CN108531448A - A kind of human mesenchymal stem cell is at chondrocyte induction differential medium and preparation method - Google Patents
A kind of human mesenchymal stem cell is at chondrocyte induction differential medium and preparation method Download PDFInfo
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Abstract
The invention discloses a kind of human mesenchymal stem cells into chondrocyte induction differential medium, it is characterised in that：There is following components to be made, α MEM/HG DMEM culture mediums, 5 50% percent by volume of fetal calf serum, 50 200mg/ml concentration of Sodium Pyruvate, 20 200 μ g/ml concentration of ascorbic acid, 10 100 μ g/ml concentration of proline, the loose 50 500nM volumes of plug rice, 20% percents by volume of ITS0.5,0.1 20ng/ml concentration of TGF β, 20.5 50nM volumes of insulin-like growth factor.Culture dish sterilizes；Addition source culture medium；Ingredient mixes, and FBS, Sodium Pyruvate mother liquor, ascorbic acid mother liquor, proline mother liquor, dexamethasone mother liquor, 2 mother liquor of ITS mother liquors, 3 mother liquors of TGF β and insulin-like growth factor is added by the concentration, is uniformly mixed；Mixed culture solution is used 0.22 μm of membrane filtration degerming by filtration sterilization.Induced velocity of the present invention is fast, and all there is preferably induction differentiation effect to the differentiation raw material of the separate sources such as human marrow mesenchymal stem cell, human umbilical cord mesenchymal stem cells and human adipose mesenchymal stem cells, culture medium preparation method is convenient, easy to use, and process is stablized.
Description
Technical field
The present invention relates to culture medium technical field, specially a kind of human mesenchymal stem cell is at chondrocyte induction differential medium
And preparation method.
Background technology
Mescenchymal stem cell is present in the Various Tissues such as marrow, umbilical cord, fat, can be acquired by detaching, in vitro greatly
Amount 1 research of amplification cultivation has shown that mescenchymal stem cell has good self-renewing and multidirectional point as a kind of multipotential stem cell
Change potential, induced through specified conditions, can be broken up as multiple types groups such as fat thin, bone, cartilage, flesh, liver, nerve, skins
Cell to be knitted, this characteristic is based on, mescenchymal stem cell has been attempted applied in Various Tissues and Multi-organ dysfunction repairing research,
Wherein, the cartilaginous tissue for cartilage differentiation characteristic being oriented based on mescenchymal stem cell reproduces repairing research, in animal and clinic
Satisfactory curative effect is obtained in the research of early period, has great clinical conversion potential, application market huge.
The special construction (no vascular distribution) of cartilaginous tissue, determines that it is difficult to the characteristic of self-regeneration after injury,
Mesenchymal stem cells are induced as seed cell through specified conditions, can be thin by that will be done by mesenchyma to cartilage cell's directed differentiation
The cartilaginous tissue that born of the same parents differentiate is transplanted, and clinical cartilage damage reparation and specific shaping operation demand can be met, and curative effect is aobvious
It writes, but under existence conditions, human mesenchymal stem cell is at cartilage differentiation limited efficacy, it is therefore, dry to obtain enough mesenchymas
The cartilage cell of cell origin carries out clinical application, need to lead to optimization Incubation Condition, be oriented induction, to short
Sufficient and uniform cartilaginous tissue cell is efficiently obtained in phase and carries out injury repair, and existing human mesenchymal stem cell is at chondrocyte induction
Differential medium ingredient is generally：DMEM culture mediums, 10% fetal calf serum (FBS), 1mM Sodium Pyruvates, 20ug/mL proline,
1XITS, 50 μ g/mL ascorbic acid, 100nM dexamethasone and 10ng/mL TGF-β.
