CN105112359A - Separating and cultivating method of mouse umbilical cord mesenchymal stem cells - Google Patents
Separating and cultivating method of mouse umbilical cord mesenchymal stem cells Download PDFInfo
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Abstract
The invention relates to a separating and cultivating method of mouse umbilical cord mesenchymal stem cells, and belongs to the field of cell culture in vitro. The method provided by the invention is an improvement and innovation of a tissue adherent culture method, a beaded pregnant uterus of a pregnant mouse whose gestation age is 15-19 days is taken out and placed in a PBS containing antibiotics consisting of both penicillin and streptomycin for immersion; a umbilical cord tissue is separated and placed into the PBS for cleaning and immersing, the tissue is placed in a medium, and is cut and cultured; when P0 generation cell grows and 80-90% of the cells fuse, 0.25% of pancreatin is used for digestion and subculture, a continuous subculture is carried out, until the cells are in an uniform state; the medium used in the invention is F12/DMEM, and a fetal calf serum is added; a lot of mouse umbilical cord mesenchymal stem cells of high activity and high purity can be obtained according to the separating and culture method provided by the invention, and activity of stem cells can be maintained in a long-term subculture; the invention solves the problem of transplant method limitation in the mouse experiment animal model construction, and also provides basis for researches of cell molecular mechanisms of related diseases.
Description
Technical field
The present invention relates to a kind of isolation cultivation method of mouse umbilical cord mesenchymal stem cells, belong to the vitro culture field of cell.
Background technology
Mescenchymal stem cell (mesenchymalstemcells, MSC) is the multipotent adult stem cell that a class originates from embryo, and success is separated and obtains from Various Tissues at present, common as marrow, umbilical cord, placenta, fat etc.The outstanding advantage of mescenchymal stem cell is: the immunoregulation function of the self-renewal capacity of height, powerful multi-lineage potential and broadness, thus becomes the first-selected cell source in " stem cell therapy ".
Mescenchymal stem cell is seek that it is desirable tissue-derived in the key of clinical application.Mescenchymal stem cell finds in marrow, but the mescenchymal stem cell of derived from bone marrow exists the restriction of donor age, tissue sampling and immunological rejection.Umbilical cord belongs to embryo to be organized outward, include more original mescenchymal stem cell, immunogenicity is lower, not only there is the gene expression profile (HsiehJY very similar to embryonic stem cell, FuYS, ChangSJ, etal.Functionalmoduleanalysisrevealsdifferentialosteogen icandstemnesspotentialsinhumanmesenchymalstemcellsfrombo nemarrowandWharton'sjellyofumbilicalcord.Stemcellsanddev elopment.2010, 19:1895-910.), and the mescenchymal stem cell tissue-derived compared with other possesses higher proliferation potential, stronger amplification ability and shorter population doubling time (LiWW, WeiYH, LiH, etal.Isolationandcharacterizationofanovelstrainofmesench ymalstemcellsfrommouseumbilicalcord:potentialapplication incell-basedtherapy.PloSone.2013, 8:e74478.).Therefore, the mescenchymal stem cell in umbilical cord source compensate for the shortcoming of derived from bone marrow well, more not by the constraint of ethics, can be used as desirable cell source.
In preclinical research, the mescenchymal stem cell in people's umbilical cord source has been applied in some rodent model, as apoplexy (ZhuJ, LiuQ, JiangY, etal.Enhancedangiogenesispromotedbyhumanumbilicalmesench ymalstemcelltransplantationinstrokedmouseisNotch1signali ngassociated.Neuroscience.2015, 290C:288-99.), liver injury (BurraP, ArcidiaconoD, BizzaroD, etal.Systemicadministrationofanovelhumanumbilicalcordmes enchymalstemcellspopulationacceleratestheresolutionofacu teliverinjury.BMCgastroenterology.2012, 12:88.), injury of the kidney (LiW, ZhangQ, WangM, etal.Macrophagesareinvolvedintheprotectiveroleofhumanumb ilicalcord-derivedstromalcellsinrenalischemia-reperfusio ninjury.Stemcellresearch.2013, 10:405-16.) etc.But, research is had to point out, the mescenchymal stem cell in people and mouse source is except general character, really there is the difference (BernardoME in biology and function assessment characteristic, FibbeWE.Mesenchymalstromalcells:sensorsandswitchersofinf lammation.Cellstemcell.2013,13:392-402.), if human umbilical cord mesenchymal stem cells xenotransplantation is entered in rodent body, likely can change host immune response, bring the complicacy of result.It is be clinical study service that animal model builds, and pattern layout ought to simulate clinical practice as much as possible.So, the design that in mouse model, homogeneous variant cell is transplanted will be more reasonable.
