CN106190966A - A kind of preparation method of the cell preparation for defying age - Google Patents
A kind of preparation method of the cell preparation for defying age Download PDFInfo
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- CN106190966A CN106190966A CN201610569971.7A CN201610569971A CN106190966A CN 106190966 A CN106190966 A CN 106190966A CN 201610569971 A CN201610569971 A CN 201610569971A CN 106190966 A CN106190966 A CN 106190966A
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Abstract
The invention discloses a kind of preparation method for defying age cell preparation, carry out in accordance with the following steps: the bone marrow of extraction is added to culture fluid, abandon supernatant after Li Xin and be inoculated in culture dish cultivation, liquid is changed for the first time after original cuiture 24 48h, every 3d changes liquid once afterwards, third time is changed after liquid 12 18h with 1:2 ratio Secondary Culture, and the third generation mesenchymal stem cells MSCs taking cultivation afterwards joins in normal saline resuspended, prepares mesenchymal stem cells MSCs preparation.The inventive method is by by mesenchymal stem cells MSCs separation and Culture, and the mesenchymal stem cells MSCs of In vitro culture is joined in normal saline resuspended, prepare mesenchymal stem cells MSCs preparation, found that this cell preparation can improve the damage of aging rats each histiocyte form, there is certain effect reversing senescense and damnification, for studying mesenchymal stem cells MSCs mechanism of resisting senility, and flight against senium of human body research is given a clue.
Description
Technical field
The invention belongs to cell preparation technical field, particularly relate to a kind of preparation side for defying age cell preparation
Method.
Background technology
China the most quickly steps into aging society, at present China year above old people have about hundred million.Estimate the year two thousand fifty China 60
Above old people will account for three one-tenth in year, and all puzzlements and problem that aging brings are more serious.Old and feeble and death is any species
The final final result that is all difficult to avoid that of individuality, but we are the most fairly limited to the understanding of old and feeble mechanism so far.In view of it is heavy
The property wanted, the focus of old and feeble always biological study.
Mesenchymal stem cells MSCs, is derived from a fetal development mesoblastic class pluripotent stem cell in early days, is whole body knot
Form the stem cell of tissue.Great many of experiments confirms under the conditions of suitable vitro differentiation, is possible not only to be divided into osteoblast, fat
The Interstitial cells such as cell, chondrocyte, endotheliocyte, muscle cell, and can across differentiation of germinal layers be ectodermic neuron,
Neurogliocyte and endoblastic hepatocyte.And there is not the feature of rejection after there is transplanting.Along with to go deep into
Research, it is now recognized that except having multi-lineage potential, moreover it is possible at damage local response secretion many neurotrophic.
Summary of the invention
It is an object of the invention to provide a kind of preparation method for defying age cell preparation, by by medulla mesenchyma
Stem cell separation and Culture, and the mesenchymal stem cells MSCs of In vitro culture is joined in normal saline resuspended, prepare between bone marrow
Mesenchymal stem cells preparation, this cell preparation can improve the damage of aging rats each histiocyte form, have certain reverse
The effect of senescense and damnification.
The present invention is achieved by the following technical solutions:
A kind of preparation method for defying age cell preparation, is carried out in accordance with the following steps:
Step (1), preparation medulla mesenchyma culture fluid；
Under step (2), aseptic condition, the bone marrow of extraction is added to sterile culture flask, culture bottle adds described step
Suddenly the medulla mesenchyma culture fluid of configuration in (1), volume ratio is 1:1, fully dispels medullary cell with sterile pipette, prepares bone
Myelocyte suspension；
Step (3), after centrifugal for the bone marrow cell suspension obtained in described step (2), abandon supernatant, obtain marrow stem thin
Born of the same parents group；
Step (4), take the bone marrow stem cell group obtained in described step (4) and add the cultivation of configuration in described step (1)
Resuspended rear filtration in liquid, after filtering, the culture fluid containing bone marrow stem cell group is with 1 × 109Individual/mL is inoculated in culture dish training
Support；
Changing liquid for the first time after step (5), original cuiture 24-48h, every 3d changes liquid once afterwards, and third time changes liquid 12-18h
After with 1:2 ratio Secondary Culture；
Step (6), take the third generation mesenchymal stem cells MSCs cultivated in described step (5) and join weight in normal saline
Outstanding, prepare mesenchymal stem cells MSCs preparation.
Further, medulla mesenchyma culture fluid described in step (1) is addition penicillin concn in L-DMEM culture medium
For 100U/mL, adding streptomycin concentration is 100U/mL.
Further, in described step (3), centrifugation rate is 800-1500r/min, and centrifugation time is 5-10min.
Further, with 70 μm sterile nylon filters in described step (4).
Further, described culture medium is placed in by step (4) 37 DEG C, CO2Saturated humidity incubator is cultivated.
Further, in described step (6), mesenchymal stem cells MSCs concentration is (2-5) × 107Individual/mL.