The Chinese invention patent of 105695402 A of Publication No. CN discloses a kind of mescenchymal stem cell into chondrocyte induction
Induce realignment and regrouping object and application method, the culture medium on DMEM in high glucose medium base, 10% fetal calf serum of addition,
50ug/mL L-AA -2-2 phosphates, 5ng/mL TGFb2,100nM dexamethasone, 25ug/mL vitamin Cs, 20ug/
ML proline, 2.5ug/mL Indomethacins, 1XITS+1premix, 1ug/ml chlormadinone esters, after 14-21 days Fiber differentiations
It carries out cartilage cell and dyes identification, which just for umbilical cord derived mesenchymal stem cell at chondrocyte induction break up, lure
It is undesirable to lead differentiation efficiency, specificity is relatively low, Osteoblast Differentiation sign occurs, and its contained chlormadinone ingredient is not easy to obtain, at
This is higher, lacks actual application value.
In conclusion existing can not fill at chondrocyte induction differential medium between efficient stable induction people's Various Tissues source
Matter stem cell breaks up to chondroblast, still needs to the differentiation condition more optimized and improves human mesenchymal stem cell into chondrocyte induction point
Change efficiency, to provide advantageous item at the identification of cartilage differentiation ability and clinical cartilage damage treatment and beauty for mescenchymal stem cell
Part.
Invention content
To solve existing in the prior art to ask at chondrocyte induction efficiency and specificity are not ideal enough, induction time is longer etc.
Topic, the present invention provide it is a kind of can efficient stable induce a variety of people's tissue-derived mesenchymal stem cell directionals at the culture of cartilage differentiation
Base and preparation method thereof.
A kind of human mesenchymal stem cell is at chondrocyte induction differential medium, it is characterised in that：There is following components to be made, α-
MEM/HG-DMEM culture mediums, fetal calf serum 5-50% percents by volume, Sodium Pyruvate 50-200mg/ml concentration, ascorbic acid
20-200 μ g/ml concentration, proline 10-100 μ g/ml concentration, plug rice pine 50-500nM volumes, ITS0.5-20% volume basis
Than, TGF-β 0.1-20ng/ml concentration, insulin-like growth factor -20.5-50nM volumes.
It is preferred that being made of following components, α-MEM/HG-DMEM culture mediums, fetal calf serum 5-50% percents by volume, acetone
Sour sodium 50-200mg/ml concentration, ascorbic acid 20-100 μ g/ml concentration, proline 10-500 μ g/ml concentration, plug rice pine 50-
200nM volumes, ITS0.5-5% percents by volume, TGF-β 0.1-10ng/ml concentration, insulin-like growth factor -20.5-50nM
Volume.
It is preferred that being made of following components, HG-DMEM culture mediums, 10% percent by volume of fetal calf serum, Sodium Pyruvate
100mg/ml concentration, 50 μ g/ml concentration of ascorbic acid, 20 μ g/ml concentration of proline, plug rice pine 100nM volumes, ITS1% volumes
Percentage, TGF-β 5ng/ml concentration, insulin-like growth factor -220nM volumes.
Human mesenchymal stem cell provided by the invention is existing at the efficient inductive differentiation medium of cartilage and operational version
At addition insulin-like growth factor -2 in cartilage differentiation culture medium.Dexamethasone in the existing differential medium at chondrocyte induction can
The aggrecan of human mesenchymal stem cell is raised by activating WNT/ β-catenin signal paths；Ascorbic acid can pass through
ERK signals are activated, to activate Runx2, Sox9 etc. to be transcribed at chondrocyte gene, promote Type Ⅱ collagen expression.TGF-β 3 can pass through
Smad2/3/4 signals are activated, activation is transcribed at cartilage related gene, promotes mescenchymal stem cell at cartilage differentiation 6.Proline can
It is maintained by forming the α collagen helix stable structure that proline ring promotes mescenchymal stem cell induction to generate.Containing insulin, turn
The ITS of ferritin and selenium equally can be by raising Sox9 gene transcript expressions, to promote the expression of Type Ⅱ collagen.The present invention
Insulin-like growth factor -2 is added in the existing differential medium ingredient at chondrocyte induction.We have found under study for action, insulin
Growth factor-2 can be dialled further up the mRNA transcriptions of Sox9 and BMP2 in mescenchymal stem cell, promote Type Ⅱ collagen expression heavy
Product, positive regulation aggrecan, cartilage oligo-substrate protein and mucopolysaccharide expression, to further efficiently the human world be promoted to fill
Matter stem cell is to cartilage cell's specific differentiation.
