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CN106497872B - Skeletal muscle stem Cells serum free medium and its preparation method and application - Google Patents

Skeletal muscle stem Cells serum free medium and its preparation method and application Download PDF

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CN106497872B
CN106497872B CN201611087240.5A CN201611087240A CN106497872B CN 106497872 B CN106497872 B CN 106497872B CN 201611087240 A CN201611087240 A CN 201611087240A CN 106497872 B CN106497872 B CN 106497872B
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skeletal muscle
stem cells
muscle stem
vitamin
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CN106497872A (en
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刘谋元
刘麟
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Beijing Xin Feng Cmx Medical Technology Development Co Ltd
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Abstract

The invention discloses skeletal muscle stem Cells serum free mediums and its preparation method and application.The culture medium includes following ingredient: DMEM:F12, serum substitute, cell factor, vitamin, amino acid, biosynthetic human insulin, helping strong element and 6- chondroitin sulfate；The pH value of the culture medium is 7.2~7.4.The present invention further discloses the methods for preparing the skeletal muscle stem Cells serum free medium.Serum substitute of the present invention is non-animal derived ingredient; composition determines; avoid the pathogenic risk of serum bring; the factors such as batch is unstable; culture medium of the present invention is suitable for Human Skeletal Muscle stem cell primary and scale amplification cultivation, has purity is high, and cell Proliferation is fast; amplification efficiency is high, can stablize the advantages that passage 20 is more than generation.The present invention effectively increases skeletal muscle stem Cells amplification efficiency and purity, can be as scale, good skeletal muscle stem Cells cultivating system to meet clinical research and application.

Description

Skeletal muscle stem Cells serum free medium and its preparation method and application
Technical field
The present invention relates to a kind of cell culture medium more particularly to a kind of Human Skeletal Muscle stem cell serum-free culture medium, this hairs Bright preparation method further to Human Skeletal Muscle stem cell serum-free culture medium and in culture Human Skeletal Muscle stem cell Using belonging to the culture medium field of Human Skeletal Muscle stem cell.
Background technique
Skeletal muscle stem Cells (also known as sarcoblast, myoblast, English name: myoblast) can only directed differentiation be flesh Meat tissue has natural cell fusion, is damaged in muscle there are in skeletal muscle tissue in the form of skeletal muscle satellite cell After wound, skeletal muscle stem Cells are activated, and are largely proliferated and generate contractile protein, repair and maintain skeletal muscle tissue.Skeletal muscle is dry Cell transplant techniques treatment amyotrophia, heart disease, diabetes, tumour, pain, depression, in terms of bone and joint diseases in recent years By domestic and international follow-up story, skeletal muscle stem Cells implantation technique is as a kind of New Type of Diseases solution increasingly by the whole world Concern.Gusso ni etc. treats 8 DMD patients using double blind control ruling by law, proves that skeletal muscle stem Cells transplanting has DMD patient Certain curative effect.The clinical research of the whole world 20 countries, 230 patients at present shows to inject Human Skeletal Muscle to myocardial infarction patient Infarct perilesional regional myocardial function can be improved after stem cell, increase Left Ventricular Ejection Fraction, perfusion, survival ability, power, wall The blood volume of thickness and diastasis and end-systole is not accompanied by arrhythmia cordis.This treatment means will likely become the treatment heart Flesh infarct and the most important new way of heart failure.Skeletal muscle stem Cells transplanting has also been unfolded for treating type-2 diabetes mellitus. All the above therapeutic effects rely heavily on cell transplantation amount, and according to relevant report, skeletal muscle stem Cells treat the heart Popular name for, the cell quantity that the cardiomyopathys such as myocardial infarction use are 2 × 109, the quantity that treatment DMD patient uses is 5 × 1010.Institute With, explore it is a kind of being capable of high-purity, scale amplification skeletal muscle stem Cells and to be applied to clinical cultivating system extremely urgent.
Currently, most of research institutions are with DMEM:F12/1:1, the basal mediums such as α-MEM add 10%-20%'s Fetal calf serum cultivates skeletal muscle stem Cells.But fetal calf serum culture medium contains animal source component, ingredient do not know, there are risk and The differences between batches of serum are big, are not easy to control, skeletal muscle stem Cells are difficult to purify, amplification efficiency it is low become it is difficult in clinical application With the difficult point gone beyond.
