CN103525761A - Method for separating and culturing animal bone marrow mesenchymal stem cell - Google Patents
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Abstract
The invention discloses a method for separating and culturing an animal bone marrow mesenchymal stem cell (MSC). The method comprises the following steps: inoculating an inoculum into an animal cell culturing medium for primary culture, and subcultring cells obtained after the primary culture to obtain the animal MSC, wherein the inoculum is a precipitate obtained after centrifuging a long bone breaking liquid, and the long bone breaking liquid is a mixture obtained by breaking in-vitro animal long bones in a buffer solution to 0.5-2mm<3> particles. The method does not comprise a collagenase digestion step or an animal long bone marrow removal step. The method has the advantages of full utilization of the marrow and sclerite, simple processing, and obtaining of enough-quantity, good-growth-state and strong-proliferation-capability MSCs within a short time, and is a convenient, fast and efficient MSC separating and culturing method.
Description
Technical field
The present invention relates to a kind of method of separation and Culture marrow mescenchymal stem cell.
Background technology
Mescenchymal stem cell (mesenchymal stem cell, MSC) be in marrow except hemopoietic stem cell another important stem cell.MSC has the ability of self, Multidirectional Differentiation, immunoregulation and hematopoiesis support, and wide material sources, easily cultivates in vitro and increases, and has been applied at present clinical and has shown good therapeutic action.Although MSC clinical application is extensive, many key issues are as concrete mechanism of immunoregulation etc. unresolved.Mouse is one of important laboratory animal, is widely used in Basic Experiment Study.Successful separation and Culture MSC in vitro, and the MSC that obtains large-scale purification is the prerequisite of its differentiation of research and function controlling mechanism.MSC is mainly derived from marrow, but in marrow, the content of MSC seldom, every 10<sup TranNum="63">4</sup>-10<sup TranNum="64">5</sup>individual monocyte is approximately containing 1 MSC(Morikawa S, Mabuchi Y, Kubota Y, et al.Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow.J Exp Med.2009.206.2483-2496).The method of separating mouse marrow MSC comprises full marrow adherent method, osteocomma digestion method, streaming or magnetic bead sorting method etc. at present.Full marrow adherent method, according to the adherent property of MSC, regularly changes liquid and removes not attached cell, thereby reaches object (Soleimani, the M. ， & of purifying MSC; Nadri, S.A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow.Nat.Protoc.2009.4.102-106; Sun S, Guo Z, Xiao X, et al.Isolation of mouse marrow mesenchymal progenitors by a novel and reliable method.Stem Cells.2003.21.527-535).Yet the MSC that the method obtains often contains more stroma cell and hematopoietic cell pollutes.Osteocomma digestion method, first marrow to be rinsed, femur and shin bone are shredded, then carry out collagenase digesting (Zhu, H., Guo, Z.K., Jiang, X.X., et al..A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone.Nat.Protoc.2010.5.550-560; Guo, Z.Li, H., Li, X., et al.In vitro characteristics and in vivo immunosuppressive activity of compact bone-derived murine mesenchymal progenitor cells.Stem Cells2006.24.992-1000).Although the MSC purity that the method obtains is higher, program is complicated, and the asynchronism(-nization) of different mouse osteocomma in age collagenase digesting, is not easy to grasp.Along with going deep into MSC surface antigen understanding, there is investigator to utilize immunization method it to be carried out to separation and purification (Houlihan as flow cytometer method, immunomagnetic beads method, D.D., Mabuchi, Y., Morikawa, S., et al.Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1and PDGFR-α .Nat.Protoc.2012.7.2103-2111; Baddoo M, Hill K, Wilkinson R, et al.Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection.J Cell Biochem.2003.89.1235-1249).Although these two kinds of methods can obtain the cell that purity is higher, sepn process is larger to the activity influence of cell, even causes cell to lose activity completely.And requirement for experiment condition is high, need marrow amount large, expend large and technical difficulty in addition, limited to a certain extent the widespread use of these two kinds of methods.Therefore need to set up a kind of from marrow the high efficiency method of separation and Culture MSC.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of method of acquisition high purity and high Board Lot (the every MSC that long bone obtains) separation and Culture marrow mescenchymal stem cell, the method also have MSC time of occurrence early, do not need to remove marrow, do not need digestion, easy and simple to handle and advantage efficiently.
