CN108676772A - It is a kind of at chondrocyte induction culture medium and at cartilage differentiation method - Google Patents
It is a kind of at chondrocyte induction culture medium and at cartilage differentiation method Download PDFInfo
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- CN108676772A CN108676772A CN201810523292.5A CN201810523292A CN108676772A CN 108676772 A CN108676772 A CN 108676772A CN 201810523292 A CN201810523292 A CN 201810523292A CN 108676772 A CN108676772 A CN 108676772A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0655—Chondrocytes; Cartilage
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
- C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
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- C12N2513/00—3D culture
Abstract
The present invention relates to biotechnologies more particularly to a kind of at chondrocyte induction culture medium and at cartilage differentiation method.This includes conditioned medium, dexamethasone, vitamin C, insulin, transferrins and transforminggrowthfactor-β1 at chondrocyte induction culture medium, it is carried out into chondrocyte induction yield higher using the culture medium, more cartilage cells can be obtained, and cartilage cell's survival rate is higher.The present invention also provides the method broken up at chondrocyte induction, using at chondrocyte induction three-dimensional environment instead of conventional two-dimensional culture environment, it is easy to operate, success rate is high, cost is lower.
Description
Technical field
The present invention relates to biotechnologies more particularly to a kind of at chondrocyte induction culture medium and at cartilage differentiation method.
Background technology
China has been enter into aging Rapid development stage, and the morbidity of osteoarthropathy is very extensive in the elderly, seriously affects
Gerontal patient take care of oneself and ability to act, and accelerate many diseases of old people such as cardiovascular and cerebrovascular disease, dysbolism disease etc.
Disease process.Seed cell source is always the main bottleneck for limiting cartilage tissue engineered development.Chondrocyte proliferation ability has
It limits, aging is easy to happen after external large amplification and dedifferentes, lose the Forming ability of cartilage.And the proliferative capacity of cartilage cell
Gradually decline with the growth at age and further limits its application in organizational project.Therefore, seek suitable kind a kind of
Daughter cell source is particularly important.
For source for mesenchymal stem cells in mesoderm, research has shown that it is a kind of cartilage tissue engineered seed cell well.
Parodontium is a kind of the one of tissue-derived of mescenchymal stem cell, has convenient material drawing, the low feature of immunogenicity therefore can
Using the membrane derived mescenchymal stem cell of periodontal as a kind of new cartilage tissue engineered seed cell.
Periodontal ligament stem cell based on chondrocyte induction generally use DMEM/F12 high glucose mediums at cultivating in the prior art
The traditional of base composition induces the quantity of chondroblast few at chondrocyte induction culture medium, and cell survival rate is low, far from full
The requirement of the modern cartilage tissue engineered seed cell of foot.On the other hand, the prior art still uses traditional two-dimentional abductive approach,
Traditional two-dimentional abductive approach induction is relatively low at cartilage differentiation success rate, and needs timbering material that could form tissue, and operation is multiple
Miscellaneous, cost is higher.
Invention content
In view of this, the present invention provides a kind of at chondrocyte induction culture medium and at cartilage differentiation method.The invention improves
Formula of the stem cell at chondrocyte induction culture medium, adds conditioned medium, and has replaced into chondrocyte induction device, has behaviour
Make the high advantage of simplicity, success rate height, high income, cell survival rate.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical schemes：
The present invention provides a kind of into chondrocyte induction culture medium, including conditioned medium, dexamethasone, vitamin C, pancreas islet
Element, transferrins and transforming growth factor-beta 1.
In cell cultivation process, in cell meeting secretion activity substance to culture solution, wherein cell factor is main work
One of property substance, it is this cultivated cell or tissue after, the culture solution containing cell secreta is conditioned medium.Condition
Culture medium may participate in various kinds of cell reaction, and cell growth, cell culture effect is promoted to be better than traditional basal medium.
Preferably, the dosage of each component is in the chondroblast culture medium：
Dexamethasone：0.1-10μM；
Vitamin C：10-100μg/mL；
Insulin：1-10μg/mL；
Transferrins：1-10μg/mL；
Transforming growth factor-beta 1：1-20ng/mL；
Conditioned medium：It supplies.
