CN109097331A - A kind of NK cell non-serum culture medium - Google Patents
A kind of NK cell non-serum culture medium Download PDFInfo
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Abstract
The present invention relates to the technical fields of cell culture, disclose a kind of NK cell non-serum culture medium, including basal medium and additive, and the additive includes albumin, amino acid, buffer, oleic acid, linoleic acid and insulin；The present invention provides a kind of NK cell non-serum culture medium, have the advantages that (1), medium component are clear, stability is high, and Commercial sources are substantially unrestricted, are easy to buy.(2), it using culture medium prepared by the present invention, promotes the amplification efficiency of cell and guarantees the specificity of NK cell, the cell of high-quality can be obtained in the case where simplifying Cell isolation and culture step.(3), serum free medium prepared by the present invention, the unknown heterologous protein of no ingredient avoid cell contamination and reduce the security risk of cell preparation, be conducive to clinical application.
Description
Technical field
The present invention relates to the technical field of cell culture, especially a kind of NK cell non-serum culture medium.
Background technique
NK cell (Natural killer cells, NK cells) i.e. natural killer cells, is body innate immunity system
The important component of system and the important composition cell of antiviral and antitumor.Studies have shown that in most of tumor patient bodies
The ability decline of the removing cancer cell of the decline of NK cell quantity and/or NK cell, NK cell surface activation receptor and inhibition receptor
Expression determines the power of its anti-tumor capacity.The quantity and activity of tumor patient and tumor post-operation patient's body NK cell all occur
Certain change.Generation, development and the transfer of tumour and the antineoplastic immune function of NK cell, which are suppressed, close contact.
NK cell is primarily present in Peripheral Circulation in normal human, accounts for the 90% of NK total number of cells.But NK cell exists
Proportional amount in peripheral blood is few (5%-7% for only accounting for about lymphocyte), therefore regardless of in experimental study or clinic
Treatment use, NK cell must all carry out in vitro culture and amplification, to reach ideal research and treat required state.
Currently, in the scheme of common NK cell expansion ex vivo and activation, it is dynamic using being added in order to improve expanding effect
The culture medium of object serum (such as FBS).But animal blood serum complicated component, and the animal blood serum of different batches is difficult to consistency
And stability, it is also possible to mycoplasma, bacterium, virus or albumen communicable disease etc. can be brought to pollute.In addition animal blood serum is brought
Foreign protei, also will increase the risk of NK cell in clinical application, foreign protei can cause immune response, allergic reaction and
The immunological rejections such as inflammatory reaction.Therefore the cell culture medium containing serum limits the application of NK cell clinically.
For the needs for adapting to clinical application, (or blood serum substituting is added in basal medium using serum free medium
Object) culture NK cell method used.But serum free medium currently used in the market, which tends not to reach, serum
The effect of culture medium, while most of serum free mediums are the collective medias for cultivating immunocyte, without especially effectively
Special culture NK cell serum free medium.
Summary of the invention
The purpose of the present invention is to provide a kind of NK cell non-serum culture mediums, it is intended to which solution lacks one kind in the prior art
The problem of serum free medium of particularly effective special culture NK cell.
The invention is realized in this way a kind of NK cell non-serum culture medium, including basal medium and additive, it is described
Additive includes albumin, amino acid, buffer, oleic acid, linoleic acid and insulin.
Further, the concentration of the component in the additive are as follows: albumin (1g/L), oleic acid (0.05-0.2mg/L),
Linoleic acid (0.2-0.5mg/L), insulin (5-10mg/L).
Further, the amino acid in the additive includes l-tyrosine (30mg/L), l-cysteine (70mg/L), L-
Glutamy amino acid (50mg/L), L-Glutamine (350mg/L), glycine (20mg/L), Valine (60mg/L), L- color ammonia
Sour (20mg/L), L-phenylalanine (30mg/L), Serine (20mg/L) and L-threonine (60mg/L).
Further, the buffer includes L- phosphoglycerol disodium salt hydrate (1g/L) and 4- hydroxyethyl piperazine second sulphur
Sour (1g/L).
Further, the concentration of the component in the additive are as follows: albumin (1g/L), oleic acid (0.12mg/L), sub- oil
Acid (0.35mg/L), insulin (7.5mg/L).
Further, the albumin is human serum albumin.
