CN103525761B - Method for separating and culturing animal bone marrow mesenchymal stem cell - Google Patents
Method for separating and culturing animal bone marrow mesenchymal stem cell Download PDFInfo
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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 to clinical at present and has shown good therapeutic action.Although MSC clinical application is extensive, the concrete mechanism of many key issues as immunoregulation waits and 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 obtaining 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 is little, and every 10
4-10
5individual 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 current separating mouse marrow MSC comprises full marrow adherent method, osteocomma digestion method, streaming or magnetic bead sorting method etc.Full marrow adherent method is namely according to the adherent property of MSC, regularly change liquid and remove not attached cell, thus reach the object (Soleimani of purifying MSC, M., & 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).But the MSC that the method obtains often pollutes containing more stroma cell and hematopoietic cell.Osteocomma digestion method, first marrow is rinsed, femur and shin bone are shredded, then collagenase digesting (Zhu, H., Guo is carried out, 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 different mouse osteocomma in the age collagenase digesting time is different, is not easy to grasp.Along with going deep into of being familiar with MSC surface antigen, investigator is had to utilize immunization method such as Flow cytometry, immunomagnetic beads method to carry out separation and purification (Houlihan to it, 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 higher cell of purity, sepn process is comparatively large 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 comparatively large and technical difficulty in addition, limit the widespread use of these two kinds of methods to a certain extent.Therefore the high efficiency method setting up a kind of separation and Culture MSC from marrow is needed.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of method obtaining high purity and high Board Lot (MSC that every root long bone obtains) separation and Culture marrow mescenchymal stem cell, the method also have MSC time of occurrence early, do not need removing 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 to be inoculated in Zooblast culture medium and carry out original cuiture, then the cell obtained by original cuiture carries out Secondary Culture, obtains marrow mescenchymal stem cell; Wherein, described inoculum is carry out the centrifugal precipitation obtained to the broken liquid of long bone, and the broken liquid of described long bone in vitro animal long bone is crushed in buffered soln the mixture that 0.5-1.5 cubic millimeter particle (as 1 cubic millimeter of particle) obtains;
Described method does not comprise the step with collagenase digesting, and described method does not remove the marrow of described animal long bone.
In aforesaid method, described long bone is made up 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 is that femur is or/and shin bone is or/and derive from the long bone of precursor data.
In aforesaid method, describedly centrifugally can be the centrifugal 5-10 of 300g-500g, as centrifugal in 400g 5 minutes.
Described buffered soln can be the liquid making zooblast keep active, 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 cultivated 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 Secondary Culture can be and carries out at least 2 Secondary Culture, as carried out 2 Secondary Culture.
Present invention also offers the method for the above-mentioned inoculum for separating of cultivation marrow mescenchymal stem cell of preparation.
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 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) obtained carries out centrifugal, and get precipitation, this precipitation is the inoculum for separating of cultivating marrow mescenchymal stem cell.
This prepares above-mentioned for separating of cultivating in the method for inoculum of 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 is or/and derive from the long bone of precursor data.
Describedly centrifugally can be the centrifugal 5-10 of 300g-500g, as centrifugal in 400g 5 minutes.
Described buffered soln can be the liquid making zooblast keep active, as the phosphate buffered saline buffer of 0.01M, pH7.2-7.4.
Experiment proves, the described marrow mescenchymal stem cell that the present invention obtains is spindle cell, expresses Sca1, CD44, CD29, does not express endothelial cell marker CD31 and hematopoietic cell mark CD45.The described marrow mescenchymal stem cell that the present invention obtains is cultivated and can be divided into scleroblast and adipocyte in skeletonization and adipogenic induction system.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 experimental result display of the application, marrow of the present invention adds the MSC clone-time of osteocomma method appearance 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, marrow of the present invention adds osteocomma method and obtains cell quantity at most, is the twice of full marrow adherent method and osteocomma digestion method.