At present, the mescenchymal stem cell of mouse is mainly derived from mouse bone marrow cells (ShiratsukiS, TeraiS, MurataY, etal.Enhancedsurvivalofmiceinfusedwithbonemarrow-derived ascomparedtoadipose-derivedmesenchymalstemcells.Hepatolo gyresearch:theofficialjournaloftheJapanSocietyofHepatolo gy.2015.), secondly be mouse adipose (LiQ, ZhouXM, ShiYQ, etal.InVivoTrackingandComparisonoftheTherapeuticEffectso fMSCsandHSCsforLiverInjury.PloSone.2013, 8.), and 1 example is only reported in the mouse umbilical cord whole world.The separation method of this research is classical organize adherent method, and program is many, more additionally with the addition of many commercialization factors, as dexamethasone, Urogastron, PDGF, leukaemia inhibitory factor etc. in cell culture medium.Therefore, cultivate in view of mouse umbilical cord mesenchymal stem cells and may become the Important Platform of illustrating numerous disease molecular mechanism, a kind of newly, simple and the foundation of cost-saving isolation cultivation method is very necessary and significant.
Summary of the invention
The object of the invention is to overcome the defect existed in prior art, the isolation cultivation method that a kind of mouse umbilical cord mesenchymal stem cells is new is provided.
Cultivate an isolation cultivation method for mouse umbilical cord mesenchymal stem cells, comprise the following steps:
(1) the pregnant mouse the being reached 15-19 days pregnant age neck that breaks is put to death, and put into ethanol for disinfection and soak 5 minutes, take out in Bechtop, belly is placed in the large ware of aseptic 10cm upward;
(2) cut with Sterile ophthalmic and with tweezers, pregnant mouse abdominal skin and mucous layer are successively cut off, take out beading sample and to become pregnant uterus, put into and soak 5-10 minute containing 500U-1000U high strength penicillin and the dual anti-PBS of Streptomycin sulphate;
(3) with Sterile ophthalmic tweezers, every tire mouse is stripped out, pick clean rete layer, coordinate eye scissors to isolate the umbilical cord tissue connecting tire mouse navel and placenta again, put into and wash containing 500U-1000U high strength penicillin and the dual anti-PBS of Streptomycin sulphate and soak 5-10 minute;
(4) umbilical cord tissue is dipped in containing in dual anti-substratum, with eye scissors and tweezers, it is shredded by root;
(5) by umbilical cord tissue block (together with substratum) uniform spreading in Tissue Culture Dish (3.5cm capsule), be placed in 37 DEG C of cell culture incubators and cultivate;
(6) namely umbilical cord tissue block has cell to climb out of after cultivating 24h to 48h, within the 3rd day, changes normal incubation medium, within later every 3 days, changes a not good liquor, when P0 reaches 80-90% for Growth of Cells fusion, with the trysinization Secondary Culture of 0.25%, continue to go down to posterity until cell presents homogeneous form, do next step qualification.