The method have the advantages that
The inventive method is by by mesenchymal stem cells MSCs separation and Culture, and is done by the medulla mesenchyma of In vitro culture
Cell joins in normal saline resuspended, prepares mesenchymal stem cells MSCs preparation, found that this cell preparation can improve
The damage of aging rats each histiocyte form, has certain effect reversing senescense and damnification, does for research medulla mesenchyma
Cell mechanism of resisting senility, and flight against senium of human body research give a clue.
Certainly, the arbitrary product implementing the present invention it is not absolutely required to reach all the above advantage simultaneously.
Detailed description of the invention
Technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is only
The a part of embodiment of the present invention rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art
The all other embodiments obtained under not making creative work premise, broadly fall into the scope of protection of the invention.
The present invention is a kind of cell preparation for defying age, and the method specifically comprises the following steps that
Embodiment 1
Step (1), in L-DMEM culture medium add penicillin concn be 100U/mL, streptomycin concentration be 100U/mL,
It is configured to medulla mesenchyma culture fluid；
Under step (2), aseptic condition, rinse rat bone pulp cavity with the medulla mesenchyma culture fluid of configuration in step (1), will
Go out bone marrow add in sterile culture flask, culture bottle adds the medulla mesenchyma culture fluid of configuration, body in step (1)
Long-pending ratio is 1:1, fully dispels medullary cell with sterile pipette, prepares bone marrow cell suspension；
Step (3), the bone marrow cell suspension obtained in step (2) is centrifuged 5min with 800r/min, abandons supernatant, obtain
Bone marrow stem cell group；
Step (4), take the bone marrow stem cell group obtained in step (4) add in step (1) prepare culture fluid in resuspended
Filtering with 70 μm sterile nylon filters afterwards, after filtering, the culture fluid containing bone marrow stem cell group is with 1 × 109Individual/mL inoculation
In 5cm × 5cm culture dish, it is placed in 37 DEG C, CO2Saturated humidity incubator is cultivated；
For the first time changing liquid after step (5), original cuiture 24h, every 3d changes liquid once afterwards, and third time is changed after liquid 12h with 1:2
Ratio Secondary Culture；
Step (6), take the third generation mesenchymal stem cells MSCs cultivated in step (5) and join in normal saline resuspended,
Preparing mesenchymal stem cells MSCs preparation, wherein, mesenchymal stem cells MSCs concentration is 2 × 107Individual/mL；
Step (7), the mesenchymal stem cells MSCs preparation of preparation in step (6) is entered little by tail vein injection infusion
To reach the purpose of defying age in Mus body.
Embodiment 2
Step (1), in L-DMEM culture medium add penicillin concn be 100U/mL, streptomycin concentration be 100U/mL,
It is configured to medulla mesenchyma culture fluid；
Under step (2), aseptic condition, rinse rat bone pulp cavity with the medulla mesenchyma culture fluid of configuration in step (1), will
Go out bone marrow add in sterile culture flask, culture bottle adds the medulla mesenchyma culture fluid of configuration, body in step (1)
Long-pending ratio is 1:1, fully dispels medullary cell with sterile pipette, prepares bone marrow cell suspension；
Step (3), the bone marrow cell suspension obtained in step (2) is centrifuged 10min with 1500r/min, abandons supernatant,
To bone marrow stem cell group；
Step (4), take the bone marrow stem cell group obtained in step (4) add in step (1) prepare culture fluid in resuspended
Filtering with 70 μm sterile nylon filters afterwards, after filtering, the culture fluid containing bone marrow stem cell group is with 1 × 109Individual/mL inoculation
In 5cm × 5cm culture dish, it is placed in 37 DEG C, CO2Saturated humidity incubator is cultivated；
For the first time changing liquid after step (5), original cuiture 48h, every 3d changes liquid once afterwards, and third time is changed after liquid 18h with 1:2
Ratio Secondary Culture；
Step (6), take the third generation mesenchymal stem cells MSCs cultivated in step (5) and join in normal saline resuspended,
Preparing mesenchymal stem cells MSCs preparation, wherein, mesenchymal stem cells MSCs concentration is 5 × 107Individual/mL；
Step (7), the mesenchymal stem cells MSCs preparation of preparation in step (6) is entered little by tail vein injection infusion
To reach the purpose of defying age in Mus body.