Summary of the invention
The technical problem to be solved by the present invention is to add fetal calf serum for bone stem cell media, contain animal sources Ingredient, lead to problems such as skeletal muscle stem Cells be difficult to purify, amplification efficiency it is low, provide the skeletal muscle stem Cells of optimization a kind of without Blood serum medium, be used for the culture of Human Skeletal Muscle stem cell primary and scale amplification cultivation, for clinical research and apply provide it is excellent The skeletal muscle stem Cells of matter.
The technical problem to be solved by the present invention is to what is be achieved through the following technical solutions:
A kind of skeletal muscle stem Cells serum free medium includes following ingredient:
The mixture that DMEM and F12 is formed according to the mass ratio of 1:1, serum substitute, cell factor, vitamin, amino Acid, biosynthetic human insulin, transferrins, Pidolidone amide, thiamine hydrochloride, inositol, niacinamide, cholesterol, P68 are spat Temperature 80, ferric citrate and NaHCO3；PH value is 7.2~7.4.
The serum substitute is Ultroser G Xue Cheongju substitute；
The cell factor is insulin-like growth factor, in epithelical cell growth factor or Basic Fibroblast Growth Factor It is any one or more of；
The vitamin is the combination of any one of vitamin C, vitamin B1 or vitamin B6 or both.
The amino acid is 1 water altheine, L-Aspartic acid, L-Histidine, l-Isoleucine, L-Leu, L- Lysine, L-Methionine, L- phenylalanine, Serine, L-Trp, L-cysteine, L-Orn hydrochloric acid or to ammonia Any one or more of yl benzoic acid.
In order to reach better effect, in every liter of culture medium, the content of each ingredient are as follows:
DMEM:F12 content is 15.6g/L, wherein the mass ratio of DMEM:F12 is 1:1；
The serum substitute content is 20mL/L；
The vitamin B6 content is 0.95~1.05mg/L, preferably 0.975mg/L；The vitamin B1 content is 1.0~1.03mg/L, preferably 1.03mg/L；The Vitamin C content is 6~6.9mg/L, preferably 6.45mg/L；
The insulin-like growth factor content is 80~100 μ g/L, preferably 100 μ g/L；Epithelical cell growth factor Content is 18~22 μ g/L, preferably 20 μ g/L；Basic Fibroblast Growth Factor content is 18~22 μ g/L, preferably 20 μ g/ L；
Biosynthetic human insulin's content is 0.01~0.02mg/L, preferably 0.02mg/L；
The 1 water altheine content is 48.45~53.25mg/L, preferably 51.25mg/L；L-Aspartic acid contains Amount is 30.35~31.35mg/L, preferably 30.75mg/L；L-Histidine content is 85.12~89.12mg/L, preferably 87.12mg/L；L-Isoleucine content is 38.55~42.13mg/L, preferably 41.13mg/L；L-Leu content is 33.55~36.55mg/L, preferably 34.55mg/L；L-lysine content is 165.44~172.44mg/L, preferably 168.44mg/L；L-Methionine content is 4.9~5.1mg/L, preferably 5.0mg/L；L- phenyl alanine content be 21~ 23mg/L, preferably 22mg/L；Serine content is 38.5~40.35mg/L, preferably 39.5mg/L；L-Trp content For 12.3~14.46mg/L, preferably 13.6mg/L；L-cysteine content is 108.7~115.3mg/L, preferably 113.7mg/L；L-Orn content of hydrochloric acid is 27.3~30.3mg/L, and preferably 29.3mg/L, p-aminobenzoic acid content are 1.0~1.03mg/L, preferably 1.03mg/L；
The transferrin content is 11.5~12.5mg/L, preferably 12.5mg/L；Pidolidone amide content is 0.2mM；Thiamine hydrochloride cellulose content is 1.0~1.03mg/L, and preferably 1.03mg/L, inositol content is 34.5~37.01mg/L, Preferably 35.51mg/L；Niacinamide content is 1.0~1.03mg/L, preferably 1.03mg/L；Cholesterol level is 0.015 ~0.019mg/L, preferably 0.019mg/L；Pluronic F68 content is 97~100mg/L, preferably 98mg/L；Tween 80 Content is 5.1~5.4mg/L, preferably 5.25mg/L；Ammonium ferric citrate content is 179.1~186.6mg/L, preferably 181.6mg/L；NaHCO3Content is 1.2g/L.