Further, the basal medium is RPMI1640 serum free medium.
Further, the additive further includes D glucose.
Further, the concentration of the D glucose are as follows: 1-5g/L.
Compared with prior art, it the present invention provides a kind of NK cell non-serum culture medium, has the advantages that
(1), medium component is clear, and stability is high, and Commercial sources are substantially unrestricted, are easy to buy.
(2), it using culture medium prepared by the present invention, promotes the amplification efficiency of cell and guarantees the specificity of NK cell, it can
To obtain the cell of high-quality in the case where simplifying Cell isolation and culture step.
(3), serum free medium prepared by the present invention, the unknown heterologous protein of no ingredient, avoids cell contamination and reduction
The security risk of cell preparation, is conducive to clinical application.
Detailed description of the invention
Fig. 1 is the NK cell amplification effect provided in an embodiment of the present invention using serum free medium culture provided by the invention
Rate result figure；
Fig. 2 is provided in an embodiment of the present invention thin using the NK cell streaming of serum free medium culture provided by the invention
Born of the same parents' instrument testing result figure；
Fig. 3 is that the NK cell killing provided in an embodiment of the present invention using serum free medium culture provided by the invention is examined
Survey result figure.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
The same or similar label correspond to the same or similar components in the attached drawing of the present embodiment；In description of the invention
In, it is to be understood that if there is the orientation or positional relationship of the instructions such as term " on ", "lower", "left", "right" for based on attached drawing institute
The orientation or positional relationship shown, is merely for convenience of description of the present invention and simplification of the description, rather than the dress of indication or suggestion meaning
It sets or element must have a particular orientation, be constructed and operated in a specific orientation, therefore describe the use of positional relationship in attached drawing
Language only for illustration, should not be understood as the limitation to this patent, for the ordinary skill in the art, can be with
The concrete meaning of above-mentioned term is understood as the case may be.
Realization of the invention is described in detail below in conjunction with specific embodiment.
Referring to Fig.1 shown in -3, preferred embodiment is provided for the present invention.
A kind of NK cell non-serum culture medium, including basal medium and additive, additive include albumin, amino
Acid, buffer, oleic acid, linoleic acid and insulin.
The present invention provides a kind of NK cell non-serum culture medium, NK cell non-serum culture medium include definite ingredients
The additive of alternative serum, additive include albumin, amino acid, buffer, oleic acid, linoleic acid and insulin.Albumin is
Surviving and expanding in vitro on NK cell influences maximum nutriment, is the basis for constructing cell membrane and other important substances.
Entrained fatty acid influences the various aspects such as growing multiplication, the cell function of NK cell in albumin.In culture medium oleic acid and
Linoleic concentration is also an important factor for influencing NK cell Proliferation and killing activity.Insulin can stimulate cell to uridine and Portugal
The absorption of grape sugar synthesizes RNA, protein and lipid with this, and insulin can also be adjusted in conjunction with the insulin receptor on cell membrane
Intracellular multiple metabolic pathways are controlled, the synthesis of fatty acid and glucose is increased, are played an important role to cell growth.
A kind of NK cell non-serum culture medium of above-mentioned offer, has the advantages that
(1), medium component is clear, and stability is high, and Commercial sources are substantially unrestricted, are easy to buy.
(2), it using culture medium prepared by the present invention, promotes the amplification efficiency of cell and guarantees the specificity of NK cell, it can
To obtain the cell of high-quality in the case where simplifying Cell isolation and culture step.
(3), serum free medium prepared by the present invention, the unknown heterologous protein of no ingredient, avoids cell contamination and reduction
The security risk of cell preparation, is conducive to clinical application.
Preferably, the concentration of the component in additive are as follows: albumin (1g/L), oleic acid (0.05-0.2mg/L), linoleic acid
(0.2-0.5mg/L), insulin (5-10mg/L).
It is further preferred that the concentration of additive each component are as follows: albumin (1g/L), oleic acid (0.12mg/L), linoleic acid
(0.35mg/L), insulin (7.5mg/L).
Furthermore the amino acid in additive includes l-tyrosine (30mg/L), l-cysteine (70mg/L), L-Glutamine
Acid (50mg/L), L-Glutamine (350mg/L), glycine (20mg/L), Valine (60mg/L), L-Trp (20mg/
L), L-phenylalanine (30mg/L), Serine (20mg/L) and L-threonine (60mg/L).
