CN106244550A - A kind of method that cell culture fluid and application and induction skeletal muscle stem Cells neurad like cell thereof break up - Google Patents
A kind of method that cell culture fluid and application and induction skeletal muscle stem Cells neurad like cell thereof break up Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1392—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from mesenchymal stem cells from other natural sources
Abstract
The present invention relates to technical field of stem cell culture, particularly relate to a kind of cell culture fluid and application thereof and the method for induction skeletal muscle stem Cells neurad like cell differentiation.The culture fluid that the present invention provides includes basic culture solution, FBS, B27, BDNF, bFGF and NGF.The culture fluid that the present invention provides can improve the ratio of skeletal muscle stem Cells neurad like cell differentiation and shorten the time of differentiation.Experiment shows, the culture fluid induction skeletal muscle stem Cells provided with the present invention, i.e. visible part cell attachment have short and small projection to stretch out after 24 hours, visible most cells adherent growth after 3 days, form is irregular, projection constantly increases thick and elongation, be divided into after 1 week that form differs, disperse sheet of many projections sternzellen and neuron cell, projection is interleaved with each other and reticulates.
Description
Technical field
The present invention relates to technical field of stem cell culture, particularly relate to a kind of cell culture fluid and application thereof and induction skeleton
The method of flesh stem cell neurad like cell differentiation.
Background technology
Central nervous system, after damage, the recovery of function of nervous system is extremely difficult, and main cause is the regeneration of neuron in brain
Ability extreme difference.Although with the presence of neural stem cell in research proves brain at present, and neuron and neurogliocyte can be divided into,
But the position existed due to neural stem cell is limited to very much, and quantity is the most considerably less, therefore uses it for neuronal damage reparation still
Extremely difficult.Along with the research of stem cell field deepens continuously, find that the stem cell at other positions can also be divided into nerve
Cell.By in embryonic neural or Umbilical Cord Blood Transplant to the Mus brain of damaged, appropriateness its function of nervous system can be improved, but
Being due to ethics, the factor such as immunologic rejection limits the extensive application clinically of these stem cell.Medulla mesenchyma is done
Cell in autologous acquisition and amplification can be stablized in vitro, and can induce differentiation into neurocyte in vitro, is expected to become neural
The seed cell of systemic disease transplantation treatment.But induce the neural-like cells differentiated can't effectively stablize earth's surface at present
Reaching neurocyte characteristic, whether this type of neural-like cells possesses the function of real neuron, however it remains dispute, and from normal person
Internal bone marrow extraction, also can cause a certain degree of actual bodily harm to donor.
Skeletal Muscle derived stem cells (muscle-derived stem cells, MDSCs) is adults or people's muscle
In tissue distinctive dry carefully as a kind of adult mesenchymal stem cells of mesoderm origin, there is self-renewal capacity and multinomial point
The potential changed, and, muscle derived cell, in draw materials convenience, muscular tissue, cell content is big, value-added speed is fast.Research table
Bright, MDSCs can be divided into the cell that hemocyte, osteocyte, endotheliocyte etc. are in vitro, studies have found that, is lured by external
MDSCs also can be induced to differentiate into neurocyte and neurogliocyte by the method led.
This functional characteristic of skeletal muscle stem Cells has greatly attracted, from preclinical medicine and clinical medical researcher, to give
The researchers of basic science provide a kind of biological pattern of research and development, reparation that skeletal muscle stem Cells is had, replace
Generation and regeneration capacity also provide the chance of a development new therapy for clinical medicine.But, in current cultural method, skeleton
The transformation efficiency of flesh stem cell neurad like cell is the lowest, it would be highly desirable to research and develop new promotion skeletal muscle stem Cells neurad
The method of like cell differentiation.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of cell culture fluid and application thereof and induction bone
Bone flesh stem cell neurad like cell differentiation the method present invention provide cell culture fluid can promote skeletal muscle stem Cells to
Neural-like cells breaks up, and its differentiation cycle is short, and differentiation efficiency is high.
The cell culture fluid that the present invention provides, including basic culture solution, FBS, B27, BDNF, bFGF and NGF.
