, coating buffer is gone, cell is uniformly transferred to respectively in 3 T75 culture bottles in T75 bottles by before, often
Bottle plus 14mL GT-T581 culture medium, by 20ml culture medium, system adds IL-2, IL-12, IL-15 and FBS respectively, ibid.
7) 3 T175 culture bottles of the 10th day coating, add 30 μ gCD16 monoclonal antibodies, are put into 37 DEG C of cultures by every bottle plus 10mLD-PBS
Luggage is by 4h.T175 bottles are taken out, coating buffer is gone, cell is uniformly transferred to 3 T175 culture bottles respectively in T75 bottles by before
In, IL-2, IL-12, IL-15 and FBS is added by 60mL final volume, ibid in every bottle plus 40mL GT-T581 culture medium.
8) the 14th day harvest cell counts cell quantity.
It is counted and is detected, total number of cells and NK cells expanded are almost the same with embodiment 1.
The placental blood NK cell of this method culture, flow cytometer detection is as a result, NK cell proportion is normal, immunosuppressant cell ratio
It is very low, it can satisfy criterion for clinical use.
Embodiment 3
A kind of cultural method of placental blood NK cell, steps are as follows:
1.1 placental blood NK cell culture mediums
1) basal medium: VIVO-15, GT-T581；
2) additive: FBS, IL-2, IL-12, IL-15, CD16 monoclonal antibody；
1.2 placental blood NK cellular processes
1) it is coated with: taking T25 culture bottle, add 1mLD-PBS, add 3 μ gCD16 monoclonal antibodies, be put into 37 DEG C of incubator coating 4h.
2) PBMC is separated: extracting PBMC cell with Ficoll density gradient separation.
It 3) is about 2 × 10 with VIVO-15 culture medium adjustment cell density6/ mL is inoculated into the T25 culture bottle being coated with,
5mL/ bottles.
4) the 0th day plus 250 μ L of FBS, add IL-2:2500IU, IL-12:12.5ng, IL-15:100ng.
5) the 4th day, it is coated with 1 T75 culture bottle, adds 3mLD-PBS, add 9 μ g CD16 monoclonal antibodies, be put into 37 DEG C of incubator coatings
4h.T75 bottles are taken out, coating buffer is gone, cell in T25 bottles is transferred in T75 culture bottle, supplement 15mL GT-T 581 is cultivated
Base makes culture medium total amount 20mL, adds IL-2:10000U, IL-12:50ng, IL-15:1 μ g, FBS by final volume of culture:
1mL。
6) 3 T75 culture bottles of the 7th day coating, add 9 μ gCD16 monoclonal antibodies, are put into 37 DEG C of culture luggages by every bottle plus 3mLD-PBS
By 4h.T75 bottles are taken out, coating buffer is gone, cell is uniformly transferred to respectively in 3 T75 culture bottles in T75 bottles by before, often
Bottle plus 14mL GT-T581 culture medium, by 20ml culture medium, system adds IL-2, IL-12, IL-15 and FBS respectively.
7) 3 T175 culture bottles of the 10th day coating, add 30 μ gCD16 monoclonal antibodies, are put into 37 DEG C of cultures by every bottle plus 10mLD-PBS
Luggage is by 4h.T175 bottles are taken out, coating buffer is gone, cell is uniformly transferred to 3 T175 culture bottles respectively in T75 bottles by before
In, IL-2, IL-12, IL-15 and FBS is added by 60mL final volume in every bottle plus 40mL GT-T581 culture medium.
8) the 14th day harvest cell.
It is counted and is detected, total number of cells and NK cells expanded are almost the same with embodiment 1.
By the placental blood NK cell of this method culture, flow cytometer detection is as a result, NK cell proportion is normal, immunosuppressant cell
Ratio is very low, can satisfy criterion for clinical use.
Comparative example 1
Unlike the first embodiment, in the spreading cultivation of 581 culture medium of the initial incubation of VIVO-15 culture medium and GT-T,
Additional component only adds IL-2.
Different cell numbers is counted and detects, it is as a result as follows.
2 cell of table totally expands information
| 0 day placental blood PBMC sum (× 106) | 5 |
| 14th day harvest total number of cells (× 106) | 105 |
| Total number of cells amplification times | 21 |
Comparative example 2
Unlike the first embodiment, in the spreading cultivation of 581 culture medium of the initial incubation of VIVO-15 culture medium and GT-T,
Additional component only adds IL-2 and IL-12.
Different cell numbers is counted and detects, it is as a result as follows.
3 cell of table totally expands information
| 0 day placental blood PBMC sum (× 106) | 5 |
| 14th day harvest total number of cells (× 106) | 115 |
| Total number of cells amplification times | 23 |
Comparative example 3
Unlike the first embodiment, in the spreading cultivation of 581 culture medium of the initial incubation of VIVO-15 culture medium and GT-T,
Additional component only adds IL-15 and IL-21, and the concentration of IL-21 is 500IU/mL.
Different cell numbers is counted and detects, it is as a result as follows.
4 cell of table totally expands information
| 0 day placental blood PBMC sum (× 106) | 5 |
| 14th day harvest total number of cells (× 106) | 150 |
| Total number of cells amplification times | 30 |
Comparative example 4
Unlike the first embodiment, in the spreading cultivation of 581 culture medium of the initial incubation of VIVO-15 culture medium and GT-T,
Additional component only adds IL-2, IL-15 and IL-21, and the concentration of IL-21 is 500IU/mL.
Different cell numbers is counted and detects, it is as a result as follows.
