CN109777774A - The preparation method of macrophage derived extracellular matrix - Google Patents
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- 238000002360 preparation method Methods 0.000 title claims abstract description 16
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- 239000001963 growth medium Substances 0.000 claims description 21
- 210000004027 cell Anatomy 0.000 claims description 13
- 210000000689 upper leg Anatomy 0.000 claims description 9
- 210000004979 bone marrow derived macrophage Anatomy 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 7
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
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- 239000012530 fluid Substances 0.000 claims description 5
- 210000002303 tibia Anatomy 0.000 claims description 5
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 claims description 3
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Abstract
The invention discloses a kind of preparation method for the M2 type macrophage derived extracellular matrix for belonging to tissue engineering technique field, separation, culture including macrophage, macrophage continue three steps of preparation of culture, macrophage derived extracellular matrix.The timbering material that can be used for subsequent stem cells behavioral implications research using host material prepared by method of the invention, new selection is provided for Tissue Engineering Biomaterials.
Description
Technical field
The invention belongs to tissue engineering technique fields, and in particular to a kind of system of M2 type macrophage derived extracellular matrix
Preparation Method.
Background technique
Recently as the development of organizational project, the tissue and device of disease damage are repaired or substituted using cell and timbering material
The method of official is increasingly by the attention of researcher.Organizational project includes that seed cell, timbering material, growth regulator three are wanted
Element, wherein suitable timbering material is successfully the precondition of repair tissue.In the past few years, a heat of organizational project
Door research be exactly manufacture by tissue or organ take off from cell-derived extracellular matrix (extracellular matrix,
ECM) biological support.The method for the de- cell epimatrix extracted from donor tissue has been applied to skin, bladder, heart valve
The positions such as film and submucous layer of small intestine.Although the biological support in non-autologous tissue source has been used to clinic, related organization
Engineering studies have shown that the biological support of autologous tissue is better than the bracket in non-autologous tissue source.In order to find autologous tissue
Biological support, use cell-derived extracellular matrix as the brand-new method of one kind that bracket is in recent years.Tissue derived
Extracellular matrix causes to be very different in different structural constituents, natural structure is non-since the function of different tissues is different
It is often complicated.And bracket is made of cell-derived extracellular matrix, compared with the extracellular matrix bracket of tissue derived, have
Extremely strong biomimetic features, plasticity, without immunogenicity the features such as.In conclusion cell-derived extracellular matrix bracket is group
Weaver's journey provides new direction.
Distributed pole is wide in vivo for macrophage, has highly plastic, and in the balance of the development of body and interior environment
It plays an important role.Under stressed condition, macrophage polarizes, and the macrophage subsets after polarization careful can be adjusted and be returned
A variety of different stimulations are answered, the reparation degree of development and histoorgan to inflammatory reaction or disease plays critical effect,
With important researching value.Make at present the universal thinking of cell-derived epimatrix bracket be using mescenchymal stem cell,
Fibroblast, stroma cell etc., the extracellular matrix derived from macrophage has not been reported.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation methods of M2 type macrophage derived extracellular matrix, for a group weaver
Journey biomaterial provides new selection.
A kind of preparation method of M2 type macrophage derived extracellular matrix, comprising the following steps:
S1: separation, the culture of macrophage: extracting bone marrow cell, and Single cell suspensions are made, and centrifugation discards supernatant liquid,
Erythrocyte cracked liquid is added to mix, stands cracking, 1640 culture mediums are added and terminate reaction；It is centrifuged again, abandons supernatant, lower layer is thin
Born of the same parents are precipitated to be resuspended with culture medium, and then bed board enters to obtain within steril cell culture dish culture 6-10 days mature bone marrow derived macrophage thin
Born of the same parents；
S2: macrophage continues to cultivate: discarding culture medium used in S1 step, is changed to continuation culture medium, cultivates
For 24 hours, M2 type macrophage is obtained；
S3: the preparation of macrophage derived extracellular matrix: continuing culture 5 days for macrophage in continuing culture medium,
PBS cleaning down after pretreatment fluid impregnates 1 hour, obtains derived extracellular matrix.
The method of bone marrow cell is extracted described in step S1 of the present invention are as follows: neck marrow detachment puts to death experimental animal, 75% ethyl alcohol
5min is impregnated, bilateral femur, shin bone are taken under aseptic condition, muscle is removed, clean femur will be separated, shin bone is put into 1640 cultures
In base, femur, tibial metaphysis are cut off, exposure ossis draws 1640 culture medium repeated flushing, goes out marrow.
