CN105368779A - Macrophage induction culture medium and culture method - Google Patents
Macrophage induction culture medium and culture method Download PDFInfo
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- CN105368779A CN105368779A CN201510891639.8A CN201510891639A CN105368779A CN 105368779 A CN105368779 A CN 105368779A CN 201510891639 A CN201510891639 A CN 201510891639A CN 105368779 A CN105368779 A CN 105368779A
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- cell
- scavenger cell
- scavenger
- inducing culture
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Abstract
The invention discloses a macrophage induction culture medium and a culture method. The macrophage induction culture medium contains GM-CSF, plasma and serum-free basic culture medium. The culture method of the macrophage comprises the step of stimulating the macrophage by using the supernatant of the tumor cell lysate. The invention adopts a certain concentration of GM-CSF and plasma to CD14+The cells are induced and stimulated by the tumor cell lysis supernatant, thereby promoting the proliferation of the macrophage and simultaneously improving the purity of the macrophage.
Description
Technical field
The present invention relates to field of cell culture, particularly relate to a kind of scavenger cell inducing culture and cultural method.
Background technology
Scavenger cell is that one is positioned at in-house white cell, is derived from monocyte.Scavenger cell is one of three large antigen presenting cells, has biological effect widely, as anti-infective, antitumor, participation immunne response and immunomodulatory etc., is participate in important cells that is non-specific and specific immunity.The major function of scavenger cell carries out phagocytosis (namely engulf and digest) with the form of fixed cell or free cell to cell debris and pathogenic agent, and activate lymph corpuscle or other immunocytes, makes it react to pathogenic agent.Scavenger cell belongs to immunocyte, has several functions, and its easily obtain, be convenient to cultivate, can purifying, be the important object of research cytophagy, cellular immunization and molecular immunology.
Scavenger cell belongs to not propagated cell group, can life 2-3 week under condition is suitable, multiplexly does original cuiture, is difficult to long-term survival.Prior art is directly separated the peripheral blood mononuclear cell (PBMC) in peripheral blood, then it is cultivated 7-8 days in the RPMI1640 substratum containing finite concentration GM-CSF (granulocyte and macrophage colony stimulating factor), results attached cell is scavenger cell.But it is very low to cultivate the scavenger cell purity obtained in prior art, and cell amplification is not obvious.
Summary of the invention
In view of this, be necessary for above-mentioned problem, a kind of scavenger cell inducing culture and cultural method are provided.
To achieve these goals, the present invention adopts following technical scheme:
A kind of scavenger cell inducing culture, comprises GM-CSF, blood plasma and serum-free basic medium.
Preferably, described scavenger cell inducing culture is made up of the following component of following content: 300-1000U/mLGM-CSF, volume fraction 3%-10% blood plasma, and surplus is X-VIVO15 serum free medium.
More preferably, in described scavenger cell inducing culture, Plasma volumes mark is 4%-8%.
Still more preferably, in described scavenger cell inducing culture, Plasma volumes mark is 5%.
Preferably, described blood plasma is autologous plasma.
More preferably, in described scavenger cell inducing culture, the content of GM-CSF is 500U/mL.
A cultural method for scavenger cell, comprises the step using tumor cell lysis liquid supernatant stimulating expression of macrophage.Tumor cell lysis liquid can promote propagation and the maturation of scavenger cell, and the scavenger cell after maturation has stronger kill capability.
Preferably, the cultural method of described scavenger cell, comprises the following steps:
S1, use scavenger cell inducing culture cultivate CD14
+cell, consisting of of described scavenger cell inducing culture: 300-1000U/mLGM-CSF, volume fraction 3%-10% autologous plasma, surplus is X-VIVO15 serum free medium;
S2, be cultured to the 5th day, adding tumor cell lysis liquid supernatant stimulates; Continue to be cultured to 8-21 days results scavenger cells.
Preferably, in tumor cell lysis liquid supernatant, the final concentration of albumen is 50 μ g/mL.
Preferably, described CD14
+the CD14 of cell behaviour derived from peripheral blood or derived from cord blood
+cell.
The present invention is by CD14
+cell sorts out from mononuclearcell, then adopts certain density GM-CSF to induce it, and stimulates it with tumor cell lysis liquid supernatant, thus promotes the propagation of scavenger cell, also improves the purity of scavenger cell simultaneously.