It is a discovery of the invention that being added in above-mentioned culture medium equal to the proliferation activity of skeletal muscle stem Cells when 6- chondroitin sulfate There is certain facilitation；Preferably, the additive amount of 6- chondroitin sulfate is 50-500mg/L, wherein 6- chondroitin sulfate Additive amount is near 200 μ g/ml, and skeletal muscle stem Cells proliferation activity is optimal.
The present invention strengthens element it has furthermore been found that adding helping for certain dosage into above-mentioned skeletal muscle stem Cells serum free medium (N, N- dimethylpiperidine chloride) can effectively facilitate the growth of skeletal muscle stem Cells, wherein helping the additive amount for strengthening element is 8.7 The growth of skeletal muscle stem Cells can be obviously promoted when~10.7mg/L, it can be significant when the additive amount of strong element being helped to be 9.0mg/L Promoting the growth of skeletal muscle stem Cells, skeletal muscle stem Cells proliferation activity reaches highest, when being greater than this concentration, skeletal muscle stem Cells Moderate tone is presented in proliferation activity, shows that the concentration of 9.0mg/L is optimum addn amount.
μ g/L, mg/L and g/L in the present invention are the weight based on ingredient described in every liter of culture medium, and mL/L is based on every The volume of ingredient described in culture medium is risen, mM is the molal quantity based on ingredient described in every liter of culture medium.
Invention further provides a kind of method for preparing Human Skeletal Muscle stem cell serum-free culture medium, the culture medium by Following steps are made:
(1) dry powder of DMEM:F12 (1:1) is dissolved in water for injection and obtains basal medium；
(2) remaining its each ingredient is added in basal medium, stirring is to being completely dissolved；
(3) pH value is adjusted to 7.2~7.4, Osmo detection range 290-320mOsm/kg；
(4) with 0.22 μm of membrane filtration degerming to get.
Serum substitute in skeletal muscle stem Cells serum free medium of the present invention is non-animal derived ingredient, and composition determines, It avoids serum bring to cause a disease risk, the factors such as batch is unstable；Skeletal muscle stem Cells serum free medium of the present invention is applicable in In Human Skeletal Muscle stem cell primary and scale amplification cultivation.There is purity is high using culture medium of the present invention, cell Proliferation is fast, expands Increasing Efficiency is high, can stablize the advantages that passage 20 is more than generation, the skeletal muscle stem Cells turned out can be used for clinical research and answer With.Skeletal muscle stem Cells serum free medium of the present invention effectively increases skeletal muscle stem Cells amplification efficiency and purity, for clinic Research and application provide good skeletal muscle stem Cells, can be as scale, good skeletal muscle stem Cells cultivating system To meet clinical research and application.
Detailed description of the invention
Growth curve contrast and experiment of Fig. 1 skeletal muscle stem Cells in three kinds of culture mediums；
Fig. 2 P20For skeletal muscle stem Cells immunohistochemical staining result；
Fig. 3 P20Result is redyed for skeletal muscle stem Cells immunohistochemistry；
Fig. 4 P20Differentiation myotube is induced to form figure (10 ×) for skeletal muscle stem Cells；
Fig. 5 P20Differentiation myotube is induced to form figure (40 ×) for skeletal muscle stem Cells；
The 6- chondroitin sulfate of Fig. 6 various concentration promotes the experimental result of skeletal muscle stem Cells growth.
The experimental result for helping strong element that skeletal muscle stem Cells is promoted to grow of Fig. 7 various concentration.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art Member it should be understood that under without departing from the spirit and scope of the invention can details to technical solution of the present invention and form into Row modifications or substitutions, but these modifications and replacement are fallen within the protection scope of the present invention.
