CN106916782A - A kind of cell culture fluid and induction method of the skeletal muscle stem Cells to Chondrocyte Differentiation - Google Patents
A kind of cell culture fluid and induction method of the skeletal muscle stem Cells to Chondrocyte Differentiation Download PDFInfo
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Abstract
The present invention relates to stem cell culture field, and in particular to a kind of cell culture fluid and induction method of the skeletal muscle stem Cells to Chondrocyte Differentiation.Transforming growth factor β 3, IGF, dexamethasone, L ascorbic acid and ITS are included in the nutrient solution that the present invention is provided+Premix, rationally, collaboration promotes induction skeletal muscle stem Cells to Chondrocyte Differentiation to each component compatibility, improves skeletal muscle stem Cells to the quantity of Chondrocyte Differentiation and shortens the time of differentiation.
Description
Technical field
The present invention relates to stem cell culture field, and in particular to a kind of cell culture fluid and induction skeletal muscle stem Cells are to soft
The method of bone cell differentiation.
Background technology
Osteoarthritis is the common disease of Orthopedic Clinical, due to the articular cartilage defect that retrogression pathological changes, wound etc. are caused,
The major reason for causing maimed influence quality of life is turned into.Articular cartilage is a kind of vesselless tissue, cartilage cell mainly by
The matrix of knuckle synovia and cell peripheral supplies nutrition with anerobic glycolysis energy supply, and the repair ability of its own is very limited.Joint is soft
Bone will cause arthralgia, unstable and stiff once damaging, and can accelerate the ossified of joint, the speed of extracellular matrix degradation
More than the synthesis of new matrix, then permanent lesion is left, so as to cause arthritic generation.Under traditional treatment method such as cartilage
The transplanting of drilling of bone, cartilage transplantation, periosteum or perichondrium, chondrocyte cell transplantation etc. lack because repair tissue is based on fibrocartilage
The mechanical property and durability of few normal hyaline, therefore promising result can not be obtained.Therefore, how it is minimally invasive repair damage it is soft
Bone, alleviates the pain of patient, to improve the quality of living be exactly for a long time one of emphasis direction of Osteopathic Medicine area research.
It is the new hand for being used to substitute above-mentioned traditional therapy in recent years that the cartilaginous tissue for damaging is repaired using cartilage cell
Section, but the source of cartilage cell is the research emphasis of those skilled in the art.Skeletal Muscle derived stem cells (Muscle-
Derived Stem Cells, MDSCs) it is that distinctive dry spy is the one of mesoderm origin in adults or people's musculature
Adult mesenchymal stem cells are planted, the potential with self-renewal capacity and multinomial differentiation can in vitro be divided into cartilage cell, blood
Cell, endothelial cell and nerve cell, this functional characteristic have greatly attracted from preclinical medicine and clinical medical research
Person, the researchers to basic science provide a kind of pattern of research and development biology, and what skeletal muscle stem Cells had repaiies
Multiple, replacement and power of regeneration are also for clinical medicine provides a chance for development new treatment.Therefore it provides a kind of induction
MDSCs, into the method for cartilage differentiation, is those skilled in the art's technical problem urgently to be resolved hurrily.
The content of the invention
In view of this, it is thin to cartilage it is an object of the invention to provide a kind of cell culture fluid and induction skeletal muscle stem Cells
The method of born of the same parents' differentiation, the cell culture fluid can effectively facilitate skeletal muscle stem Cells to Chondrocyte Differentiation, its differentiation cycle
Short, differentiation efficiency is high.
The invention provides a kind of cell culture fluid, including the life of basal medium, Transforming growth factor-β3, Insulin-Like
The factor long, dexamethasone, L-AA and ITS+Premix。
Preferably, the concentration of the Transforming growth factor-β3 is 5~15 μ g/L；The IGF it is dense
It is 50~100 μ g/L to spend.
Preferably, the concentration of the dexamethasone is 0.5 × 10-7~1 × 10-7mol/L；The L-AA it is dense
It is 50~100 μm of ol/L to spend.
Preferably, the ITS+The volume fraction of Premix is 1%.
Preferably, the concentration of the Transforming growth factor-β3 is 10 μ g/L；
The concentration of the IGF is 100 μ g/L；
The concentration of the dexamethasone is 1 μM；
The concentration of the L-AA is 100 μM；
The ITS+The volume fraction of Premix is 1%.
