CN106085961A - A kind of culture medium cultivating macrophage and cultural method - Google Patents
A kind of culture medium cultivating macrophage and cultural method Download PDFInfo
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Abstract
The invention belongs to cell biology, be specifically related to a kind of culture medium cultivating macrophage and cultural method.The present invention cultivates the culture medium of macrophage, including component 1 and component 2；Described component 1 is made up of DMEM culture medium, methylcellulose, hyclone, GM CSF and lipopolysaccharide；Described component 2 is made up of DMEM culture medium, hyclone, interleukin-11 4 and GM CSF.The present invention cultivates the method for macrophage, by mononuclearcell by DMEM culture medium according to 2 × 106The density of/mL is resuspended, after then the component 1 with described culture medium mixes according to the volume ratio of 1:1, and 37 DEG C, 5%CO2Incubator is cultivated；Every 3 days of period added the component 2 of described culture medium according to 1/2nd of cumulative volume.The present invention of the present invention uses the mode of dimensional culture, makes macrophage suspension culture, simulated in vivo environment, can effectively improve the rate of increase and the phagocytic function of macrophage.
Description
Technical field
The invention belongs to cell biology, be specifically related to a kind of culture medium cultivating macrophage and cultural method.
Background technology
Macrophage is one of three big antigen presenting cells, is to participate in the non-specific and important cells of specific immunity.It
Major function be that the form with fixing cell or free cell carries out phagocytosis to cell debris and pathogen and (i.e. swallows
And digestion), and activate lymph corpuscle or other immunocytes, make it that pathogen is reacted.Owing to macrophage has relatively
Strong phagocytic function and antigen presentation function, so having bigger researching value in immunology, but bite carefully due to huge
Born of the same parents' multiplication capacity is poor, so substantial amounts of macrophage cannot be obtained, also limit its application clinically.
The mononuclearcell (PBMC) that the cultivation of macrophage is mainly directly separated in peripheral blood at present, then by PBMC
Cell is cultivated 7 days in RPMI 1640 culture medium containing 30ng/mLGM-CSF, 10%FBS, gathers in the crops attached cell, is huge
Phagocyte.
But prior art cultivation macrophage proliferation multiple is fairly limited.And macrophage adherent growth is huge with it bites
Cell growing environment in vivo is different, and the characteristic of macrophage can be caused to change, and weakens its phagocytic function, reduces and increases
Grow, and also be difficult to digestion.
Summary of the invention
In view of this, present invention aims to the problem that prior art exists, it is provided that a kind of cultivation macrophage
Culture medium and cultural method.
In order to realize the purpose of the present invention, the present invention adopts the following technical scheme that
A kind of culture medium cultivating macrophage, including component 1 and component 2；Described component 1 is by DMEM culture medium, methyl
Cellulose, hyclone, GM-CSF and lipopolysaccharide composition；Described component 2 is by DMEM culture medium, hyclone, interleukin-11 4 and
GM-CSF forms.
Described in culture medium of the present invention, component 1 is by DMEM culture medium, methylcellulose, hyclone, GM-CSF and fat
Polysaccharide forms.Wherein, methylcellulose is white or off-white color is fibrous or particulate powder；Odorless, tasteless.Methylcellulose
In water, it is swelled into clarification or slightly cloudy colloid solution, presents semi-solid.Macrophage grows in component 1 and presents three-dimensional
The mode cultivated, will not be adherent, but suspension growth, simulated in vivo environment, the characteristic of macrophage can be kept well, have
The rate of increase improving macrophage of effect and phagocytic function.
As preferably, described component 1 is made up of following compositions:
Described component 2 is made up of following compositions:
In some embodiments, described in described culture medium, component 1 is made up of following compositions:
In some embodiments, described in described culture medium, component 1 is made up of following compositions:
In some embodiments, described in described culture medium, component 1 is made up of following compositions:
In some embodiments, described in described culture medium, component 2 is made up of following compositions:
In some embodiments, described in described culture medium, component 2 is made up of following compositions:
In some embodiments, described in described culture medium, component 2 is made up of following compositions:
Present invention also offers a kind of method cultivating macrophage, mononuclearcell (PBMC) is pressed by DMEM culture medium
According to 2 × 106The density of/mL is resuspended, after then the component 1 with described culture medium mixes according to the volume ratio of 1:1, and 37 DEG C, 5%CO2
Incubator is cultivated；Every 3 days of period added the component 2 of described culture medium according to 1/2nd of cumulative volume.
Wherein, cultural method incubation time of the present invention is preferably 17 days.
Further, in some embodiments, described cultural method is cultivated to adding after the 17th day identical with cumulative volume
PBS, 800g be centrifuged 10 minutes, remove supernatant, add harvesting after PBS.
Mononuclearcell described in cultural method of the present invention can be by peripheral blood or Cord blood isolated.
