CN102424813A - Simple extraction method of high-purity mouse skeletal muscle satellite cells - Google Patents
Simple extraction method of high-purity mouse skeletal muscle satellite cells Download PDFInfo
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Abstract
The invention provides a simple extraction method of high-purity mouse skeletal muscle satellite cells, which is characterized by comprising the concrete steps that: step 1, bupivacaine hydrochloride is injected at the limb muscle of a mouse; step 2, the mouse is killed, the limb muscle is winkled, vessels, fat and fascias are removed, the muscle is sheared into small granules being 0.5 to 1.5mm<3>; step 3, mixed digestive enzyme is added for incubation, the cell suspension liquid is filtered, the obtained filter liquid is centrifuged, the supernate is abandoned, and a growth culture medium for cell precipitation is suspended again; step 4, 60 percent percoll separation liquid, 20 percent percoll separation liquid and cell suspension liquid are respectively injected along the tube wall, a long-head suction tube is used for sucking the cell suspension liquid at the interface of the 20 percent percoll separation liquid and the 60 percent percoll separation liquid, and cell precipitates are obtained through centrifugation; and step 5, the cell precipitates are cultured, and the mouse skeletal muscle satellite cells are obtained. The method is simple, easy and fast, the purification rate and the yield of the skeletal muscle satellite cells are high, higher multiplication and differentiation capability is realized, and the external gene modification or the internal transplantation can be carried out.
Description
Technical field
The invention provides a kind of process for extracting of simple, highly purified mice skeletal satellite cell, regenerating for gene transfection, Skelettmuskel and the cardiac muscle of the mediation of Skelettmuskel satellite cell provides seed cell.
Background technology
Along with the development of microsurgical technique and the people understanding to the mouse model meliority, as breed that fast (only needing 21 days), litter size are many, keeping consumption, to reach gene, signalling channel and human similarity less high, mouse model is widely used in present scientific research.The mice skeletal satellite cell is the muscle-derived stem cell that has differentiation and proliferation potential in the Skelettmuskel.A kind of as adult stem cell, high purity Skelettmuskel satellite cell has been showed good potential applicability in clinical practice as adult seed cell and gene therapy engineering cell in the treatment of ischemic heart disease.Yet; Muscle satellite cell content in the Skelettmuskel of growing up is lower; About 1%-5%; And its quantity reduces owing to the influence of various factors in the vital process of individuality gradually, and is in the G0 phase of cell cycle in the ordinary course of things, and this has just determined that the Skelettmuskel satellite cell quantity that can obtain through the primary cell stripping technique must be limited.Therefore, its separation and culture technique are always in constantly groping and improving.Traditional method adopts the mode that collagenase and pancreatin digest step by step and differential is adherent to extract purifying more; Perhaps separate satellite cell through silica gel particle (Percoll) gradient centrifugation that is surrounded by V-Pyrol RC; In these methods, because to digestion and difficult assurance of adherent time of differential, and do not form unified standard; Thereby the satellite cell that obtains still contains a large amount of impurity cells such as inoblast, and purity is lower; And fluidic cell sorting art, immunological magnetic bead sorting art and plane are sticked methods such as partition method and can be obtained the high purity satellite cell; But part unit lacks the corresponding apparatus condition; And testing reagent such as required antibody costs an arm and a leg; The antibody that uses also has corresponding species specificity, makes the widespread use of this method limited.
Summary of the invention
The purpose of this invention is to provide a kind of method that obtains highly purified mice skeletal satellite cell fast, efficiently, economically.
In order to achieve the above object, the invention provides a kind of simple and easy process for extracting of high purity mice skeletal satellite cell, it is characterized in that concrete steps are:
The first step: get 5～7 the week age C57BL/6 mouse, penetrate 0.75%, 0.05～0.15mL in the intramuscular injection of mouse four limbs;
Second step: after 36～60 hours, put to death mouse, with the alcohol-pickled 10～20min of 75vol%, in Bechtop, pick out limb muscle 0.5～1.5g, remove blood vessel, fat and manadesma, aseptic PBS liquid flushing is cut into 0.5～1.5mm with muscle
3Granule adds PBS liquid and leaves standstill 3～7min, and is centrifugal, abandons supernatant;
The 3rd step: add 1～5mL mixture slaking enzyme, place 37 ℃, CO
2Volumetric concentration is to hatch in 5% the incubator; Whenever shake 3～7min at a distance from 10～20min with suction pipe piping and druming, repeat 3 times, add the F12/DMEM substratum termination digestion that 3～7mL contains the 15vol% foetal calf serum; The filtration cell suspension; It is centrifugal that gained is filtrated, and abandons supernatant, and cell precipitation is resuspended for use to 2～4mL with growth medium;
The 4th step: behind the wetting centrifuge tube tube wall of foetal calf serum; Successively 60%Percoll parting liquid 2～4mL, 20%Percoll parting liquid 2～4mL, cell suspension 2～4mL are injected along tube wall; Centrifugal; With careful sucking-off 20% Percoll parting liquid of long head straw and the interfacial cell suspension 0.5～1.5mL of 60% Percoll parting liquid, centrifugal, get cell precipitation;
The 5th step: in the culturing bottle that cell precipitation encapsulates with the resuspended back immigration mouse of growth medium tail glue, in CO
2Cultivate 0.5h in the incubator; Called after PP1, after substratum together transferred to together with non-adherent cell cultivate 0.5h, called after PP2 in the new culturing bottle again; Afterwards substratum is changed in the new culturing bottle together with not adherent cell and cultivate 24h; Called after PP3 changes liquid with full dose behind the PP3 continuation cultivation 72h, obtains the mice skeletal satellite cell.
