CN107384860A - The cultural method of cell culture fluid and NK cells - Google Patents
The cultural method of cell culture fluid and NK cells Download PDFInfo
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- CN107384860A CN107384860A CN201710876924.1A CN201710876924A CN107384860A CN 107384860 A CN107384860 A CN 107384860A CN 201710876924 A CN201710876924 A CN 201710876924A CN 107384860 A CN107384860 A CN 107384860A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C12N5/0646—Natural killers cells [NK], NKT cells
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- C12N2501/20—Cytokines; Chemokines
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- C12N2501/2302—Interleukin-2 (IL-2)
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- C12N2501/20—Cytokines; Chemokines
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- C12N2501/2305—Interleukin-5 (IL-5)
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Abstract
The application belongs to immunocyte culture technique field, and in particular to the cultural method of cell culture fluid and NK cells.Cell culture fluid provided by the present invention includes a variety of nutrient solutions, including the ALyS505NK AC nutrient solutions containing IL 2 and IL 5, and the ALyS505NK EX nutrient solutions only containing IL 2；Present invention also offers a kind of cultural method of NK cells, obtains the bioactivity height of NK cells using this method, amplification quantity is more, therefore, cultural method provided by the invention is not only simple to operate, and NK Cell viabilities are high, amplification quantity is more, can meet clinical or academic research a variety of application demands.
Description
Technical field
The invention belongs to immunocyte culture technique field, and in particular to the cultural method of cell culture fluid and NK cells.
Background technology
NK (Natural killer cells, NK cells) is that a group is different from T, bone-marrow-derived lymphocyte
Large granular lymphocyte, each lymphoid organ in periphery and blood circulation system are distributed in, advance stimulation and the activation without antigen are
Cellulotoxic effect can be played, and cytokine profiles and chemotactic factor (CF) can be secreted, is the main undertaker of the body innate immunity,
It is the core regulation cell of acquired cellular resistance, is played in tumour immunity, viral infection resisting and the non-own cell of removing important
Effect.NK cell adoptive immunotherapies are the important means of current clinically tumour cell immunization therapy.Due in peripheral blood
NK cell contents are few (account for lymphocyte 5%~10%), moreover, cell concentration, culture medium system, combination of cytokines with
And many factors such as derived from peripheral blood (age, health status) directly affect the effect of NK cell expansion ex vivos.Therefore, one
The efficient NK cell culture fluids of kind amplification in vitro and cultural method are then as one of key issue of NK cell therapies.
At present, in the amplification in vitro incubation of NK cells, frequently with the basal mediums of RPMI 1640 as main training
Base is supported, IL-2, IL-12 and IL-15 are as the cell factor for stimulating NK cell expansion ex vivos.However, a variety of researchs at present
As a result the less stable of above-mentioned cultivating system is shown, cell expanding effect is poor or even fails, and it is thin to amplify the NK come
The bioactivity of born of the same parents is generally relatively low.
The content of the invention
In order to solve the technical problem that prior art expanding effect is poor, NK cell bio-activities are low, the invention provides one
The cultural method of kind cell culture fluid and NK cells.
The concrete technical scheme of the present invention is as follows：
Cell culture fluid, including：First nutrient solution, first nutrient solution include：Hyclone, IL-2, IL-5 and
ALyS505NK-AC nutrient solutions.
Preferably, the percent by volume of the hyclone is 5%~10%；
The concentration of the IL-2 is 500~1500U/mL；
The concentration of the IL-15 is 500~1500U/mL.
Preferably, the cell culture fluid also includes：Second nutrient solution, second nutrient solution include：IL-2 and
ALyS505NK-AC nutrient solutions.
It is highly preferred that the concentration of the IL-2 is 500~1500U/mL.
Preferably, the cell culture fluid also includes：3rd nutrient solution, the 3rd nutrient solution include：IL-2 and
ALyS505NK-EX nutrient solutions.
It is highly preferred that the concentration of the IL-2 is 500~1500U/mL.
Preferably, the cell culture fluid also includes：4th nutrient solution, the 4th nutrient solution include：IL-2 and
ALyS505NK-EX nutrient solutions.
The concentration of the IL-2 is 1000U/mL.