Marrow of the present invention adds osteocomma method and femur and/or shin bone muscle is gone totally, and do not need to rush marrow, do not need digestion, directly shred, damping fluid is resuspended in mammalian cell culture after washing and directly inoculates.Compare full marrow adherent method and osteocomma digestion method, marrow adds osteocomma method and occurs morning time that mouse MSC clones, and quantity is many.Although first-generation peptic cell still has a small amount of CD45 positive cell, to the third generation, MSC purity can reach more than 95%, and the MSC sum obtained is 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, and CD44, CD29 etc., do not express endothelial cell marker CD31 and hematopoietic cell mark CD45.The MSC that the method obtains cultivates and can be divided into scleroblast and adipocyte in skeletonization and adipogenic induction system.In sum, marrow of the present invention adds osteocomma method and takes full advantage of marrow and osteocomma, process is simple, can obtain sufficient amount in the short period of time, growth conditions is good, multiplication capacity is strong mescenchymal stem cell, is a kind of convenient, fast, efficient MSC isolation cultivation method.
Accompanying drawing explanation
Fig. 1 is the original cuiture of MSC.
A is the cellular form being inoculated in 6 orifice plate 72h.
B is the inoculation cell quantity of the 4th day.
C is original cuiture 5 days CD45 positive cell ratios.
Fig. 2 is cellular form after going down to posterity and cell count.
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 qualification of osteocomma group (BM+Bone) respectively.
Fig. 4 is the differentiation capability that marrow adds the third generation cell of osteocomma group (BM+Bone).
The third generation cell that A, marrow add osteocomma group (BM+Bone) cultivates the ALP coloration result after 1 week through HG-DMEM nutrient solution (containing 10% foetal calf serum).
The third generation cell that B, marrow add osteocomma group (BM+Bone) cultivates the ALP coloration result after 1 week through Osteoinductive differentiation complete culture solution.
The third generation cell that C, marrow add osteocomma group (BM+Bone) cultivates Von Kossa coloration result after 2 weeks through HG-DMEM nutrient solution (containing 10% foetal calf serum).
The third generation cell that D, marrow add osteocomma group (BM+Bone) cultivates Von Kossa coloration result after 2 weeks through Osteoinductive differentiation complete culture solution.
To be the marrow third generation cell that adds osteocomma group (BM+Bone) cultivate oil red O stain result after 1 week through HG-DMEM nutrient solution (containing 10% foetal calf serum) to E.
To be the marrow third generation cell that adds osteocomma group (BM+Bone) cultivate oil red O stain result after 1 week through adipogenic induction differentiation complete culture solution to F.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Main agents in following embodiment and instrument 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 (Shanghai Excell Biology Product Co., Ltd. product cat.no.FSP500); The PBS of 0.01M, pH7.2-7.4, dexamethasone, phosphorylated Vit C, β-phospho-glycerol, II Collagenase Type, pancreatin, oil red O dye liquor, Alkaline Phosphatase Kit is Sigma Products; Detect 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 Products; 25cm
2, 75cm
2culturing bottle (costar), 6 hole/48 well culture plate (Falcon, nunc); Flow cytometer (U.S. company BD), cell culture incubator (Thermo Forma), biopurification worktable (system is closed by Zhonghua Biological Technology Inst., Beijing/Wei Da purification techniques institute, 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 have employed 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: shred to 1 cubic millimeter of particle by remaining femur after removing marrow in full marrow adherent method (after being removed by the marrow of complete femur remaining part) in the PBS of 0.01M, pH7.2-7.4, after adding 0.1% II Collagenase Type, be positioned over 37 DEG C, 5%CO
2, saturated humidity cell culture incubator in hatch 1h, the osteocomma obtained is inoculum.The inoculum that marrow adds osteocomma method is carry out the centrifugal precipitation obtained to the broken liquid of femur, the broken liquid of described femur another root femur (be complete femur, do not remove marrow) in vitro for same C57BL/6 mouse is crushed in damping fluid the mixture that 1 cubic millimeter of particle obtains.