A kind of human mesenchymal stem cell includes the following steps at the preparation method of chondrocyte induction differential medium,
(1), culture dish sterilizes, and is rinsed well with clear water after culture dish is scrubbed with suds, and paper or cloth bag are used after drying
It is good, or in metal box, be placed in high-pressure steam sterilizing pan and sterilize, sterilising temp should be 121 DEG C, and sterilization time should be
3min is dried for standby after sterilizing；
(2), source culture medium is added, HG-DMEM culture mediums are added in culture dish, adding procedure should wear sterile hand
Set；
(3), ingredient mix, by the concentration addition FBS, Sodium Pyruvate mother liquor, ascorbic acid mother liquor, proline mother liquor,
Dexamethasone mother liquor, -2 mother liquor of ITS mother liquors, 3 mother liquor of TGF-β and insulin-like growth factor are uniformly mixed；
(4), mixed culture solution is used 0.22 μm of membrane filtration degerming by filtration sterilization.
The human mesenchymal stem cell breaks up by the following method at chondrocyte induction,
1) stem cell is obtained：Human mesenchymal stem cell is expanded using the low sugar DMEM medium cultures containing 10%FBS；
2) it pre-processes：Using trypsin digestion cell, with the human mesenchymal stem cell at chondrocyte induction differential medium weight
Outstanding cell, a concentration of 2X107/mL；
3) grouping culture：10 μ L cell suspensions are added drop-wise on six orifice plates, are dripped per hole 5；
4) differentiation culture：Cell is placed in the incubator 2 hours, the human mesenchymal stem cell is added per hole into cartilage
Induction medium culture 2 weeks.
It is dry that the human mesenchymal stem cell is selected from mesenchymal stem cell, umbilical cord mesenchymal stem cells or fat mesenchymal
Cell.
The TGF-β is TGF-β 3.
The Sodium Pyruvate is that life companies article No. is 11360070.
The ITS is that invitrogen companies article No. is 41400-045.
The human mesenchymal stem cell can add other compositions according to actual needs at the efficient inductive differentiation medium of cartilage,
Such as the nonessential amino acid of 1% percent by volume or the antibiotic of 1% percent by volume.
Human mesenchymal stem cell provided by the invention is at chondrocyte induction differential medium, it can be achieved that including human bone marrow mesenchymal
Various Tissues source human mesenchymal stem cell including stem cell, umbilical cord mesenchymal stem cells and fat mesenchymal stem cell to
The efficient induction of chondroblast is broken up, high at cartilage differentiation specificity.Induce human mesenchymal stem cell at soft with the prior art
It is compared within 3-4 weeks needed for bone differentiation, human mesenchymal stem cell provided by the invention can use 3 weeks at chondrocyte induction differential medium
It is interior, so that human mesenchymal stem cell the largely cartilaginous tissue extracellular matrix with Type Ⅱ collagen (collagen II) for representative is occurred,
Meanwhile making and significantly being raised at the closely related mucin of cartilage effect (Glycosaminoglycan, GAG) protein expression level,
Shorten human mesenchymal stem cell into cartilage differentiation induction time.In addition, human mesenchymal stem cell provided by the invention is lured at cartilage
It is easy to lead differential medium preparation method, easy to use, it can be achieved that stablizing, efficient human mesenchymal stem cell is at chondrocyte induction point
Change.
The beneficial effects of the invention are as follows：The present invention adapts to a variety of culture raw materials, and induction differentiation degree is high, and speed is fast, training
It is convenient to support base preparation method, easy to use, process is stablized.
Description of the drawings
Fig. 1 be different formulations of the present invention human mesenchymal stem cell at chondrocyte induction differential medium induction 2 weeks when pair
It is influenced at the expression of chondrocyte induction differentiation associated gene Collagen II, Aggrecan.
Fig. 2 be different formulations of the present invention human mesenchymal stem cell at chondrocyte induction differential medium induction 3 weeks when pair
It is influenced at the expression of chondrocyte induction differentiation associated gene CollagenII, Aggrecan.
Fig. 3 is that P3 generation human marrow mesenchyme stem cells of the present invention induce 3 in the present invention is at chondrocyte induction differential medium
The expression of Zhou Shicheng chondrocyte induction differentiation associated genes CollagenII, Aggrecan.