Present invention also offers a kind of cultural method of NK cells, including：PMNC is inoculated in first
Cultivated in nutrient solution, supplement second nutrient solution every other day；Culture at the 5th~8 day supplement the 3rd nutrient solution containing IL-2 after
Continuous culture, supplements the 4th nutrient solution every other day；
First nutrient solution includes：Hyclone, the 500~1500U/mL IL- that percent by volume is 5%~10%
2nd, 500~1500U/mL IL-5 and ALyS505NK-AC nutrient solutions；
Second nutrient solution includes：500~1500U/mL IL-2 and ALyS505NK-AC nutrient solutions；
3rd nutrient solution includes：500~1500U/mL IL-2 and ALyS505NK-EX nutrient solutions；
4th nutrient solution includes：1000U/mL IL-2 and ALyS505NK-EX nutrient solutions.
Preferably, the inoculum density of the PMNC is 1 × 106~1 × 107Individual/mL.
Preferably, the cell density of the NK cells is up to 5 × 108~1 × 109During individual/mL, stop culture.
In summary, cell culture fluid provided by the present invention includes a variety of nutrient solutions, including containing IL-2's and IL-5
ALyS505NK-AC nutrient solutions, and the ALyS505NK-EX nutrient solutions only containing IL-2, ALyS505NK-AC nutrient solutions can be used for
Induced NK cell is activated, ALyS505NK-EX nutrient solutions can be used for amplification NK cells, contained a small amount of IL-2 in cell culture fluid
And/or IL-5 can also further promote the fast breeding of cell, a stable cell culture system is provided for cell culture.This
Invention additionally provides a kind of method using above-mentioned cell culture fluid culture NK cells, and the biology that NK cells are obtained by this method is living
Property it is high, amplification quantity is more, up to 5 × 108More than, therefore, cultural method provided by the invention is not only simple to operate, and
The NK Cell viabilities arrived are high, and amplification quantity is more, can meet clinical or academic research a variety of application demands.Experiments verify that this
The there is provided cell culture fluid of invention and cultural method can carry out induced amplification, peripheral blood to the NK cells of a variety of derived from peripheral blood
Sample age size, whether the factor such as chemotherapy does not influence the propagation quantity and bioactivity of NK cells.
Embodiment
In order to solve the technical problem that prior art expanding effect is poor, NK cell bio-activities are low, the invention provides one
The external evoked amplification cultivation method of kind cell culture fluid and NK cells.
Below in conjunction with the specific embodiment of the invention, technical scheme is clearly and completely described, shown
So, described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.Based on the reality in the present invention
Example is applied, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made, is all belonged to
In the scope of protection of the invention.
Embodiment 1
1. the preparation of PMNC
(1) peripheral blood 30mL is taken, after anti-freezing is handled, according to peripheral blood and physiological saline 1:1 ratio adds physiology
Salt solution, it is well mixed；
(2) it is another to take a 50mL centrifuge tube, 12.5mL lymphocyte separation mediums are added, the blood after dilution is slowly shifted
To the surface of lymphocyte separation medium, make to form clearly interface (blood dilution liquid therebetween：Lymph separating liquid=2:1),
800g centrifuges 15min；
(3) take upper strata to contain the liquid of PMNC (PBMC) layer, be careful not in the lump inhale red blood cell layer
Go out, PMNC crude extract is transferred in another 50mL centrifuge tube；
(4) PMNC crude extract is resuspended with the culture mediums of 40mL RPMI 1640,500g centrifugation 5min, abandons
Clearly, then with RPMI 1640 peripheral hematopoietic stem cells are resuspended, obtain the peripheral hematopoietic stem cells, adjust the concentration of peripheral hematopoietic stem cells
For 1 × 106cells/mL。
The in vitro culture of 2.NK cells
(1) by peripheral hematopoietic stem cells according to 1 × 106Cells/mL density, which is inoculated in Tissue Culture Flask, cultivates, induction
Culture medium be 10mL ALyS505NK-AC activation cultures liquid (purchased from Beijing up to section be Bioisystech Co., Ltd), 1mL tire ox bloods
(it is purchased from GIBCO) clearly, and IL-2 and IL-15 is added according to 1000U/mL concentration, meter PMNC starts to cultivate
For the 0th day；
(2) in the 3rd day and difference fluid infusion in the 5th day, and continue to cultivate, add in right amount containing 1000U/mL IL-2 and
The inducing culture of ALyS505NK-AC activation culture liquid；
(3) liquid was changed in the 7th day, adds 500mL amplification culture mediums, and cell is transferred in 1L cell culture bags
Continue to cultivate, wherein amplification culture medium is ALyS505NK-EX amplification cultivation liquid, and adds IL-2 according to 1000U/mL concentration；
(4) in the 9th day and difference fluid infusion in the 11st day, continue to cultivate, add containing 1000U/mL IL-2 and ALyS505NK-
The amplification culture medium of EX amplification cultivation liquid, when the cell density of the NK cells in nutrient solution is up to 5 × 108~1 × 109During individual/mL,
Stop culture.