Hyclone (fetal calf serum, FBS) is the one in serum, using the teaching of the invention it is possible to provide maintain cell index growth
Hormone, in basal medium not or measure little nutrient, and main low molecule nutrient.
B27 is that a kind of cell cultivates additive, for hippocampal neuron and other central nervous system (CNS) neuron
Grow and keep its short-term or long period of activity.
Brain Derived Neurotrophic Factor (brain derived neurophic factor, BDNF) synthesizes in brain
A kind of protein, it is distributed widely in central nervous system, during development of central nervous system, deposits neuron
Work, differentiation, growth promoter play an important role, and can prevent neuronal damage wound death, improve the pathological state of neuron, promotion
The biological effects such as damaged neuron regeneration and differentiation, and be the neuron maintenance of ripe maincenter and peripheral nervous system
Existence and normal physiological function institute are required.
Basic fibroblast growth factor (fibroblast growth factor-basic, bFGF) its primary biological
Effect has: as angiogenesis factor；Promote wound healing and tissue repair；Promote tissue regeneration；Participate in neuranagenesis etc..
Nerve growth factor (Nerve growth factor, NGF) nerve growth factor can regulate around and maincenter god
Through the growth promoter of unit, maintain the survival of neuron.
The present invention utilizes FBS, B27, BDNF, bFGF and NGF induction skeletal muscle stem Cells to be divided into neural-like cells.Induction
The cell obtained not only is provided with the representative configuration of neurocyte, and expresses neuronal marker: nestin (Nestin),
Neuronspecific enolase (neuron specific enolase, NSE), neurogliocyte mark gtelatinous fibre
Acidic protein (glial fibrilamentacidic protein, GFAP)；Show that skeletal muscle stem Cells remains to divide across germinal layer
Turn to the ability of non-mesenchymal cell, can be neuron cell across differentiation of germinal layers.
The culture medium that the present invention provides improves differentiation rate the shortening point of skeletal muscle stem Cells neurad like cell differentiation
The time changed.Experiment shows, the culture fluid induction skeletal muscle stem Cells provided with the present invention, is visible part cell after 24 hours
Adherent and have short and small projection to stretch out, visible most cells adherent growth after 3 days, form is irregular, and projection constantly increases thick and stretches
Long, be divided into after 1 week that form differs, disperse sheet of many projections sternzellen and neuron cell, projection is interleaved with each other
Reticulate.Calculating the differentiation rate of each group of cell, the differentiation rate of result display experimental group cell is significantly higher than matched group, (p
< 0.05, or p < 0.01).
In an embodiment of the present invention, the volume fraction of FBS is 10%；The volume fraction of B27 is 5%.
In an embodiment of the present invention, the concentration of BDNF is 10ng/mL～30ng/mL；The concentration of bFGF be 10ng/mL～
30ng/mL；The concentration of NGF is 10ng/mL～30ng/mL.
In certain embodiments, cell culture fluid includes FBS, the volume fraction that basal medium, volume fraction are 10%
Be 5% B27, concentration be the BDNF of 10ng/mL, concentration is the bFGF of 10ng/mL and concentration is the NGF of 10ng/mL.
In certain embodiments, cell culture fluid includes FBS, the volume fraction that basal medium, volume fraction are 10%
Be 5% B27, concentration be the BDNF of 20ng/mL, concentration is the bFGF of 20ng/mL and concentration is the NGF of 20ng/mL.
In certain embodiments, cell culture fluid includes FBS, the volume fraction that basal medium, volume fraction are 10%
Be 5% B27, concentration be the BDNF of 30ng/mL, concentration is the bFGF of 30ng/mL and concentration is the NGF of 30ng/mL.
In certain embodiments, basal medium is DMEM/F12.
Basic culture solution used by the present invention can be that acquisition is alternatively bought in self-control, and this is not limited by the present invention, in fact
Execute all within protection scope of the present invention.The human stem cell serum-free medium that the present invention uses is the training of DMEM/F12 serum-free
Nutrient solution.The culture fluid that the present invention provides does not contains antibiotic, but by more suitable condition of culture, promotes that skeletal muscle is done
The differentiation of cell neurad like cell, strengthens the growth potential of target cell, reduces infection risk.