Embodiment 3
Step (1), in L-DMEM culture medium add penicillin concn be 100U/mL, streptomycin concentration be 100U/mL,
It is configured to medulla mesenchyma culture fluid；
Under step (2), aseptic condition, rinse rat bone pulp cavity with the medulla mesenchyma culture fluid of configuration in step (1), will
Go out bone marrow add in sterile culture flask, culture bottle adds the medulla mesenchyma culture fluid of configuration, body in step (1)
Long-pending ratio is 1:1, fully dispels medullary cell with sterile pipette, prepares bone marrow cell suspension；
Step (3), the bone marrow cell suspension obtained in step (2) is centrifuged 7min with 1200r/min, abandons supernatant,
To bone marrow stem cell group；
Step (4), take the bone marrow stem cell group obtained in step (4) add in step (1) prepare culture fluid in resuspended
Filtering with 70 μm sterile nylon filters afterwards, after filtering, the culture fluid containing bone marrow stem cell group is with 1 × 109Individual/mL inoculation
In 5cm × 5cm culture dish, it is placed in 37 DEG C, CO2Saturated humidity incubator is cultivated；
For the first time changing liquid after step (5), original cuiture 36h, every 3d changes liquid once afterwards, and third time is changed after liquid 15h with 1:2
Ratio Secondary Culture；
Step (6), take the third generation mesenchymal stem cells MSCs cultivated in step (5) and join in normal saline resuspended,
Preparing mesenchymal stem cells MSCs preparation, wherein, mesenchymal stem cells MSCs concentration is 3 × 107Individual/mL；
Step (7), the mesenchymal stem cells MSCs preparation of preparation in step (6) is entered by tail vein injection infusion
To reach the purpose of defying age in Mice Body.
The inventive method is by by mesenchymal stem cells MSCs separation and Culture, and by dry for the medulla mesenchyma of In vitro culture thin
Born of the same parents join in normal saline resuspended, prepare mesenchymal stem cells MSCs preparation, decline found that this cell preparation can improve
The damage of Mus each histiocyte form greatly, has certain effect reversing senescense and damnification, dry thin for research medulla mesenchyma
Born of the same parents' mechanism of resisting senility, and flight against senium of human body research give a clue..
Above content is only citing made for the present invention and explanation, and affiliated those skilled in the art are to being retouched
The specific embodiment stated makes various amendment or supplements or use similar mode to substitute, without departing from inventing or super
More scope defined in the claims, all should belong to protection scope of the present invention.
Claims (6)
1. the preparation method for defying age cell preparation, it is characterised in that carry out in accordance with the following steps:
Step (1), preparation medulla mesenchyma culture fluid；
Under step (2), aseptic condition, the bone marrow of extraction is added to sterile culture flask, culture bottle adds described step (1)
The medulla mesenchyma culture fluid of middle configuration, volume ratio is 1:1, fully dispels medullary cell with sterile pipette, prepares bone marrow thin
Born of the same parents' suspension；
Step (3), after centrifugal for the bone marrow cell suspension obtained in described step (2), abandon supernatant, obtain bone marrow stem cell group；
Step (4), take the bone marrow stem cell group obtained in described step (4) and add in described step (1) in the culture fluid of configuration
Filtering after resuspended, after filtering, the culture fluid containing bone marrow stem cell group is with 1 × 109Individual/mL is inoculated in culture dish cultivation；
For the first time changing liquid after step (5), original cuiture 24-48h, every 3d changes liquid once afterwards, third time change after liquid 12-18h with
1:2 ratio Secondary Culture；
Step (6), take the third generation mesenchymal stem cells MSCs cultivated in described step (5) and join in normal saline resuspended,
Prepare mesenchymal stem cells MSCs preparation.
A kind of preparation method for defying age cell preparation the most according to claim 1, it is characterised in that: step (1)
Described in medulla mesenchyma culture fluid be in L-DMEM culture medium add penicillin concn be 100U/mL, add streptomycin concentration
For 100U/mL.
A kind of preparation method for defying age cell preparation the most according to claim 1, it is characterised in that: described step
(3) in, centrifugation rate is 800-1500r/min, and centrifugation time is 5-10min.
A kind of preparation method for defying age cell preparation the most according to claim 1, it is characterised in that: described step
(4) with 70 μm sterile nylon filters in.
A kind of preparation method for defying age cell preparation the most according to claim 1, it is characterised in that: step (4)
Middle described culture medium is placed in 37 DEG C, CO2Saturated humidity incubator is cultivated.
A kind of preparation method for defying age cell preparation the most according to claim 1, it is characterised in that: described step
(6) in, mesenchymal stem cells MSCs concentration is (2-5) × 107Individual/mL.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107041894A (en) * | 2016-12-26 | 2017-08-15 | 湖南新起源医疗技术有限公司 | A kind of stem cell liquid for skin delication preparation method for anti-aging |
| CN110613736A (en) * | 2019-10-30 | 2019-12-27 | 广州陈运贤生命科技有限公司 | Composition and preparation for treating bone marrow failure diseases, preparation method and application |
| CN111996163A (en) * | 2020-09-04 | 2020-11-27 | 安徽惠恩生物科技股份有限公司 | Skin anti-aging method based on mesenchymal stem cell disrupted cytoplasm |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107041894A (en) * | 2016-12-26 | 2017-08-15 | 湖南新起源医疗技术有限公司 | A kind of stem cell liquid for skin delication preparation method for anti-aging |
| CN110613736A (en) * | 2019-10-30 | 2019-12-27 | 广州陈运贤生命科技有限公司 | Composition and preparation for treating bone marrow failure diseases, preparation method and application |
| CN111996163A (en) * | 2020-09-04 | 2020-11-27 | 安徽惠恩生物科技股份有限公司 | Skin anti-aging method based on mesenchymal stem cell disrupted cytoplasm |
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