In some embodiments, the preparation method of described mononuclearcell is peripheral blood or Cord blood addition normal saline
Dilution, is slowly added in the centrifuge tube containing lymphocyte separation medium, and the blood making dilution is clear with lymphocyte separation medium layering
Clear, 500-800g is centrifuged 20-30min.
As shown from the above technical solution, the invention provides a kind of culture medium cultivating macrophage and cultural method.This
The culture medium of macrophage is cultivated in invention, including component 1 and component 2；Described component 1 is by DMEM culture medium, methylcellulose, tire
Ox blood serum, GM-CSF and lipopolysaccharide composition；Described component 2 is by DMEM culture medium, hyclone, interleukin-11 4 and GM-CSF group
Become.The present invention cultivates the method for macrophage, and mononuclearcell DMEM culture medium is resuspended according to the density of 2 × 106/mL,
Then, after the component 1 with described culture medium mixes according to the volume ratio of 1:1,37 DEG C, 5%CO2 incubator is cultivated；Period every 3
It is according to 1/2nd of the cumulative volume components 2 adding described culture medium.The present invention of the present invention uses the side of dimensional culture
Formula, makes macrophage suspension culture, simulated in vivo environment, can effectively improve the rate of increase and the phagocytic function of macrophage.
Macrophage proliferation quantity and stream data that the experimental result display present invention cultivates are higher than existing cultural method.Due to the present invention
Cultivation macrophage is closer to internal milieu, and cell state is more preferable, and phagocytosis effect is better than existing cultural method.Existing cultural method
Macrophage is adherent the tightest, it is very difficult to digestion, and the present invention need not use trypsinization when gathering in the crops macrophage, operation
Simplicity, and decrease the mortality rate of cell.
Detailed description of the invention
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described,
Obviously, described embodiment is only a part of embodiment of the present invention rather than whole embodiments.Based in the present invention
Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under not making creative work premise, all
Belong to the scope of protection of the invention.
In order to be better understood from the present invention, below in conjunction with specific embodiment, the present invention will be described in detail.
Embodiment 1:
Culture medium prescription: component 1
Component 2
Cultural method:
Extraction peripheral blood, obtains PBMC therein with Ficoll method is centrifugal.
By PBMC by DMEM culture medium according to 2 × 106The density of/mL is resuspended, then with component 1 according to the volume ratio of 1:1
Mixing, period avoids the occurrence of larger bubble.
Cell is put into 37 DEG C, 5%CO2Incubator is cultivated.
Within every 3 days, adding component 2 culture medium according to 1/2nd of cumulative volume, and rock mixing, period avoids the occurrence of relatively
Air pocket.
Within 17th day, add the PBS, 800g identical with cumulative volume to be centrifuged 10 minutes, remove supernatant, addition PBS one time with
Rear harvesting.
Embodiment 2:
Culture medium prescription: component 1
Component 2
Cultural method:
Harvesting after cultivating according to cultural method described in embodiment 1.
Embodiment 3
Culture medium prescription: component 1
Component 2
Cultural method:
Harvesting after cultivating according to cultural method described in embodiment 1.
Comparative example:
Extract a certain amount of peripheral blood, obtain PBMC therein with Ficoll method is centrifugal.
By PBMC cell by RPMI 1640 culture medium containing 30ng/mL GM-CSF, 10%FBS according to 1 × 106/ML
Density resuspended.
Cell is put into 37 DEG C, 5%CO2Incubator is cultivated.
Within every 3 days, supplement the RPMI 1640 containing 30ng/mL GM-CSF, 10%FBS according to 1/2nd of cumulative volume to train
Support base.
Add the pancreatin of 0.25% after within 17th day, removing culture supernatant, PBS 2 times to digest, add after digesting 5 minutes
The FBS entering 5 times of pancreatin volumes terminates digestion, centrifugal segregation supernatant, time later harvesting of PBS.
Test example 1: cell counting, viability examination and flow cytometer detection
The cell collecting embodiment 1-3 and comparative example carries out cell counting, viability examination and flow cytometer detection.The results are shown in Table
1-3。
Count results after the 17th day harvesting of table 1
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example | |
| Cell number (million) | 113.2 | 107.5 | 109.6 | 24.3 |
The cell number that the cell number of table 1 result display embodiment 1-3 cultural method results to be cultivated far more than comparative example.
Cell viability testing result after the 17th day harvesting of table 2
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example | |
| Cell viability | 96.3% | 93.8% | 91.3% | 76.5% |
Cell viability represent survivaling cell number, numerical value is the highest shows that the cell of survival is the most, numerical value is low then represent dead
The cell died is many.The cell that the cell viability comparative example to be far above of table 2 result display embodiment 1-3 cultural method results is cultivated
Vigor, shows that the cell viability that cultural method of the present invention is gathered in the crops is high.