Fig. 4 is that P3 generation human marrow mesenchyme stem cells of the present invention induce 3 in the present invention is at chondrocyte induction differential medium
Zhou Shicheng chondrocyte inductions break up A Erxin indigo plant coloration results.
Fig. 5 is that P3 of the present invention induces 3 for human umbilical cord mesenchymal stem cells in the present invention is at chondrocyte induction differential medium
The expression of Zhou Shicheng chondrocyte induction differentiation associated genes CollagenII, Aggrecan.
Fig. 6 is that P3 of the present invention induces 3 for human umbilical cord mesenchymal stem cells in the present invention is at chondrocyte induction differential medium
Zhou Shicheng chondrocyte inductions break up A Erxin indigo plant coloration results.
Fig. 7 is that P3 of the present invention induces 3 for human adipose mesenchymal stem cells in the present invention is at chondrocyte induction differential medium
The expression of Zhou Shicheng chondrocyte induction differentiation associated gene Collagen II, Aggrecan.
Fig. 8 is that P3 of the present invention induces 3 for human adipose mesenchymal stem cells in the present invention is at chondrocyte induction differential medium
Zhou Shicheng chondrocyte inductions break up A Erxin indigo plant coloration results.
Specific implementation mode
In order to make the technical means, the creative features, the aims and the efficiencies achieved by the present invention be easy to understand, tie below
Specific embodiment is closed, the present invention is further explained.
Involved in the present invention is market conventional products on sale to each component.It is embodied involved in case into cartilage
Inductive differentiation medium information is as follows：
Inductive differentiation medium 1：The human mesenchymal stem cell provided in the embodiment of the present invention two breaks up at chondrocyte induction to be trained
Support base.
Inductive differentiation medium 2：The human mesenchymal stem cell provided in the embodiment of the present invention three breaks up at chondrocyte induction to be trained
Support base.
Inductive differentiation medium 3：The human mesenchymal stem cell provided in the embodiment of the present invention four breaks up at chondrocyte induction to be trained
Support base.
Inductive differentiation medium 4：Compared with inductive differentiation medium 1, insulin-like growth factor -2 is not included.
Inductive differentiation medium 5：The human mesenchymal stem cell of Thermo Fisher Scientific companies is lured at cartilage
Lead differential medium, article No. A1007101.
Embodiment one：Human mesenchymal stem cell at chondrocyte induction differential medium recipe determination
Inventor screens human mesenchymal stem cell ru inductive differentiation medium ingredients by many experiments.
Human mesenchymal stem cell is expanded using the low sugar DMEM medium cultures containing 10%FBS.Trypsin digestion cell is wanted with patent right
Cell, a concentration of 2X10 is resuspended in chondrocyte induction differential medium described in asking7A/mL；10 μ L cell suspensions are added drop-wise to six holes
On plate, dripped per hole 5；Cell is placed in the incubator 2 hours, inductive differentiation medium 1, induction differentiation training are separately added into per hole
Base 2, inductive differentiation medium 3 and inductive differentiation medium 4 are supported, is changed the liquid once within every 3 days.Respectively when inducing 2 weeks and 3 weeks, profit
Chondroblast related gene CollagenII, Aggrecan expression is detected with realtime PCR.As a result such as Fig. 1 and figure
Shown in 2, by existing human mesenchymal stem cell at chondrocyte induction differential medium in simultaneously add certain density insulin
Growth factor-2 (IGF-2), 2 weeks or 3 weeks either after Fiber differentiation, all can it is notable on be tuned into cartilage related gene
The expression of CollagenII, Aggrecan are (with inductive differentiation medium 2, inductive differentiation medium 3, and induction differentiation training
Support base 4, p<0.01 or p<0.05).