5 cell of table totally expands information
| 0 day placental blood PBMC sum (× 106) | 5 |
| 14th day harvest total number of cells (× 106) | 245 |
| Total number of cells amplification times | 49 |
Comparative example 5
Unlike the first embodiment, in entire incubation, basal medium is VIVO-15 culture medium.
Different cell numbers is counted and detects, it is as a result as follows.
6 cell of table totally expands information
| 0 day placental blood PBMC sum (× 106) | 5 |
| 14th day harvest total number of cells (× 106) | 265 |
| Total number of cells amplification times | 53 |
Comparative example 6
Unlike the first embodiment, in entire incubation, basal medium is GT-T581 culture medium.
Different cell numbers is counted and detects, it is as a result as follows.
7 cell of table totally expands information
| 0 day placental blood PBMC sum (× 106) | 5 |
| 14th day harvest total number of cells (× 106) | 240 |
| Total number of cells amplification times | 58 |
From the above, it is seen that the cultural method of placental blood NK cell provided by the invention, can effectively expand NK
Cell, method is easy, better meets clinical demand.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of cultural method of placental blood NK cell, which comprises the following steps:
It is coated with the PBMC cell that inoculation is isolated by placental blood in the culture vessel of CD16 monoclonal antibody, is cultivated, cultivating system is
Following component is added based on VIVO-15 basal medium: in terms of the volume of cultivating system, FBS 50 ± 2 μ L/mL, IL-2
20 ± 2ng/mL of 500 ± 10IU/mL, IL-12 2.5 ± 0.2ng/mL, IL-15；
Culture 4-4.5 days, is inoculated in the culture vessel for being coated with CD16 monoclonal antibody and spreads cultivation, the cultivating system that spreads cultivation is with GT-T581
Following component is added based on basal medium: in terms of the volume for the cultivating system that spreads cultivation, FBS 50 ± 2 μ L/mL, IL-2 500 ±
10IU/mL, IL-12 2.5 ± 0.2ng/mL, IL-1550 ± 2ng/mL；
It is described to spread cultivation at least once.
2. the cultural method of placental blood NK cell according to claim 1, which is characterized in that described to be coated with CD16 monoclonal antibody
Culture vessel be all made of following methods preparation:
It is 25cm with culture area2Meter, the D-PBS of addition the CD16 monoclonal antibody containing 3 ± 1 μ g and 1 ± 0.2mL, coating 3.5h~
4.5h。
3. the cultural method of placental blood NK cell according to claim 1, which is characterized in that be coated with the training of CD16 monoclonal antibody
It supports and is inoculated in container by the isolated PBMC cell step of placental blood, density of the PBMC cell in culture vessel is 0.5
×106A/mL-2.0 × 106A/mL.
4. the cultural method of placental blood NK cell according to claim 1, which is characterized in that the placental blood uses
Ficoll density gradient separation obtains PBMC cell.
5. the cultural method of placental blood NK cell according to claim 1-4, which is characterized in that spread cultivation every time
Multiple is 2-5 times.
6. the cultural method of placental blood NK cell according to claim 1-4, which is characterized in that spread cultivation every time
Multiple is 2-3 times.
7. the cultural method of placental blood NK cell according to claim 1-4, which is characterized in that described to spread cultivation
Number is 2-4 times.
8. the cultural method of placental blood NK cell according to claim 1-4, which is characterized in that described to spread cultivation
Number is 3-4 times.
9. the cultural method of placental blood NK cell according to claim 1-4, which is characterized in that cultivating system is equal
It is 25cm with culture area2Meter, the volume of cultivating system are 5-9mL.
10. the cultural method of placental blood CIK cell according to claim 1-4, which is characterized in that spread cultivation every time
Incubation time is 3-3.5 days.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021022229A1 (en) * | 2019-07-31 | 2021-02-04 | Celularity Inc. | Populations of natural killer cells comprising a cleavage resistant cd16 |
| CN113025572A (en) * | 2021-04-09 | 2021-06-25 | 太东(镇江)生物科技有限公司 | Amplification culture medium and application thereof in NK cell culture |
| CN114591904A (en) * | 2022-03-09 | 2022-06-07 | 河南省组织细胞库有限公司 | A method for extracting natural killer cells from the placenta |
| EP4245847A4 (en) * | 2020-11-11 | 2024-10-30 | Hanbio Co., Ltd. | METHOD FOR CULTIVATION OF NK CELLS FOR MASS PROLIFERATION |
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| CN106967682A (en) * | 2017-05-25 | 2017-07-21 | 北京焕生汇生物技术研究院 | A kind of NK cell expansion ex vivos method |
| CN107488631A (en) * | 2017-10-09 | 2017-12-19 | 天津长和生物技术有限公司 | The amplification cultivation method of NK culture matrix and NK |
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| CN106967682A (en) * | 2017-05-25 | 2017-07-21 | 北京焕生汇生物技术研究院 | A kind of NK cell expansion ex vivos method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021022229A1 (en) * | 2019-07-31 | 2021-02-04 | Celularity Inc. | Populations of natural killer cells comprising a cleavage resistant cd16 |
| EP4245847A4 (en) * | 2020-11-11 | 2024-10-30 | Hanbio Co., Ltd. | METHOD FOR CULTIVATION OF NK CELLS FOR MASS PROLIFERATION |
| CN113025572A (en) * | 2021-04-09 | 2021-06-25 | 太东(镇江)生物科技有限公司 | Amplification culture medium and application thereof in NK cell culture |
| CN114591904A (en) * | 2022-03-09 | 2022-06-07 | 河南省组织细胞库有限公司 | A method for extracting natural killer cells from the placenta |
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