The method of separation and Culture marrow mescenchymal stem cell provided by the present invention, comprises inoculum is inoculated in and in Zooblast culture medium, carries out former culture, then the cell that former culture the is obtained cultivation of going down to posterity, and obtains marrow mescenchymal stem cell; Wherein, described inoculum is for carrying out the centrifugal precipitation obtaining to the broken liquid of long bone, and the broken liquid of described long bone is that in vitro animal long bone is crushed to the mixture that 0.5-1.5 cubic millimeter particle (as 1 cubic millimeter of particle) obtains in buffered soln;
Described method does not comprise the step with collagenase digesting, and described method is not removed the marrow of described animal long bone.
In aforesaid method, described long bone is comprised of key and epiphysis two portions, and surface is covered with periosteum and joint cartilage, and inside is medullary space, is wherein full of marrow.
In aforesaid method, described animal can be vertebrates.Described vertebrates can be Mammals, as mouse.
In aforesaid method, described long bone be femur or/and shin bone or/and derive from the long bone of forelimb bone.
In aforesaid method, the described centrifugal centrifugal 5-10 of 300g-500g that can be, as centrifugal in 400g 5 minutes.
Described buffered soln can be and makes zooblast keep active liquid, as the phosphate buffered saline buffer of 0.01M, pH7.2-7.4.
In aforesaid method, described Zooblast culture medium can be the mammalian cell culture of cultivating for attached cell, as MEM substratum, DMEM substratum.In one embodiment of the invention, described Zooblast culture medium is α-MEM complete culture solution.
In aforesaid method, described in go down to posterity to cultivate can be and carry out the cultivation of going down to posterity at least 2 times, as carry out the cultivation of going down to posterity for 2 times.
The present invention also provides and has prepared the above-mentioned method for separating of cultivating the inoculum of marrow mescenchymal stem cell.
The method of the inoculum for the preparation of separation and Culture marrow mescenchymal stem cell provided by the present invention, comprising:
1) in vitro animal long bone is crushed in buffered soln to 0.5-1.5 cubic millimeter particle (as 1 cubic millimeter of particle) and obtains the broken liquid of long bone;
2) the broken liquid of long bone step 1) being obtained carries out centrifugal, gets precipitation, and this precipitation is for separating of the inoculum of cultivating marrow mescenchymal stem cell.
This is prepared in the method for the above-mentioned inoculum for separating of cultivation marrow mescenchymal stem cell, and described animal can be vertebrates.Described vertebrates can be Mammals, as mouse.Described long bone can be femur or/and shin bone or/and derive from the long bone of forelimb bone.
The described centrifugal centrifugal 5-10 of 300g-500g that can be, as centrifugal in 400g 5 minutes.
Described buffered soln can be and makes zooblast keep active liquid, as the phosphate buffered saline buffer of 0.01M, pH7.2-7.4.
Experiment showed, that the described marrow mescenchymal stem cell that the present invention obtains is spindle cell, express Sca1, CD44, CD29, does not express endothelial cell marker CD31 and hematopoietic cell sign CD45.At skeletonization with in becoming fat induction system, cultivation can be divided into scleroblast and adipocyte to the described marrow mescenchymal stem cell that the present invention obtains.The application adds osteocomma method by more full marrow adherent method, osteocomma digestion method and marrow of the present invention, finds that marrow of the present invention adds a kind of method that osteocomma method is convenient, fast and effective separation and Culture MSC.The application's experimental result demonstration, the MSC that marrow of the present invention adds the appearance of osteocomma method clones the time the earliest, and clone's quantity is maximum, and within the 4th day, clone's number is 20 ± 4/root femur; Osteocomma digestion method is 11.5 ± 2.5/root femur; Full marrow adherent method is minimum, is 9.5 ± 1.5/root femur; During to the third generation, it is maximum that marrow of the present invention adds osteocomma method acquisition cell quantity, is the twice of full marrow adherent method and osteocomma digestion method.