Embodiment two：Human mesenchymal stem cell is prepared at chondrocyte induction differential medium
Human mesenchymal stem cell at chondrocyte induction differential medium, including HG-MEM culture mediums, also following components and its
Concentration：
| Fetal calf serum (FBS) | 10% (percent by volume) |
| Sodium Pyruvate | 100mg/ml |
| Ascorbic acid | 50μg/ml |
| Proline | 20μg/ml |
| Dexamethasone | 100nM |
| ITS | 1% (percent by volume) |
| TGF-β3 | 5ng/ml |
| Insulin-like growth factor -2 (IGF-2) | 20nM |
Preparation method：Into HG-MEM culture mediums, it is female that FBS, Sodium Pyruvate mother liquor, ascorbic acid is added by above-mentioned concentration
Liquid, proline mother liquor, dexamethasone mother liquor, ITS mother liquors, TGF-β3Mother liquor and IGF-2 mother liquors are uniformly mixed, 0.22 μm of filter
Membrane filtration degerming to obtain the final product.
Ascorbic acid mother liquor：50mg ascorbic acid is taken, is dissolved with α-MEM culture mediums, is made into the mother liquor of 50 μ g/ μ L ,-
20 DEG C of preservations.Proline mother liquor mother liquor：20mg proline is taken, is dissolved using α-MEM culture mediums, it is female to be configured to 20 μ g/ μ L
Liquid, -20 DEG C of preservations,.With dexamethasone mother liquor：10g dexamethasone is taken, is dissolved with 95% ethyl alcohol, is made into 100 μm of mother liquors ,-
20 DEG C of preservations.TGF-β3Mother liquor is matched：Take 5mg TGF-β3, dissolved with α-MEM culture mediums, be made into 5 μ g/ μ L.IGF-2 mother liquors processed are matched
System：2mg IGF-1 are taken, are dissolved with α-MEM culture mediums, 2 μM of mother liquor, -20 DEG C of preservations are made into.
Embodiment three：Human mesenchymal stem cell is at chondrocyte induction differential medium
Human mesenchymal stem cell at chondrocyte induction differential medium, including HG-MEM culture mediums, also following components and its
Concentration：
Preparation method：It is similar with preparation method provided in embodiment two.
Example IV：Human mesenchymal stem cell is at chondrocyte induction differential medium
Human mesenchymal stem cell at chondrocyte induction differential medium, including HG-MEM culture mediums, also following components and its
Concentration：
| Fetal calf serum (FBS) | 10% (percent by volume) |
| Sodium Pyruvate | 100mg/ml |
| Ascorbic acid | 20μg/ml |
| Proline | 10μg/ml |
| Dexamethasone | 50nM |
| ITS | 1% (percent by volume) |
| TGF-β3 | 1ng/ml |
| Insulin-like growth factor -2 (IGF-2) | 40nM |
Preparation method：It is similar with preparation method provided in embodiment two.
Embodiment five：Human marrow mesenchymal stem cell breaks up at chondrocyte induction
Human mesenchymal stem cell is expanded using the low sugar DMEM medium cultures containing 10%FBS.Trypsin digestion cell, with special
Cell, a concentration of 2X10 is resuspended in chondrocyte induction differential medium described in sharp claim7A/mL；10 μ L cell suspensions are dripped
It is added on six orifice plates, is dripped per hole 5；Cell is placed in the incubator 2 hours, 1 He of inductive differentiation medium is separately added into per hole
Inductive differentiation medium 5 changes the liquid once for every 3 days.After inducing 2 weeks, chondroblast related gene is detected with realtime PCR
The expression of CollagenII, Aggrecan, as shown in Figure 3 the present invention in human mesenchymal stem cell break up at chondrocyte induction
In the induced noble cells of culture medium at cartilage related gene CollagenII, Aggrecan expression higher (with lure
It leads the effect of differential medium 5 to compare, p<0.01)；It is dyed to being identified at cartilage degree, as a result such as using A Erxin indigo plants simultaneously
Shown in Fig. 4, the human mesenchymal stem cell in the present invention has more rich at the induced noble cells of chondrocyte induction differential medium
Rich cartilage cell epimatrix (compared with inductive differentiation medium 5 acts on).The above results illustrate that the human mesenchyme in the present invention is dry
Cell has for human marrow mesenchymal stem cell preferably at chondrocyte induction differentiation effect at chondrocyte induction differential medium.