300 × g is centrifuged 5min under the conditions of centrifugation described in step S1 of the present invention is 4 DEG C；Culture used when the resuspension
Base is to include 20ng/ml M-CSF, 10%FBS, 2mM L-Glutamine, 1% dual anti-1640 culture medium of RMPI.
Continuation culture medium described in step S2 and step S3 of the present invention is to include 10ng/ml IL-4,10%FBS, 1% couple
Anti- 1640 culture medium of RMPI.
Treatment fluid described in step S3 of the present invention includes 1M NaCl, 10mM Tris and 5mM EDTA.
Compared with prior art, the present invention has following excellent effect: it is derivative that the present invention relates to a kind of M2 type macrophages
The preparation method of extracellular matrix can be used for subsequent stem cells behavioral implications using host material prepared by this method and study
Timbering material, new selection is provided for Tissue Engineering Biomaterials.
Detailed description of the invention
The mature bone marrow derived macrophage morphology figure that Fig. 1 is；Wherein A is bone marrow derived primary macrophage light microscopic
(10X) figure；B is that immunofluorescence dyeing detects bone marrow derived macrophage morphology (20X) figure.
Fig. 2 is the purity figure of the resulting mature bone marrow derived macrophage of Flow cytometry culture.
Fig. 3 is to continue culture cellular morphology figure for 24 hours；Wherein A is bone marrow derived primary macrophage light microscopic (10X) figure；B is
Immunofluorescence dyeing detects bone marrow derived macrophage morphology (20X) figure.
Fig. 4 is that Flow cytometry continues to cultivate the purity figure of resulting bone marrow derived M2 type macrophage for 24 hours.
Fig. 5 is the derivative ECM immunofluorescence dyeing of M2 type macrophage, and laser co-focusing is taken pictures (20X) result figure, wherein A:
Collagen I；B:Collagen III；C:Fibronectin.
Specific embodiment
The present invention is described in detail with reference to the accompanying drawings and examples, but implementation of the invention is not limited only to this.
Separation, the culture of 1 mouse bone marrow cells source property macrophage of embodiment
Neck marrow detachment puts to death C57BL/6 mouse, and 75% ethyl alcohol impregnates 5min, takes mouse bilateral femur, shin under aseptic condition
Bone, it is sterile to cut blunt separation muscle, clean femur will be separated, shin bone is put into 1640 culture mediums, cuts off femur, tibial shaft epiphysis
End, exposure ossis are gone out marrow and are centrifuged in 50ml with 1640 culture medium repeated flushing of 10ml syringe and 25G needle aspirate
Guan Zhong, uses 23G needle instead bone marrow cell is transferred to in new 50ml pipe repeatedly pressure-vaccum and be made single cell suspension, 300 under the conditions of 4 DEG C
× g is centrifuged 5min, abandons supernatant, and 2ml erythrocyte cracked liquid is added and softly mixes, and after standing cracking 5min, 4ml1640 training is added
Base terminates reaction.It is centrifuged again, condition is the same.Abandon supernatant, by lower layer's cell precipitation M-CSF containing 20ng/ml, 10%FBS,
2mM L-Glutamine, 1% dual anti-1640 culture medium of RMPI are resuspended, and then bed board enters steril cell culture dish, and every 48h is changed
Liquid.To get the bone marrow derived macrophage for arriving maturation after culture 8 days.
As shown in Figure 1, light microscopic observation cell growth state is good, passed through after carrying out immunofluorescence dyeing as antibody using CD68
Observation cellular morphology is satisfied after Laser Scanning Confocal Microscope is taken pictures.The macrophage that culture obtains is collected, using F4/80 antibody as mark
Note, the purity through the resulting macrophage of Flow cytometry culture, as shown in Fig. 2, the positive expression rate of F4/80 antibody can
Reach 99%.
The culture of 2 bone marrow derived M2 type macrophage of embodiment
Behind the separating obtained culture of bone marrow derived macrophage 8 days, former culture medium is discarded, IL- containing 10ng/ml is changed to
4,10%FBS, 1% dual anti-1640 culture medium of RMPI continue culture and obtain M2 type macrophage afterwards for 24 hours.
As shown in figure 3, microscopic observation colony and cell growth state are good in incubation.It is carried out by antibody of CD206
It is obvious that cell pseudopodium is observed after immunofluorescence dyeing after Laser Scanning Confocal Microscope is taken pictures, form satisfaction.Collect the M2 that culture obtains
Type macrophage, using F4/80 antibody and CD206 antibody as label, through the resulting M2 type macrophage of Flow cytometry culture
The purity of cell.As shown in figure 4, being not added with F4/80 antibody and the double positive expression rates of CD206 antibody before stimulant is 15.1%,
F4/80 antibody and the double positive expression rates of CD206 antibody are 69.8% after stimulation 24 hours, after stimulation 5 days F4/80 antibody and
The double positive expression rates of CD206 antibody can be ideal to 90% or more, M2 type macrophage purity.