Preferably, the dosage of each component is in the culture medium at chondrocyte induction：
Dexamethasone：0.1μM；
Vitamin C：50μg/mL；
Insulin：6.25μg/mL；
Transferrins：6.25μg/mL；
Transforming growth factor-beta 1：10ng/mL；
Conditioned medium：It supplies.
In embodiment provided by the invention, conditioned medium is the DMEM in high glucose training for cultivating remarkable knee cartilage cell
Support base.
Preferably, the human knee joint cartilage cell is the second generation to the third generation.
The present invention also provides a kind of inducing mesenchymal stem cells into the method for cartilage differentiation, using above-mentioned chondroblast
Medium culture mescenchymal stem cell.
Preferably, the mescenchymal stem cell is parodontium mescenchymal stem cell.
It is highly preferred that the parodontium mescenchymal stem cell is the second generation to the 5th generation.
Preferably, the apparatus for deivation of the mescenchymal stem cell is three-dimensional cultivation device.
It is highly preferred that the three-dimensional cultivation device is 15mL centrifuge tubes.
The present invention provides a kind of chondroblast culture medium including conditioned medium and a variety of inducible factors composition, compared with
Traditional can using culture medium provided by the invention progress at chondrocyte induction cell yield higher at chondrocyte induction culture medium
More cartilage cells are obtained, and cell survival rate is higher, disclosure satisfy that the requirement of modern cartilage tissue engineered seed cell.
On the other hand, the present invention also provides the method at chondrocyte induction, at using 15mL in chondrocyte induction atomization
Centrifuge tube constructs one simply at chondrocyte induction three-dimensional environment, instead of the two-dimentional culture environment of traditional six orifice plates, the party
Method is easy to operate, cost is relatively low, success rate is high.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings.
Fig. 1 is that the cell of 40 times of amplification when 1 periodontal film mescenchymal stem cell of embodiment reaches 80%-90% fusions is given birth to
Long aspect graph；
Fig. 2 is cell mass size figure of the cartilage cell of embodiment 1 after centrifugation；
Fig. 3 is cell mass size figure of the cartilage cell of comparative example 1 after centrifugation；
Fig. 4 is the result that cellular identification is carried out using toluidine blue dye liquor.
Specific implementation mode
Below in conjunction with the specific embodiment of the invention, technical scheme of the present invention is clearly and completely described, is shown
So, described embodiment is a part of the embodiment of the present invention, instead of all the embodiments.Those skilled in the art should
Understand, modifies to specific embodiments of the present invention or some technical characteristics are replaced on an equal basis, without departing from this hair
The spirit of bright technical solution should all cover in the scope of protection of the invention.
Embodiment 1
1, the preparation at chondrocyte induction culture medium that this patent provides：
1) the human knee joint cartilage in P2 generations is pressed 5 × 106~10 × 106The inoculum density of/cell is inoculated in DMEM high sugar
3d is cultivated in culture medium, is filtered, is obtained conditioned medium；
2) it is configured to chondrocyte induction culture medium according to composition and dosage shown in table 1：
Table 1
2, parodontium mescenchymal stem cell at cartilage differentiation method：
1) the parodontium mescenchymal stem cell of 2nd generation is obtained from laboratory；
2) parodontium mescenchymal stem cell is taken out, is placed in inverted microscope and observes, when parodontium mescenchymal stem cell converges
It is right up to 80% when, carry out passage processing；
3) with old culture medium is abandoned, the cleaning of cell cleaning solution is added, is repeated 1 times, 2mL is added in each culture dish
0.25% trypsin solution makes trypsase infiltrate entire ware bottom and is incubated 1~2min, observed under inverted microscope, 80%
Cell shrinkage when being rounded floating, adds 5mL stem cell medias to terminate digestion；Wherein, the stem cell media used is containing 10%
The DMEM/F12 culture mediums of FBS；
4) cell suspension is transferred in centrifuge tube, mixes well rear 1500rpm centrifugations 5min；
5) after centrifuging, centrifuged supernatant is outwelled, a certain amount of stem cell media is added and carries out cell count, adjusts
Whole cell-seeding-density is 1 × 105Cell/mL adds 1 μ g/mL EGF to final concentration of 10ng/mL, by postdigestive cell
Inoculated and cultured；
6) when parodontium mescenchymal stem cell reaches 80%~90% fusion, its cell growth shape is observed under the microscope
State, the results are shown in Figure 1 for observation；Former culture medium is discarded, with 0.25% trypsin digestion 1~2 minute, 1000rpm centrifugations 5
New stem cell media is added after minute, is inoculated into novel apparatus for deivation 15mL centrifuge tubes (being purchased from CORNING companies of the U.S.)