Buffer includes L- phosphoglycerol disodium salt hydrate (1g/L) and 4- hydroxyethyl piperazineethanesulfonic acid (1g/L).
Specifically, albumin is human serum albumin.
Furthermore basal medium is RPMI1640 serum free medium.
In the present embodiment, additive further includes D glucose.
Specifically, the concentration of D glucose are as follows: 1-5g/L.
The following are specific embodiments provided by the invention:
NK cell non-serum culture medium includes the additive of basal medium and the specific alternative serum of other compositions, the training
The basal medium for supporting base is RPMI1640 serum free medium, and additive includes albumin, amino acid, buffer, oleic acid, Asia
Oleic acid and insulin.The concentration of additive is albumin 1g/L, oleic acid 0.12mg/L, linoleic acid 0.35mg/L, insulin
7.5mg/L, albumin are human serum albumin, and the amino acid in additive includes l-tyrosine (30mg/L), l-cysteine
(70mg/L), L-Glutamine acid (50mg/L), L-Glutamine (350mg/L), glycine (20mg/L), Valine
(60mg/L), L-Trp (20mg/L), L-phenylalanine (30mg/L), Serine (20mg/L) and L-threonine (60mg/
L), buffer includes L- phosphoglycerol disodium salt hydrate (1g/L) and 4- hydroxyethyl piperazineethanesulfonic acid (1g/L).
Using the serum free medium amplification in vitro culture NK cell of above-mentioned configuration, specific implementation step is as follows:
(1) Tissue Culture Flask is coated with:
Culture bottle coating buffer used in the present invention using the following antibody of addition and is diluted to preferred concentration Heceptin
The PBS of (800ug/ml), CD16 (0.5ug/ml) and NKP46 (0.5ug/ml).Coating buffer is added in culture bottle, keeps liquid uniform
Spread out, 37 DEG C, be incubated for 1h after inhale abandon coating buffer.
(2) PBMC is separated:
The NK cell origin that the present invention uses utilizes following steps and the single core of the isolated peripheral blood of method in peripheral blood
Cell (Peripheral blood mononuclear cell, PBMC): the peripheral blood of patient is acquired, and has been transferred to anticoagulant
In the bottle of agent, then the blood being collected into gently poured into containing (not mixing) in lymphocyte separation medium test tube.At room temperature with
2000rpm/min is centrifuged 20min.Blood is divided into four layers, at the monocyte between the blood plasma on upper layer, blood plasma and separating liquid
In the second layer, the lymphocyte separation medium of third layer and the 4th layer of red blood cell.Second layer monocyte and three layers of separating liquid are taken,
Physiological saline centrifugation is cleaned twice, and purpose PBMC is obtained.
(3) cell-stimulating
Cell-stimulating culture solution used in the present invention, for the free serum culture for being added to cell stimulation factor Krestin
Base, the preferred concentration of stimulating factor Krestin are 50ug/ml.
The PBMC that step (2) are obtained is resuspended with cell-stimulating culture solution, and adjusts cell concentration 1 × 106/ ml inoculation
It is wrapped in processed culture bottle to step (1).
Addition volume ratio is 10% autologous plasma, and 500IU/mlIL-2,50ng/mlIL-12 and 50ng/mlIL- is added
15.Cultivating system is controlled in 10-15ml.
37 DEG C of temperature, 5%CO2It is cultivated under environment.
(4) cell induced amplification culture
In the present invention culture of cell induced amplification use induction broth, induction broth be added to IL-2, IL-12
With the serum free medium of one of IL-15 or cytokine profiles.The cell factor preferred concentration of addition are as follows: IL-2
(1000IU/ml, 50ng/mlIL-12 and 50ng/mlIL-15).
The cell of above-mentioned steps (3) activation, after activation culture for 24 hours, directly supplement adds induction broth, by cultivating system
It is supplemented to 40ml.And the autologous plasma of certain proportion and concentration (10%) is added, and at 37 DEG C of temperature, 5%CO2Continue to train under environment
It supports；Centre was according to cell growth status every 2-3 days primary supplement culture solutions, and double cultivating system.