Preferably, described mixture slaking enzyme compound method is: with II Collagen Type VI enzyme 0.11g, and II type dispease enzyme 0.124g, CaCl
214mg and PBS50mL mix.
Principle of the present invention is:
Muscle satellite cell in the Skelettmuskel is present between myofibrillar sarolemma layer and the basilar membrane; Satellite cell content is few in the adult Skelettmuskel; When skeletal muscle tissue sustained damage, the muscle satellite cell of these quiescent stages just can be activated, a large amount of divisions, propagation, differentiation.Therefore, after with pre-treatment Skelettmuskel, can cause the muscular tissue damage,, thereby activate satellite cell and make its a large amount of propagation with a large amount of inflammatory cell infiltrations.Concrete mechanism maybe for, a large amount of monokaryons that muscular tissue damage back produces and scavenger cell can be through discharging activation and proliferation and the differentiation that some cytokine with MA such as IL-6 etc. and growth factor promote muscle satellite cell.In addition; There is the scholar to find; Contact activation, propagation and the differentiation that can suppress muscle satellite cell between being in contact with one another between the myofiber of muscle satellite cell and survival; Bupivacaine can the selective injury myofiber and muscle satellite cell is not had damaging action, and the downright bad weakening that can cause this inhibition relation of myofiber dissolving causes the activation and proliferation of muscle satellite cell.With after the bupivacaine pre-treatment, the cell concentration of being gathered in the crops obviously increases in this experiment, for keeping the satellite cell of capacity behind the purifying guarantee is provided.
Traditional method adopts the extraction that collagenase and pancreatin digest step by step, repeatedly the centrifugal mode is carried out satellite cell more.The present invention optimizes on the basis of traditional separation method and improves, and through the mixed enzyme Digestive system that adopts autogamy skeletal muscle tissue is carried out disposable digestion, only needs a low-speed centrifugal just can obtain cell.This kind modification method has been avoided repeatedly digesting the centrifugal troublesome operation in the traditional method, has saved the time of cell extraction, thereby has alleviated the damage of digestive ferment pair cell, has reduced losing of cell, has increased the results of cell.
Separate in the amplification cultivation process at satellite cell; Inoblast in the skeletal muscle tissue can influence the purity and the growth conditions of satellite cell; Exist competitive growth characteristics of a kind of these those length that disappear between the two; Therefore, how removing the fibroblastic pollution that is mingled with wherein is the constantly major objective of improvement of purifying mode.Methods such as partition method are sticked on fluidic cell sorting art, immunological magnetic bead sorting art and plane can obtain the high purity satellite cell; But part unit lacks the corresponding apparatus condition; And testing reagent such as required antibody costs an arm and a leg; The antibody that uses also has corresponding species specificity, makes the widespread use of this method limited.Therefore, how fast the emphasis of research concentrates on, obtains efficiently, economically highly purified satellite cell at present.
Most of inoblast is adherent in 10-30min, and beginning is adherent about satellite cell 30min, but adheres to not firmly, rocks promptly gently and floats.Common differential is adherent can't to make a part of inoblast adherent, and satellite cell purity is lower; And repeatedly repeatedly the best differential time of the adherent purifying of differential still be not sure of, therefore cause the purity of extracting satellite cell to guarantee; Percoll is a kind of silica gel particle that is surrounded by V-Pyrol RC; Do not penetrate microbial film; The pair cell nontoxicity, its diffusion constant is low, and two discontinuous density gradients of 60% and 20% of laying are more stable in advance; In several minutes to tens of minutes, can reach satisfied cellular segregation effect under the low-speed centrifugal power, therefore be widely used in the experiment of isolated cell, subcellular component, bacterium and virus.Yet,, can not avoid sneaking into of non-muscle-derived cell fully though the Percoll partition method comes the satellite cell purity of isolated cell results to be higher than simple differential adherent method.Therefore; For further reducing non-muscle-derived cell contamination, the present invention has optimized the adherent time of a series of differentials through comparative study repeatedly; And the Percoll partition method combined with it; The PP3 purity of its acquisition is apparently higher than other purification process, and the purification time span is merely two days, avoided repeatedly permanent consuming time of differential adherent method.