The cell culture fluid that the present invention provides application in induction skeletal muscle stem Cells neurad like cell differentiation.
The method that present invention also offers induction skeletal muscle stem Cells neurad like cell differentiation, skeletal muscle stem Cells is with base
Basal culture medium cultivates the cell culture fluid induction provided with the present invention after merging to 70%～80%.
In an embodiment of the present invention, the time of induction is 7d.
In an embodiment of the present invention, skeletal muscle stem Cells is the skeletal muscle stem Cells in 3～5 generations.
In some embodiments, skeletal muscle stem Cells is the skeletal muscle stem Cells in the 3rd generation.
The container of described induction skeletal muscle stem Cells neurad like cell differentiation is aseptic six orifice plates, it is preferred that described six
Orifice plate processes through poly-D-lysine.
In the present invention, the separation method of skeletal muscle stem Cells includes:
Step 1: after broken for skeletal muscle, after Dispase II and type i collagen enzymic digestion, with trypsinization, obtain
Obtain cell resuspended with culture medium；
Step 2: gained cell in step 1 is separated by differential attachment method, it is thus achieved that skeletal muscle stem Cells.
In the present invention, skeletal muscle is temporalis.
Differential velocity adherent particularly as follows: by cell suspension inoculation in culture bottle, be positioned over 37 DEG C, volume fraction be the CO of 5%2
Incubator is cultivated.Attached cell after 2h is designated as PP1, moves in new culture bottle and train after the most non-attached cell sucking-off
Support, and be designated as PP2.Cultivate moving into after cell sucking-off the most adherent in PP2 in new culture bottle after 24h, be labeled as PP3.With
Rear every 24h carries out differential velocity adherent cultivation, until obtaining PP6, is designated as skeletal muscle stem Cells.
Skeletal muscle stem Cells changes liquid after cultivating 3 days, often within 2-3 days, changes liquid later, after cell growth is fused to 70%-80%
Secondary Culture.
Skeletal muscle stem Cells (MDSCs) original cuiture 8-10 days, treats that cell covers with 80-90%, inhales with suction pipe and abandons old cultivation
Liquid, adds 0.25% trypsin-EDTA, digests 1-3 minute, when Microscopic observation cellular contraction becomes round, adds containing 20%FBS's
DMEM/F12 culture fluid terminates digestion, collects cell, and 1000r/min is centrifuged 5min, abandons supernatant.Add appropriate containing 20%FBS's
DMEM/F12 culture fluid, carries out cell count, by 8 × 104Cell/mL density is inoculated in culture dish and carries out Secondary Culture, and 5%
CO2Incubator 37 DEG C cultivation, changed liquid once every 2-3 days.
In the present invention, before induction skeletal muscle stem Cells neurad like cell differentiation, Skeletal Muscle Cell is with basal medium
The inoculum density cultivated is 1 × 104cell/mL。
Basal medium is DMEM/F12 culture fluid.
The culture fluid that the present invention provides includes basic culture solution, FBS, B27, BDNF, bFGF and NGF.The present invention provides
Culture fluid can become, as skeletal muscle stem Cells, the induction broth that neural-like cells breaks up.Improve skeletal muscle stem Cells to
The ratio of neural-like cells differentiation also shortens time of differentiation.Experiment shows, the culture fluid induction skeletal muscle provided with the present invention
Stem cell is visible part cell attachment after 24 hours and has short and small projection to stretch out, the visible adherent life of most cells after 3 days
Long, form is irregular, and projection constantly increases thick and elongation, be divided into after 1 week that form differs, disperse sheet of many projections starlike carefully
Born of the same parents and neuron cell, projection is interleaved with each other and reticulates.After inducing 7 days, the differentiation rate of each group of cell is calculated, result
The differentiation rate of display experimental group cell is significantly higher than matched group, (p < 0.05, or p < 0.01).