In；
7) change within second day liquid be added according to 1 method of table prepares at chondrocyte induction culture medium, replace within every 3 days thereafter once new
It is fresh at induction inducing culture, obtain cartilage cell after 21 days of induction.
Comparative example 1
1, traditional preparation at chondrocyte induction culture medium
Table 2 is traditional formula at chondrocyte induction culture medium, according to preparation culture medium shown in table 2：
Table 2
| Ingredient | Dosage |
| DMEM/F12 high glycosyl basal culture mediums | 95mL |
| Fetal calf serum | 5mL |
| Dexamethasone | 0.1μM |
| Vitamin C | 50μg/mL |
| Insulin | 6.25μg/mL |
| Transferrins | 6.25μg/mL |
| TGF-β1 | 10ng/mL |
2, induce parodontium mescenchymal stem cell at cartilage differentiation using the formula of table 2
Specific Induction Process and embodiment 1 are identical, and the biography prepared according to 2 method of table is added after changing liquid difference lies in step 7)
Unite into chondrocyte induction culture medium.
Comparative example 2
What is used is identical at chondrocyte induction culture medium and Induction Process and embodiment 1, and difference lies in by the tooth in step 6)
All film mescenchymal stem cell enzymolysis centrifugations are followed by kind to six orifice plates.
Embodiment 2
Example 1 and comparative example 1 induce obtained chondroblast, centrifugation to observe the cell mass formed after its precipitation
Block size.Fig. 2 is cell mass size figure of the cartilage cell of embodiment 1 after centrifugation, and Fig. 3 is the cartilage of comparative example 1
Cell mass size figure of the cell after centrifugation, black arrow meaning is cartilage cell's agglomerate, and Fig. 2 and Fig. 3 is carried out
Comparison, cell mass ratio Fig. 3's of Fig. 2 is big, and the cell mass naked eyes of Fig. 2 can obviously observe, and show that embodiment 1 provides
At chondrocyte induction culture medium yield higher.
The chondroblast of Example 1, and cellular identification is carried out using toluidine blue dye liquor：
1) cartilage cell after 21 days of induction is taken, old culture medium is discarded, is cleaned 5 times with PBS solution；
2) 4% paraformaldehyde solution is added and fixes 10~15min, cleaned 5 times with PBS solution；
3) prepared toluidine blue dye liquor is added and is protected from light room temperature 30~60min of dyeing, is cleaned 5 times, is set with PBS solution
It is observed, and is taken pictures under inverted microscope.
Fig. 4 is using toluidine blue dye liquor progress cellular identification as a result, most cell has been dyed to purple in Fig. 4
Color illustrates that the most cells have induced chondroblast.
Claims (10)
1. a kind of at chondrocyte induction culture medium, which is characterized in that including conditioned medium, dexamethasone, vitamin C, insulin,
Transferrins and transforming growth factor-beta 1.
2. according to claim 1 at chondrocyte induction culture medium, which is characterized in that each in the chondroblast culture medium
The dosage of component is：
Dexamethasone：0.1~10 μM；
Vitamin C：10~100 μ g/mL；
Insulin：1~10 μ g/mL；
Transferrins：1~10 μ g/mL；
Transforming growth factor-beta 1：1~20ng/mL；
Conditioned medium：It supplies.
3. according to claim 1 at chondrocyte induction culture medium, which is characterized in that each in the culture medium at chondrocyte induction
The dosage of component is：
Dexamethasone：0.1μM；
Vitamin C：50μg/mL；
Insulin：6.25μg/mL；
Transferrins：6.25μg/mL；
Transforming growth factor-beta 1：10ng/mL；
Conditioned medium：It supplies.
