CN102002478B - Adipose-derived stem cell separation culture method - Google Patents
Adipose-derived stem cell separation culture method Download PDFInfo
- Publication number
- CN102002478B CN102002478B CN 201010568873 CN201010568873A CN102002478B CN 102002478 B CN102002478 B CN 102002478B CN 201010568873 CN201010568873 CN 201010568873 CN 201010568873 A CN201010568873 A CN 201010568873A CN 102002478 B CN102002478 B CN 102002478B
- Authority
- CN
- China
- Prior art keywords
- cell
- stem cell
- fat
- fat stem
- obtains
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an adipose-derived stem cell separation culture method comprising the following steps of: separating and acquiring from adipose tissues; and culturing SVF (Stromal-vascular fraction) cells. The separation culture method is characterized by also comprising the following steps of: removing Lin+cells included in the SVF cells to obtain an Lin-cell mass by using a magnetic-activated cell sorting method; gathering CD271+Sca-1+cells from the obtained Lin-cell mass to obtain the adipose-derived stem cells by using a fluorescent-activated cell sorting method; and culturing the obtained adipose-derived stem cells by using a culture medium containing LIF (Leukemia Inhibitory Factor) and FGF2 (Fibroblast Growth Factor). The invention can efficiently and fast obtain the high-purity adipose-derived stem cells by combining FACS (Fluorescent-Activated Cell Sorting) with MACS (Magnetic-Activated Cell Sorting), wherein the high-purity adipose-derived stem cells have higher clone forming ability, self-renewal ability and multi-directional differentiation potentiality; P16 generation adipose-derived stem cells obtained by the separation culture method still have higher adipose forming ability and bone forming ability; in addition, the invention provides a new method for acquiring a large quantity of seed cells with dryness in vitro to apply to tissue regeneration.
Description
Technical field
The present invention relates to the isolation cultivation method of a kind of stem cell, specifically, relate to a kind of combine method of separation and Culture high purity fat stem cell of fluorescence-activation fluidic cell classification and Magnetic activated cell sorting that adopts.
Background technology
Fat stem cell (Adipose-derived Stem Cells, ASCs) is a kind of adult stem cell (Cowan CM .Nat Biotechnol, 2004. 22:560-567) that is widely used at present organizational project and regenerative medicine research field.Fat stem cell is the same with mesenchymal stem cells MSCs to have multi-lineage potential, under given conditions can be to a plurality of directions differentiation such as adipocyte, scleroblast, chondroblast, sarcoplast, one-tenth endotheliocyte and neuroblast.In addition, fat stem cell also has the not available advantage of many other types adult stem cells, damage slightly, dispute on without ethics such as adipose tissue-derived abundance, the convenience of drawing materials, acquisition process, on average can obtain about 1 * 10 in the fatty tissue of per 100 mL
6Individual stem cell, the cell of adipose tissue-derived have 2% to have cells and characteristic of stem approximately, far above mesenchymal stem cells MSCs (bone marrow mesenchymal stem cells, BMSCs) (about 0.02% cell has cells and characteristic of stem).And fat stem cell has stable population doublings rate, self potential and good immune compatibility (Strem BM,. Trends Biotechnol, 2005.24:1246-1253), its efficiency gene transfection is high, energy stable expression of exogenous gene, research (the Gimble JM that has been widely used in regenerative medicine,. Circ Res, 2007:1249-1260).And ASCs to be divided into the efficiency ratio BMSCs of adipocyte higher.Therefore, fat stem cell is one of desirable seed cell of Tissue Engineering Study, promises to be the seed cell of adipose tissue engineering most.
At present fat stem cell isolation cultivation method commonly used is to separate from fatty tissue to obtain cell, after machinery or method of enzymatically treating separate mature cells such as removing red corpuscle, adopts the substratum that contains 10% foetal calf serum to carry out succeeding transfer culture.Though the substratum that contains 10% foetal calf serum can promote the propagation of fat stem cell to be difficult to keep its undifferentiated state, so that the fat stem cell of succeeding transfer culture is aging easily.Machinery or method of enzymatically treating only can be removed the mature cells such as red corpuscle, except this part the cell essence after red be between matter blood vessel fragment cell (Stromal-vascular fraction, the SVF cell), still contain a large amount of mature cells in this part cell, thereby, the existing fat stem cell purity that obtains is not high, because the growth of cell is relevant with the signal traffic between the cell, the stem cell that purity is not high is easier to be aging, often external cultivate fat stem cell through going down to posterity for 5-6 time after, fat stem cell just occurs aging.Stem cell aging (or claiming old and feeble) refers to that the stem cells hyperplasia ability descends, and multi-lineage potential (or claiming dryness) reduces or disappears.Wearing out means the minimizing of stem cell population and going down of function, also can be described as and can't regenerate.Fat stem cell aging limited its further investigation of using in adipose tissue engineering and hetero-organization engineering thereof.