Wherein, in step (1), the pregnant age of pregnant mouse was advisable with 15-19 days, and pregnant age is too little, and tire mouse is not yet ripe, and its umbilical cord is not only soft but also tender, and it is more difficult to be separated;
Should be put into containing high strength antibiotic P BS immersion and washing by the uterus of separating in Mice Body and umbilical cord tissue in step (2) and (3): penicillin and the applicable concentration of Streptomycin sulphate are 500U-1000U, and the time is 5-10 minute;
In step (4), the composition of " containing dual anti-substratum " and content thereof are: F12/DMEM86%-84%(V/V), FBS14-16%(V/V), 90-110U penicillin and 90-110U Streptomycin sulphate, this concentration dual anti-not only effectively antibacterial, and to cell without any side effect;
Umbilical cord tissue shreds to 0.5-1.0mm in (4) by step
3, tissue block is less, and the efficiency that after adherent, its cell climbs out of is higher;
In step (5) by fine tissue block suspension uniform spreading in capsule, substratum is advisable on a small quantity.Cultivation base unit weight is many, and fine tissue block suspends not easily adherent, greatly reduces primary cell yield;
In step (5), primary capsule is steadily put into 37 DEG C, 5%CO lightly
2saturated humidity incubator, keeps flat, and push-and-pull of should not exerting oneself, affects tissue block adherent;
To climb out of the efficiency of cell higher for umbilical cord tissue block in step (6), soon then 24h, and then namely 48h has cell to climb out of slowly, and cultivate the normal incubation medium that changes for the 3rd day containing dual anti-, composition is F12/DMEM86-84%(V/V), FBS14-16%(V/V);
The P0 climbed out of in step (6) presents unhomogeneity for cellular form, digested when growing fusion and reaching 80-90%, the cell digested should pass P2 generation with the high-density ratio of 4-2:1, after this can go down to posterity in 1:1-2 ratio, until cell present homogeneous after can ratio Secondary Culture routinely.
When in step (6), P0 is for cell dissociation, because umbilical cord tissue block is too small, before digestion, mechanicalness rejects the loss probably bringing a large amount of cellular layer, therefore tissue block should be rejected as much as possible when resuspended, takes " weight wall built-up method ".
Beneficial effect of the present invention:
The present invention establishes the method that a set of mouse umbilical cord mesenchymal stem cells is separated, cultivates and identify.The method is " the tissue adherent method " of improvement innovation version, and its advantage has following 6 aspects:
(1) simple, workable;
(2) compared with prior art, indispensable factor component is normally cultivated without the need to additionally adding any commercial, acellular in the cell culture medium that this law adopts, save cost significantly, and had original creation point in tissue " inoculation ", " rejecting " and " primary digestion ";
(3) amplification efficiency of cell is high, and after organizing adherent 24-48h, visible cell climbs out of, general growth feasible primary digestion in 8-12 days.Different because of individual mice, cell reach continuously P5-P7 for time morphologic appearance homogeneous, by the aspects such as immunophenotype, growth characteristic, Multidirectional Differentiation confirm this law separation and Culture obtain cell be mescenchymal stem cell;
(4) multiplication capacity of cell is strong, energy Long Term Passages (at least 30 generation), and cell still has Stem Cell Activity after reaching for 20 generations, maintains propagation and differentiation capability, therefore in the cell P8-P20 generation after recommendation purifying, in visible experiment, operational cell quantity is very huge;
(5) compared with the mescenchymal stem cell technology of the bone marrow derived of current widespread use, the advantage of the mescenchymal stem cell in mouse umbilical cord source is: separation method is easy, and cost is low; Heteroproteose cell kind is little, and purifying is comparatively simple, and purification cycle is short; Generation time is long, and cell concentration is large;
(6) for Transplanted cells in mouse model provides desirable cell derived.
In sum, the mouse method for isolation and culture of umbilical mesenchymal stem cells that the present invention sets up has application prospect and value significantly.
Accompanying drawing explanation
Fig. 1 is the method schematic diagram of separation and Culture mouse umbilical cord mesenchymal stem cells of the present invention;
Fig. 2 is the cellular form (40 times) that inverted phase contrast microscope observes mouse umbilical cord mesenchymal stem cells; Wherein, A is schemed for climb out of primary cell P0 for growth figure from tissue block; Figure B be cell cultures to P6 for growth figure;
Fig. 3 is the growth curve of the mouse umbilical cord mesenchymal stem cells of separation and Culture of the present invention;
Fig. 4 is the detection of flow cytometer to the mouse umbilical cord mesenchymal stem cells cell cycle of separation and Culture of the present invention;
Fig. 5 is the analysis (CD29, CD44, CD34 and CD45) of flow cytometer to the mouse umbilical cord mesenchymal stem cells immunophenotype of separation and Culture of the present invention;
Fig. 6 is the Osteoblast Differentiation ability (200 times) that osteogenic induction detects the mouse umbilical cord mesenchymal stem cells of separation and Culture of the present invention;
Fig. 7 is the one-tenth fat differentiation capability (200 times) that adipogenic induction detects the mouse umbilical cord mesenchymal stem cells of separation and Culture of the present invention.