The method MSC that the present embodiment compares three kinds of separation and Culture marrow mescenchymal stem cells clones time of occurrence, size and quantity; Flow cytometry Immunophenotyping; And carry out skeletonization, one-tenth fat differentiation qualification.Result shows that marrow adds the MSC clone-time of osteocomma method appearance 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, marrow adds osteocomma method and obtains cell quantity at most, 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.Streaming result shows, and marrow adds one of the third generation cell high expression level stem-cell marker 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 osteocomma method obtain third generation cell can respectively to scleroblast and Adipocyte Differentiation in osteogenic induction system and adipogenic induction system, the typical MSC characteristic of tool.
Specific experiment method and experimental result as follows:
1 materials and methods
1.1 laboratory animal
Healthy C57BL/6 mouse, in 3 ~ 4 week age, male and female are not limit, and are provided by Military Medical Science Institute's animal center.
The separation and Culture of 1.2MSC
Experiment in triplicate, repeat to get 1 neck marrow from disconnected execution C57BL/6 mouse at every turn, 75% alcohol immersion 5min, mouse bilateral femur is got under aseptic condition, asepticly cut blunt separation muscle, muscle is separated the PBS(phosphate buffered saline buffer that clean femur (whole femur) puts into 0.01M, pH7.2-7.4).Get side femur and cut off Distal femoral metaphysis, expose medullary space, 0.01M is drawn with 1ml syringe, the PBS of pH7.2-7.4 rinses this side marrow cavity of femur repeatedly, the marrow gone out all is put in a 1.5mlEP pipe, blow and beat gently with rifle point and make single cell suspension, by this EP pipe with the centrifugal 5min of 400g, abandon supernatant liquor, precipitation is the inoculum of full marrow adherent method (being called for short BM), get all precipitation 1ml α-MEM complete culture solutions and (in α-MEM nutrient solution, add foetal calf serum, penicillin and Streptomycin sulphate, concentration expressed in percentage by volume to foetal calf serum is 10% foetal calf serum, the concentration of penicillin is that 100,000 units often rise, the concentration of Streptomycin sulphate is that 100,000 units often rise the liquid obtained) the full marrow group (BM) of the Kong Zhongwei of re-suspended cell kind in 6 orifice plates.After this being gone out marrow, remaining femur (after being removed by the marrow of complete femur remaining part) all shreds to 1 cubic millimeter of particle in the 1.5mlEP pipe of PBS that 0.01M, pH7.2-7.4 are housed, after adding 1ml0.1% II Collagenase Type, be positioned over 37 DEG C, 5%CO
2, saturated humidity cell culture incubator in hatch 1h and obtain osteocomma, this osteocomma is the inoculum of osteocomma digestion method (being called for short Bone), then after washing whole osteocomma three times with the PBS of 0.01M, pH7.2-7.4, by whole osteocomma with in second hole of the resuspended rear kind of above-mentioned 1ml α-MEM complete culture solution in 6 orifice plates, it is osteocomma digestion group (Bone); By the opposite side femur (whole femur of this same mouse, do not remove marrow) all shred to 1 cubic millimeter of particle in the 1.5mlEP pipe of PBS that 0.01M, pH7.2-7.4 are housed, by this EP pipe with the centrifugal 5min of 400g, sucking-off supernatant, precipitation is the inoculum that marrow adds osteocomma method (being called for short BM+Bone), by all precipitating 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 respectively does three multiple holes, in 37 DEG C, 5%CO