Preferably, basal medium is DMEM in high glucose nutrient solution.
Present invention also offers a kind of induction method of the skeletal muscle stem Cells to Chondrocyte Differentiation, using above-mentioned any one
Cell culture fluid described in induces skeletal muscle stem Cells to Chondrocyte Differentiation as induction liquid.
Preferably, the skeletal muscle stem Cells are skeletal muscle stem Cells of the third generation to the 5th generation.
In sum, the invention provides a kind of cell culture fluid and induction skeletal muscle stem Cells to Chondrocyte Differentiation
Method.It is anti-bad comprising Transforming growth factor-β3, IGF, dexamethasone, L- in the nutrient solution that the present invention is provided
Hematic acid and ITS+Premix, rationally, collaboration promotes induction skeletal muscle stem Cells to Chondrocyte Differentiation to each component compatibility, improves
Quantity from skeletal muscle stem Cells to Chondrocyte Differentiation and shorten time of differentiation.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing to be used needed for having technology description is briefly described, it should be apparent that, drawings in the following description are only this
Inventive embodiment, for those of ordinary skill in the art, on the premise of not paying creative work, can also basis
The accompanying drawing of offer obtains other accompanying drawings.
Fig. 1 is second generation skeletal muscle stem Cells aspect graph, and wherein Fig. 1 a are the second generation skeletal muscle stem Cells of 40 times of amplification
Aspect graph, Fig. 1 b are the second generation skeletal muscle stem Cells aspect graph of 100 times of amplification；
Fig. 2 is the experimental result of safranin O-fast green dyeing in embodiment 2；
Fig. 3 is the experimental result of II Collagen Type VI SABCs in embodiment 2；
Fig. 4 is the experimental result of safranin O-fast green dyeing in comparative example 1；
Fig. 5 is the experimental result of II Collagen Type VI SABCs in comparative example 1.
Specific embodiment
Technical scheme is clearly and completely described below in conjunction with description of the invention accompanying drawing, it is clear that
Described embodiment is a part of embodiment of the invention, rather than whole embodiments.Those skilled in the art should manage
Solution, modifies to specific embodiment of the invention or some technical characteristics is replaced on an equal basis, without deviating from the present invention
The spirit of technical scheme, all should cover in the scope of protection of the invention.
The material and instrument that the present invention is used are all common commercially available product, can all be bought in market.Wherein, ITS+Premix bags
Contain：Insulin (5 μ g/mL), Transferrin (5 μ g/mL) and Selenious Acid (5ng/mL), its brand are life,
Article No. is 354350.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1
1st, draw materials
Adult normal temporalis sample (being taken from operation of opening cranium patient) is taken, is transferred to after being rinsed containing dual anti-PBS
In sterile petri dish, 1mm is cut into eye scissors3The fragment of left and right size, then moves into 50mL centrifuge tubes, and PBS blows
Beat and rinse 3 times, upper liquid and floating tissue are abandoned after standing 1 minute.
2nd, digest
2 times of mixed enzymes of volume, including 2.4mg/mL Dispase II, 1%I are added to the muscle fragment of above-mentioned acquisition
Collagenase Type and 2.5mM CaCl2, 60min or so is digested in the rearmounted 37 DEG C of constant-temperature tables of mixing, until the muscle in test tube is broken
Untill block digestion is for flesh gruel, the flesh block that is invisible to the naked eye.After digestion terminates, 1000r/min centrifugation 5min abandon supernatant.Add 2~3
0.25% trypsase-EDTA of times volume, to mix and digest 15min in rearmounted 37 DEG C of constant-temperature tables, after digestion terminates, 1000r/
Min is centrifuged 5min, abandons supernatant.Add appropriate PBS resuspended, 1000r/min centrifugation 5min abandon supernatant.Add about 3 times of flesh gruel volumes
PBS blow and beat repeatedly after filtered through 200 mesh filter screens, the filtrate of collection 1000r/min centrifugations 5min again abandons supernatant.Again using life
Culture medium (DMEM/F12 containing 20%FBS) re-suspended cell long.