The preparation of 1 Human Skeletal Muscle stem cell serum-free culture medium of embodiment
Cell Basal Medium (DMEM:F12 (1:1) dry powder, article No.: SH30004 is purchased from Thermo company), cell because Sub (being purchased from PEPROTECH company) helps strong element (being purchased from Sigma company, purity is 98% or more), and biosynthetic human insulin (is purchased from Sigma company, purity are 98% or more), 6- chondroitin sulfate (is purchased from Hainan Gourd Doll Pharmaceutical Co., Ltd.), vitamin C (purchase In, Sigma company, purity is 98% or more), (product name is " Ultroser G ", brand Lonza to serum substitute；? Crescent Chemical Co., Inc. can be purchased from), NaHCO3(being purchased from Sigma company, purity is 98% or more) and amino Acid (is purchased from Sigma company, purity is 98% or more), and transferrins (is purchased from Sigma company, purity is 98% or more), L- paddy Glutamine (is purchased from Sigma company, purity is 98% or more), and vitamin B6 (is purchased from Sigma company, purity is 98% or more), Thiamine hydrochloride (is purchased from Sigma company, purity is 98% or more), and inositol (is purchased from Sigma company, purity is 98% or more), Niacinamide (is purchased from Sigma company, purity is 98% or more), and cholesterol (is purchased from Sigma company, purity is 98% or more), P68 (Pluronic F68 is purchased from Sigma, the mainly addition polymers of polypropylene glycol and ethylene oxide), Tween 80 (is purchased from Sigma Company, purity are 98% or more) and ferric citrate (being purchased from Sigma company, purity is 98% or more).
Each ingredient and content of the skeletal muscle stem Cells serum free medium of the present invention of table 1
The preparation of Human Skeletal Muscle stem cell serum-free culture medium:
(1) 15.6g DMEM:F12 (1:1) dry powder is accurately weighed, is dissolved in 800ml water for injection.
(2) remaining each ingredient is weighed according to dosage in table 1, be gradually added in above-mentioned solution, stirring is to being completely dissolved.
(3) 1.2g NaHCO is added3, continue to stir, be settled to 1L, adjust pH to 7.2-7.4, Osmo detection range 290- 320mOsm/kg。
(4) with 0.22 μm of membrane filtration degerming to get.
Growth curve comparative experiments of 1 skeletal muscle stem Cells of experimental example in three kinds of culture mediums
Three kinds of used culture mediums of this experiment be respectively the embodiment of the present invention 1 prepare serum free medium (hereinafter referred to as from Culture medium processed), the DMEM:F12 1:1 culture medium (hereinafter referred to as culture medium A) of 20% fetal calf serum is purchased from the bone of Lonza company The dedicated low blood serum medium SKGM-2 (hereinafter referred to as culture medium B) of flesh stem cell.
1. skeletal muscle stem Cells isolate and purify
(1) open surgical biopsy is sterile takes young healthy people voluntarily to contribute rectus femoris or deltoid muscle 2-5g, is transferred to and contains equipped with 20ml In the 50ml centrifuge tube of the PBS of 1% mycillin, 4 DEG C of transports to laboratory.
(2) musculature is taken out from 50ml centrifuge tube, is put into sterilized petri dishes, removal fat and connective tissue, use are sterile Operating scissors are cut into about 1mm3Fritter is flushed three times with containing 1% dual anti-PBS.
(3) the II Collagenase Type that 10-15ml concentration is 0.1% is added, is blown and beaten and is mixed with pipette, in 37 DEG C of constant temperature oscillations The digested 2h of device.
(4) 0.25% trypsase of 10-20ml is added, is blown and beaten and is mixed with pipette, it is digested in 37 DEG C of constant temperature oscillators 20-30min。
(5) 10ml is added and terminates digestion containing 10% serum substitute culture medium.
(6) it successively is sieved through filter with 400 mesh of preparatory moist heat sterilization, 200 mesh Stainless Steels, removes residue.
(7) filtrate is abandoned supernatant and is collected cell with 2000rpm, 5min centrifugation.
(8) it is cleaned with PBS, 2000rpm, 5min centrifugation, in triplicate.
(9) with 4ml gentamicin containing 160IU/ml, the DMEM:F12 1:1 of 10% serum substitute is inoculated into T25 culture Bottle, density 104/ml。
(10) be put into 37 DEG C, saturated humidity, 5% carbon dioxide incubator in cultivate.