Marrow of the present invention add osteocomma method by femur and/or shin bone muscle go clean after, do not need to rush marrow, do not need digestion, directly shred, damping fluid is resuspended in directly inoculation in mammalian cell culture after washing.Compare full marrow adherent method and osteocomma digestion method, marrow adds morning time that mouse MSC clone appears in osteocomma method, and quantity is many.Although first-generation peptic cell still has a small amount of CD45 positive cell, to the third generation, MSC purity is more than 95%, and the MSC sum obtaining will be higher than full marrow adherent method and osteocomma digestion method.Similar to the MSC that additive method obtains, marrow of the present invention adds the MSC high expression level Sca1 that osteocomma method obtains, CD44, and CD29 etc., do not express endothelial cell marker CD31 and hematopoietic cell sign CD45.At skeletonization with in becoming fat induction system, cultivation can be divided into scleroblast and adipocyte to the MSC that the method obtains.In sum, marrow of the present invention adds osteocomma method and takes full advantage of marrow and osteocomma, the mescenchymal stem cell that processing is simple, can obtain sufficient amount in the short period of time, growth conditions is good, multiplication capacity is strong is a kind of convenient, fast, efficient MSC isolation cultivation method.
Accompanying drawing explanation
Fig. 1 is the former culture of MSC.
A is the cellular form that is inoculated in 6 orifice plate 72h.
B is the inoculation cell quantity of the 4th day.
C is 5 days CD45 positive cell ratios of former culture.
Fig. 2 is cellular form and the cell count after going down to posterity.
A is the form of third generation cell.
B is the cell count of s-generation cell and third generation cell.
Fig. 3 is for full marrow group (BM), osteocomma digestion group (Bone) and marrow add the third generation cell phenotype evaluation of osteocomma group (BM+Bone) respectively.
Fig. 4 is the differentiation capability that marrow adds the third generation cell of osteocomma group (BM+Bone).
A, marrow add the third generation cell of osteocomma group (BM+Bone) and cultivate the ALP coloration result after 1 week through HG-DMEM nutrient solution (containing 10% foetal calf serum).
B, marrow add the third generation cell of osteocomma group (BM+Bone) and cultivate the ALP coloration result after 1 week through Osteoinductive differentiation complete culture solution.
C, marrow add third generation cell Von Kossa coloration result after HG-DMEM nutrient solution (containing 10% foetal calf serum) is cultivated 2 weeks of osteocomma group (BM+Bone).
D, marrow add third generation cell Von Kossa coloration result after Osteoinductive differentiation complete culture solution is cultivated 2 weeks of osteocomma group (BM+Bone).
E is third generation cell oil red O stain result after HG-DMEM nutrient solution (containing 10% foetal calf serum) is cultivated 1 week that marrow adds osteocomma group (BM+Bone).
F is third generation cell oil red O stain result after becoming fat induction differentiation complete culture solution to cultivate 1 week that marrow adds osteocomma group (BM+Bone).