3rd, isolation and purification
MDSCs is isolated and purified using differential velocity adherent isolation technics, by cell suspension inoculation in 25mL blake bottles,
Be positioned over 37 DEG C, the CO that volume fraction is 5%2Cultivated in incubator.Attached cell after 2h is designated as PP1, wherein not adherent thin
Born of the same parents are cultivated during new blake bottle is moved into after suctioning out, and are designated as PP2.Moved into after cell not adherent in PP2 is suctioned out after 24h
Cultivated in new blake bottle, labeled as PP3.Differential velocity adherent culture is carried out per 24h later, until obtaining PP6, period is according to cell
Growing state carry out changing liquid.PP6 changes liquid after cultivating 3 days, changes liquid once every 3 days afterwards, observed and recorded under inverted microscope
Cell growth and the formation collection daily amplification situation of backwardness, the Secondary Culture after cell growth is fused to 70~80%.
4th, the Secondary Culture of MDSCs
When cell fusion degree is 80~90%, is inhaled with suction pipe and abandon old nutrient solution, appropriate 0.25% trypsase of addition-
EDTA, digests 1~3min, and Microscopic observation cellular contraction becomes bowlder, and the appropriate DMEM/F12 nutrient solutions containing 20%FBS are added immediately
Terminate digestion, 1000r/min centrifugation 5min abandon supernatant, collect cell.Add the appropriate DMEM/F12 nutrient solutions containing 20%FBS
It is resuspended, cell count is carried out, by 8 × 104Cell/mL density carries out Secondary Culture, 5%CO in being inoculated in culture dish2Incubator 37
DEG C culture, changed liquid once every 3 days.
5th, the identification of MDSCs
(1) cell climbing sheet makes：Sterile cover slips are positioned in 6 well culture plates, exponential phase cell is prepared into carefully
Born of the same parents' suspension, by 5 × 104Cell/mL density is inoculated in 6 well culture plates, and per hole 2mL, growth medium is containing 20%FBS's
DMEM/F12, cultivates 24~48 hours, makes cell growth on cover glass.
(2) Desmin, Sca-1 immunocytochemical stain：Nutrient solution in the well culture plate of cover glass 6 will be placed with to suction out, PBS
Buffer solution rinsing cell tile 5 minutes, is repeated 3 times.15 minutes are fixed with 4% paraformaldehyde, are dried after PBS rinsing,
Neutral gum is adhered on slide, 4 DEG C of refrigerator overnights.3% hydrogen peroxide is added dropwise to be incubated 15 minutes, endogenous peroxidating is eliminated
The activity of thing enzyme, PBS is rinsed 5 minutes, is repeated 3 times.Primary antibody rabbit-anti people desmin (Desmin) antibody (1 is added dropwise respectively:
200 dilution), rabbit-anti people Sca-1 antibody (1:100 dilutions), it is incubated 1 hour in 37 DEG C of wet box, 4 DEG C are overnight.PBS after next day taking-up
Buffer solution is rinsed 5 minutes, and secondary antibody is added dropwise, and is incubated 40 minutes in 37 DEG C of wet box, and PBS is rinsed 5 minutes, is repeated 3 times, and is added dropwise
The DAB solution of Fresh develops the color 5-10 minutes, in light Microscopic observation, color development stopping is rinsed with running water.Slice, thin piece is got rid of
After dry, haematoxylin is redyed 1 minute, and indigo plant is returned in differentiation, and gradient alcohol dehydration, dimethylbenzene is transparent, neutral gum mounting.Under the microscope
, there is brown yellow granule in after birth and/or endochylema in observation, illustrates to be successfully separated acquisition MDSCs, and its result is as shown in Figure 1.
The differentiation of embodiment 2MDSCs
The 3rd generation MDSCs is taken, by 5 × 105Cell/ pipes are inoculated in 15mL centrifuge tubes, 1000rpm centrifugation 5min, make cell
Agglomerating to be gathered in centrifugation bottom of the tube, lid unscrews half, is put into 37 DEG C, 5%CO2Incubator quiescent culture.
Inhaled after 24h and abandon old liquid, addition 1mL positive control composition chondrocyte induction liquid：Containing 10 μ g/L TGF-βs 3,100 μ g/L pancreases
The plain like growth factor in island, 10-7Mol/L dexamethasone, 100 μm of ol/L ascorbic acid, 1vol%ITS+The DMEM in high glucose of Premix
Nutrient solution.Flicking centrifuge tube floats cell mass, and lid unscrews half, is put into 37 DEG C, 5%CO2Incubator quiescent culture.