Compared with prior art, scavenger cell cultural method provided by the invention has following advantage:
1) contain autologous plasma in the scavenger cell inducing culture that the present invention uses, autologous plasma can promote the propagation of scavenger cell, and the macrophage proliferation ability of acquisition is stronger;
2) the present invention is only to CD14
+cell is induced, and the scavenger cell purity obtained is higher;
3) the present invention adopts tumor cell lysis liquid supernatant to stimulate scavenger cell, and tumor cell lysis liquid supernatant can promote propagation and the maturation of scavenger cell, and the scavenger cell obtained has stronger HLA-II antigen and kill capability.
Accompanying drawing explanation
Fig. 1 is the schema of the cultural method of the present inventor's peripheral blood scavenger cell.
Fig. 2 is cell microscopic morphology.Wherein Fig. 2 A is cultivation the 3rd day cellular form figure, Fig. 2 B is cultivation the 8th day cellular form figure.
Fig. 3 is the growth curve figure of scavenger cell.
Fig. 4 is the flow cytometer detection figure of scavenger cell.Wherein Fig. 4 A is the flow cytometer detection figure of the scavenger cell of comparative example 1 acquisition, Fig. 4 B is the flow cytometer detection figure of the scavenger cell that embodiment 1 obtains.
Embodiment
In order to better the present invention is described, be described further below in conjunction with the drawings and specific embodiments.In the present invention, agents useful for same or instrument all can be buied by market, and the detection method of use etc. are all known in the art, do not repeat them here.
Embodiment 1, human peripheral scavenger cell inducing culture and cultural method
A kind of scavenger cell inducing culture, comprises the following component of following content: 500U/mLGM-CSF, volume fraction 5% blood plasma, surplus is X-VIVO15 serum free medium.
Use the CD14 that PBMC sub-elects by above-mentioned scavenger cell inducing culture
+cell induction is the method for scavenger cell, comprises the following steps:
S1, in PBMC, sub-elect CD14 with CD14 sorting test kit (purchased from STEMCELL company)
+cell; With the resuspended CD14 of X-VIVO15 serum free medium
+cell, by 5 × 10
5the density of cells/mL is seeded in T25 culturing bottle, uses scavenger cell inducing culture at 37 DEG C, 5%CO
2cultivate under condition, regularly observe, carried out cell fluid infusion every 2-3 days.
S2, be cultured to the 5th day, adding K562 cell lysate supernatant stimulates, and in preferred K562 cell lysate supernatant, the final concentration of albumen is 50 μ g/mL; Continue to be cultured to the 8th day harvested cell.
CD14
+cell comprises scavenger cell and monocyte, and in culturing process of the present invention, monocyte induction is for breeding after scavenger cell, and original scavenger cell is also bred.Be cultured to the 5th day monocyte substantially to have induced into scavenger cell, add the specificity that tumor cell lysis liquid supernatant can strengthen Macrophages For Tumor, then continue cultivation and make macrophage proliferation.
As shown in Figure 2, wherein Fig. 2 A is cultivation the 3rd day cellular form figure, Fig. 2 B is cultivation the 8th day cellular form figure to basis of microscopic observation cellular form.As shown in Figure 2, cell is spindle shape, and volume is comparatively large, meets the cellular form of scavenger cell.
The present embodiment can continue to be cultured to 14-21 days and gather in the crops scavenger cell again after adding the stimulation of K562 cell lysate supernatant at the 5th day.Be cultured to the 8th day and namely gather in the crops compared with scavenger cell, be cultured to 14-21 days, the difference that the purity of scavenger cell is too large, but the increasing number of scavenger cell.
Use tumor cell lysis liquid supernatant standby by multigelation legal system in the present invention, PBMC and autologous plasma are prepared by the following method:
20mL peripheral blood is transferred in centrifuge tube from anticoagulant tube, the centrifugal 5-10min of 400-500g, upper plasma is collected in another centrifuge tube after centrifugal end, hot deactivation is carried out in the water-bath of 56 DEG C, then filter with the disposable filter of 0.22 μM, obtain autologous plasma, mark is placed on 4 DEG C of refrigerators, for subsequent use.