Behind the passage, its miscellaneous inoblast will form growth vigor because of rate of propagation faster, thereby cause satellite cell purity to descend rapidly.So when going down to posterity, must remove inoblast as far as possible, at every turn to keep the certain purity of the satellite cell that goes down to posterity through preparatory adherent 30min.
The inventive method is simple and easy, quick, and Skelettmuskel satellite cell purifying rate and productive rate are high, through present method extract, the mice skeletal satellite cell vitro culture of purifying has higher propagation and differentiation capability, can carry out that outer-gene is modified or body in transplant.Present method is through pre-treatment muscle tissue; Single stage method mixed enzyme digestion combines not isopycnic gradient centrifugation of Percoll, and through the optimization adherent time of differential; Extracted highly purified mice skeletal satellite cell, for further gene transfection and Transplanted cells provide the foundation.
Description of drawings
Fig. 1 is Percoll synoptic diagram after the isopycnic gradient centrifugation not: the Skelettmuskel satellite cell is 20% and 60%Percoll liquid intersection.
Fig. 2 is the back Skelettmuskel satellite cell desmin immunofluorescence dyeing figure that purifies: desmin expresses strong positive.
Fig. 3 is l cell desmin immunostaining (negative control) figure: no desmin expresses.
Fig. 4 identifies figure for flow cytometer satellite cell purity: red curve is the positive purity (92.59 ± 1.29%) of Skelettmuskel satellite cell desmin, the l cell of the negative contrast of green curve.
Fig. 5 is back the 4th sky maps of purifying: Skelettmuskel satellite cell logarithmic phase, cell are fusiformis, the form homogeneous.
Fig. 6 forms figure for myotube: after inducing differentiation with horse serum, a plurality of adjacent satellite cells can merge can form myotube.
Embodiment
Specify the present invention below in conjunction with embodiment.It is 7.4 phosphate buffer soln that PBS described in the present invention, PBS liquid are all the pH value.Concentration all among the present invention are volumetric concentration.
Embodiment
1. laboratory animal: 6 age in week the C57BL/6 mouse, provide by Shanghai Slac Experimental Animal Co., Ltd..
2. the position pre-treatment of drawing materials: penetrate 0.75% 0.1mL in the intramuscular injection of mouse four limbs with 100 μ L microsyringes.
3. draw materials: after 48 hours, the cervical vertebra dislocation method is put to death mouse, with whole mouse with 75% alcohol-pickled 15min; In Bechtop, pick out the about 1g of limb muscle; Careful blood vessel, fat and the manadesma that adheres on the muscle of removing, aseptic PBS liquid flushing 3 times is cut into about 1mm with muscle
3The meat gruel granule adds aseptic PBS liquid and soaks, and leaves standstill 5min, and is centrifugal, abandons supernatant.
4. single stage method mixture slaking enzymic digestion: (compound method is: with II Collagen Type VI enzyme (Sigma Aldrich to add 3mL mixture slaking enzyme; Catalog No.:C6885) 0.11g; II type dispease enzyme (Roche, Catalog No.:4942078001) 0.124g, CaCl
214mg, PBS50mL mixes), place 37 ℃, 5%CO
2Hatch in the incubator, every piping and druming with suction pipe at a distance from 15min shaken 5min repetition 3 times, after one hour; Adding 5mL contains the F12/DMEM substratum of 15% foetal calf serum and ends digestion; 200 order filtering net filtration cell suspensions 2 times, the centrifugal 10min of 1400r/min abandons supernatant; Cell precipitation is resuspended for use to 3mL with DMEM/F12 growth medium (Gibco, Catalog No.:11330032).
5. Percoll discontinuous density gradient centrifugation: with 2mL foetal calf serum (Gibco; Catalog No.:10099141) behind the wetting 15mL centrifuge tube tube wall; Successively with 60%Percoll parting liquid (GE Healthcare; Catalog No.:17-0891-01) the cell suspension 3mL in 3mL, 20%Percoll parting liquid 3mL, the step 4 slowly injects the centrifugal 20min of 1800r/min along tube wall.Careful sucking-off 20% Percoll parting liquid of long head straw and the about 1mL of the interfacial cell suspension of 60% Percoll parting liquid, the centrifugal 5min of 1000r/min gets cell precipitation.