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment providing is only in order to illustrate the present invention, rather than in order to limit the scope of the invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Main agents and instrument in following embodiment are as follows:
α-MEM nutrient solution (Gibco company, cat.no.12000-022), DMEM-HG nutrient solution (Gibco company, cat.no.10566-024), foetal calf serum (the product cat.no.FSP500 of Shanghai Excell Biology Product Co., Ltd.); The PBS of 0.01M, pH7.2-7.4, dexamethasone, phosphorylation vitamins C, β-phospho-glycerol, II Collagenase Type, pancreatin, oil red O dye liquor, alkaline phosphatase enzyme reagent kit is Sigma company product; Detection MSC phenotypic correlation antibody: FITC anti-mouse CD45, APC anti-mouse I-A/I-E, PerCP anti-mouse/human CD44, PE anti-mouse CD31, APC anti-mouse/rat CD29, PerCP anti-mouse Ly-6A/E(Sca-1) be BD company product; 25cm
2, 75cm
2culturing bottle (costar), 6 hole/48 well culture plates (Falcon, nunc); Flow cytometer (U.S. company BD), cell culture incubator (Thermo Forma), (system is closed to biopurification worktable by /Wei Da purification techniques institute of Zhonghua Biological Technology Inst., Beijing, X90-3), inverted microscope (Japanese Olympus company), low speed centrifuge (in good SC-3610).
The method of embodiment 1, separation and Culture marrow mescenchymal stem cell
This enforcement has adopted the method for three kinds of separation and Culture marrow mescenchymal stem cells, is respectively full marrow adherent method (being called for short BM), osteocomma digestion method (being called for short Bone) and marrow and adds osteocomma method (being called for short BM+Bone).The inoculum of these three kinds of methods all derives from the femur of same C57BL/6 mouse.The inoculum of full marrow adherent method is the marrow of in vitro mouse femur, the inoculum of osteocomma digestion method is the osteocomma of mouse femur, osteocomma is prepared as follows: remaining femur (remaining part after the marrow of complete femur is removed) after removing marrow in full marrow adherent method is shredded to 1 cubic millimeter of particle in the PBS of 0.01M, pH7.2-7.4, add after 0.1% II Collagenase Type, be positioned over 37 ℃, 5%CO
2, saturated humidity cell culture incubator in hatch 1h, the osteocomma obtaining is inoculum.Marrow adds the inoculum of osteocomma method for the broken liquid of femur is carried out to the centrifugal precipitation obtaining, the broken liquid of described femur is that same in vitro another root femur (be complete femur, do not remove marrow) of C57BL/6 mouse is crushed to the mixture that 1 cubic millimeter of particle obtains in damping fluid.
The present embodiment has compared method MSC clone time of occurrence, size and the quantity of three kinds of separation and Culture marrow mescenchymal stem cells; Flow cytometry Immunophenotyping; And carry out skeletonization, become fat differentiation to identify.Result shows that marrow adds MSC clone's time that osteocomma method occurs the earliest, and clone's quantity is maximum, and within the 4th day, clone's number is 20 ± 4/root femur; Osteocomma digestion method is 11.5 ± 2.5/root femur; Full marrow adherent method is minimum, is 9.5 ± 1.5/root femur.During to the third generation, it is maximum that marrow adds osteocomma method acquisition cell quantity, is the twice of full marrow adherent method and osteocomma digestion method.The third generation cell that marrow adds the acquisition of osteocomma method is spindle cell.The demonstration of streaming result, marrow adds one of the third generation cell high expression level stem cell sign of osteocomma method acquisition Sca-1, and high expression level CD44, CD29, do not express Swine lymphocyte antigen CD45 and endothelial cell marker CD31 etc.; Marrow add third generation cell that osteocomma method obtains osteogenic induction system with become fat induction system in can be respectively to scleroblast and Adipocyte Differentiation, the typical MSC characteristic of tool.
Specific experiment method and experimental result are as follows:
1 materials and methods
1.1 laboratory animal
Healthy C57BL/6 mouse, in 3～4 week age, male and female Bu Xian，You Military Medical Science Institute animal center provides.
