CN108504636A - A kind of efficient NK cell culture processes - Google Patents
A kind of efficient NK cell culture processes Download PDFInfo
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Abstract
The invention discloses a kind of efficient NK cell culture processes, the quantity foot for the NK cells that cultural method of the invention is separately cultured, amplification is fast, and general Peripheral blood culture 15 days can reach 7,000,000,000 cells；The present invention is used as activation transfer agent by adding acetylation alpha-glucan in culture medium; successful activation NK cells; it is promoted to secrete IL2, IL12 and IFN γ; and IL2, IL12 and IFN γ maintain its activity and rate of amplification to play crucial effect after NK cell activations, it is made to have more clinical value；In addition, the purity that the method for the present invention obtains NK cells is high, the testing result of flow cytometer is analyzed, and finds CD3‑CD56+NK cell numbers reach 80% or more, meet the requirement of clinical application.
Description
Technical field
The present invention relates to field of cell culture more particularly to a kind of efficient NK cell culture processes.
Background technology
Natural killer cells (natural killer cell, NK) is the important immunocyte of body, is distributed mainly on outer
In all blood, the 5%-10% of PBMC is accounted for, also has NK activity in lymph node and marrow, but horizontal low compared with peripheral blood.NK cells are one
Group is heterogeneous, multi-functional immunocyte, be the mankind numerous immunocytes in one kind, show as nature cell cytotoxic activity
Kill cell.Main function eliminates virus, bacterium, the cancer cell etc. of attacks body.The mode to play a role is divided into：Directly
Killing, release perforin, NK cytotoxic factors and TNF etc..At least 5,000,000,000 NK cells, at most reach in everyone body
100000000000.But the quantity of NK cells and its energy are not directly proportional.Even if NK cells show enough quantity, still
Them are not activated, their working efficiency is also low-down.
Currently, having there is a large amount of researcher to carry out the culture of NK cells of human beings and grinding in terms of biological therapy both at home and abroad
Study carefully, but most researchs are all the simple knots for going to cultivate, but being obtained by using interleukins type cytokines such as IL-2 and IL-12
Fruit is not achieved the amount needed for therapeutic feedback or is that purity does not increase often there are two drawback or be that quantity does not increase,
Due effect is not achieved.Because interleukin type cytokines are not strong NK cell activators, lack strong work
Change acts on.But after NK cells are sufficiently activated, interleukin type cytokines are maintained to the activity of NK cells and proliferation plays
Particularly important effect.Therefore, the NK cells of high quantity high-purity are obtained, it is necessary first to abundant activated NK.
Invention content
The present invention provides a kind of NK cells of human beings being different from culture-based method to solve the above problem in the prior art
Cultural method is allowed to simple and practicable.The NK cells of human beings that acquisition is cultivated in the present invention is activated, NK cells secretion IL2, IL12 and
The expression higher of IFN-γ, cell purity are high, well-grown, and faster, and cell yield and survival rate are big for cell Proliferation
It is big to improve.
In order to achieve the above technical purposes, the technical measures of the invention taken are：
A kind of efficient NK cell culture processes, include the following steps：
Step 1：Antibody diluent is obtained with PBS dilution monoclonal antibodies CD16 and CD2, is then stayed overnight with antibody diluent
Coated cell culture vessel；
Step 2：Detach mononuclearcell；
Step 3：The mononuclearcell that step 2 obtains is inoculated into culture vessel, the timing since inoculation time, every
Fresh cell culture medium is replaced within 3 days, culture collects cell after 18 days；It is poly- containing the Portugals acetylation α in the cell culture medium
Sugar.
In order to optimize above-mentioned technical proposal, the technical measures that the present invention is taken further include：
Preferably, above-mentioned a concentration of 5-15mg/ml of acetylation alpha-glucan.
Preferably, above-mentioned cell culture medium, the also IL-2 containing 10%knockout serum replacements and 500U/ml.
Preferably, above-mentioned 10%knockout serum replacements can also be substituted for human blood platelets lysate.
Preferably, above-mentioned cell culture medium is CellGro SCGM or RPMI-1640 culture mediums.
Preferably, above-mentioned culture vessel is Tissue Culture Flask or tissue culture plate, more preferably T75 Tissue Culture Flasks.
Preferably, the inoculating cell density of above-mentioned steps 2 is 2 × 106/ml。
On the other hand, it the present invention also provides the NK cells obtained according to above-mentioned cell culture processes culture and its is preparing
Application in antitumor drug.