The method of separation and purification stem cell has fluidic cell separating method, immunological magnetic bead sorting method etc. at present, from amniotic fluid, obtain the fetus mescenchymal stem cell such as utilization fluidic cell separating methods such as In ' t Anker, the utilization immunomagnetic beads methods such as Coppi are isolated AFS cell (amniotic fluid stem cells, AFS) from amniocyte.But these in-vitro separation technology to stem cell are to be based upon on the basis that cell surface marker is identified mostly, c-Kit or CD117(STEM CELL FACTOR acceptor such as isolated AFS cells such as Coppi) express positive cell, and various adult stem cell has unique separately marker.Rodeheffer MS etc. are separated to Lin with cell airflow classification (Fluorescent-activated cell sorting, FACS) from fatty tissue
-CD29
+CD34
+Sca-1
+CD24
+Cell mass, but this group cell can only be to Adipocyte Differentiation, and gained quantity lower (0.08% ± 0.015)).Up to the present, the specific marker of fat stem cell does not also find, also do not find adipose stromal cells (Adipose-derived stromal cells, ADSCs) inner cell mass with dryness, research both domestic and external all can not obtain highly purified fat stem cell (the high purity fat stem cell refers to the stem cell that the cell-surface antigens sign is identical, have homogeneity, the self a group adipose tissue-derived consistent with the Multidirectional Differentiation ability).
The existing problem that separative fat stem cell purity is not high, the fat stem cell vitro culture is difficult to keep self and multi-lineage potential has greatly limited contrast and repetition between the domestic and international result of study.
Summary of the invention
The object of the invention is to overcome in the prior art deficiency that fat stem cell under the condition of in vitro culture is difficult to keep self and multi-lineage potential, a kind of isolation cultivation method and effectively new substratum thereof of fat stem cell is provided.Through according to the method separation and Culture fat stem cell, through more than 10 for succeeding transfer culture after fat stem cell still can keep the potential of its self and Multidirectional Differentiation.
The present invention realizes that the technical scheme of foregoing invention purpose is:
A kind of isolation cultivation method of fat stem cell comprises from fatty tissue separating and obtains, cultivates the SVF cell, also comprises with the immunological magnetic bead sorting method removing Lin in the SVF cell
+Cell obtains Lin
-Cell mass; With the Lin of fluidic cell separating method from obtaining
-Enrichment CD271 in the cell mass
+Sca-1
+Cell obtains fat stem cell; With the fat stem cell culture medium culturing that contains LIF, FGF2 that obtains.
In the isolation cultivation method of above-mentioned fat stem cell, the marker that adopts in the immunological magnetic bead sorting method is CD5, CD45R, CD11b, Anti-Gr-1 and Ter-119.
In the isolation cultivation method of above-mentioned fat stem cell, the marker that uses in the fluidic cell separating method is Lin, CD271 and Sca-1.
In the isolation cultivation method of above-mentioned fat stem cell, the used substratum of fat stem cell that cultivate to obtain is the α that contains LIF, FGF2-MEM substratum.
In the isolation cultivation method of above-mentioned fat stem cell, cultivating the used substratum of fat stem cell that obtains is to contain 10% volume ratio foetal calf serum, 10
3-10
5The α of U/mL LIF, 10-100 ng/ mL FGF2-MEM substratum.
In the isolation cultivation method of above-mentioned fat stem cell, cultivating the used substratum of fat stem cell that obtains is to contain 10% foetal calf serum, 10
3The α of U/mL LIF, 20 ng/mL FGF2-MEM substratum.
In the isolation cultivation method of above-mentioned fat stem cell, also comprise the fat stem cell that the obtains cultivation of going down to posterity.
In the isolation cultivation method of above-mentioned fat stem cell, the described cultivation of going down to posterity is adherent culture.
In the isolation cultivation method of above-mentioned fat stem cell, when the stand density of the fat stem cell that obtains in the culture dish bottom reaches 70-90%, the cultivation of going down to posterity.
Preferably, in the isolation cultivation method of above-mentioned fat stem cell, when the stand density of the fat stem cell that obtains in the culture dish bottom reaches 80%, the cultivation of going down to posterity.