The separation and Culture of 1.2MSC
Experiment in triplicate, repeat to get 1 neck marrow from the disconnected C57BL/6 of putting to death mouse at every turn, 75% alcohol immersion 5min, under aseptic condition, get mouse bilateral femur, the aseptic blunt separation muscle of cutting, puts into the separated clean femur (complete femur) of muscle the PBS(phosphate buffered saline buffer of 0.01M, pH7.2-7.4).Get a side femur and cut off Distal femoral metaphysis, expose medullary space, with 1ml syringe, draw 0.01M, the PBS of pH7.2-7.4 rinses this side marrow cavity of femur repeatedly, the marrow of going out is all put in a 1.5mlEP pipe, with rifle point, blow and beat gently and make single cell suspension, by this EP pipe with the centrifugal 5min of 400g, abandon supernatant liquor, precipitation is the full marrow adherent method inoculum of (being called for short BM), get whole precipitations and (in α-MEM nutrient solution, add foetal calf serum with 1ml α-MEM complete culture solution, penicillin and Streptomycin sulphate, concentration expressed in percentage by volume to foetal calf serum is 10% foetal calf serum, the concentration of penicillin is every liter of 10Wan unit, the concentration of Streptomycin sulphate is the liquid that every liter of 10Wan unit obtains) the Kong Zhongwei full marrow group (BM) of re-suspended cell kind in 6 orifice plates.After this is gone out to marrow, remaining femur (remaining part after the marrow of complete femur is removed) all shreds to 1 cubic millimeter of particle in the 1.5mlEP pipe of PBS that 0.01M, pH7.2-7.4 are housed, add after 1ml0.1% II Collagenase Type, be positioned over 37 ℃, 5%CO
2, saturated humidity cell culture incubator in hatch 1h and obtain osteocomma, this osteocomma is the osteocomma digestion method inoculum of (being called for short Bone), then with the PBS of 0.01M, pH7.2-7.4, wash after whole osteocommas three times, by whole osteocommas, by the resuspended rear kind of above-mentioned 1ml α-MEM complete culture solution, in second hole in 6 orifice plates, be osteocomma digestion group (Bone); By opposite side femur (the complete femur of this same mouse, do not remove marrow) in the 1.5mlEP pipe of PBS that 0.01M, pH7.2-7.4 are housed, all shred to 1 cubic millimeter of particle, by this EP pipe with the centrifugal 5min of 400g, sucking-off supernatant, precipitation is marrow and adds the osteocomma method inoculum of (being called for short BM+Bone), to all precipitate by the resuspended rear kind of above-mentioned 1ml α-MEM complete culture solution in the 3rd hole of this 6 orifice plate, for marrow adds osteocomma group (BM+Bone).Method of the same race is respectively done three multiple holes, in 37 ℃, 5%CO
2, saturated humidity cell culture incubator in cultivate.The later half amount of 48h is changed liquid (changing liquid with above-mentioned α-MEM complete culture solution), after 72h, full dose is changed liquid (changing liquid with above-mentioned α-MEM complete culture solution), within later every 2-3 days, change liquid and once (with above-mentioned α-MEM complete culture solution, change liquid), observation of cell growing state day by day under inverted microscope, at the bottom of Growth of Cells is paved with plate, use 0.25% trypsin 80～90% time containing 0.02%EDTA) to cell (primary cell, first-generation cell) digest counting, in 1:2 ratio, be inoculated in 6 new orifice plates and go down to posterity for the first time.The cell going down to posterity is for the first time in 37 ℃, 5%CO
2, saturated humidity cell culture incubator in cultivate.The later half amount of 48h is changed liquid (changing liquid with above-mentioned α-MEM complete culture solution), after 72h, full dose is changed liquid (changing liquid with above-mentioned α-MEM complete culture solution), within later every 2-3 days, change liquid and once (with above-mentioned α-MEM complete culture solution, change liquid), observation of cell growing state day by day under inverted microscope, at the bottom of Growth of Cells is paved with plate, use 0.25% trypsin 80～90% time containing 0.02%EDTA) cell (s-generation cell) is digested to counting, in 1:2 ratio, be inoculated in and in 6 new orifice plates, carry out second pass generation.The cell in second pass generation is in 37 ℃, 5%CO
2, saturated humidity cell culture incubator in cultivate.The later half amount of 48h is changed liquid (changing liquid with above-mentioned α-MEM complete culture solution), after 72h, full dose is changed liquid (changing liquid with above-mentioned α-MEM complete culture solution), within later every 2-3 days, change liquid and once (with above-mentioned α-MEM complete culture solution, change liquid), observation of cell growing state day by day under inverted microscope, at the bottom of being paved with plate, Growth of Cells 80～90% time, obtains third generation cell.