The present invention is had the following technical effect that compared with prior art using above-mentioned technical proposal：
(1) amplification of traditional method NK cells is slow, and quantity is difficult the cultural method for meeting clinical demand, and utilizing the present invention
The quantity foot for the NK cells being separately cultured, amplification is fast, and general Peripheral blood culture 15 days can reach 7,000,000,000 cells；
(2) traditional method NK cell activity is low, and the quantity of NK cells and its activity be not directly proportional, and the present invention passes through training
Support in base addition acetylation alpha-glucan and transfer agent as activation, successful activation NK cells, promote its secrete IL2, IL12 and
IFN-γ, and IL2, IL12 and IFN-γ maintain its activity and rate of amplification to play crucial effect after NK cell activations, make
It has more clinical value；
(3) compared with conventional method, the purity that the method for the present invention obtains NK cells is high, flow cytometer testing result into
Row analysis, finds CD3-CD56+NK cell numbers reach 80% or more.
Description of the drawings
Fig. 1 is the microscope figure (10X) for the NK passage cells being separately cultured in embodiment one；
Fig. 2 is the microscope figure (20X) for the NK passage cells being separately cultured in embodiment one；
Specific implementation mode
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments be only used for the present invention without
For limiting the scope of the invention.
Embodiment 1
1. the comparison of the cells expanded of this method and conventional method
Experimental group is separately cultured the cell culture processes of NK cells in the present embodiment, is as follows：
The venous blood 60ml of step 1. taking heparin anti-freezing divides 2 to draw mononuclearcell (PBMC).It is as follows：To
15ml lymphocyte separation mediums are added in 50ml centrifuge tubes, then close to tube wall, is slowly added to 30ml venous blood, avoids damage to lymph
The water phase of cell separating liquid.400g is centrifuged 20 minutes.Upper plasma is carefully sucked out into new 50ml centrifuge tubes, 56 DEG C add
Heat 25 minutes, fully inactivates complement, for use.By the tunica albuginea layer of middle layer, i.e. mononuclearcell layer is sucked out to new 15ml centrifugations
Guan Zhong is fully washed 3 times with the PBS of 0.01M, and when washing, 200g is centrifuged 5 minutes, abandons supernatant, gained is PBMC.
The above-mentioned culture mediums of PBMC for centrifuging gained are resuspended step 2., i.e.,：With serum replacement containing 10%knockout
It is resuspended with 1640 culture mediums of acetylation alpha-glucan, cell density is adjusted to 2 × 106Cell re-suspension liquid is transferred to T75 by/ml
In culture bottle, every 3 days culture mediums more renewed, and utilize micro- sem observation cellular morphology (such as attached drawing 1-2).
The present embodiment experimental subjects is divided into experimental group and conventional two groups of group.Wherein, conventional group presses traditional technology culture.Point
Culture cell was not taken from two groups at the 0th, 6,12 and 18 day, with being counted after Trypan Blue, the total number of cells for counting the same day are removed
With the cell number (i.e. the 0th day) before culture, numerical value is the amplification times of cell.It in this approach can be with two groups of cells of Dynamic comparison
Amplification situation, the results are shown in Table 1：At the 6th, 12,18 day of culture, the cells expanded of this law was all remarkably higher than routine
Group, P<0.05.
| Cultivated days (day) | 0 | 6 | 12 | 18 |
| Conventional group | 1 | 11.58±2.27 | 42.59±4.09 | 79.25±5.78 |
| Experimental group | 1 | 22.28±4.34 | 71.43±4.56 | 150.05±5.54 |
The comparison of the cells expanded of 1 this method of table and conventional method
It can be analyzed and be obtained by table 1, traditional method culture NK cells, of high cost, complex process, effect is undesirable, and
And cell amplification is slow, quantity is difficult to meet clinical demand, and utilize the cultural method technique of the present invention relatively easy, is separately cultured
NK cells quantity foot, amplification is fast, disclosure satisfy that clinical demand.
Embodiment 2
1. the comparison of the cell purity of this method and conventional method
The present embodiment experimental subjects is divided into experimental group and conventional two groups of group.Wherein, experimental group is by the cell training in embodiment 1
The method of supporting culture NK cells.Conventional group presses traditional technology culture.At the 18th day cell culture was taken from two groups of cultivating systems respectively
Liquid, PBS are washed twice, and it is 2X10 that cell concentration is adjusted after washing5Streaming antibody is added in/ml, and 4C is protected from light 30min, and it is extra to wash off
After PBS is resuspended, flow cytometer detection is carried out to cell phenotype for antibody.Antibody used comes from U.S. company BD, and the results are shown in Table 2, often
The CD3 of rule group-CD56+NK cells account for 50.16 ± 1.02%, the CD3 of this method-CD56+NK cells account for 82.86 ±
1.83%, two groups of significant differences (P ＜ 0.05).
| Group | CD3-CD56+ |
| Conventional group | 49.16 ± 1.02% |
| Experimental group | 79.86 ± 1.91% |
The comparison of the cell purity of 2 this method of table and conventional method
It can be analyzed and be obtained by table 2, the purity for the NK cells that conventional method obtains is low, largely limits NK cells
Application, and the purity that the method for the present invention obtains NK cells is high, passes through the detection of flow cytometer, CD3-CD56+NK cell numbers
Reach 80% or more, purity has reached the requirement of clinical application, has important directive significance to its application clinically.