Embodiment 3 prepares bone marrow derived M2 type macrophage derived extracellular matrix
M2 type macrophage is continued in IL-4 containing 10ng/ml, 10%FBS, 1% dual anti-1640 culture medium of RMPI
After culture 5 days, with 1M NaCl is contained, the pretreatment fluid of 10mM Tris and 5mM EDTA impregnate, PBS cleaning down after 1 hour.
Then it is handled under the conditions of 37 DEG C using the treatment fluid containing 0.15% (volume/volume) Triton X-100 and 10mM NH4OH
1 minute, Immunofluorescence test is carried out after PBS washing, observes the form of cell-derived matrix.As shown in figure 5, on silk-fibroin bracket
There is the expression of a large amount of matrix, including Collagen I, Collagen III and fibronection.Antibody is purchased from
abcam。
Disclosed above is only specific embodiments of the present invention, and still, the present invention is not limited to this, any this field
What technical staff can think variation should all fall into protection scope of the present invention.
Claims (6)
1. a kind of preparation method of M2 type macrophage derived extracellular matrix, which comprises the following steps:
S1: separation, the culture of macrophage: extracting bone marrow cell, and Single cell suspensions are made, and centrifugation discards supernatant liquid, is added
Erythrocyte cracked liquid mixes, and stands cracking, and 1640 culture mediums are added and terminate reaction；It is centrifuged again, abandons supernatant, lower confluent monolayer cells are sunk
Shallow lake is resuspended with culture medium, and then bed board, which enters, obtains mature bone marrow derived macrophage for steril cell culture dish culture 6-10 days；
S2: macrophage continues to cultivate: discarding culture medium used in S1 step, is changed to continuation culture medium, culture for 24 hours, obtains
To M2 type macrophage；
S3: the preparation of macrophage derived extracellular matrix: continuing culture 5 days for macrophage in continuing culture medium, pre- to locate
PBS cleaning down after liquid impregnates 1 hour is managed, derived extracellular matrix is obtained.
2. the preparation method of M2 macrophage derived extracellular matrix according to claim 1, which is characterized in that step S1
Described in extract the method for bone marrow cell are as follows: neck marrow detachment puts to death experimental animal, and 75% ethyl alcohol impregnates 5min, takes under aseptic condition
Bilateral femur, shin bone remove muscle, will separate clean femur, shin bone is put into 1640 culture mediums, cut off femur, tibial shaft
Epiphysis end, exposure ossis, draws 1640 culture medium repeated flushing, goes out marrow.
3. the preparation method of M2 macrophage derived extracellular matrix according to claim 1, which is characterized in that in step S1
300 × g is centrifuged 5 min under the conditions of the centrifugation is 4 DEG C.
4. the preparation method of M2 macrophage derived extracellular matrix according to claim 1, which is characterized in that in step S1
Culture medium used is to include 20ng/ml M-CSF, 10%FBS, 2 mM L-Glutamines, 1% dual anti-RMPI when the resuspension
1640 culture mediums.
5. the preparation method of M2 macrophage derived extracellular matrix according to claim 1, which is characterized in that step S2 and
Continuation culture medium described in step S3 is to include 10ng/ml IL-4,10%FBS, 1% dual anti-1640 culture medium of RMPI.
6. the preparation method of M2 macrophage derived extracellular matrix according to claim 1, which is characterized in that in step S3
The treatment fluid includes 1M NaCl, 10mM Tris and 5mM EDTA.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111961649A (en) * | 2019-09-27 | 2020-11-20 | 云南洛宇生物科技有限公司 | Rat macrophage culture method |
| CN114891718A (en) * | 2022-06-08 | 2022-08-12 | 天康制药(苏州)有限公司 | Culture medium for suspension culture of bone marrow cells, preparation method, application and method for inducing differentiation of bone marrow-derived cells into macrophages |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111961649A (en) * | 2019-09-27 | 2020-11-20 | 云南洛宇生物科技有限公司 | Rat macrophage culture method |
| CN114891718A (en) * | 2022-06-08 | 2022-08-12 | 天康制药(苏州)有限公司 | Culture medium for suspension culture of bone marrow cells, preparation method, application and method for inducing differentiation of bone marrow-derived cells into macrophages |
| CN114891718B (en) * | 2022-06-08 | 2024-01-30 | 天康制药股份有限公司 | Culture medium for suspension culture of bone marrow cells, preparation method, application and method for inducing differentiation of bone marrow-derived cells into macrophages |
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Application publication date: 20190521 |