In the isolation cultivation method of above-mentioned fat stem cell, separating the process of obtaining the SVF cell from fatty tissue is: cut mouse inguinal region fat pad under conventional aseptic condition, through rinsing, shred, except after blood etc. processes, in fatty tissue, add and the isopyknic 0.1% NTx enzyme of fat, be the SVF cell through digesting, filter, split the cell that obtains after red, centrifugal.
In the isolation cultivation method of above-mentioned fat stem cell, the α contain 10% volume ratio foetal calf serum-MEM substratum is adopted in the cultivation of SVF cell, and culture condition is 37 ℃, 5%(v/v) CO
2
The fat stem cell of the isolation cultivation method of the fat stem cell of any one institute separation and Culture as previously mentioned is used for the production of purifying or clone stem cell, transgenosis stem cell line or for the production of becoming fat/scleroblast.
Fat stem cell is for the production of becoming fat/osteoblastic method to be as previously mentioned:
(1), the classification of fluorescence-activation fluidic cell combines to separate with Magnetic activated cell sorting and obtains ASCs;
(2), with ASCs respectively conjugated fibre albumen and BCP timbering material, be inoculated in the damaged place of nude mice back or mouse femur.
6-12 took out graft and carries out HE section statining, immunohistochemistry detection etc. after week.
The cultivation of going down to posterity of fat stem cell occurs aging easily in the prior art, for this problem, the contriver conducts extensive research, found that, to separate the SVF cell that obtains from fatty tissue combines with immunological magnetic bead sorting method and fluidic cell separating method, can remove as much as possible the mature cell in the SVF cell, can obtain thus the antigen sign identical, have homogeneity, a self high purity fat stem cell consistent with the Multidirectional Differentiation ability.The contriver has compared in substratum and to have added the factor LIF that suppresses differentiation of stem cells and the factor FGF2 that promotes stem cells hyperplasia and the clonality under the cellar culture condition, Multidirectional Differentiation ability, found that to add the factor LIF that suppresses cytodifferentiation and promote the growth factor FGF-2 2 of stem cells hyperplasia can keep the dryness of the high purity stem cell of acquisition in substratum.
Therefore, the present invention separates from fatty tissue and obtains, cultivates the SVF cellular fat, adopts the MACS method to remove CD5 from the SVF cell again
+, CD45R
+, CD11b
+And Ter
-119
+These hematopoiesis and mature cell, enrichment Lin
-Cell; Then adopt FACS from Lin
-Sorting CD271, Sca-1 express positive cell, enrichment CD271 in the cell mass
+Sca-1
+Cell (being fat stem cell ASCs); After obtaining fat stem cell, by in substratum, adding the factor LIF that suppresses cytodifferentiation and promoting the growth factor FGF-2 2 of stem cells hyperplasia to keep the dryness of stem cell.The present invention has compared in substratum the factor LIF that suppresses differentiation of stem cells and the factor FGF2 that promotes stem cells hyperplasia and the clonality under the cellar culture condition, the Multidirectional Differentiation ability of adding, proved in substratum, to add LIF and FGF2, helped to keep the dryness of fat stem cell.The present invention has measured cultivation and the clonality of ASCs, and confirms that from aspects such as morphology, immunocytochemistry, mRNA and protein levels the ASCs obtain has a function such as multi-lineage potential with external in vivo.
The present invention adopts FACS and MACS to combine, and can efficiently obtain fast highly purified fat stem cell, and compared with prior art, the present invention has the following advantages and positively effect:
1, the present invention adopts the method that the fluorescence-activation fluidic cell is classified (FACS) and Magnetic activated cell sorting (MACS) combines to use first Lin at home and abroad
-: CD271
+: Sca-1
+Be enriched to highly purified fat stem cell subgroup, this group cell has stronger clonality, self-renewal capacity and multi-lineage potential, and other cell does not possess this ability is arranged.
2, the present invention uses the conditioned medium culturing cell that contains LIF and FGF2, and P16 fat subsitutes stem cell still has stronger one-tenth fat ability, and P16 fat subsitutes stem cell still has the ability of skeletonization.
3, the present invention uses scleroproein and biophasic calcium phosphate ceramic (BCP) as the carrier of fat stem cell, adopt the ASCs of marrow of transgenic GFP mouse as spike, proved that ASCs becomes in fat/skeletonization microenvironment in vivo can be converted into adipocyte and scleroblast.