2, saturated humidity cell culture incubator in cultivate.The later half amount of 48h changes liquid (carrying out changing liquid with above-mentioned α-MEM complete culture solution), after 72h, full dose changes liquid (carrying out changing liquid with above-mentioned α-MEM complete culture solution), often within 2-3 days, change liquid once (to carry out changing liquid with above-mentioned α-MEM complete culture solution) later, observation of cell growing state day by day under inverted microscope, 0.25% trypsin is used containing 0.02%EDTA when Growth of Cells to be paved with at the bottom of plate 80 ~ 90%) to cell (primary cell, first-generation cell) carry out digestion counting, be inoculated in 6 new orifice plates in 1:2 ratio and carry out first time and go down to posterity.The cell that first time goes down to posterity is in 37 DEG C, 5%CO
2, saturated humidity cell culture incubator in cultivate.The later half amount of 48h changes liquid (carrying out changing liquid with above-mentioned α-MEM complete culture solution), after 72h, full dose changes liquid (carrying out changing liquid with above-mentioned α-MEM complete culture solution), often within 2-3 days, change liquid once (to carry out changing liquid with above-mentioned α-MEM complete culture solution) later, observation of cell growing state day by day under inverted microscope, 0.25% trypsin is used containing 0.02%EDTA when Growth of Cells to be paved with at the bottom of plate 80 ~ 90%) digestion is carried out to cell (s-generation cell) count, be inoculated in 6 new orifice plates in 1:2 ratio and carry out second pass generation.The cell in second pass generation is in 37 DEG C, 5%CO
2, saturated humidity cell culture incubator in cultivate.The later half amount of 48h changes liquid (carrying out changing liquid with above-mentioned α-MEM complete culture solution), after 72h, full dose changes liquid (carrying out changing liquid with above-mentioned α-MEM complete culture solution), often within 2-3 days, change liquid once (to carry out changing liquid with above-mentioned α-MEM complete culture solution) later, observation of cell growing state day by day under inverted microscope, when Growth of Cells to be paved with at the bottom of plate 80 ~ 90%, obtain third generation cell.
1.3MSC morphological observation
The upgrowth situation of observation of cell and metamorphosis feature day by day on inverted microscope, before first time goes down to posterity, manifold falls to counting and takes pictures, also Taking Pictures recording before later going down to posterity.
The immunophenotypic characterization of 1.4MSC
Get growth conditions well postdigestive 3rd generation cell, PBS washes a rear overhang and becomes 1x10
6individual/ml single cell suspension, 200 μ l/EP manage, and add fluorescent-labeled antibody respectively: 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 DEG C 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
Getting third generation cell is seeded in 48 orifice plates, and every porocyte number is 2 × 10
4individual, often organize and all establish 3 multiple holes, every hole adds the above-mentioned α of 400 μ l-MEM complete culture solution, puts into 37 DEG C, 5%CO
2, saturated humidity cell culture incubator in cultivate.After cell pellet overnight is adherent, 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 μMs of phosphorylated Vit C).Control group is with HG-DMEM nutrient solution (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
Getting third generation cell is seeded in 48 orifice plates, and every porocyte number is 2 × 10
4individual, often organize and all establish 3 multiple holes, every hole adds the above-mentioned α of 400 μ l-MEM complete culture solution, puts into 37 DEG C, 5%CO
2, saturated humidity cell culture incubator in cultivate.After cell pellet overnight is adherent, suck nutrient solution, the adipogenic induction adding 400 μ l/ holes breaks up complete culture solution, and (HG-DMEM nutrient solution is containing 10% foetal calf serum and 10
-6m dexamethasone, 10ng/ml Regular Insulin, 0.5 μM of IBMX).Control group is with HG-DMEM nutrient solution (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, data acquisition mean ± standard deviation
represent, comparing between group and adopt t inspection, is that difference has statistical significance with P<0.05.