(11) supernatant liquid after preculture 20min is transferred to a new culture bottle, removes adherent fibroblast, so Afterwards by 2h, 8h, for 24 hours, the cell suspension of 48h, 72h are updated to new culture bottle.
(12) attached cell after 72h is collected, self-made medium is added and formally cultivates.
(13) passage when covering with bottom of bottle face 70%-80% to cell passes on ratio 1:2, operating method: pours out culture in glassware Liquid is added PBS and washes 2-3 times, and 0.25% trypsase 2ml is added, and 37 DEG C of constant temperature oscillators digest 3min, fresh culture is added 3ml terminate digestion, mix, year 2000rpm, 5min, abandon supernatant, be added fresh culture 8ml, assign in 2 culture bottles, in 37 DEG C, saturated humidity, 5%CO2Under the conditions of continue to cultivate.
2. the P5 collected according to the above method for skeletal muscle stem Cells is used culture medium of the present invention respectively, and (embodiment 1 is made It is standby), the skeletal muscle stem Cells of DMEM:F12 1:1, the Lonza company of 20% fetal calf serum (being purchased from Lanzhou Min Hai company) are dedicated SKGM-2 culture medium equivalent is resuspended, by 105/ hole is seeded to 24 orifice plates, changes liquid every three days, totally 3 blocks of plates, counts three holes, note daily Record growth curve.
2 three kinds of culture medium cultivation results of table compare
Experimental result is shown in Table 2 and Fig. 1.As seen from Table 2, skeletal muscle stem Cells culture medium prepared by the present invention expands skeletal muscle The efficiency of stem cell is apparently higher than DMEM:F121:1 culture medium and SKGM-2 culture medium containing 20% fetal calf serum.
2 skeletal muscle stem Cells Purity of experimental example and differentiation capability evaluation
Desmin antibody and immunohistochemical kit and auxiliary reagent are purchased from Wuhan doctor's moral company；PBS is pH 7.2- 7.4 phosphate buffer；SABC reagent, DAB color developing agent, DAB color developing agent (being purchased from Wuhan doctor's moral company).
1. P20 skeletal muscle stem Cells are digested, collects and press 2 × 10 with self-made medium5The density in a/hole is added to 1:10 The diluted coated 6 orifice plates of poly-D-lysine are cultivated, real for immunohistochemistry when cell covers with 90% or more 6 orifice plates It tests.
2. being cleaned cell 3 times, 2min/ times with PBS solution.
3. 4% hole paraformaldehyde 1ml/ is added, 30min is fixed under room temperature.
4. being cleaned cell 3 times, 2min/ times with PBS solution.
5. the hole 0.5%triton x-100 1ml/ is added, 10min enhances the permeability of cell.
6. being cleaned cell 3 times, 2min/ times with PBS solution.
7. the H after methanol dilution is added2O2(1 part of H2O2+ 50 parts of methanol) 10 drops/hole, 10min.
8. being cleaned cell 3 times, 2min/ times with PBS solution.
10. lowlenthal serum (lowlenthal serum: PBS=1:10) hole 1ml/ after dilution, 20min is added.
11. primary antibody Desmin antibody (primary antibody: PBS=1:100) hole 1ml/ after dilution is added, it is protected from light, 4 DEG C of overnight (16- 18h) or 37 DEG C, 1 hour.
12. being cleaned cell 3 times, 2min/ times with PBS solution.
13. secondary antibody (secondary antibody: PBS=1:100) hole 1ml/ after dilution is added, 37 DEG C, 30min or room temperature 1h.
14. being cleaned cell 3 times, 2min/ times with PBS solution.
15. SABC reagent is added dropwise, the hole 1ml/, 37 DEG C, 20min.PBS cleans 5min × 4 time.
16. DAB color developing agent (1ml adds A, each drop of B, C) is added, the hole 1ml/ is protected from light, 10min, and microscopically observation is thin There is sepia and distilled water color development stopping are used to react in born of the same parents.Distilled water cleans 5min × 3 time.
17. redying 0.5-1min with haematoxylin, then indigo plant is returned with PBS.It observes under the microscope.5 visuals field are taken at random, are recorded Positive cell number and negative cells number calculate positive rate.Experimental result is shown in Fig. 2, Fig. 3 and tables 3.