4, the present invention found a kind of feasible, efficiently obtain the method for fat stem cell, for obtaining a large amount of seed cells with dryness and apply to tissue regeneration new method is provided external.
Description of drawings
The Lin of Fig. 1 for obtaining through MACS
-Then cell mass dyes rear fluidic cell sorting altogether with CD271 and two antibody of Sca-1, obtains Lin
-: CD271
+: Sca-1
+Cell mass (being fat stem cell).
Fig. 2 is for adopting limiting dilution assay that ASCs is cloned cultivation: wherein A is Lin
-: CD271
+: Sca-1
+Cell mass has formed clone (the 3rd day), Scale bars=100 μ m; B is Lin
-: CD271
+: Sca-1
+The clone (the 5th day) that cell mass forms, Scale bars=100 μ m.
Fig. 3 A, B are the light microscopic figure of fat stem cell growth conditions, and cell is for becoming the fiber-like fusiformis, visible typical whirlpool shape growth, the Scale bars=40 μ m of A figure wherein, the Scale bars=100 μ m of B figure.
Fig. 4 is the external Multidirectional Differentiation figure of ASCs, wherein A: after becoming fat to induce, oil red O dyes lipid for orange red, Scale bars=200 μ m; B: behind the Osteoinductive differentiation, Alizarin red staining shows that the calcium tubercle is that dye the red of sheet, Scale bars=100 μ m; C: after becoming the chondrocyte induction differentiation, Toluidine blue staining shows that cartilage matrix is blue, has cartilage cavities to form Scale bars=400 μ m; D: after becoming flesh to induce differentiation, α-SMA immunocytochemical stain is positive, and DAPI is blue with nuclei dyeing, Scale bars=400 μ m.
Fig. 5 is that RT-PCR detects ASCs after becoming fat (PPAR γ 2, C/EBP-α, LPL), skeletonization (Runx2, Opn), becoming cartilage (Acan, Sox9) and become flesh (Myog, Myod1) to induce, and the expression of genes involved, confidential reference items are GAPDH.The result shows, after multidirectional the inducing of process, the expression level of PPAR γ 2, C/EBP-α, LPL, Acan, Sox9, Myog, Myod1 gene raises, and has proved absolutely that from the mRNA level fat stem cell that we cultivate can be to fat, bone, cartilage, the differentiation of myocyte's direction.
Fig. 6 FGF2 and LIF are on the impact of ASCs multiplication capacity.The substratum that adds FGF2 and LIF has been kept the fast breeding ability of ASCs, has been stablized the cell colony doubling time; In the contorl group without FGF2, LIF substratum, along with passage number increases, the cell colony doubling time prolongs.
Fig. 7 is FGF2 and LIF form ability on the CFU-F of ASCs impact.FGF2 and LIF substratum group CFU-F form ability without obvious decline; And form obvious minimizing without CFU-F in the contorl group of FGF2, LIF substratum.
Fig. 8 is FGF2 and LIF become the fat differentiation potential on keeping ASCs impact.A be FGF2 with the LIF substratum in P16 become fat to induce oil red O stain result after 10 days for ASCs, B becomes fat to induce oil red O stain result after 10 days for the P16 that cultivates without FGF2 and LIF for ASCs, ASCs one-tenth fat ability obviously reduces Scale bars=200 μ m.
Fig. 9 glycerol-3-phosphate dehydrogenase active level is measured.From P6 for, FGF2, LIF are arranged and two groups of the ASCs that cultivate without FGF2, LIF between become fat to induce rear glycerol-3-phosphate dehydrogenase activity significant difference to occur, data represent (n=3) with mean ± SE, (P ﹤ 0.01).
Figure 10 A: have under the culture condition of FGF2 and LIF, P16 forms for the visible mineralising tubercle of ASCs osteogenic induction hystazarin red colouring; B: under the culture condition without FGF2 and LIF, P16 has no the mineralising tubercle after for the ASCs osteogenic induction and forms.Scale?bars=?100μm。
The GFP fat stem cell of Figure 11 C57 mouse is fibroblast-like fusiformis, drips without fat in the born of the same parents, and egfp expression is all arranged.Figure 11 A is the image of ordinary optical microscope, and Figure 11 B is the image that inverted fluorescence microscope is observed.Scale?bars=?40μm。
Figure 12 cell-scleroproein mixture one-tenth fat situation behind the 6w that implants.The A:HE demonstration of dyeing, rounded or Polygons, kytoplasm contains cavity, and cavity is dissolved the position that fat drips for making in the slicing processes, and karyon is dripped by fat and presses to a side, is oblateness, Scale bars=200 μ m.B: oil red O stain shows that fat drips and is that orange, nucleus are pressed against on one side, are blue, Scale bars=200 μ m.C: the anti-GFP immunohistochemistry of newborn adipocyte is positive, Scale bars=100 μ m.