Embodiment six：Human umbilical cord mesenchymal stem cells break up at chondrocyte induction
Human mesenchymal stem cell is expanded using the low sugar DMEM medium cultures containing 10%FBS.Trypsin digestion cell, with special
Cell, a concentration of 2X10 is resuspended in chondrocyte induction differential medium described in sharp claim7A/mL；10 μ L cell suspensions are dripped
It is added on six orifice plates, is dripped per hole 5；Cell is placed in the incubator 2 hours, 1 He of inductive differentiation medium is separately added into per hole
Inductive differentiation medium 5 changes the liquid once for every 3 days.After inducing 2 weeks, chondroblast related gene is detected with realtime PCR
The expression of CollagenII, Aggrecan, as shown in Figure 5 the present invention in the induced noble cells of human mesenchymal stem cell
In at cartilage related gene CollagenII, Aggrecan expression higher (with inductive differentiation medium 5 act on phase
Than p<0.01)；It is dyed simultaneously using A Erxin indigo plants to identifying that the results are shown in Figure 6 at cartilage degree, in the present invention
Human mesenchymal stem cell at the induced noble cells of chondrocyte induction differential medium have more horn of plenty cartilage cell epimatrix (with
The effect of inductive differentiation medium 5 is compared).The above results illustrate that the human mesenchymal stem cell in the present invention breaks up at chondrocyte induction and train
Supporting base has for human umbilical cord mesenchymal stem cells preferably at chondrocyte induction differentiation effect.
Embodiment seven：Human adipose mesenchymal stem cells break up at chondrocyte induction
Human mesenchymal stem cell is expanded using the low sugar DMEM medium cultures containing 10%FBS.Trypsin digestion cell, with special
Cell, a concentration of 2X10 is resuspended in chondrocyte induction differential medium described in sharp claim7A/mL；10 μ L cell suspensions are dripped
It is added on six orifice plates, is dripped per hole 5；Cell is placed in the incubator 2 hours, 1 He of inductive differentiation medium is separately added into per hole
Inductive differentiation medium 5 changes the liquid once for every 3 days.After inducing 2 weeks, chondroblast related gene is detected with realtime PCR
The expression of CollagenII, Aggrecan, as shown in Figure 7 the human mesenchymal stem cell culture medium in the present invention induce point
The expression higher at cartilage related gene CollagenII, Aggrecan changed in cell (is made with inductive differentiation medium 5
With compared to p<0.01)；It is dyed simultaneously using A Erxin indigo plants to identifying that the results are shown in Figure 8 at cartilage degree, the present invention
In human mesenchymal stem cell at the induced noble cells of chondrocyte induction differential medium have more horn of plenty cartilage cell outside base
Matter (compared with inductive differentiation medium 5 acts on).The above results illustrate the human mesenchymal stem cell in the present invention at chondrocyte induction
Differential medium has for human adipose mesenchymal stem cells preferably at chondrocyte induction differentiation effect.
It is that human mesenchymal stem cell commonly in the market is compared at chondrocyte induction differential medium, the present invention has apparent
The fast feature of induced velocity, and to human marrow mesenchymal stem cell, human umbilical cord mesenchymal stem cells and people's fat mesenchymal
The differentiation raw material of the separate sources such as stem cell all has preferably induction differentiation effect, and culture medium preparation method is convenient, user
Just, process is stablized.
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (10)
1. a kind of human mesenchymal stem cell is at chondrocyte induction differential medium, it is characterised in that：There is following components to be made, α-MEM/
HG-DMEM culture mediums, fetal calf serum 5-50% percents by volume, Sodium Pyruvate 50-200mg/ml concentration, ascorbic acid 20-200
μ g/ml concentration, proline 10-100 μ g/ml concentration, plug rice pine 50-500nM volumes, ITS0.5-20% percents by volume, TGF-
β 0.1-20ng/ml concentration, insulin-like growth factor -20.5-50nM volumes.
2. a kind of human mesenchymal stem cell according to claim 1 is at chondrocyte induction differential medium, which is characterized in that by
Following components is made, α-MEM/HG-DMEM culture mediums, fetal calf serum 5-50% percents by volume, Sodium Pyruvate 50-200mg/ml
Concentration, ascorbic acid 20-100 μ g/ml concentration, proline 10-500 μ g/ml concentration, plug rice pine 50-200nM volumes, ITS0.5-
5% percent by volume, TGF-β 0.1-10ng/ml concentration, insulin-like growth factor -20.5-50nM volumes.