6. optimize the adherent time of differential: cell precipitation moves into to the 5mL in the culturing bottle that mouse tail glue encapsulates, in CO with the DMEM/F12 growth medium that contains 15% foetal calf serum is resuspended
2Cultivate 0.5h (for the first time adherent) in 37 ℃ of the incubators; Called after PP1; After substratum together transferred in the new culturing bottle together with non-adherent cell again cultivate 0.5h (for the second time adherent) at 37 ℃; Called after PP2 changes substratum in the new culturing bottle 37 ℃ of cultivation 24h (adherent for the third time) together with not adherent cell afterwards, obtains PP3.Full dose was changed liquid after PP3 continued to cultivate 72h.
7. the Skelettmuskel satellite cell is identified: get the PP3 cell, 4g/L trypan blue dyeing counting viable cell.Fix with 4% Paraformaldehyde 96, identify myoblastic purity by the routine immunization fluorescent staining method.One anti-main muscle satellite cell specificity marker thing desmin (the desmin) (abcam that adopts; Catalog No.:ab15200), Cy3 mark two anti-(Beyotime, Catalog No.:A0516); And employing hoechst33258 (Beyotime; Catalog No.:C1017) redye nucleus, observe the immunofluorescence result, flow cytometer detects satellite cell purity (doing negative control with the l cell system of expression of desmin not).
8. Skelettmuskel satellite cell multiplication by culture, differentiation: cell after the purification of step 6 gained is cultivated with the F12/DMEM substratum that contains 15% foetal calf serum; Every day observation of cell form and growing state; Treat to go down to posterity after cell grows to 80% contact; The all preparatory adherent 30min that at every turn goes down to posterity, preparatory adherent step is adherent identical with the above-mentioned first time, changes in the new culturing bottle substratum over to 37 ℃ together with not adherent cell afterwards and cultivates and accomplish cell attachments; After cell cultures is adherent; Substratum is replaced by differentiation liquid (compound method is: 100:2 adds horse serum by volume in the DMEM/F12 substratum, and 100:1 adds mycillin by volume again) induces differentiation culture, observe the state that is differentiated to form myotube.
9. result: improvement present method is extracted, Percoll does not see Fig. 1 after the isopycnic gradient centrifugation in the purifying mode; The final cell that obtains is through the trypan blue dyeing counting behind the purifying; There is the cell more than 95% not painted approximately, points out the cell survival rate of this method gained higher.Behind immunofluorescence dyeing, desmin is strong positive and expresses (Fig. 2,3), and flow cytometer identifies that its purity is: 92.59 ± 1.29% (Fig. 4).
Adherent speed was obviously accelerated after former generation, the Skelettmuskel satellite cell went down to posterity, and 0.5h begins adherent, and the satellite cell in the 2h more than 90% is accomplished adherent.Be 1-2d latent period during Skelettmuskel satellite cell vitro culture, and from 3d entering logarithmic phase, 5-6d begins to get into the plateau of cell proliferation.Get into the logarithmic growth after date, along with the propagation of cell, form the cell colony that is dispersed in distribution (
Fig. 5); Cell usually elongates and attenuates, and trends towards simultaneously being arranged in parallel.After cell converges above 60%-70%, fusion phenomenon appears between the Skelettmuskel satellite cell, and there is the myotube cell that is dispersed in to form.To the visible large stretch of myotube cell formation of 7-8d, also can merge each other between the myotube cell.When culture condition is good, can be observed the spontaneous contraction of myotube cell under the inverted phase contrast microscope.
Be replaced by division culture medium behind the cell attachment, find that division, the rate of propagation of cell significantly descends, cell maintains lower level of density.Begin to have a small amount of bone muscle satellite cell to merge each other behind the 24h, the myotube cell that is dispersed in begin to form (
Fig. 6), visible most Skelettmuskel satellite cell merges behind the 72h, and petridish is interior to be main with the myotube cell.