Skeletonization situation after Figure 13 cell-BCP timbering material mixture implants.A, B are HE and Masson dyeing, and newly bone mainly forms in the hole of BCP timbering material, are the bone island sample and distribute, and new bone periphery is scleroblast, and the visible rose pink bone trabecula of HE dyeing reaches just at the collegen filament of mineralising, can see the formation of bone lacuna; The visible new bone trabecula that forms of Masson dyeing and be blueness at the collegen filament of mineralising just.Scale?bars=200μm。C is that new fracture GFP immunohistochemistry detects, wherein the positive cell of light brown person, the as seen anti-GFP of scleroblast positive (shown in the red arrow).Scale?bars=200μm。
Embodiment
The present invention is described in further detail below in conjunction with test example and embodiment.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following embodiment, all technology that realizes based on content of the present invention all belong to scope of the present invention.
The adipose tissue-derived of mentioning in the embodiment of the invention is in mouse, and used main raw, reagent and equipment are as follows in the embodiment of the invention:
C57 mouse (being provided by Sichuan University's West China medical experiment animal center);
Gfp transgene C57 mouse (biotherapy National Key Laboratory is so kind as to give the stem cell biology research department);
Nude mouse (available from Institute of Experimental Animals, Chinese Academy of Medical Sciences);
Fibrin Glue support (the general Ji in Hangzhou, China);
Porous biophasic calcium phosphate ceramic (BCP) timbering material (being provided by country of Sichuan University biomedical material Engineering Technical Research Centre).
α-MEM substratum (Hyclone, the U.S.);
Foetal calf serum (Hyclone, the U.S.);
Trypsinase-ethylenediamine tetraacetic acid (EDTA) (EDTA) (Gibco, the U.S.);
FGF2 (Peprotech Inc, the U.S.);
LIF, Anti-GFP antibody (Millipore, the U.S.);
Type i collagen enzyme (COLLAGENASE TYPE I), dimethyl sulfoxide (DMSO) (DMSO), dexamethasone (Dex), L-AA (L-ascorbic acid), 1,25-dihydroxyvitamin D3 (1,25-(OH) 2VitD3), 3-isobutyl-1-methylxanthine (IBMX), indomethacin, oil red O is all available from U.S. Sigma company;
RNA extraction agent box (Shanghai Hua Shun, China);
TaKaRa one step RNA PCR Kit (AMV), DL2000 are all available from Japanese TaKaRa company.
Constant water bath box (Heto-Hoten, Denmark); Micropipet (Gilson company, France); Culturing bottle (Corning, the U.S.), electronic balance (Strtorius, the U.S.), 0.22pm Millex Syringe Filters (Millipor, the U.S.), ultrapure water machine UNIQUE-R30(Millipore, the U.S.); CO2 incubator (Thermo, Germany, desk centrifuge SORVALLR LEGEND RM(Thermo, Germany), temperature control desk centrifuge SORVALLR LEGEND T(Thermo, Germany), Thermo KS12 Bechtop (Thermo, Germany), Thermo-86 ℃ of HERA freeze, (Thermo, Germany); OLYMPUS IX71 is inverted and differs fluorescent microscope (Olympus, Japan), OLYMPUS CKX41 inverted microscope (Olympus, Japan), OLYMPUS CX41 is just putting microscope (Olympus, Japan) ,-152 ℃ of Ultralow Temperature Freezer (SANYO, Japan), scanning electronic microscope (JSM-5900LV) (Jeol Ltd.); Tanon-2500 gel imaging instrument (sky, Shanghai energy, China); PCR instrument (BIO-RAD, the U.S.), gel imaging analysis software Quantity One 4.6.2 (BIO-RAD, the U.S.).
The isolation cultivation method of the fat stem cell described in the embodiment that the present invention enumerates comprises from the mouse fatty tissue separating and obtains, cultivates the SVF cell, also comprises with the immunological magnetic bead sorting method removing Lin in the SVF cell
+Cell obtains Lin
-Cell mass; With the Lin of fluidic cell separating method from obtaining
-Enrichment CD271 in the cell mass
+Sca-1
+Cell obtains fat stem cell; The fat stem cell usefulness that obtains is contained the culture medium culturing of LIF, FGF2 and the cultivation of going down to posterity.