1.3MSC morphological observation
The upgrowth situation of observation of cell and metamorphosis feature day by day on inverted microscope, before going down to posterity for the first time, manifold falls to counting and takes pictures, also Taking Pictures recording before later going down to posterity.
The immunophenotype of 1.4MSC is identified
Get growth conditions good postdigestive the 3rd generation cell, PBS washes a rear overhang and becomes 1x10
6individual/ml single cell suspension, 200 μ l/EP pipes, add respectively fluorescent-labeled antibody: FITC anti-mouse CD45, APC anti-mouse I-A/I-E, PerCP anti-mouse/human CD44APC anti-mouse/rat CD29 and PerCP anti-mouse Ly-6A/E(Sca-1).4 ℃ hatch 30min after PBS wash away unmarked antibody, the positive expression rate of respective markers antigen in application flow cytometry analysis sample.
The external Osteoblast Differentiation induction of 1.5MSC
Get third generation cell and be seeded in 48 orifice plates, every porocyte number is 2 * 10
4individual, all establish 3 ，Mei holes, multiple hole for every group and add the above-mentioned α-MEM of 400 μ l complete culture solution, put into 37 ℃, 5%CO
2, saturated humidity cell culture incubator in cultivate.Cell spend the night adherent after, suck nutrient solution, (HG-DMEM nutrient solution is containing 10% foetal calf serum and 10 to add the Osteoinductive differentiation complete culture solution in 400 μ l/ holes
-7m dexamethasone, 10mM β-phospho-glycerol, 50 μ M phosphorylation vitamins Cs).HG-DMEM nutrient solution for control group (containing 10% foetal calf serum).Within every 3 days, change liquid, after 1 week, carry out ALP(alkaline phosphatase) dyeing, after 2 weeks, carry out Von Kossa dyeing.
The external one-tenth fat induction of 1.6MSC
Get third generation cell and be seeded in 48 orifice plates, every porocyte number is 2 * 10
4individual, all establish 3 ，Mei holes, multiple hole for every group and add the above-mentioned α-MEM of 400 μ l complete culture solution, put into 37 ℃, 5%CO
2, saturated humidity cell culture incubator in cultivate.Cell spend the night adherent after, suck nutrient solution, (HG-DMEM nutrient solution is containing 10% foetal calf serum and 10 to add the one-tenth fat induction differentiation complete culture solution in 400 μ l/ holes
-6m dexamethasone, 10ng/ml Regular Insulin, 0.5 μ M IBMX).HG-DMEM nutrient solution for control group (containing 10% foetal calf serum).Within every 3 days, change liquid, after 1 week, carry out oil red O stain.
1.7 statistical method
Use SPSS16.0 software, mean ± standard deviation for data acquisition<img TranNum="138" file="BDA0000392294810000071.GIF" he="65" img-content="drawing" img-format="GIF" inline="yes" orientation="portrait" wi="184"/>represent, relatively adopt t check between group, take P<0.05 has statistical significance as difference.