Claims (2)
1. the simple and easy process for extracting of a high purity mice skeletal satellite cell is characterized in that, concrete steps are:
The first step: get 5～7 the week age C57BL/6 mouse, penetrate 0.75%, 0.05～0.15mL in the intramuscular injection of mouse four limbs;
Second step: after 36～60 hours, put to death mouse, with the alcohol-pickled 10～20min of 75vol%, in Bechtop, pick out limb muscle 0.5～1.5g, remove blood vessel, fat and manadesma, aseptic PBS liquid flushing is cut into 0.5～1.5mm with muscle
3Granule adds PBS liquid and leaves standstill 3～7min, and is centrifugal, abandons supernatant;
The 3rd step: add 1～5mL mixture slaking enzyme, place 37 ℃, CO
2Volumetric concentration is to hatch in 5% the incubator; Whenever shake 3～7min at a distance from 10～20min with suction pipe piping and druming, repeat 3 times, add the F12/DMEM substratum termination digestion that 3～7mL contains the 15vol% foetal calf serum; The filtration cell suspension; It is centrifugal that gained is filtrated, and abandons supernatant, and cell precipitation is resuspended for use to 2～4mL with growth medium;
The 4th step: behind the wetting centrifuge tube tube wall of foetal calf serum; Successively 60%Percoll parting liquid 2～4mL, 20%Percoll parting liquid 2～4mL, cell suspension 2～4mL are injected along tube wall; Centrifugal; With careful sucking-off 20% Percoll parting liquid of long head straw and the interfacial cell suspension 0.5～1.5mL of 60% Percoll parting liquid, centrifugal, get cell precipitation;
The 5th step: in the culturing bottle that cell precipitation encapsulates with the resuspended back immigration mouse of growth medium tail glue, in CO
2Cultivate 0.5h in the incubator; Called after PP1, after substratum together transferred to together with non-adherent cell cultivate 0.5h, called after PP2 in the new culturing bottle again; Afterwards substratum is changed in the new culturing bottle together with not adherent cell and cultivate 24h; Called after PP3 changes liquid with full dose behind the PP3 continuation cultivation 72h, obtains the mice skeletal satellite cell.
2. the simple and easy process for extracting of high purity mice skeletal satellite cell as claimed in claim 1 is characterized in that, described mixture slaking enzyme compound method is: with II Collagen Type VI enzyme 0.11g, and II type dispease enzyme 0.124g, CaCl
214mg and PBS50mL mix.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105505861A (en) * | 2015-12-29 | 2016-04-20 | 邬江 | Separation culture technology for animal muscle satellite cells |
| CN108048396A (en) * | 2017-12-29 | 2018-05-18 | 山西农业大学 | A kind of separation method of Sheep Muscle stem cell |
| CN110760475A (en) * | 2019-12-04 | 2020-02-07 | 扬州大学 | Isolated culture method of chicken muscle satellite cells |
| CN111117953A (en) * | 2020-01-08 | 2020-05-08 | 上海市口腔病防治院 | In-vitro culture method of aged mouse genioglossus muscle-derived myoblasts |
| CN111735673A (en) * | 2019-03-25 | 2020-10-02 | 广州医科大学附属第一医院 | Liquid-based thin-layer film preparation for pathogenic bacteria detection and application thereof |
| CN112011502A (en) * | 2020-09-09 | 2020-12-01 | 扬州大学 | Method for efficiently separating and purifying porcine skeletal muscle satellite cells |
| CN113355281A (en) * | 2021-06-19 | 2021-09-07 | 浙江大学 | Method for efficiently separating mouse intramuscular fiber-adipogenic progenitor cells |
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2011
- 2011-12-25 CN CN2011104386411A patent/CN102424813A/en active Pending
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105505861A (en) * | 2015-12-29 | 2016-04-20 | 邬江 | Separation culture technology for animal muscle satellite cells |
| CN108048396A (en) * | 2017-12-29 | 2018-05-18 | 山西农业大学 | A kind of separation method of Sheep Muscle stem cell |
| CN111735673A (en) * | 2019-03-25 | 2020-10-02 | 广州医科大学附属第一医院 | Liquid-based thin-layer film preparation for pathogenic bacteria detection and application thereof |
| CN110760475A (en) * | 2019-12-04 | 2020-02-07 | 扬州大学 | Isolated culture method of chicken muscle satellite cells |
| CN111117953A (en) * | 2020-01-08 | 2020-05-08 | 上海市口腔病防治院 | In-vitro culture method of aged mouse genioglossus muscle-derived myoblasts |
| CN111117953B (en) * | 2020-01-08 | 2023-09-19 | 上海市口腔病防治院 | In-vitro culture method of myoblasts derived from genioglossus muscles of aged mice |
| CN112011502A (en) * | 2020-09-09 | 2020-12-01 | 扬州大学 | Method for efficiently separating and purifying porcine skeletal muscle satellite cells |
| CN113355281A (en) * | 2021-06-19 | 2021-09-07 | 浙江大学 | Method for efficiently separating mouse intramuscular fiber-adipogenic progenitor cells |
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Application publication date: 20120425 |