In the isolation cultivation method of above-mentioned fat stem cell, from the mouse fatty tissue, separate and obtain, cultivate the SVF cell and comprise, under conventional aseptic condition, cut mouse inguinal region fat pad, through rinsing, shred, except after blood processes, in fatty tissue, add and the isopyknic 0.1% NTx enzyme of fat, be the SVF cell through digesting, filter, split the cell that obtains after red, centrifugal.
The α that contains 10% foetal calf serum-MEM substratum is adopted in the cultivation of SVF cell, and culture condition is v) CO of 37 ℃, 5%(v/
2
The marker that adopts in the immunological magnetic bead sorting method is CD5, CD45R, CD11b, Anti-Gr-1 and Ter-119.The marker that uses in the fluidic cell separating method is Lin, CD271 and Sca-1.
Cultivating the used substratum of fat stem cell that obtains is to contain 10% volume ratio foetal calf serum, 10
3The α of U/mLLIF, 20ng/mLFGF2-MEM substratum.
Carry out the adherent cultivation of going down to posterity, when the stand density of the fat stem cell that obtains in the culture dish bottom reaches 80%, cultivations of going down to posterity, and with the fat stem cell of P16, for the production of one-tenth fat/scleroblast.
1, the obtaining and cultivating of matter blood vessel fragment (SVF) cell:
1) gets female C57 mouse in 4 ages in week, with vetanarcol 0.1mg/100g intraperitoneal injection of anesthesia, under aseptic condition, get rat inguinal region fat pad after the anesthesia produce effects, with aseptic PBS solution cleansing tissue piece;
2) in Bechtop, the fatty tissue piece is shredded, on 37 ℃ of constant-temperature tables, digest 45-60 min with equal-volume 0.1% NTx enzyme, during suitably vibrate with abundant digestion;
3) add in the substratum that contains 10% volume ratio foetal calf serum and the NTx enzyme, remove not digest completely larger tissue block and the remaining residue of digestion with the cell sieving of 70 μ m, low-speed centrifugal (1200g, 5 min), the centrifuge tube bottom is cell mass;
4) absorb fatty tissue and the mature fat cell of surface flotation, cell mass is resuspended and leave standstill 10 min, centrifugal (300g, 5 min) with freshly prepared erythrocyte cracked liquid;
5) collect the cell mass (a matter blood vessel fragment cell, SVF cell) of centrifuge tube bottom, substratum washing 3 times is inoculated in the culture dish of diameter 10cm by the cell density of 1 * 105/mL, at saturated humidity, 37 ℃, 5%CO
2Incubator in cultivate, per 3 days replaced mediums, substratum are the 10% volume ratio FBS+ α-MEM substratum that contains 100u/mL penicillin and 100 μ g/mL Streptomycin sulphates;
6) cell reaches about 80% when merging at culture dish bill kept on file layer growth, the cultivation of going down to posterity.
2, enrichment fat stem cell from the SVF cell
2.1 obtain SVF, select Lineage Cell Depletion Kit the moon to select Lin
-Cell:
1) with the SVF cell counting that obtains, PBS solution is washed, and centrifugal (300 * g, 10 min) absorb supernatant fully.
2) 40 μ L damping fluids resuspended 10
7Individual cell.
3) add 10 μ L Biotin-Antibody Cocktail in 10
7In the cell.
4) abundant mixing is hatched 10 min for 4 ℃.
5) add 30 μ L damping fluids in 10
7In the cell.
6) add 20 μ L Anti-Biotin Microbeads.
7) fully behind the mixing 4 ℃ hatch 15 min.
8) 1-2 mL damping fluid is washed cell, and centrifugal (300 * g, 10 min) absorb supernatant.
9) re-suspended cell is in 500 μ L damping fluids.
10) the magnetic post is put in the correct position of magnet stand, 500 μ L damping fluids are washed pillar once.
11) 500 μ L cell suspensions are added in the magnetic post, collect the liquid that flows out, the inside is contained to be Lin
-Cell
12) 500 μ L damping fluids are washed the magnetic post 3 times, and the effluent liquid that the effluent liquid of collection and previous step are collected mixes.
13) move lower magnetic post, place new collection tube top, add 1 mL damping fluid in the magnetic post, release rapidly the cell of magnetic mark.This gained cell is Lin
+Cell (CD5, CD45R, CD11b, Anti-Gr-1 or Ter-119 are positive).