When cultivating system is more than 400ml, cell suspension is transferred to and continues to cultivate in cell culture bags, and adjusts blood plasma
Ratio is 1%, maintains IL-2 and IL-12 constant, but no longer adds cell factor IL-15.After culture 11 days, cultivating system fluid infusion
To 1.6L.Take out cell from culture bag within 13rd day, harvest the NK cell of amplification, counted, flow cytometer showed detection and
Cytoactive detection.
(5) NK cell amplification efficiency detects
Step (3) (4) records cell growth condition, in the difference of culture during cytositimulation and amplification cultivation
Between section (being counted the 0th, 2,4,6,8,10,12,14 day from inoculation time), carry out cell count simultaneously draw NK cell growth curve.
(6) NK cell flow cytometer showed
The NK cell of step (4) harvest analyzes cell surface antigen phenotype using flow cytomery, detects cell
Purity.
Method is as follows: collecting cell to be measured, with table-type low-speed autobalance centrifuge centrifuge cell, relative centrifugal force is
1200g, delaying the slow drop of liter is 5, abandons supernatant after being centrifuged 5min.Cell is resuspended in PBS, and washing cell is three times.With cell counter into
Row counts, and is again 1.0 × 10 with PBS adjustment cell density6A/ml.It takes in 100 microlitres of addition streaming dedicated pipes, is added not
Synantibody (AntiCD3 McAb and CD56 antibody).It is placed in ice chest and is protected from light incubation 30 minutes, loading is analyzed respectively.
(7) NK cell killing activity detects
The NK cell of step (4) harvest detects NK cell to target cell using lactic dehydrogenase (LDH) method for releasing
K562 fragmentation effect.Lactic dehydrogenase is present in into the cell, cannot penetrate cell membrane under normal circumstances.When cell is damaged
When, LDH can be from intracellular release into culture solution.The LDH released is detected during being catalyzed lactic acid generation pyruvic acid,
It can produce the compound for having absorption peak at specific wavelength (450nm), therefore can be when enzyme detector reads 450nm
OD value calculates the killing activity of NK cell.
As a result:
Cell amplification efficiency result is as shown in Fig. 1；
Respectively at the 0th, 2,4,6,8,10,12,14 day, culture cell is counted, cell growth curve figure is drawn.Such as
Figure obtains 100-200 times of growth as it can be seen that cell growth efficiency height within the amplification period.
FCM analysis result is as shown in Fig. 2；
Fig. 2 is the NK cell flow cytometer testing result figure of the present embodiment amplification, image left upper quadrant: CD56 is positive,
CD3 is negative, represents NK cell, accounts for 84%；As shown in the figure, it can be seen that the NK cell purity that the amplification of the present embodiment obtains is high.
NK cell killing testing result is as shown in Fig. 3；
It is made pair using the killing activity of NK cell and target cell K562 the method detection NK cell co-cultured, while of PBMC
According to group, effector cell (E) and target cell (T) are mixed by 0.156,0.625,2.5,10,40 effect target ratio.As shown, with
PBMC is compared, and the killing activity of NK cell has obtained significant raising.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (9)
1. a kind of NK cell non-serum culture medium, which is characterized in that including basal medium and additive, the additive includes
Albumin, amino acid, buffer, oleic acid, linoleic acid and insulin.
2. a kind of NK cell non-serum culture medium as described in claim 1, which is characterized in that component in the additive
Concentration are as follows: albumin (1g/L), oleic acid (0.05-0.2mg/L), linoleic acid (0.2-0.5mg/L), insulin (5-10mg/L).
3. a kind of NK cell non-serum culture medium as claimed in claim 2, which is characterized in that the amino acid in the additive
Including l-tyrosine (30mg/L), l-cysteine (70mg/L), L-Glutamine acid (50mg/L), L-Glutamine (350mg/
L), glycine (20mg/L), Valine (60mg/L), L-Trp (20mg/L), L-phenylalanine (30mg/L), L- ammonia
Sour (20mg/L) and L-threonine (60mg/L).
4. a kind of NK cell non-serum culture medium as claimed in claim 3, which is characterized in that the buffer includes L- phosphoric acid
Glycerol disodium salt hydrate (1g/L) and 4- hydroxyethyl piperazineethanesulfonic acid (1g/L).