2 results
The separation and Culture of 2.1MSC
Full marrow group (BM) medullary cell is inoculated in 6 orifice plate 48h and in nutrient solution, take suspension round cell as main, and the hematopoiesis that is mixed with a large amount of more tiny circles is cell; Osteocomma digestion group (Bone) osteocomma is inoculated in 48h in 6 orifice plates and has no cell suspension; Marrow adds osteocomma group (BM+Bone) and is inoculated in 6 orifice plate 48h and in nutrient solution, take suspension round cell as main, and the hematopoiesis that is mixed with a large amount of more tiny circles is cell.48h half amount is changed after liquid visible three groups all a small amount of cell attachment, be colony shape, cellular form is irregular, there is spindle shape, there is polygon etc., full marrow group (BM) and marrow add osteocomma group (BM+Bone) and are also shown in some circular attached cells, and in full marrow group (BM), circular attached cell is particularly many.Being inoculated in 6 orifice plate 72h(and changing for the first time 24h after liquid) visible every group of attached cell colony number all have increase, but marrow adds osteocomma group (BM+Bone), increases more remarkable (A in Fig. 1); Add up every group of colony number, full marrow group (BM), osteocomma digestion group (Bone), marrow add osteocomma group (BM+Bone) and are respectively 9.5 ± 1.5/root femur, 11.5 ± 2.5/root femur, 20 ± 4/root femur (B in Fig. 1).Former culture 5 days (inoculating 5 days), marrow adds osteocomma group (BM+Bone) cell can reach 80%-90% fusion, and osteocomma digestion group (Bone) is taken second place, and full marrow group (BM) is minimum, at this moment cell is mainly strip fusiform, and overall alignment is tending towards rule and is whirlpool shape.Peptic cell is also got same cell amount and is detected CD45 expression level, find that CD45 positive cell is maximum in full marrow group (BM), reach 78.6%, it is 10.6% that marrow adds that osteocomma group (BM+Bone) takes second place, and osteocomma digestion group (Bone) is minimum, C in 2.66%(Fig. 1 only).Along with passage increased frequency, cell, by cultivating early stage Combination cell mass, gradually becomes the spindle cell (A in Fig. 2) of the relative homogeneous of form, collecting cell counting, and marrow adds osteocomma group (BM+Bone) harvested cell number (B in Fig. 2) at most.In s-generation cell (P2) and third generation cell (P3), the cell quantity that marrow adds osteocomma group (BM+Bone) is the twice of full marrow group (BM) and osteocomma digestion group (Bone), the s-generation cell count that marrow adds osteocomma group (BM+Bone) is 5.3 ± 0.14/root femur, and the third generation cell count that marrow adds osteocomma group (BM+Bone) is 12.5 ± 3.5/root femur.
The phenotypic evaluation of 2.2MSC
Collect respectively the third generation cell that full marrow group (BM), osteocomma digestion group (Bone) and marrow adds osteocomma group (BM+Bone) and carry out phenotypic evaluation.Streaming result shows (Fig. 3), and each organizes one of cell high expression level stem cell sign Sca-1, high expression level adhesion molecule CD44 and CD29; Do not express MHC II quasi-molecule IA/IE, and one of endothelium sign CD31.Osteocomma digestion group (Bone) and marrow add the third generation cell of osteocomma group (BM+Bone) and do not express one of hematopoietic cell sign CD45, and the third generation cell of full marrow group (BM) still has 5% CD45 positive cell.
The skeletonization of 2.3MSC and one-tenth fat Function Identification
The third generation cell that collection marrow adds osteocomma group (BM+Bone) carries out skeletonization and becomes fat differentiation to identify, as Fig. 4 shows, in osteogenic induction system, induce after one week, alkaline phosphatase staining positive (B in Fig. 4), induce VonKossa stained positive (D in Fig. 4) after two weeks, illustrate that the third generation cytodifferentiation that marrow adds osteocomma group (BM+Bone) is scleroblast; Becoming fat induction system to induce after one week, cell is the oil red O stain positive (F in Fig. 4), illustrates that the third generation cytodifferentiation that marrow adds osteocomma group (BM+Bone) is adipocyte; Their control group does not add the dyeing negative (A in Fig. 4, C, E) of inductor.