3 skeletal muscle stem Cells culture purity result of table
From table 3 as the result is shown: three groups it is parallel the result shows that, obtained using skeletal muscle stem Cells culture medium culture of the present invention Human Skeletal Muscle stem cell purity reach 97% or more.
The differentiation myotube identification experiment of 3 skeletal muscle stem Cells of experimental example
By skeletal muscle stem Cells to contain 2%FBS, (100ng/ml) containing IGF-1, the DMEM:F12 1:1 culture without BFGF Base presses 2 × 105/ hole is inoculated into 6 orifice plates, is changed the liquid once within the 4th day, observes that myotube was formed in the 7th day.Experimental result is shown in figures 4 and Fig. 5.
4 6- chondroitin sulfate of experimental example promotes the experiment of skeletal muscle stem Cells growth
The skeletal muscle stem Cells culture medium used in this experiment, formula are shown in Table 4:
The skeletal muscle stem Cells culture medium used in 4, table experiments
The 6- chondroitin sulfate of various concentration is added in the culture medium of above-mentioned table 4, experiment is divided into six groups of 6- chondroitin sulfates Plain intervention group, it may be assumed that the 1st, 2,3,4,5,6 group, the addition concentration of the 6- chondroitin sulfate of each group is respectively as follows: 0 μ g/ml, 50 μ g/ ml,100μg/ml,200μg/ml,300μg/ml,500μg/ml.Microscopically observation each group is imaged in inversion within 4th day, the 5th day The proliferative conditions of skeletal muscle stem Cells sarcoblast prepare 6 parts of skeletal muscle stem Cells cells (1 × 10 according to grouping on the 6th day8L-1) Suspension is inoculated in 12 orifice plates, every group of 3 holes respectively, and every hole 1.5mL is incubated at 37 DEG C, the CO of volume fraction 5%2Incubator, the 6th day 160 μ L of MTT (thiazolyl blue) solution is added and is protected from light incubation, terminates culture after 4h, removes supernatant, it is sub- that 1.6mL dimethyl is added dropwise in every hole Sulfone, oscillation mix 10min, so that crystallization sufficiently dissolution, measures the absorbance (OD value) at 490nm using microplate reader, calculate Cell survival rate, this experiment repeat 3 plates.Influence of the comparative analysis 6- chondroitin sulfate to skeletal muscle stem Cells proliferation function.
Experimental result: MTT experiment increases absorbance value with 6- chondroitin sulfate concentration as the result is shown and increases, and in concentration Proliferation activity highest when 200 μ g/ml.Skeletal muscle stem Cells number known to various concentration cell activity curve graph is drawn to become in growth Gesture is shown in Fig. 6.
Statistical analysis show that the 1st group and the 2nd, 3,4,5,6 group has significant difference, P < 0.05；Between 2nd, 3,5,6 group No significant difference, P > 0.05；4th group and the 2nd, 3,5,6 group there is also significant difference, P < 0.05；(table 5).
5 various concentration 6- chondroitin sulfate of table is intervened skeletal muscle stem Cells activity analysis result and is proliferated to skeletal muscle stem Cells It is active influence (N=3)
Experimental result shows that 6- chondroitin sulfate has facilitation, and concentration 200 to the proliferation activity of skeletal muscle stem Cells Near μ g/ml, skeletal muscle stem Cells proliferation activity is optimal.