Transferred in 50mL centrifuge tube by lower floor's hemocyte, the physiological saline adding two volumes dilutes.Separately get a new sepmate centrifuge tube (purchased from STEMCELL company), add Ficoll lymphocyte separation medium (purchased from STEMCELL company) 12mL, hemocyte after dilution is transferred to sepmate centrifuge tube, the centrifugal 15-20min of 600-800g.After centrifugal, supernatant liquid is poured out, add physiological saline and carry out washing 1 time, obtain PBMC.
Comparative example 1, by the direct inducing macrophage of PBMC
This comparative example uses ordinary method inducing macrophage, and the scavenger cell inducing culture namely used cultivates PBMC, at 37 DEG C, 5%CO
2under condition, cultivation is gathered in the crops attached cell for 8 days afterwards and is scavenger cell.In this comparative example, scavenger cell inducing culture is made up of the following component of following content: volume fraction 10% foetal calf serum, 1000U/mLGM-CSF, and surplus is RPMI1640 substratum.
Effect example 1, macrophage proliferation detect
Use CCK8 method to detect embodiment 1 and the cell proliferative conditions of comparative example 1 in the process of inducing culture scavenger cell, detection method is specific as follows:
(1) in PBMC, CD14 is sub-elected
+cell, preparation CD14
+cell suspension, and carry out cell counting;
(2) with 2 × 10
3the density of cells/mL is by above-mentioned CD14
+cell suspension inoculation, in 96 orifice plates, inoculates 28 holes, every hole 100 μ L cell suspension altogether;
(3), after cultivating 24h, get 4 multiple holes and add 10 μ LCCK8 solution continuation cultivation 1-4 hour;
(4) detect its absorbance at 450nm wavelength place, reference wavelength is 600-650nm;
(5) get the detection that absorbance is carried out in 4 multiple holes every day, continuous detecting 7 days later.
(6) absorbance of gained is depicted as growth curve.
The growth curve of scavenger cell as shown in Figure 3.As shown in Figure 3,1-3 days after inoculation, two groups of cell growth status are similar.The scavenger cell rapid development that after 4th day, embodiment 1 obtains, exceed the rate of growth of scavenger cell in comparative example 1, the scavenger cell rate of growth after the 6th day in embodiment 1 improves further, the rate of growth of scavenger cell in comparative example 1.
Effect example 2, scavenger cell flow cytometer detection
Ordinary method is adopted to detect the expression of surface antigen CD14 and CD11b of the scavenger cell of embodiment 1 and comparative example 1 acquisition.Result as shown in Figure 4.
As shown in Figure 4, the CD14 of the scavenger cell obtained in embodiment
-cD11b
+ratio be 96.1%, and the CD14 of scavenger cell obtained in comparative example 1
-cD11b
+ratio be 13.1%, difference has significant statistical significance.Illustrate that the inventive method and substratum induce the scavenger cell purity obtained higher.
Effect example 3, macrophage biology Function detection
Collection, concentrated embodiment 1 and comparative example 1 cultivate the macrophage-conditioned media of 8 days, and use Elisa standard measure to detect its TNF-α and IL-10 content, result is as shown in table 1.
Table 1TNF-α and IL-10 is containing scale
| Project | TNF-α | IL-10 |
| Embodiment 1 | 17.4μg | 10.8μg |
| Comparative example 1 | 5.8μg | 7.8μg |
As shown in Table 1, the TNF-α that the scavenger cell that embodiment 1 obtains produces and IL-10 content are respectively 17.4 μ g, 10.8 μ g, the TNF-α (5.8 μ g) that the scavenger cell obtained higher than comparative example 1 far away produces and IL-10 (7.8 μ g) content.Therefore, the scavenger cell better effects if that the inventive method and scavenger cell inducing culture obtain is used.
Effect example 4, killing activity detect
Collect, concentrated embodiment 1 and comparative example 1 cultivate the macrophage-conditioned media of 8 days, use serum lactic dehydrogenase (LDH) method for releasing to detect its killing activity to K562 cell.Result is as shown in table 2.