Flow cytometer detection result after the 17th day harvesting of table 3
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example | |
| Flow cytometer detection (CD11+CD80+) | 78.3% | 71.2% | 72.4% | 18.9% |
CD11+CD80+ is Macrophage Surface mark, and numerical value is the highest, shows that in cell, the content of macrophage is the most.Table
The cell that the cell CD11+CD80+ numerical value comparative example to be far above of 3 result display embodiment 1-3 cultural method results is cultivated, table
In the cell of bright cultural method of the present invention results, the content of macrophage is high.
Test example 2: macrophage phagocytic is tested:
Take 1mL 1 × 10 respectively6Embodiment 1-3 of/mL and the macrophage suspension of comparative example results and 1mL 1 ×
107Put into after the mixing of/mL/mL chicken erythrocyte suspension, in aseptic centrifuge tube, cultivate 30,45,60 and 90min respectively for 37 DEG C
(period, every 5min shook up 1 time), then take 300 μ L to clean wave carrier piece 37 DEG C attach 30min, with 37 DEG C of preheatings after taking-up
PBS rinse the chicken red blood cell that do not swallowed, wash unnecessary dye liquor, dry high power lens or oil Microscopic observation and counting calculates
Phagocytic percentage.The results are shown in Table 4.The computing formula of phagocytic percentage is:
The MF number of phagocytic percentage=phagocytosis chicken red blood cell/MF sum
Table 4 macrophage phagocytic experimental result
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example | |
| Phagocytic percentage | 70.0% | 66.9% | 68.3% | 54.2% |
The phagocytic function of phagocytic percentage the highest explanation macrophage is the strongest.Table 4 result display embodiment 1-3 cultural method
The cell phagocytic percentage of results is higher than the cell that comparative example is cultivated, and shows in the cell that cultural method of the present invention is gathered in the crops
The phagocytic function of macrophage is higher.
Claims (10)
1. cultivate a culture medium for macrophage, including component 1 and component 2；Described component 1 is fine by DMEM culture medium, methyl
Dimension element, hyclone, GM-CSF and lipopolysaccharide composition；Described component 2 is by DMEM culture medium, hyclone, interleukin-11 4 and GM-
CSF forms.
Culture medium the most according to claim 1, it is characterised in that described component 1 is made up of following compositions:
Described component 2 is made up of following compositions:
Culture medium the most according to claim 2, it is characterised in that described component 1 is made up of following compositions:
Culture medium the most according to claim 2, it is characterised in that described component 1 is made up of following compositions:
Culture medium the most according to claim 2, it is characterised in that described component 1 is made up of following compositions:
Culture medium the most according to claim 2, it is characterised in that described component 2 is made up of following compositions:
Culture medium the most according to claim 2, it is characterised in that described component 2 is made up of following compositions:
Culture medium the most according to claim 2, it is characterised in that described component 2 is made up of following compositions:
9. the method cultivating macrophage, by mononuclearcell by DMEM culture medium according to 2 × 106The density of/mL is resuspended,
Then after the component 1 with culture medium described in claim 1 mixes according to the volume ratio of 1:1,37 DEG C, 5%CO2Incubator is trained
Support；Every 3 days of period added the component 2 of culture medium described in claim 1 according to 1/2nd of cumulative volume.
Method the most according to claim 9, incubation time is 17 days.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007143794A1 (en) * | 2006-06-15 | 2007-12-21 | Murray Goulburn Co-Operative Co. Limited | Formulation comprising whey protein and hydrolysates for improving muscle recovery |
| CN104232579A (en) * | 2014-09-28 | 2014-12-24 | 武汉大学 | Culture method and application thereof for rhesus peripheral blood mononuclear macrophages |
| CN104640974A (en) * | 2012-07-24 | 2015-05-20 | 日产化学工业株式会社 | Culture medium composition, and method for culturing cell or tissue using said composition |
| CN105779392A (en) * | 2016-05-20 | 2016-07-20 | 广州赛莱拉干细胞科技股份有限公司 | Macrophage culture medium and culture method |
-
2016
- 2016-07-28 CN CN201610616747.9A patent/CN106085961A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007143794A1 (en) * | 2006-06-15 | 2007-12-21 | Murray Goulburn Co-Operative Co. Limited | Formulation comprising whey protein and hydrolysates for improving muscle recovery |
| CN104640974A (en) * | 2012-07-24 | 2015-05-20 | 日产化学工业株式会社 | Culture medium composition, and method for culturing cell or tissue using said composition |
| CN104232579A (en) * | 2014-09-28 | 2014-12-24 | 武汉大学 | Culture method and application thereof for rhesus peripheral blood mononuclear macrophages |
| CN105779392A (en) * | 2016-05-20 | 2016-07-20 | 广州赛莱拉干细胞科技股份有限公司 | Macrophage culture medium and culture method |
Non-Patent Citations (1)
| Title |
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