Accompanying drawing explanation
Fig. 1 shows that MDSCs P2 is for form；Wherein, the amplification of Fig. 1-a be 40 ×；The amplification of Fig. 1-b is 100
×。
Detailed description of the invention
The invention provides a kind of cell culture fluid and application thereof and the differentiation of induction skeletal muscle stem Cells Cardiocytes
Method, those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realize.Special needs to be pointed out is,
All similar replacements and change apparent to those skilled in the art, they are considered as being included in this
Bright.Method and the application of the present invention are described by preferred embodiment, and related personnel substantially can be without departing from this
In bright content, spirit and scope, methods herein and application it is modified or suitably changes and combine, realizing and apply this
Inventive technique.
Below in conjunction with embodiment, the present invention it is expanded on further:
The separation of embodiment 1 MDSCs and passing on
1, draw materials:
Take adult normal temporalis specimen (being taken from operation of opening cranium patient), proceed to after rinsing containing dual anti-PBS
In sterile petri dish, it is cut into 1mm with eye scissors3The fragment of left and right size, then moves in 50ml centrifuge tube, and PBS blows
Beat and rinse 3 times, after standing 1 minute, abandon upper liquid and floating tissue.
2, enzymolysis:
The mixed enzyme of the volume of 2 times is added, including 2.4u/ml Dispase II, 1%I to the muscle fragment of above-mentioned acquisition
Collagenase Type, 2.5mmol/L CaCl2, mix digestion about 60min in rearmounted 37 DEG C of constant-temperature tables, until the muscle in test tube
Fragment digestion is rotten for flesh, and be invisible to the naked eye flesh block.After digestion terminates, 1000r/min is centrifuged 5min, abandons supernatant.Add 2～3 times
0.25% trypsin-EDTA of volume, mixes digestion 15min in rearmounted 37 DEG C of constant-temperature tables, after digestion terminates, 1000r/
Min is centrifuged 5min, abandons supernatant.Adding appropriate PBS resuspended, 1000r/min is centrifuged 5min, abandons supernatant.Add about 3 times of flesh gruel volumes
PBS repeatedly blow and beat after through 200 mesh filter screens filter, the filtrate 1000r/min of collection is centrifuged 5min, abandons supernatant.Growth medium
(DMEM/F12 containing 20%FBS) re-suspended cell.
3, isolation and purification:
Use differential velocity adherent isolation technics that MDSCs is separated.By cell suspension inoculation in 25ml culture bottle, place
In 37 DEG C, volume fraction be 5% CO2 incubator in cultivate.Attached cell after 2h is designated as PP1, and the most non-attached cell inhales
Move into after going out in new culture bottle and cultivate, and be designated as PP2.After 24h new by moving into after cell sucking-off the most adherent in PP2
Culture bottle is cultivated, is labeled as PP3.The most every 24h carries out differential velocity adherent cultivation, until obtaining PP6, period is according to the life of cell
Long situation carries out changing liquid.PP6 changes liquid after cultivating 3 days, often within 2-3 days, changes liquid later, observed and recorded cell growth under inverted microscope
And formation collection expands situation backward every day, Secondary Culture after cell growth is fused to 70-80%.
4, the Secondary Culture of MDSCs:
MDSCs original cuiture 8-10 days, treats that cell covers with 80-90%, inhales with suction pipe and abandons old culture fluid, adds appropriate
0.25% trypsin-EDTA, digests 1-3 minute, when Microscopic observation cellular contraction becomes round, adds appropriate containing 20%FBS immediately
DMEM/F12 culture fluid terminate digestion, collect cell, 1000r/min is centrifuged 5min, abandons supernatant.Add appropriate containing 20%FBS
DMEM/F12 culture fluid, carry out cell count, by 8 × 104Cell/mL density is inoculated in culture dish and carries out Secondary Culture,
5%CO2Incubator 37 DEG C cultivation, changed liquid once every 2-3 days.
5, the qualification of MDSCs:
1) cell climbing sheet makes: being positioned over by sterile cover slips in 6 well culture plates, exponential phase cell is prepared as cell
Suspension, by 5 × 104Cell/mL density is inoculated in 6 well culture plates, every hole 2ml, and growth medium is containing 20%FBS's
DMEM/F12, cultivates 24-48 hour, makes cell be grown on coverslip.