2 results
The separation and Culture of 2.1MSC
Full marrow group (BM) medullary cell is inoculated in 6 orifice plate 48h in nutrient solution based on suspension round cell, is mixed with the hematopoietic lineage cells of a large amount of more tiny circle; 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 in nutrient solution based on suspension round cell, is mixed with the hematopoietic lineage cells of a large amount of more tiny circle.After 48h half amount changes liquid, visible three groups all have a small amount of cell attachment, in 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.Be inoculated in 6 orifice plate 72h(first times change liquid after 24h) visible attached cell colony number of often organizing all has increase, but marrow adds osteocomma group (BM+Bone) increases more remarkable (in Fig. 1 A); Statistics often organizes colony number, and full marrow group (BM), osteocomma digestion group (Bone), marrow add osteocomma group (BM+Bone) and is respectively 9.5 ± 1.5/root femur, 11.5 ± 2.5/root femur, 20 ± 4/root femur (in Fig. 1 B).Original cuiture 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 in strip fusiform, and it is swirling that overall alignment is tending towards rule.Peptic cell also gets same cell amount detection CD45 expression level, find that CD45 positive cell is maximum in full marrow group (BM), reaches 78.6%, it is 10.6% that marrow adds that osteocomma group (BM+Bone) takes second place, and osteocomma digestion group (Bone) is minimum, only C in 2.66%(Fig. 1).Along with passage increased frequency, cell, by cultivating early stage Combination cell mass, gradually becomes the relatively homogeneous spindle cell of form (in Fig. 2 A), and collecting cell also counts, and marrow adds osteocomma group (BM+Bone) harvested cell number at most (in Fig. 2 B).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 the third generation cell that full marrow group (BM), osteocomma digestion group (Bone) and marrow adds osteocomma group (BM+Bone) respectively and carry out phenotypic evaluation.Streaming result display (Fig. 3), one of each group cell high expression level stem-cell marker Sca-1, high expression level adhesion molecule CD44 and CD29; Do not express MHC II quasi-molecule IA/IE, and one of endothelium mark CD31.The third generation cell that osteocomma digestion group (Bone) and marrow add osteocomma group (BM+Bone) does not express one of hematopoietic cell mark CD45, and the third generation cell of full marrow group (BM) still has the CD45 positive cell of 5%.
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 qualification, as Fig. 4 display, after osteogenic induction system induces one week, the alkaline phosphatase staining positive (in Fig. 4 B), induce VonKossa stained positive (in Fig. 4 D) after two weeks, illustrate that the third generation cytodifferentiation that marrow adds osteocomma group (BM+Bone) is scleroblast; After adipogenic induction system induces one week, cell is the oil red O stain positive (in Fig. 4 F), illustrates that the third generation cytodifferentiation that marrow adds osteocomma group (BM+Bone) is adipocyte; The dyeing that namely their control group does not add inductor is negative (in Fig. 4 A, C, E).
The experiment of the present embodiment proves 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 have employed 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 replaces with except shin bone except by femur, 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 (12)
1. the method for separation and Culture animal mescenchymal stem cell, comprises inoculum to be inoculated in Zooblast culture medium and carry out original cuiture, then the cell obtained by original cuiture carries out Secondary Culture, obtains animal mescenchymal stem cell; It is characterized in that: described inoculum is carry out the centrifugal precipitation obtained to the broken liquid of long bone, the broken liquid of described long bone in vitro animal long bone is crushed in buffered soln the mixture that 0.5-2 cubic millimeter particle obtains;
Described method does not comprise the step with collagenase digesting, and described method does not remove 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 described method arbitrary in claim 1-3, it is characterized in that: described inoculum is according to the method preparation comprised the steps: 1) in vitro animal long bone is crushed in buffered soln 0.5-2 cubic millimeter particle and obtains the broken liquid of long bone;
2) by step 1) the broken liquid of the long bone that obtains carries out centrifugal, and get precipitation, this precipitation is the inoculum for separating of cultivating animal mescenchymal stem cell.
5. method according to claim 4, is characterized in that: described animal is vertebrates.
6. method according to claim 5, is characterized in that: described vertebrates is Mammals.
7. method according to claim 4, is characterized in that: described long bone is that femur is or/and shin bone.
8., 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 0.5-2 cubic millimeter particle and obtains the broken liquid of long bone;
2) by step 1) the broken liquid of the long bone that obtains carries out centrifugal, and get precipitation, this precipitation is the inoculum for separating of cultivating animal mescenchymal stem cell.
9. method according to claim 8, is characterized in that: described animal is vertebrates.
10. method according to claim 9, is characterized in that: described vertebrates is Mammals.
11. methods according to claim 8 or claim 9, is characterized in that: described long bone is that femur is or/and shin bone.
12. inoculums for separating of cultivation animal mescenchymal stem cell prepared by described method arbitrary in claim 8-11.
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| 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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| A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow;Masoud Soleimani et al;《NATURE PROTOCOLS》;20090108;第4卷(第1期);102-106 * |
| A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone;Heng Zhu et al;《nature protocols》;20100225;第5卷(第3期);550-560 * |
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