concrete embodiment
The main raw that following instance uses is as follows respectively with source:
Kunming white adult mice (female/male) (Jiangsu University's Experimental Animal Center, this experiment is ratified by Ethics Committee of Jiangsu University);
0.25% trypsin Amresco company); Autogamy phosphate buffered saline buffer PBS; F12/DMEM substratum (Hyclone company); Foetal calf serum FBS(Gibco company);
Tissue Culture Dish (3.5cm capsule), cell cultures 24 orifice plate (Corning company); Tissue Culture Flask (Nunc company);
The streaming antibody (BD company) of CD29, CD34, CD44, CD45, IgG-FITC and IgG-PE contrast;
Interstital stem cell adipogenic induction liquid (preparing voluntarily); Interstital stem cell osteogenic induction liquid (preparing voluntarily);
Alkaline phosphatase (ALP) staining kit (Shanghai sun biotech firm); Oil red O(Sigma company);
Below in conjunction with concrete embodiment and accompanying drawing, the present invention will be further described.Unreceipted actual conditions person in example, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Material therefor or the unreceipted production firm person of instrument, be commercial obtainable conventional item.
embodiment 1:
Combination technology route map 1, is described below the concrete implementation step of the present invention:
(1) healthy adult kunming mice (female/male) is bought in Jiangsu University's Experimental Animal Center, when mouse rutting sedson, 2 female mouse and 1 male mouse are spent the night in same mouse case, check female mouse vaginal tract early morning on next day, naked eyes are shown in that seminal fluid bolt person represents and become pregnant, and start to calculate pregnant age (the 0th day).If check negative, then continue to mate until check the positive;
(2) be that the pregnant mouse of the 17 days neck that breaks is put to death by pregnant age, put into ethanol for disinfection and soak 5 minutes, take out in Bechtop, belly is placed in the large ware of aseptic 10cm upward;
(3) cut with Sterile ophthalmic and with tweezers, pregnant mouse abdominal skin and mucous layer are successively cut off, fully expose uterus of becoming pregnant, with eye scissors and tweezers, beading appearance palace is taken out, put into and soak 8 minutes containing 800U penicillin and the dual anti-PBS of Streptomycin sulphate;
(4) Sterile ophthalmic tweezer is used by each tire mouse (containing placenta, number 8-20 is not etc.) separate, remove the rete layer on surface, eye scissors is coordinated to isolate the umbilical cord tissue connecting tire mouse navel and placenta from two ends again, put into and remove remaining blood containing 800U penicillin and the dual anti-PBS washing of Streptomycin sulphate, soak about 8 minutes;
(5) umbilical cord tissue is immersed rapidly containing the dual anti-85%F12/DMEM of 100U, in 15%FBS substratum, with eye scissors and tweezers, it is shredded to 0.5-1.0mm by root
3fine tissue block;
(6) by umbilical cord tissue block (together with a small amount of substratum) uniform spreading in Tissue Culture Dish (3.5cm capsule), steadily put into 37 DEG C of constant temperature, 5%CO lightly
2cultivate in saturated humidity cell culture incubator;
(7) namely umbilical cord tissue block has cell to climb out of after cultivating 24h to 48h, within the 3rd day, changes normal incubation medium, within later every 3 days, changes a not good liquor.P0 presents unhomogeneity for cell, when Growth of Cells fusion reaches 85%, with the trysinization of 0.25%, passes P2 generation, after this in the ratio continuous passage of 1:1, until cellular form homogeneous (to P5-P7 generation), can do next step qualification with the inoculation of the ratio of 3:1.