2) Desmin, Sca-1 immunocytochemical stain: culture fluid sucking-off in coverslip 6 well culture plate, PBS will be placed with
Buffer rinsing cell tile 5 minutes, is repeated 3 times.Fix 15 minutes with 4% paraformaldehyde, dry after PBS rinsing,
Neutral gum adheres on microscope slide, 4 DEG C of refrigerator overnight.Drip 3% hydrogen peroxide to hatch 15 minutes, eliminate endogenous peroxidating
The activity of thing enzyme, PBS rinses 5 minutes, is repeated 3 times.Drip respectively an anti-rabbit anti-human desmin (Desmin) antibody (1:
200 dilution), rabbit anti-human Sca-1 antibody (1:100 dilution), hatch in 37 DEG C of wet boxes 1 hour, 4 DEG C overnight.PBS after taking out next day
Buffer rinses 5 minutes, and dropping two resists, and hatches 40 minutes in 37 DEG C of wet boxes, and PBS rinses 5 minutes, is repeated 3 times, dropping
Freshly prepared DAB solution develops the color 5-10 minute, at light Microscopic observation, is rinsed color development stopping with tap water.Sheet will be dried
After son, haematoxylin is redyed 1 minute, and indigo plant, gradient alcohol dehydration are returned in differentiation, and dimethylbenzene is transparent, neutral gum mounting.At Jing Xiaguan
Examining, brown yellow granule occur in after birth and/or endochylema, (Fig. 1 is that MDSCs P2 is for form to illustrate to be successfully separated acquisition MDSCs
Figure).
The differentiation of embodiment 2 MDSCs
The formula of each tested group of culture fluid such as table 1:
Induction formula of liquid respectively organized by table 1
| Group | Basal medium | FBS | B27 | BDNF | bFGF | NGF |
| Experimental group 1 | DMEM/F12 | 10% | 5% | 10ng/ml | 10ng/ml | 10ng/ml |
| Experimental group 2 | DMEM/F12 | 10% | 5% | 20ng/ml | 20ng/ml | 20ng/ml |
| Experimental group 3 | DMEM/F12 | 10% | 5% | 30ng/ml | 30ng/ml | 30ng/ml |
| Matched group 1 | DMEM/F12 | 10% | 5% | -- | -- | -- |
| Matched group 2 | DMEM/F12 | 10% | 5% | 20ng/ml | -- | -- |
| Matched group 3 | DMEM/F12 | 10% | 5% | -- | 20ng/ml | -- |
| Matched group 4 | DMEM/F12 | 10% | 5% | -- | -- | 20ng/ml |
Choose the 3rd generation MDSCs, by 1 × 104Cell/mL density is inoculated in and is placed with the sterility cover glass that poly-D-lysine processes
In six orifice plates of sheet, every hole adds the 2ml DMEM/F12 culture medium containing 10%FBS.The training that in table 1, each group is corresponding is changed after 24h
Nutrient solution, at 37 DEG C, 5%CO2, saturated humidity quiescent culture, the culture fluid more renewed every 2～3 days.
Observe cellular morphology in incubation: i.e. visible part cell attachment have short and small projection to stretch out after 24 hours, 3 days
Rear visible most cells adherent growth, form is irregular, and projection constantly increases thick and elongation, be divided into after 1 week that form differs,
Disperseing sheet of many projections sternzellen and neuron cell, projection is interleaved with each other and reticulates.
After cultivating 7 days take out cell climbing sheet, according to the description of immunohistochemical staining test kit carry out Nestin, NSE,
The immunohistochemical staining of GFAP, DAB develops the color.
Culture fluid sucking-off in coverslip 6 well culture plate, PBS rinsing cell tile 5 minutes will be placed with, be repeated 3 times.
Fixing 15 minutes with 4% paraformaldehyde, dry after PBS rinsing, neutral gum adheres on microscope slide, 4 DEG C of refrigerator mistakes
Night.Dripping 3% hydrogen peroxide to hatch 15 minutes, eliminate the activity of endogenous peroxydase, PBS rinses 5 minutes, weight
Multiple 3 times.Dripping appropriate one anti-(Nestin, NSE, GFAP) respectively, hatch 1 hour in 37 DEG C of wet boxes, 4 DEG C overnight.Next day takes out
Rear PBS rinses 5 minutes, and dropping two resists, and hatches 40 minutes in 37 DEG C of wet boxes, and PBS rinses 5 minutes, repeats 3
Secondary, drip freshly prepared DAB solution and develop the color 5-10 minute, at light Microscopic observation, be rinsed color development stopping with tap water.Will
After drying slice, thin piece, haematoxylin is redyed 1 minute, and indigo plant, gradient alcohol dehydration are returned in differentiation, and dimethylbenzene is transparent, neutral gum mounting.