Embodiment 3
1. this method is compared with the secretion level of IL2, IL12, IFN-γ that the cell of conventional method is secreted
The present embodiment experimental subjects is divided into experimental group and conventional two groups of group.Wherein, experimental group is by the cell training in embodiment 1
The method of supporting culture NK cells.Conventional group presses traditional technology culture.It was taken respectively from two groups of cultivating systems at the 18th day 100 milliliters thin
Born of the same parents' culture solution, after being concentrated with conventional method, with kit detection IL2, IL12 therein, IFN-γ content, all kits
It buys from Wuhan Boster Biological Technology Co., Ltd..Experimental result is as shown in table 3, IL2, IL12 of experimental group cell secretion,
IFN-γ content is significantly higher than conventional group content (unit μ g/ml).
| Group | IL2 | IL12 | IFN-γ |
| Conventional group | 10±1.4 | 17±1.8 | 24±2.2 |
| Experimental group | 20±1.7 | 31±0.9 | 39±1.8 |
3 this method of table is compared with the secretion level of IL2, IL12, IFN-γ that the cell of conventional method is secreted
It can be analyzed and be obtained by table 3, compared with conventional method, the present invention is by adding acetylation alpha-glucan in culture medium
Agent is transferred as activation, and successful activation NK cells promote it to secrete IL2, IL12 and IFN-γ, and IL2, IL12 and IFN-γ
It maintains its activity and rate of amplification to play crucial effect after NK cell activations, it is made to have more clinical value.
Specific embodiments of the present invention are described in detail above, but it is intended only as example, the present invention is simultaneously unlimited
It is formed on particular embodiments described above.To those skilled in the art, it is any to the equivalent modifications that carry out of the present invention and
It substitutes also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by impartial conversion and
Modification, all should be contained within the scope of the invention.
Claims (9)
1. a kind of efficient NK cell culture processes, which is characterized in that include the following steps：
Step 1：It dilutes monoclonal antibody CD16 and CD2 and obtains antibody diluent, then stay overnight coated cell with antibody diluent
Culture vessel；
Step 2：Detach mononuclearcell；
Step 3：The mononuclearcell that step 2 obtains is inoculated into culture vessel, the timing since inoculation time, every 3 days
Fresh cell culture medium is replaced, culture collects cell after 18 days；Contain acetylation alpha-glucan in the cell culture medium.
2. a kind of efficient NK cell culture processes according to claim 1, which is characterized in that the Portugals acetylation α are poly-
A concentration of 5-15mg/ml of sugar.
3. a kind of efficient NK cell culture processes according to claim 1, which is characterized in that the cell culture medium,
The also IL-2 containing 10%knockout serum replacements and 500U/ml.
4. a kind of efficient NK cell culture processes according to claim 1, which is characterized in that the cell culture medium is
CellGro SCGM or RPMI-1640 culture mediums.
5. a kind of efficient NK cell culture processes according to claim 1, which is characterized in that the culture vessel is thin
Born of the same parents' culture bottle or tissue culture plate.
6. a kind of efficient NK cell culture processes according to claim 1, which is characterized in that the culture vessel is
T75 Tissue Culture Flasks.
7. a kind of efficient NK cell culture processes according to claim 1, which is characterized in that the inoculation of the step 3 is thin
Born of the same parents' density is 2 × 106/ml。
8. the NK cells obtained according to claim 1-7 any one the method cultures.
9. NK cells application in preparation of anti-tumor drugs according to claim 8.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111500535A (en) * | 2020-04-30 | 2020-08-07 | 上海近岸生物科技有限公司 | Method and culture medium for in vitro culture of human natural killer cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907714A (en) * | 2016-04-28 | 2016-08-31 | 王晓冰 | Improved method for cultivating NK (natural killer) cells |
| CN107083361A (en) * | 2017-06-14 | 2017-08-22 | 深圳市泰华细胞工程有限公司 | A kind of cell culture processes |
-
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- 2018-04-13 CN CN201810331464.9A patent/CN108504636A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907714A (en) * | 2016-04-28 | 2016-08-31 | 王晓冰 | Improved method for cultivating NK (natural killer) cells |
| CN107083361A (en) * | 2017-06-14 | 2017-08-22 | 深圳市泰华细胞工程有限公司 | A kind of cell culture processes |
Non-Patent Citations (1)
| Title |
|---|
| C. K. UGGLA等: "Agonistic EfTects of Anti-CD2 and Anti-CD16 Antibodies on Human Natural Killer Killing", 《SCAND. J. IMMUNOL.》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111500535A (en) * | 2020-04-30 | 2020-08-07 | 上海近岸生物科技有限公司 | Method and culture medium for in vitro culture of human natural killer cells |
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