The experiment that experimental example 5 helps strong plain (N, N- dimethylpiperidine chloride) that skeletal muscle stem Cells is promoted to grow
The skeletal muscle stem Cells culture medium prescription such as the following table 6 used in this experiment:
Table 6
The strong element that helps of various concentration is added in the culture medium of above-mentioned table 6, experiment is divided into seven groups and helps strong plain intervention group, it may be assumed that 1st, 2,3,4,5,6,7 group, each group helps the addition concentration for strengthening element to be respectively as follows: 0 μ g/ml, 3 μ g/ml, 6 μ g/ml, 9 μ g/ml, 12 μg/ml,15μg/ml,20μg/ml.4th day, the 5th day proliferation in inversion imaging microscopically observation each group skeletal muscle stem Cells Situation prepares 7 parts of skeletal muscle stem Cells cells (1 × 10 according to grouping on the 6th day8L-1) suspension is inoculated in 12 orifice plates respectively, every group 3 Hole, every hole 1.5mL are incubated at 37 DEG C, the CO of volume fraction 5%2Incubator, 160 μ L of addition MTT (thiazolyl blue) solution is kept away within the 6th day Light is incubated for, and culture is terminated after 4h, removes supernatant, and 1.6mL dimethyl sulfoxide is added dropwise in every hole, and oscillation mixes 10min, so that crystallization is filled Divide dissolution, microplate reader is used to measure the absorbance (OD value) at 490nm, calculate cell survival rate, this experiment repeats 3 plates.Than Influence of the strong element to skeletal muscle stem Cells proliferation function is helped compared with analysis.
Experimental result: MTT experiment increases with helping strong plain concentration to increase absorbance value as the result is shown, and concentration is in 9 μ g/ml Proliferation activity is higher, and when being greater than this concentration, proliferation activity compares presentation moderate tone.Draw various concentration cell activity curve graph It knows that skeletal muscle stem Cells number shows a increasing trend, sees Fig. 7.Statistical analysis show that the 1st group and the 2nd, 3,4,5,6,7 group presence is aobvious Write difference, P < 0.05；No significant difference between 2nd, 3 group, P > 0.05；No significant difference between 4th, 5,6,7 group, P > 0.05； 4th group and the 1st, 2,3 group there are significant difference, P < 0.05 (is shown in Table 8).
8 various concentration of table helps strong element to intervene skeletal muscle stem Cells activity analysis result to skeletal muscle stem Cells proliferation activity Influence (N=3)
Experimental result, which is shown, helps strong element to have facilitation to the proliferation activity of skeletal muscle stem Cells, and 9 μ g/ml group of concentration is attached Closely, skeletal muscle stem Cells proliferation activity reaches highest, and is greater than this concentration skeletal muscle stem Cells proliferation activity and moderate tone is presented, Show that this concentration is optimum addn amount.

Claims (10)

1. a kind of skeletal muscle stem Cells serum free medium, which is characterized in that include following ingredient: DMEM:F12, serum replace For object, cell factor, vitamin, amino acid, biosynthetic human insulin, transferrins, Pidolidone amide, thiamine hydrochloride, flesh Alcohol, niacinamide, cholesterol, Pluronic F68, Tween 80, ferric citrate, NaHCO3, N, N- dimethylpiperidine chloride With 6- chondroitin sulfate, the N, the content of N- dimethylpiperidine chloride is 8.7~10.7mg/L, the 6- chondroitin sulfate Content is 50~500mg/L；The pH value of the culture medium is 7.2~7.4.
2. according to the skeletal muscle stem Cells serum free medium described in claim 1, which is characterized in that the serum substitute For Ultroser G Xue Cheongju substitute；
The cell factor is insulin-like growth factor, appointing in epithelical cell growth factor or Basic Fibroblast Growth Factor What is one or more；The vitamin is the group of any one of vitamin C, vitamin B1 or vitamin B6 or both It closes；
The amino acid is 1 water altheine, L-Aspartic acid, L-Histidine, l-Isoleucine, L-Leu, the bad ammonia of L- Acid, L-Methionine, L- phenylalanine, Serine, L-Trp, L-cysteine, L-Orn hydrochloric acid or p-aminophenyl Any combination of more than one in formic acid；
The Pluronic F68 is mainly the addition polymers of polypropylene glycol and ethylene oxide.