Embodiment 2
1.NK cell deriveds and packet
Experimental group 1：The step of according to embodiment 1, the NK obtained from the progress induced amplification culture of health volunteer's peripheral blood
Cell；
Experimental group 2：The step of according to embodiment 1, carry out induced amplification culture from chemotherapy of tumors subject peripheral blood and obtain
NK cells；
Experimental group 3：The step of according to embodiment 1, the NK obtained from the progress induced amplification culture of aged subjects peripheral blood
Cell；
Positive control 1：The NK cell culture mediums and NK cell culture processes provided according to patent CN104894065A, from strong
The peripheral blood of health subject carries out the NK cells that induced amplification culture obtains；
Positive control 2：The NK cell culture compositions and its cultural method provided according to patent CN105296422A, from strong
The peripheral blood of health subject carries out the NK cells that induced amplification culture obtains；
Positive control 3：The NK cell culture fluids and cell culture processes provided according to patent CN106222141A, from health
The peripheral blood of subject carries out the NK cells that induced amplification culture obtains.
2.NK cell quantities obtain and viability examination
Using trypan blue staining, the NK cell quantities of experimental group and positive group are counted respectively, and is survived
Rate detects.The NK cell quantities and survival rate of Different Extraction Method and different derived from peripheral blood are as shown in table 1.
Compare NK cell quantities and the survival rate that different derived from peripheral blood obtain according to step described in embodiment 1, as a result show
Show, the NK cells of experimental group 1 can be expanded to 1 × 109More than cells, average cell motility rate are 95.5%；The NK of experimental group 2
Cell can be expanded to 5 × 108Cells, average cell motility rate are 88.5%；The NK cells of experimental group 3 can be expanded to 7.0 ×
108Cells, average cell motility rate are 90.5%.Summary experimental result, the amplification that explainable the inventive method can be stablized is not
With the NK cells of blood sources, its quantity can also meet needed for clinical treatment, and Cell viability is high.
By more different preparation methods obtain health volunteer's peripheral blood in vitro culture obtain NK cell quantities and
Survival rate, as a result show, the NK cells of experimental group 1 can be expanded to 1 × 109More than cells, average cell motility rate are 95.5%.
The NK cells progress amplification in vitro culture that the method for patent 1~3 is cultivated health volunteer's peripheral blood in vitro is respectively adopted, carefully
Born of the same parents' quantity is respectively 6.5 × 107cells、1×108cells、5.5×107Cells, the cell number expanded far below the present invention
Amount；The NK cell average viabilities that 1~3 offer method of patent obtains are respectively 74.3%, 80.7%, 76.4%, survival rate
Also NK cell survival rates are obtained far below method of the present invention.
Table 1
3.NK cell purities are identified
The purity of the NK cells obtained according to the following steps difference identification experiment group 1~3 and control group 1~3
(1) take containing 1 × 106Cells cell suspension carries out streaming identification, 500g centrifugation 5min, abandons supernatant, with containing
Twice of 5%FBS PBS；
(2) NK cells are resuspended with 10%FBS PBS, add 2.5uL CD3, CD56 antibody, fully mix, lucifuge is incubated
30min is educated, after washing twice with 10%FBS PBS, is resuspended with the basal mediums of RPMI 1640, up flow type instrument detection CD3-CD56+
Cell content, then compare Different Extraction Method and NK cells that different derived from peripheral blood obtain dye detection CD3 through streaming-
CD56+Cell content.
Compare the NK cell purities that different derived from peripheral blood obtain according to cultural method of the present invention, experimental group 1
NK cells (CD3-CD56+) content is up to 90.1%, the NK cells (CD3 of experimental group 2-CD56+) content up to 80.0%, experimental group 3
NK cells (CD3-CD56+) up to 89.5%, data above illustrates to amplify inhomogeneity using what the inventive method can be stablized content
NK cells in the blood sample of type, and the NK cell purities obtained are high.