embodiment 2:
Combination technology route map 1, is described below the concrete implementation step of the present invention:
(1) healthy adult kunming mice (female/male) is bought in Jiangsu University's Experimental Animal Center, when mouse rutting sedson, 2 female mouse and 1 male mouse are spent the night in same mouse case, check female mouse vaginal tract early morning on next day, naked eyes are shown in that seminal fluid bolt person represents and become pregnant, and start to calculate pregnant age (the 0th day).If check negative, then continue to mate until check the positive;
(2) be that the pregnant mouse of the 15 days neck that breaks is put to death by pregnant age, put into ethanol for disinfection and soak 5 minutes, take out in Bechtop, belly is placed in the large ware of aseptic 10cm upward;
(3) cut with Sterile ophthalmic and with tweezers, pregnant mouse abdominal skin and mucous layer are successively cut off, fully expose uterus of becoming pregnant, with eye scissors and tweezers, beading appearance palace is taken out, put into and soak 5 minutes containing 500U penicillin and the dual anti-PBS of Streptomycin sulphate;
(4) Sterile ophthalmic tweezer is used by each tire mouse (containing placenta, number 8-20 is not etc.) separate, remove the rete layer on surface, eye scissors is coordinated to isolate the umbilical cord tissue connecting tire mouse navel and placenta from two ends again, put into and remove remaining blood containing 500U penicillin and the dual anti-PBS washing of Streptomycin sulphate, soak about 5 minutes;
(5) umbilical cord tissue is immersed rapidly containing the dual anti-86%F12/DMEM of 90U, in 14%FBS substratum, with eye scissors and tweezers, it is shredded to 0.5-1.0mm by root
3fine tissue block;
(6) by umbilical cord tissue block (together with a small amount of substratum) uniform spreading in Tissue Culture Dish (3.5cm capsule), steadily put into 37 DEG C of constant temperature, 5%CO lightly
2cultivate in saturated humidity cell culture incubator;
(7) namely umbilical cord tissue block has cell to climb out of after cultivating 24h to 48h, within the 3rd day, changes normal incubation medium, within later every 3 days, changes a not good liquor.P0 presents unhomogeneity for cell, when Growth of Cells fusion reaches 80%, with the trysinization of 0.25%, passes P2 generation, after this go down to posterity in 1:2 ratio, until cellular form homogeneous (to P5-P7 generation), can do next step qualification with the inoculation of the ratio of 4:1.
embodiment 3:
Combination technology route map 1, is described below the concrete implementation step of the present invention:
(1) healthy adult kunming mice (female/male) is bought in Jiangsu University's Experimental Animal Center, when mouse rutting sedson, 2 female mouse and 1 male mouse are spent the night in same mouse case, check female mouse vaginal tract early morning on next day, naked eyes are shown in that seminal fluid bolt person represents and become pregnant, and start to calculate pregnant age (the 0th day).If check negative, then continue to mate until check the positive;
(2) be that the pregnant mouse of the 19 days neck that breaks is put to death by pregnant age, put into ethanol for disinfection and soak 5 minutes, take out in Bechtop, belly is placed in the large ware of aseptic 10cm upward;
(3) cut with Sterile ophthalmic and with tweezers, pregnant mouse abdominal skin and mucous layer are successively cut off, fully expose uterus of becoming pregnant, with eye scissors and tweezers, beading appearance palace is taken out, put into and soak 10 minutes containing 1000U penicillin and the dual anti-PBS of Streptomycin sulphate;
(4) Sterile ophthalmic tweezer is used by each tire mouse (containing placenta, number 8-20 is not etc.) separate, remove the rete layer on surface, eye scissors is coordinated to isolate the umbilical cord tissue connecting tire mouse navel and placenta from two ends again, put into and remove remaining blood containing 1000U penicillin and the dual anti-PBS washing of Streptomycin sulphate, soak about 10 minutes;
(5) umbilical cord tissue is immersed rapidly containing the dual anti-84%F12/DMEM of 110U, in 16%FBS substratum, with eye scissors and tweezers, it is shredded to 0.5-1.0mm by root
3fine tissue block;
(6) by umbilical cord tissue block (together with a small amount of substratum) uniform spreading in Tissue Culture Dish (3.5cm capsule), steadily put into 37 DEG C of constant temperature, 5%CO lightly
2cultivate in saturated humidity cell culture incubator;
(7) namely umbilical cord tissue block has cell to climb out of after cultivating 24h to 48h, within the 3rd day, changes normal incubation medium, within later every 3 days, changes a not good liquor.P0 presents unhomogeneity for cell, when Growth of Cells fusion reaches 90%, with the trysinization of 0.25%, passes P2 generation, after this go down to posterity in 1:1 ratio with the inoculation of the ratio of 2:1, until cellular form homogeneous (to P5-P7 generation), next step qualification can be done.