5. a kind of NK cell non-serum culture medium as claimed in claim 4, which is characterized in that component in the additive
Concentration are as follows: albumin (1g/L), oleic acid (0.12mg/L), linoleic acid (0.35mg/L), insulin (7.5mg/L).
6. a kind of NK cell non-serum culture medium as described in claim 1, which is characterized in that the albumin is the white egg of people's blood
It is white.
7. a kind of NK cell non-serum culture medium as described in claim 1, which is characterized in that the basal medium is RPMI
1640 serum free mediums.
8. a kind of NK cell non-serum culture medium as described in claim 1-7 any one, which is characterized in that the additive
It further include D glucose.
9. a kind of NK cell non-serum culture medium as claimed in claim 8, which is characterized in that the concentration of the D glucose are as follows:
1-5g/L。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112430573A (en) * | 2020-12-23 | 2021-03-02 | 杭州中赢生物医疗科技有限公司 | NK cell culture system and culture method |
| CN114891742A (en) * | 2022-06-23 | 2022-08-12 | 杭州中赢生物医疗科技有限公司 | Culture medium and in-vitro amplification method for NK (natural killer) cells with strong killing property |
| CN117546837A (en) * | 2023-11-22 | 2024-02-13 | 深圳泽医细胞治疗集团有限公司 | Blood preservation solution suitable for NK cell culture and preparation method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090123442A1 (en) * | 2007-11-09 | 2009-05-14 | Avaris Ab | Expanded nk cells |
| CN103484429A (en) * | 2013-09-28 | 2014-01-01 | 青岛麦迪赛斯生物科技有限公司 | Method for preparing NK (natural killer) cell |
| CN104152413A (en) * | 2014-08-13 | 2014-11-19 | 赛业（苏州）生物科技有限公司 | Application of clinical applicable culture system for efficient amplification of NK cells |
| CN104911147A (en) * | 2015-06-15 | 2015-09-16 | 英普乐孚生物技术(上海)有限公司 | Serum-free medium and preparation method thereof |
| CN107674860A (en) * | 2017-07-14 | 2018-02-09 | 上海源培生物科技股份有限公司 | NK92 cell non-serum culture mediums |
| CN107686828A (en) * | 2016-08-04 | 2018-02-13 | 英普乐孚生物技术(上海)有限公司 | A kind of NK cell non-serum culture mediums |
-
2018
- 2018-09-25 CN CN201811116847.0A patent/CN109097331A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090123442A1 (en) * | 2007-11-09 | 2009-05-14 | Avaris Ab | Expanded nk cells |
| CN103484429A (en) * | 2013-09-28 | 2014-01-01 | 青岛麦迪赛斯生物科技有限公司 | Method for preparing NK (natural killer) cell |
| CN104152413A (en) * | 2014-08-13 | 2014-11-19 | 赛业（苏州）生物科技有限公司 | Application of clinical applicable culture system for efficient amplification of NK cells |
| CN104911147A (en) * | 2015-06-15 | 2015-09-16 | 英普乐孚生物技术(上海)有限公司 | Serum-free medium and preparation method thereof |
| CN107686828A (en) * | 2016-08-04 | 2018-02-13 | 英普乐孚生物技术(上海)有限公司 | A kind of NK cell non-serum culture mediums |
| CN107674860A (en) * | 2017-07-14 | 2018-02-09 | 上海源培生物科技股份有限公司 | NK92 cell non-serum culture mediums |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112430573A (en) * | 2020-12-23 | 2021-03-02 | 杭州中赢生物医疗科技有限公司 | NK cell culture system and culture method |
| CN112430573B (en) * | 2020-12-23 | 2023-03-14 | 杭州中赢生物医疗科技有限公司 | NK cell culture system and culture method |
| CN114891742A (en) * | 2022-06-23 | 2022-08-12 | 杭州中赢生物医疗科技有限公司 | Culture medium and in-vitro amplification method for NK (natural killer) cells with strong killing property |
| CN114891742B (en) * | 2022-06-23 | 2024-06-04 | 杭州中赢生物医疗科技有限公司 | Culture medium of NK cells with strong killing property and in-vitro amplification method |
| CN117546837A (en) * | 2023-11-22 | 2024-02-13 | 深圳泽医细胞治疗集团有限公司 | Blood preservation solution suitable for NK cell culture and preparation method and application thereof |
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