3. according to the skeletal muscle stem Cells serum free medium described in claim 2, which is characterized in that in every liter of culture medium, respectively The content of ingredient are as follows:
DMEM:F12 content is 15.6g/L, wherein the mass ratio of DMEM:F12 is 1:1；
The serum substitute content is 20mL/L；
The vitamin B6 content is 0.95~1.05mg/L；The vitamin B1 content is 1.0~1.03mg/L；The dimension Raw element C content is 6~6.9mg/L；
The insulin-like growth factor content is 80~100 μ g/L；Epithelical cell growth factor content is 18~22 μ g/L；Alkali Property fibroblast growth factor content be 18~22 μ g/L；
Biosynthetic human insulin's content is 0.01~0.02mg/L；
The 1 water altheine content is 48.45~53.25mg/L；L-Aspartic acid content is 30.35~31.35mg/ L；L-Histidine content is 85.12~89.12mg/L；L-Isoleucine content is 38.55~42.13mg/L；L-Leu contains Amount is 33.55~36.55mg/L；L-lysine content is 165.44~172.44mg/L；L-Methionine content be 4.9~ 5.1mg/L；L- phenyl alanine content is 21~23mg/L；
Serine content is 38.5~40.35mg/L；L-Trp content is 12.3~14.46mg/L；L-cysteine contains Amount is 108.7~115.3mg/L；L-Orn content of hydrochloric acid is 27.3~30.3mg/L；The p-aminobenzoic acid content is 1.0~1.03mg/L；
The transferrin content is 11.5~12.5mg/L；Pidolidone amide content is 0.2mM；Thiamine hydrochloride cellulose content is 1.0~1.03mg/L；Inositol content is 34.5~37.01mg/L；Niacinamide content is 1.0~1.03mg/L；Cholesterol contains Amount is 0.015~0.019mg/L；Pluronic F68 content is 97~100mg/L；Tween 80 content is 5.1~5.4mg/L； Ammonium ferric citrate content is 179.1~186.6mg/L；NaHCO3 Content is 1.2g/L.
4. according to the skeletal muscle stem Cells serum free medium described in claim 3, which is characterized in that in every liter of culture medium, respectively The content of ingredient are as follows: the vitamin B6 content is 0.975mg/L；The vitamin B1 content is 1.03mg/L；The dimension Raw element C content is 6.45mg/L；The insulin-like growth factor content is 100 μ g/L；Epithelical cell growth factor content is 20μg/L；Basic Fibroblast Growth Factor content is 20 μ g/L；
Biosynthetic human insulin's content is 0.02mg/L；
The 1 water altheine content is 51.25mg/L；L-Aspartic acid content is 30.75mg/L；L-Histidine content is 87.12mg/L；L-Isoleucine content is 41.13mg/L；L-Leu content is 34.55mg/L；L-lysine content is 168.44mg/L；L-Methionine content is 5.0mg/L；L- phenyl alanine content is 22mg/L；Serine content is 39.5mg/L；L-Trp content is 13.6mg/L；L-cysteine content is 113.7mg/L；L-Orn content of hydrochloric acid is 29.3mg/L；P-aminobenzoic acid content is 1.03mg/L；
The transferrin content is 12.5mg/L；Thiamine hydrochloride cellulose content is 1.03mg/L；Inositol content is 35.51mg/L； Niacinamide content is 1.03mg/L；Cholesterol level is 0.019mg/L；Pluronic F68 content is 98mg/L；Tween 80 Content is 5.25mg/L；Ammonium ferric citrate content is 181.6mg/L；
The content of the N, N- dimethylpiperidine chloride is 9.0mg/L.
5. skeletal muscle stem Cells serum free medium described in accordance with the claim 1, which is characterized in that the 6- chondroitin sulfate Content is 180~200mg/L.
6. skeletal muscle stem Cells serum free medium according to claim 5, which is characterized in that the 6- chondroitin sulfate Content is 200mg/L.
7. a kind of method for preparing any one of claim 1-6 skeletal muscle stem Cells serum free medium, feature exist In, comprising:
(1) dry powder of DMEM:F12 is dissolved in water for injection and obtains basal medium；
(2) remaining each ingredient is added in basal medium, stirring is to being completely dissolved；
(3) pH value is adjusted to 7.2~7.4, Osmo detection range 290-320m Osm/kg；
(4) 0.22 μm of filtration sterilizations to get.
8. according to the method for claim 7, it is characterised in that: in the dry powder of the DMEM:F12, the matter of DMEM:F12 Amount ratio is 1:1.
9. use of any one of the claim 1-6 skeletal muscle stem Cells serum free medium in culture skeletal muscle stem Cells On the way.
10. purposes according to claim 9, which is characterized in that the skeletal muscle stem Cells are Human Skeletal Muscle stem cells.
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