Paraffin section, safranin O-fast green dyeing and II Collagen Type VI SABCs are carried out after induction differentiation culture 21d to cell
Deng experiment.
Fig. 2 is the experimental result of safranin O-fast green dyeing, as illustrated, cell section has coloring, pink region is obvious,
Illustrate that cellular matrix has mucopolysaccharide to generate；Fig. 3 is the experimental result of II Collagen Type VI SABCs, display yellowish-brown region, explanation
There are II Collagen Type VIs to synthesize.
Comparative example 1
The 3rd generation MDSCs is taken, by 5 × 105Cell/ pipes are inoculated in 15mL centrifuge tubes, 1000rpm centrifugation 5min, make cell
Agglomerating to be gathered in centrifugation bottom of the tube, lid unscrews half, is put into 37 DEG C, 5%CO2Incubator quiescent culture.
Inhaled after 24h and abandon old liquid, change 1mL negative control group culture mediums：DMEM in high glucose nutrient solution containing 10%FBS.Flick
Centrifuge tube floats cell mass, and lid unscrews half, is put into 37 DEG C, 5%CO2Incubator quiescent culture.
Paraffin section, safranin O-experiment such as fast green dyeing and II Collagen Type VI SABCs are carried out after culture 21d to cell.
Fig. 4 is the experimental result of safranin O-fast green dyeing, as illustrated, cell section non-coloring, illustrate cellular matrix without
Mucopolysaccharide is generated.Fig. 5 is the experimental result of II Collagen Type VI SABCs, shows non-colored section, illustrates to be formed without II Collagen Type VIs.
Claims (8)
1. a kind of cell culture fluid, it is characterised in that including basal medium, Transforming growth factor-β3, insulin-like growth factor
Son, dexamethasone, L-AA and ITS+Premix。
2. cell culture fluid according to claim 1, it is characterised in that the concentration of the Transforming growth factor-β3 is 5~
15μg/L；The concentration of the IGF is 50~100 μ g/L.
3. cell culture fluid according to claim 1, it is characterised in that the concentration of the dexamethasone is 0.5 × 10-7~
1×10-7mol/L；The concentration of the L-AA is 50~100 μm of ol/L.
4. cell culture fluid according to claim 1, it is characterised in that the ITS+The volume fraction of Premix is 1%.
5. cell culture fluid according to claim 1, it is characterised in that the concentration of the Transforming growth factor-β3 is 10 μ
g/L；
The concentration of the IGF is 100 μ g/L；
The concentration of the dexamethasone is 0.1 μM；
The concentration of the L-AA is 100 μM；
The ITS+The volume fraction of Premix is 1%.
6. cell culture fluid according to claim 1, it is characterised in that the basal medium is DMEM in high glucose culture
Liquid.
7. it is a kind of to induce method of the skeletal muscle stem Cells to Chondrocyte Differentiation, it is characterised in that to use claim 1 to 6 times
Cell culture fluid described in meaning one induces skeletal muscle stem Cells to Chondrocyte Differentiation as induction liquid.
8. method according to claim 7, it is characterised in that the skeletal muscle stem Cells are bone of the third generation to the 5th generation
Bone flesh stem cell.
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Cited By (2)
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| CN108251357A (en) * | 2017-12-26 | 2018-07-06 | 重庆斯德姆生物技术有限公司 | A kind of induction cell culture fluid and differentiation method of the skeletal muscle stem Cells to Chondrocyte Differentiation |
| CN115089614A (en) * | 2022-06-28 | 2022-09-23 | 中国人民解放军军事科学院军事医学研究院 | Method for enhancing performance of skeletal stem cells and application of method in treatment of osteoarthritis |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108251357A (en) * | 2017-12-26 | 2018-07-06 | 重庆斯德姆生物技术有限公司 | A kind of induction cell culture fluid and differentiation method of the skeletal muscle stem Cells to Chondrocyte Differentiation |
| CN115089614A (en) * | 2022-06-28 | 2022-09-23 | 中国人民解放军军事科学院军事医学研究院 | Method for enhancing performance of skeletal stem cells and application of method in treatment of osteoarthritis |
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