Table 2, killing activity detected result
As shown in Table 2, when imitating target than 40:1,20:1,10:1, the killing activity of scavenger cell prepared by embodiment 1 is all better than the killing activity of scavenger cell prepared by comparative example 1, and difference is more obvious.Illustrate that the fragmentation effect of the Macrophages For Tumor obtained by scavenger cell inducing culture of the present invention and cultural method is better.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. a scavenger cell inducing culture, is characterized in that, comprises GM-CSF, blood plasma and serum-free basic medium.
2. scavenger cell inducing culture according to claim 1, is characterized in that, is made up of the following component of following content: 300-1000U/mLGM-CSF, volume fraction 3%-10% blood plasma, and surplus is X-VIVO15 serum free medium.
3. scavenger cell inducing culture according to claim 2, is characterized in that, Plasma volumes mark is 4%-8%.
4. scavenger cell inducing culture according to claim 3, is characterized in that, Plasma volumes mark is 5%.
5. the scavenger cell inducing culture according to any one of claim 1-4, is characterized in that, described blood plasma is autologous plasma.
6. the scavenger cell inducing culture according to any one of claim 2-4, is characterized in that, the content of GM-CSF is 500U/mL.
7. a cultural method for scavenger cell, is characterized in that, comprises the step using tumor cell lysis liquid supernatant stimulating expression of macrophage.
8. the cultural method of scavenger cell according to claim 7, is characterized in that,
S1, use scavenger cell inducing culture cultivate CD14
+cell, consisting of of described scavenger cell inducing culture: 300-1000U/mLGM-CSF, volume fraction 3%-10% autologous plasma, surplus is X-VIVO15 serum free medium;
S2, be cultured to the 5th day, adding tumor cell lysis liquid supernatant stimulates; Continue to be cultured to 8-21 days results scavenger cells.
9. the cultural method of scavenger cell according to claim 8, is characterized in that, in tumor cell lysis liquid supernatant, the final concentration of albumen is 50 μ g/mL.
10. the cultural method of scavenger cell according to claim 7, is characterized in that, described CD14
+the CD14 of cell behaviour derived from peripheral blood or derived from cord blood
+cell.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609452A (en) * | 2018-12-27 | 2019-04-12 | 青岛麦迪赛斯生物科技有限公司 | A kind of efficient macrophages in vitro preparation method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101549153A (en) * | 2008-04-02 | 2009-10-07 | 于保法 | Composite and method for self-treating tumor |
| CN103642754A (en) * | 2013-12-04 | 2014-03-19 | 深圳市合一康生物科技有限公司 | Preparation method of human D-CIK (dendritic cell activated and cytokine induced killer) cell with high toxicity and high value-adding capacity |
| CN105002140A (en) * | 2015-09-01 | 2015-10-28 | 广州赛莱拉干细胞科技股份有限公司 | Culture method for enhancing killing activity and proliferation activity of LAK (fibroblast activation kinase) cells and application |
-
2015
- 2015-12-04 CN CN201510891639.8A patent/CN105368779A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101549153A (en) * | 2008-04-02 | 2009-10-07 | 于保法 | Composite and method for self-treating tumor |
| CN103642754A (en) * | 2013-12-04 | 2014-03-19 | 深圳市合一康生物科技有限公司 | Preparation method of human D-CIK (dendritic cell activated and cytokine induced killer) cell with high toxicity and high value-adding capacity |
| CN105002140A (en) * | 2015-09-01 | 2015-10-28 | 广州赛莱拉干细胞科技股份有限公司 | Culture method for enhancing killing activity and proliferation activity of LAK (fibroblast activation kinase) cells and application |
Non-Patent Citations (4)
| Title |
|---|
| 张均田等: "《现代药理实验方法》", 31 July 2012, 中国协和医科大学出版社 * |
| 方丽等: "全肿瘤细胞抗原研究进展", 《生物技术通报》 * |
| 杨志梅等: "人外周血单核巨噬细胞诱导、培养及鉴定", 《生物学杂志》 * |
| 林炜明等: "人外周血巨噬细胞培养及功能鉴定", 《中国免疫学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609452A (en) * | 2018-12-27 | 2019-04-12 | 青岛麦迪赛斯生物科技有限公司 | A kind of efficient macrophages in vitro preparation method |
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