checking example 1:
The morphological observation of the mouse umbilical cord mesenchymal stem cells that separation and Culture of the present invention obtains:
(1) the mouse umbilical cord mesenchymal stem cells adopting embodiment 1-3 method to be separated: primary (P0 generation) cell climbs out of after organizing adherent 24-48h, continue to cultivate 3-4 days, observe visible cell form heterogeneity under inverted microscope, have spindle shape, polygon, also have circle (see Fig. 2 A);
(2) according to the propagating method described in embodiment 1-3, hold concurrently by the impact of individual mice difference, cell reach P5-P7 for time observe under inverted microscope, visible cell presents relatively homogeneous spindle shape.Reach 20 generations more than continuously, cellular form is without obviously changing (see Fig. 2 B).
The morphological outcomes of mouse umbilical cord mesenchymal stem cells shows: the characteristic that the cell that separation and Culture of the present invention goes out possesses the spindle shape Morphological Features of mescenchymal stem cell and adherent growth, repeatedly goes down to posterity.
checking example 2:
The growth curve of the mouse umbilical cord mesenchymal stem cells that separation and Culture of the present invention obtains measures:
(1) embodiment 1-3 method is selected to be separated the well-grown mouse umbilical cord mesenchymal stem cells (as P7 generation) going down to posterity and obtain, by quantitative culture medium re-suspended cell precipitation after the trysinization of 0.25%, cell counting to adjust concentration be 1 × 10
4/ mL;
(2) obtained cell suspension is inoculated in 24 orifice plates, and every hole 500 μ L, is placed in 37 DEG C of constant temperature, 5%CO
2cultivate in saturated humidity cell culture incubator;
(3) get 3 porocytes every day, the row cell counting respectively after 0.25% quantitative trysinization of every hole, indigested cell every 3 days full doses change liquid 1 time;
(4) continuous counter 7 days, and draw growth curve chart;
The measurement result display of mouse umbilical cord mesenchymal stem cells growth curve: the Growth of Cells that separation and Culture of the present invention goes out is typical S type curve: 1-2 days be latent period, within 3rd day, enter logarithmic phase, after 6th day, cell proliferation enters plateau, possesses the growth characteristics (see figure 3) of normal mesenchymal stem cell.
checking example 3:
The cell cycle of the mouse umbilical cord mesenchymal stem cells that separation and Culture of the present invention obtains:
(1) select embodiment 1-3 method to be separated the mouse umbilical cord mesenchymal stem cells (as P6 generation) going down to posterity and obtain, centrifugal after the trysinization of 0.25%, PBS washing once, then precipitates with 250 μ LPBS re-suspended cells;
(2) add 250 μ L50 μ g/mL ethidium bromides, 4 DEG C of lucifuges hatch 30 minutes;
(3) flow cytometer (BD) detects, the per-cent of ModiFIT software analysis G1, G2 and S phase.
The cell cycle detected result of mouse umbilical cord mesenchymal stem cells shows: the cell G1 phase that separation and Culture of the present invention goes out is towering, account for 78.62%, the G2 phase accounts for 6.02%, the S phase accounts for 15.36%, illustrate that most cells is in quiescent condition, having high proliferation potential, is typical interstital stem cell cycle characteristics (see figure 4).
checking example 4:
The Immunophenotype analysis of the mouse umbilical cord mesenchymal stem cells that separation and Culture of the present invention obtains:
(1) select embodiment 1-3 method to be separated the mouse umbilical cord mesenchymal stem cells (as P6 generation) going down to posterity and obtain, count after the trysinization of 0.25%, by 1 × 10
5/ pipe is packed as 6 pipes, and PBS washs 1 time, then precipitates with 50 μ LPBS re-suspended cells;
(2) in every pipe, add the corresponding streaming antibody of 1 μ L respectively: CD29, CD34, CD44, CD45, FITC-IgG, PE-IgG, 4 DEG C of lucifuges hatch 30 minutes;
(3) PBS washs 1 time, analyzes in flow cytomery.