4. according to claims 1 to 3 any one at chondrocyte induction culture medium, which is characterized in that the CMC model
Base is the DMEM in high glucose culture medium for cultivating remarkable knee cartilage cell.
5. according to claim 4 at chondrocyte induction culture medium, which is characterized in that the human knee joint cartilage cell is the
In two generations, are to the third generation.
6. a kind of inducing mesenchymal stem cell is at the method for cartilage differentiation, which is characterized in that any one using claim 1 to 5
Chondroblast medium culture mescenchymal stem cell described in.
7. according to the method described in claim 6, it is characterized in that, the mescenchymal stem cell is that parodontium mesenchyma is dry thin
Born of the same parents.
8. the method according to the description of claim 7 is characterized in that the parodontium mescenchymal stem cell is the second generation to the 5th
Generation.
9. according to the method described in claim 6, it is characterized in that, the apparatus for deivation of the mescenchymal stem cell is dimensional culture
Device.
10. according to the method described in claim 9, it is characterized in that, the three-dimensional cultivation device is 15mL centrifuge tubes.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102925408A (en) * | 2012-10-31 | 2013-02-13 | 川北医学院第二临床医学院 | Method for differentiating induced pluripotent stem (iPS) cells into cartilage cells |
| CN103223194A (en) * | 2013-01-15 | 2013-07-31 | 广州莱德尔生物科技有限公司 | Cartilage graft for cartilage injury repair and preparation method thereof |
| CN104988117A (en) * | 2015-07-03 | 2015-10-21 | 深圳中基恒润投资有限公司 | Method for separating and culturing mesenchymal stem cells from umbilical cord and inducing and differentiating mesenchymal stem cells into cartilage cells |
| CN106834223A (en) * | 2017-04-05 | 2017-06-13 | 上海逍鹏生物科技有限公司 | Method from inducing umbilical cord mesenchymal stem to Chondrocyte Differentiation |
| CN107338218A (en) * | 2017-07-28 | 2017-11-10 | 中国人民解放军总医院第附属医院 | Derivant and method of a kind of induced lipolysis stem cell to Chondrocyte Differentiation |
-
2018
- 2018-05-28 CN CN201810523292.5A patent/CN108676772A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102925408A (en) * | 2012-10-31 | 2013-02-13 | 川北医学院第二临床医学院 | Method for differentiating induced pluripotent stem (iPS) cells into cartilage cells |
| CN103223194A (en) * | 2013-01-15 | 2013-07-31 | 广州莱德尔生物科技有限公司 | Cartilage graft for cartilage injury repair and preparation method thereof |
| CN104988117A (en) * | 2015-07-03 | 2015-10-21 | 深圳中基恒润投资有限公司 | Method for separating and culturing mesenchymal stem cells from umbilical cord and inducing and differentiating mesenchymal stem cells into cartilage cells |
| CN106834223A (en) * | 2017-04-05 | 2017-06-13 | 上海逍鹏生物科技有限公司 | Method from inducing umbilical cord mesenchymal stem to Chondrocyte Differentiation |
| CN107338218A (en) * | 2017-07-28 | 2017-11-10 | 中国人民解放军总医院第附属医院 | Derivant and method of a kind of induced lipolysis stem cell to Chondrocyte Differentiation |
Non-Patent Citations (5)
| Title |
|---|
| YONG-BEOM PARK等: "Effect of chondrocyte-derived early extracellular matrix on chondrogenesis of placenta-derived mesenchymal stem cells", 《BIOMED. MATER.》 * |
| 周峰等: "人软骨细胞培养上清诱导脐血间充质干细胞向软骨细胞分化", 《中国组织工程研究》 * |
| 周继如等: "《实用临床神经病学 上》", 31 January 2015, 科学技术文献出版社 * |
| 罗二梅等: "共培养人脐带间充质干细胞(hUC-MSCs)和兔关节软骨细胞诱导hUC-MSCs分化成软骨细胞", 《基础医学与临床》 * |
| 黄建荣等: "体外诱导人骨髓间充质干细胞分化为软骨细胞的研究", 《中国临床解剖学杂志》 * |
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Application publication date: 20181019 |