Compare NK cell quantities and the survival that health volunteer's peripheral blood in vitro culture that different preparation methods obtain obtains
Rate, positive control 1 amplify the NK cells (CD3 come-CD56+) it is 64.9%；Positive control 2 amplifies the NK cells come
(CD3-CD56+) it is 57.7%；Positive control 3 amplifies the NK cells (CD3 come-CD56+) it is 70.5%；Above-mentioned data are equal
Less than the NK cell acquisition rates of the present invention, it is thin to illustrate that the present invention stably can amplify NK in different types of blood sample
Born of the same parents, and the NK cell purities obtained are higher.
4.NK cell killings are tested
Extract and contain 8 × 106Cells/mL NK cell suspensions carry out killing experiments, and table 2 is that each group NK cells are thin to K562
The lethality experimental result of born of the same parents.As shown in the experimental result of table 2, the NK cells of experimental group 1~3:K562=40:1 killing rate is equal
More than 48%, far above positive controls 1 35.85%, the 10.29% of positive controls 2, positive controls 3
19.34%；The NK cells of experimental group 1~3:K562=20:1 killing rate equal more than 32%, far above positive controls 1
16.43%th, 7.58%, the 10.46% of positive controls 3 of positive controls 2；The NK cells of experimental group 1~3:K562=10:
1 killing rate equal more than 21%, far above positive controls 1 4.15%, the 2.42% of positive controls 2, positive controls 3
5.99%.It was found from Mortaility results, the NK cells of experimental group are higher than positive controls to the killing-efficiency of target cell.
Table 2
Claims (10)
1. cell culture fluid, it is characterised in that including：First nutrient solution, first nutrient solution include：Hyclone, IL-2,
IL-5 and ALyS505NK-AC nutrient solutions.
2. cell culture fluid according to claim 1, it is characterised in that the percent by volume of the hyclone is 5%
~10%；
The concentration of the IL-2 is 500~1500U/mL；
The concentration of the IL-15 is 500~1500U/mL.
3. cell culture fluid according to claim 1, it is characterised in that also include：Second nutrient solution, second culture
Liquid includes：IL-2 and ALyS505NK-AC nutrient solutions.
4. cell culture fluid according to claim 3, it is characterised in that the concentration of the IL-2 is 500~1500U/mL.
5. cell culture fluid according to claim 3, it is characterised in that also include：3rd nutrient solution, the 3rd culture
Liquid includes：IL-2 and ALyS505NK-EX nutrient solutions.
6. cell culture fluid according to claim 5, it is characterised in that the concentration of the IL-2 is 500~1500U/mL.
7. cell culture fluid according to claim 5, it is characterised in that also include：4th nutrient solution, the 4th culture
Liquid includes：IL-2 and ALyS505NK-EX nutrient solutions.
The concentration of the IL-2 is 1000U/mL.
A kind of 8. cultural method of NK cells, it is characterised in that including：PMNC is inoculated in the first nutrient solution
Middle culture, second nutrient solution is supplemented every other day；Culture to the 3rd nutrient solution of supplement at the 5th~8 day continues to cultivate, and supplements every other day
4th nutrient solution；
First nutrient solution includes：Percent by volume be 5%~10% hyclone, 500~1500U/mL IL-2,
500~1500U/mL IL-5 and ALyS505NK-AC nutrient solutions；
Second nutrient solution includes：500~1500U/mL IL-2 and ALyS505NK-AC nutrient solutions；
3rd nutrient solution includes：500~1500U/mL IL-2 and ALyS505NK-EX nutrient solutions；
4th nutrient solution includes：1000U/mL IL-2 and ALyS505NK-EX nutrient solutions.
9. cultural method according to claim 8, it is characterised in that the inoculum density of the PMNC is
1×106~1 × 107Individual/mL.
10. cultural method according to claim 8, it is characterised in that the cell density of the NK cells is up to 5 × 108~1
×109During individual/mL, stop culture.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108753724A (en) * | 2018-05-07 | 2018-11-06 | 广州沙艾生物科技有限公司 | A kind of immunocyte cultural method and its application |
| CN109207424A (en) * | 2018-10-24 | 2019-01-15 | 河南省肿瘤医院 | A kind of cultural method of immunocyte |
| KR200495373Y1 (en) | 2020-11-05 | 2022-05-09 | 최보규 | Underware washing and depository net |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108753724A (en) * | 2018-05-07 | 2018-11-06 | 广州沙艾生物科技有限公司 | A kind of immunocyte cultural method and its application |
| CN109207424A (en) * | 2018-10-24 | 2019-01-15 | 河南省肿瘤医院 | A kind of cultural method of immunocyte |
| KR200495373Y1 (en) | 2020-11-05 | 2022-05-09 | 최보규 | Underware washing and depository net |
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