The Immunophenotype analysis result of mouse umbilical cord mesenchymal stem cells shows: cell strongly expressed CD29 and CD44 that separation and Culture of the present invention goes out, and does not express CD45 and CD34, meets the surface markers feature (see figure 5) of mescenchymal stem cell.
checking example 5:
The skeletonization of the mouse umbilical cord mesenchymal stem cells that separation and Culture of the present invention obtains, the research of one-tenth fat differentiation potential:
1, Osteoblast Differentiation research
(1) embodiment 1-3 method is selected to be separated the mouse umbilical cord mesenchymal stem cells going down to posterity and obtain, by 4 × 10
4cell count is inoculated in cultivates in capsule, attach overnight;
(2) add osteogenic induction liquid, compound method is: add 10 in basic medium
-8m dexamethasone, 10mM sodium β-glycerophosphate and 50mg/L xitix, change liquid in every 3 days;
(3) induce after 14 days and use alkaline phosphatase (ALP) staining examine, observe under inverted microscope.
2, fat differentiation research is become
(1) embodiment 1-3 method is selected to be separated the mouse umbilical cord mesenchymal stem cells going down to posterity and obtain, by 2 × 10
5cell count is inoculated in cultivates in capsule, attach overnight;
(2) adipogenic induction liquid is added, be divided into A liquid and B liquid, compound method is: add 1 μM of dexamethasone, 200 μMs of indomethacins and 2 μMs of Regular Insulin in A liquid-basic medium, add 2 μMs of Regular Insulin in B liquid-basic medium, A liquid changes B liquid and maintains 1 day after inducing 3 days;
(3) induce after 14 days and detect with oil red O stain, under inverted microscope, observe fat granule.
Mouse umbilical cord mesenchymal stem cells skeletonization, adipogenic induction differentiated result show: the cell osteogenic induction stained positive (red-purple that separation and Culture of the present invention goes out, see Fig. 6), adipogenic induction dyeing is also positive (red fat drips, and sees Fig. 7), illustrates to have mescenchymal stem cell multi-lineage potential.
The above example only have expressed several better implementation method of the present invention, and it describes comparatively concrete and detailed, but protection scope of the present invention is not limited with above-mentioned implementation method.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.
Claims (9)
1. an isolation cultivation method for mouse umbilical cord mesenchymal stem cells, is characterized in that, carries out according to following steps:
(1) neck that broken by pregnant mouse is put to death, and put into ethanol for disinfection and soak, take out in Bechtop, belly is placed in the large ware of aseptic 10cm upward;
(2) cut with Sterile ophthalmic and with tweezers, pregnant mouse abdominal skin and mucous layer are successively cut off, take out beading sample and to become pregnant uterus, put into and soak containing penicillin and the dual anti-PBS of Streptomycin sulphate;
(3) with Sterile ophthalmic tweezers, every tire mouse is stripped out, picks clean rete layer, then coordinate eye scissors to isolate the umbilical cord tissue connecting tire mouse navel and placenta, put into and wash containing penicillin and the dual anti-PBS of Streptomycin sulphate and soak;
(4) umbilical cord tissue is dipped in containing in dual anti-substratum, with eye scissors and tweezers, it is shredded by root;
(5) by umbilical cord tissue block together with substratum uniform spreading in Tissue Culture Dish (3.5cm capsule), be placed in 37 DEG C of cell culture incubators and cultivate;
(6) namely umbilical cord tissue block has cell to climb out of after cultivating 24h to 48h, within the 3rd day, changes normal incubation medium, within later every 3 days, changes a not good liquor, when P0 reaches 80-90% for Growth of Cells fusion, with the trysinization Secondary Culture of 0.25%, continue to go down to posterity until cell presents homogeneous form, do next step qualification.