2.2 FACS is from Lin
-Enrichment CD271 in the cell mass
+Sca-1
+Cell
1) Lin that previous step is collected
-Cell count places 1.5mL EP pipe.
2) PBS liquid is washed once, with the α that does not contain serum-MEM substratum re-suspended cell.
3) the CD271 primary antibodie (the anti-mouse of rabbit) of adding purified, lucifuge is hatched 30 min.
4) PBS liquid is washed, and with the α that does not contain serum-MEM substratum re-suspended cell, adds fluorescence two anti-(the anti-rabbit of monkey), hatches 30 min.
5) PBS liquid is washed, and with the α that does not contain serum-MEM substratum re-suspended cell, adds Sca-1 and 7AAD or DAPI, hatches 30 min.
6) PBS liquid is washed, and with the α that does not contain serum-MEM substratum re-suspended cell, the aseptic sorting of upflowing cell instrument obtains Lin
-: CD271
+: Sca-1
+A group cell and Lin
-: CD271
-And Lin
-: CD271
+: Sca-1
-Cell mass.
7) Lin that obtains
-: CD271
+: Sca-1
+Cell mass is cultivated.Simultaneously, with the Lin that collects
+Cell mass, Lin
-: CD271
-Cell mass and Lin
-: CD271
+: Sca-1
-Cell mass is also cultivated.
3, the mensuration of ASCs clonality (Clony-forming unit-fibroblast, CFU-F)
Adopt the limiting dilution method to clone cultivation the cell mass of fresh separated, make the suspension of individual cells with 20%FBS+ α-MEM substratum, single cell is drawn in each hole of 48 orifice plates with micropipet, add 20%FBS+ α-MEM substratum.
Detect clone's formational situation behind the 14d, cloning efficiency=(clone's formation/inoculating cell number) * 100%, cloning efficiency is 15.7% ± 0.58 after measured, cell forms clone in good condition as shown in Figure 2.
4, the detection of the external Multidirectional Differentiation ability of ASCs, this step are in order to verify the Multidirectional Differentiation ability of the fat stem cell that obtains.
4.1 becoming fat induces
P1 is inoculated in six orifice plates for ASCs, adds into the fat induced liquid behind 24 h and cultivate, the induced liquid composition sees Table 1, and amount was changed liquid in per 3 days half; Become fat to induce and use 4% Paraformaldehyde 96 fixed cell after 10 days, carry out oil red O stain, DAPI dyes nuclear.Shown in Fig. 4 A, round fat drips and takes on a red color, be distributed in cell around, illustrate that cell can be to the differentiation of fatty direction.
The various induced liquid prescriptions of table 1
RT-PCR is detected as and becomes fat genes involved peroxisome proliferation body activated receptor γ 2 (peroxisome proliferator-activated receptor-γ 2 in the fat Induction Process, PPAR γ 2), CCAAT enhancer binding protein α (CCAAT/enhancer-binding α, C/EBP-α) and the expression of lipoprotein lipase (lipoprotein lipase, Lpl).RNA extraction agent box with reference to Shanghai China Shun Bioisystech Co., Ltd extracts cell total rna, and is then according to the synthetic cDNA of the operation instructions of TaKaRa One Step RNA PCR Kit (AMV), that the cDNA template-20 ℃ storage that obtains is for subsequent use.According to gene order to be detected, adopt Primer 5.0 primer-design softwares design primer, with house-keeping gene GAPDH as confidential reference items.The forward primer (Forward primer) of design, reverse primer (Reverse primer), annealing temperature (annealing temperature, Tm) and expection product size etc. see Table 2.As shown in Figure 5, after induce, the specific gene PPAR γ 2 of adipocyte, the expression of C/EBP-α, Lpl strengthen cell significantly, further show into fat from gene level and break up successfully through fat.
4.2 osteogenic induction
P1 is inoculated in six orifice plates for ASCs, changes osteogenic induction liquid behind the 24h into and cultivate, the induced liquid composition sees Table 1, changes liquid in per 3 days.With the capable Alizarin red staining of cell climbing sheet, observe the formational situation of mineralising tubercle behind the osteogenic induction cultivation 14d.Shown in Fig. 4 B, fat stem cell after induce, is divided into scleroblast through bone, has typical red calcium tubercle to form.
RT-PCR detects Bone formation-related gene Runt associated transcription factor 2/ core-binding factor a1 (runt related transcription factor 2/ core binding factor a1 in the skeletonization culturing process, Runx2/Cbfal) and the expression of osteopontin (osteopontin, OPN).As shown in Figure 5, the expression of Runx2, OPN significantly raises, and further shows the Osteoblast Differentiation success from gene level.