The present embodiment experimental results show that the third generation cell that marrow adds osteocomma group (BM+Bone) is mescenchymal stem cell.
The method of embodiment 2, separation and Culture marrow mescenchymal stem cell
This enforcement has adopted the method for three kinds of separation and Culture marrow mescenchymal stem cells, is respectively full marrow adherent method, osteocomma digestion method and marrow and adds osteocomma method.The inoculum of these three kinds of methods all derives from the shin bone of same C57BL/6 mouse.The experimental technique of these three kinds of methods is except femur is replaced with shin bone, and other is all same with embodiment 1.The experimental result of these three kinds of methods all with embodiment 1 without significant difference.
Claims (9)
1. the method for separation and Culture animal mescenchymal stem cell, comprises inoculum is inoculated in and in Zooblast culture medium, carries out former culture, then the cell that former culture the is obtained cultivation of going down to posterity, and obtains animal mescenchymal stem cell; It is characterized in that: described inoculum is for carrying out the centrifugal precipitation obtaining to the broken liquid of long bone, and the broken liquid of described long bone is that in vitro animal long bone is crushed to the mixture that 0.5-2 cubic millimeter particle obtains in buffered soln;
Described method does not comprise the step with collagenase digesting, and described method is not removed the marrow of described animal long bone.
2. method according to claim 1, is characterized in that: described animal is vertebrates.
3. method according to claim 2, is characterized in that: described vertebrates is Mammals.
4. according to arbitrary described method in claim 1-3, it is characterized in that: the inoculum for separating of cultivation animal mescenchymal stem cell of described inoculum for preparing according to arbitrary described method in claim 5-8.
5. for the preparation of the method for the inoculum of separation and Culture animal mescenchymal stem cell, comprising:
1) in vitro animal long bone is crushed in buffered soln to 0.5-2 cubic millimeter particle and obtains the broken liquid of long bone;
2) the broken liquid of long bone step 1) being obtained carries out centrifugal, gets precipitation, and this precipitation is for separating of the inoculum of cultivating animal mescenchymal stem cell.
6. method according to claim 5, is characterized in that: described animal is vertebrates.
7. method according to claim 6, is characterized in that: described vertebrates is Mammals.
8. according to the method described in claim 5 or 6, it is characterized in that: described long bone is that femur is or/and shin bone.
9. the inoculum for separating of cultivation animal mescenchymal stem cell that in claim 5-8 prepared by arbitrary described method.
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| CN108531450A (en) * | 2018-04-02 | 2018-09-14 | 中国人民解放军陆军军医大学第二附属医院 | It is a kind of extraction mesenchymal stem cell method and application |
| CN109706115A (en) * | 2017-10-26 | 2019-05-03 | 中国医科大学 | A kind of construction method of Marrow Mesenchymal Stem Cells cell line |
| CN110592008A (en) * | 2019-09-26 | 2019-12-20 | 新疆医科大学第一附属医院 | Method for culturing bone marrow mesenchymal stem cells of canine animals |
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| HENG ZHU ET AL: "A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone", 《NATURE PROTOCOLS》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109706115A (en) * | 2017-10-26 | 2019-05-03 | 中国医科大学 | A kind of construction method of Marrow Mesenchymal Stem Cells cell line |
| CN109706115B (en) * | 2017-10-26 | 2023-05-30 | 中国医科大学 | Construction method of mouse bone marrow mesenchymal stem cell line |
| CN108531450A (en) * | 2018-04-02 | 2018-09-14 | 中国人民解放军陆军军医大学第二附属医院 | It is a kind of extraction mesenchymal stem cell method and application |
| CN110592008A (en) * | 2019-09-26 | 2019-12-20 | 新疆医科大学第一附属医院 | Method for culturing bone marrow mesenchymal stem cells of canine animals |
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