2. the isolation cultivation method of a kind of mouse umbilical cord mesenchymal stem cells according to claim 1, is characterized in that, in step (1), the pregnant age of pregnant mouse is 15-19 days; Described soak time is 5 minutes.
3. the isolation cultivation method of a kind of mouse umbilical cord mesenchymal stem cells according to claim 1, is characterized in that, be 500U-1000U containing the concentration of penicillin and Streptomycin sulphate in PBS in step (2) and (3), soak time is 5-10 minute.
4. the isolation cultivation method of a kind of mouse umbilical cord mesenchymal stem cells according to claim 1, it is characterized in that, described in step (4) containing the composition of dual anti-substratum and content thereof be: F12/DMEM86%-84%(V/V), FBS14-16%(V/V), 90-110U penicillin, 90-110U Streptomycin sulphate.
5. the isolation cultivation method of a kind of mouse umbilical cord mesenchymal stem cells according to claim 1, is characterized in that, umbilical cord tissue shreds to 0.5-1.0mm by step (4) middle eye scissors and tweezers
3.
6. the isolation cultivation method of a kind of mouse umbilical cord mesenchymal stem cells according to claim 1, is characterized in that,
Cultivating the normal incubation medium changed for the 3rd day described in step (6) does not contain dual anti-, and composition is F12/DMEM86-84%(V/V), FBS14-16%(V/V).
7. the isolation cultivation method of a kind of mouse umbilical cord mesenchymal stem cells according to claim 1, it is characterized in that, the cell of had digestive transfer culture described in step (6) passes P2 generation with the ratio of 4-2:1, after this goes down to posterity in 1:1-2 ratio, until cell present homogeneous after ratio Secondary Culture routinely.
8. the isolation cultivation method of a kind of mouse umbilical cord mesenchymal stem cells according to claim 1, is characterized in that, when P0 is for cell dissociation described in step (6), takes " weight wall built-up method " to remove umbilical cord tissue block.
9. the isolation cultivation method of a kind of mouse umbilical cord mesenchymal stem cells according to claim 1, is characterized in that, is used for the Transplanted cells in mouse experiment animal model structure according to the mouse umbilical cord mesenchymal stem cells of the method gained.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110387350A (en) * | 2018-04-18 | 2019-10-29 | 江苏齐氏生物科技有限公司 | A kind of isolation and culture of Marrow Mesenchymal Stem Cells |
| CN111454890A (en) * | 2020-01-23 | 2020-07-28 | 广州医科大学附属第二医院 | Isolated culture method of mouse adipose-derived mesenchymal stem cells |
| CN113424799A (en) * | 2021-06-28 | 2021-09-24 | 中国人民解放军陆军特色医学中心 | Construction method and application of PDX model based on osteogenic niche microenvironment modification |
| CN114369569A (en) * | 2022-01-10 | 2022-04-19 | 中国科学技术大学 | Separation method of umbilical cord Wharton jelly mesenchymal stem cells |
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2015
- 2015-03-19 CN CN201510120536.1A patent/CN105112359A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110387350A (en) * | 2018-04-18 | 2019-10-29 | 江苏齐氏生物科技有限公司 | A kind of isolation and culture of Marrow Mesenchymal Stem Cells |
| CN111454890A (en) * | 2020-01-23 | 2020-07-28 | 广州医科大学附属第二医院 | Isolated culture method of mouse adipose-derived mesenchymal stem cells |
| CN113424799A (en) * | 2021-06-28 | 2021-09-24 | 中国人民解放军陆军特色医学中心 | Construction method and application of PDX model based on osteogenic niche microenvironment modification |
| CN114369569A (en) * | 2022-01-10 | 2022-04-19 | 中国科学技术大学 | Separation method of umbilical cord Wharton jelly mesenchymal stem cells |
| CN114369569B (en) * | 2022-01-10 | 2024-04-12 | 中国科学技术大学 | Separation method of umbilical cord Walton gum mesenchymal stem cells |
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