To each group of gained cell after SABC detects, the cell of Nestin, NSE, GFAP dyeing is pressed formula:
Induction differentiation rate=staining positive cells number/total cellular score × 100%
Counting, the differentiation rate calculating each group compares.Often group sampling 3 times, respectively statistical data after detection.System
Meter result such as table 2.
Cell differentiation rate (*, * * represents p < 0.01) respectively organized by table 2
Table 2 result shows, matched group 1 cell Nestin, NSE, GFAP are showed no coloring, and differentiation rate is zero.In experimental group 2
Cell Nestin, NSE, GFAP coloring is the deepest, and positive expression rate pole is significantly higher than each matched group, is significantly higher than experimental group 1 and reality
Test group 3 (p < 0.05)；In experimental group 1 and experimental group 3, cell Nestin, NSE, GFAP coloring is relatively deep, and positive expression rate is significantly high
In each matched group (p < 0.05).Cell differentiation rate 68.93 ± 0.56% (p < 0.01) in experimental group 2, illustrates used point of the present invention
Change culture medium and can effectively induce MDSCs Differentiation into Neuron-like Cells.
Below it is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come
Saying, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (10)
1. a cell culture fluid, it is characterised in that include basic culture solution, FBS, B27, BDNF, bFGF and NGF.
Cell culture fluid the most according to claim 1, it is characterised in that
The volume fraction of described FBS is 10%；
The volume fraction of described B27 is 5%.
Cell culture fluid the most according to claim 1, it is characterised in that
The concentration of described BDNF is 10ng/mL～30ng/mL；
The concentration of described bFGF is 10ng/mL～30ng/mL；
The concentration of described NGF is 10ng/mL～30ng/mL.
Cell culture fluid the most according to claim 1, it is characterised in that include that basal medium, volume fraction are 10%
FBS, volume fraction be 5% B27, concentration be the BDNF of 10ng/mL, concentration is the bFGF of 10ng/mL and concentration is 10ng/
The NGF of mL.
Cell culture fluid the most according to claim 1, it is characterised in that include that basal medium, volume fraction are 10%
FBS, volume fraction be 5% B27, concentration be the BDNF of 20ng/mL, concentration is the bFGF of 20ng/mL and concentration is 20ng/
The NGF of mL.
Cell culture fluid the most according to claim 1, it is characterised in that include that basal medium, volume fraction are 10%
FBS, volume fraction be 5% B27, concentration be the BDNF of 30ng/mL, concentration is the bFGF of 30ng/mL and concentration is 30ng/
The NGF of mL.
7. according to the cell culture fluid described in any one of claim 1～6, it is characterised in that described basal medium is DMEM/
F12。
8. the cell culture fluid described in any one of claim 1～7 is in induction skeletal muscle stem Cells neurad like cell differentiation
Application.
9. induce the method that skeletal muscle stem Cells neurad like cell breaks up for one kind, it is characterised in that skeletal muscle stem Cells is with base
Basal culture medium is cultivated after merging to 70%～80% and is induced with the cell culture fluid described in any one of claim 1～7.
Method the most according to claim 8, it is characterised in that the time of described induction is 7d.
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| CN111944752A (en) * | 2020-08-28 | 2020-11-17 | 广州同康生物科技有限公司 | Skeletal muscle stem cell serum-free medium and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111944752A (en) * | 2020-08-28 | 2020-11-17 | 广州同康生物科技有限公司 | Skeletal muscle stem cell serum-free medium and preparation method thereof |
| CN111944752B (en) * | 2020-08-28 | 2021-09-03 | 广州同康生物科技有限公司 | Skeletal muscle stem cell serum-free medium and preparation method thereof |
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