3. a kind of human mesenchymal stem cell according to claim 1 is at chondrocyte induction differential medium, which is characterized in that by
Following components is made, HG-DMEM culture mediums, 10% percent by volume of fetal calf serum, Sodium Pyruvate 100mg/ml concentration, Vitamin C
Sour 50 μ g/ml concentration, 20 μ g/ml concentration of proline, plug rice pine 100nM volumes, ITS1% percents by volume, TGF-β 5ng/ml
Concentration, insulin-like growth factor -220nM volumes.
4. a kind of human mesenchymal stem cell according to claim 1 is at the preparation method of chondrocyte induction differential medium,
It is characterized in that, includes the following steps,
(1), culture dish sterilizes, and is rinsed well with clear water after culture dish is scrubbed with suds, is wrapped with paper or cloth after drying, or
It in metal box, is placed in high-pressure steam sterilizing pan and sterilizes, sterilising temp should be 121 DEG C, and sterilization time should be 3min, go out
It is dried for standby after bacterium；
(2), source culture medium is added, HG-DMEM culture mediums are added in culture dish, adding procedure should wear sterile gloves；
(3), ingredient mix, by the concentration be added FBS, Sodium Pyruvate mother liquor, ascorbic acid mother liquor, proline mother liquor, fill in
The loose mother liquor of rice, -2 mother liquor of ITS mother liquors, 3 mother liquor of TGF-β and insulin-like growth factor, are uniformly mixed；
(4), mixed culture solution is used 0.22 μm of membrane filtration degerming by filtration sterilization.
5. a kind of human mesenchymal stem cell according to claim 1 is at the preparation method of chondrocyte induction differential medium,
It being characterized in that, the human mesenchymal stem cell breaks up by the following method at chondrocyte induction,
1) stem cell is obtained：Human mesenchymal stem cell is expanded using the low sugar DMEM medium cultures containing 10%FBS；
2) it pre-processes：Using trypsin digestion cell, it is resuspended carefully at chondrocyte induction differential medium with the human mesenchymal stem cell
Born of the same parents, a concentration of 2X107/mL；
3) grouping culture：10 μ L cell suspensions are added drop-wise on six orifice plates, are dripped per hole 5；
4) differentiation culture：Cell is placed in the incubator 2 hours, the human mesenchymal stem cell is added per hole into cartilage differentiation
Inducing culture culture 2 weeks.
6. a kind of human mesenchymal stem cell according to claim 4 is at the preparation method of chondrocyte induction differential medium,
It is characterized in that, the human mesenchymal stem cell is selected from mesenchymal stem cell, umbilical cord mesenchymal stem cells or fat mesenchymal
Stem cell.
7. a kind of human mesenchymal stem cell according to claim 4 is at the preparation method of chondrocyte induction differential medium,
It is characterized in that, the TGF-β is TGF-β 3.
8. a kind of human mesenchymal stem cell according to claim 4 is at the preparation method of chondrocyte induction differential medium,
It is characterized in that, the Sodium Pyruvate is that life companies article No. is 11360070.
9. a kind of human mesenchymal stem cell according to claim 4 is at the preparation method of chondrocyte induction differential medium,
It is characterized in that, the ITS is that invitrogen companies article No. is 41400-045.
10. a kind of human mesenchymal stem cell according to claim 4 is at the preparation method of chondrocyte induction differential medium,
Be characterized in that, the human mesenchymal stem cell at the efficient inductive differentiation medium of cartilage can add according to actual needs other at
Point, such as nonessential amino acid of 1% percent by volume or the antibiotic of 1% percent by volume.
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| CN110592010A (en) * | 2019-10-14 | 2019-12-20 | 杨姣姣 | Application of asiatic acid in promoting in-vitro proliferation of human adipose tissue-derived mesenchymal stem cells and inducing chondrogenic differentiation of human adipose tissue-derived mesenchymal stem cells |
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