4.3 one-tenth chondrocyte induction
P1 is resuspended in the 1.2% low viscous alginate jelly with the cell concn of 1 * 107/mL for ASCs, with the CaCl2 solution crosslinking of 100mM, places into chondrocyte induction liquid and cultivates, and the induced liquid composition sees Table 1, changes liquid in per 3 days.Become chondrocyte induction to cultivate after 3 weeks sample fixed, cuts into slices the rear Toluidine blue staining of use, the formation of observation cartilage matrix.Shown in Fig. 4 C, fat stem cell is after becoming chondrocyte induction, and Toluidine blue staining is positive, illustrates that fat stem cell can break up to the cartilage direction.
RT-PCR is detected as the expression that cartilage genes involved SRY box comprises gene 9 (SRY-box containing gene 9, Sox9)) and aggrecan (aggrecan, Acan).As shown in Figure 5, the genetic expression of these 2 cartilage specificities strengthens, and further shows into the cartilage differentiation success from gene level.
4.4 becoming flesh induces
P1 is inoculated in six orifice plates for ASCs, changes into the flesh induced liquid behind the 24h into and cultivate, the induced liquid composition sees Table the hydrocortisone that 1(contains 50 μ mol/L, 5%HS, the DMEM/F12 of 10%FBS).4% Paraformaldehyde 96 is fixed α-SMA antibody mediated immunity fluorescent dye after 3 weeks.Shown in Fig. 4 D, the immunocytochemistry fluorescent dye of smooth muscle cell specific proteins α-SMA be positive (green).
RT-PCR is detected as the expression of myocyte's genes involved Myog and Myod1.As shown in Figure 5, the expression amount of Myog and Myod1 significantly increases, and confirms that further ASCs is through becoming flesh to induce backward one-tenth flesh direction differentiation on the mRNA level.
The primer sequence of gene to be detected is as shown in table 2:
The primer of table 2 testing gene and expection product situation
Annotate: PPAR-γ 2: peroxisome proliferation body activated receptor γ 2; C/EBP-α: CCAAT enhancer binding protein α; Lpl: the white enzyme of fat; Runx2:Runt associated transcription factor 2; Opn: osteopontin; The Sox9:SRY box comprises gene 9; Acan: aggrecan; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.
5, add FGF2 and LIF to the impact of ASCs propagation and Multidirectional Differentiation ability
5.1 FGF2 and LIF are on the impact of ASCs multiplication capacity
1) ASCs is divided into without FGF2 and LIF group and FGF2 is arranged and the LIF group, FGF2 and LIF group add the FGF2 of 20 ng/mL and the LIF of 1000 units/mL in 10% FBS (v/ v)+α-MEM substratum.Get respectively the ASCs in the 1st, 6,11 and 16 generations in two groups, with 5 * 103/ holes the cell kind is entered in 12 orifice plates, 2,4,6,8 and 10d take out respectively the cell that adds and do not add FGF2, LIF, detect cell number with mtt assay, draw growth curve, calculate the population doubling time of cell.
2) with 10/cm
2Inoculating cell, detecting respectively behind 14 d has and without the clone's formational situation among two groups of FGF2, the LIF, calculates cloning efficiency.
5.2 FGF2 and LIF are to keeping the effect of ASCs multi-lineage potential
5.2.1 on becoming the impact of fat differentiation potential
Get respectively the ASCs in the 1st, 6,11 and 16 generations in two groups, with identical density inoculating cell.Add into the fat induced liquid behind the 24h and cultivate, become fat to induce after 10 days and carry out oil red O stain.By glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate dehydrogenase, GPDH) the adipocyte formation volume is judged in determination of activity, the GPDH activity determination method is with reference to the method for the reports such as Hauner: PBS washes the purpose cell, the 25 mM Tris-HCl damping fluid collecting cells (pH 7.5) that comprise 1 mM EDTA of precooling, get supernatant behind the ultrasonic degradation cell, UV spectrophotometer measuring GPDH is active, reaction system: 100 mM trolamine HCl damping fluids (pH 7.5), 2.5 mM EDTA, 0.12mM NADH, 0.1 mM mercaptoethanol and 0.2 mM phosphodihydroxyacetone.
5.2.2 the impact on Osteoblast Differentiation potential
1) gets respectively the ASCs in the 1st, 3,6,11 and 16 generations in two groups, with identical density inoculating cell.Add osteogenic induction liquid behind 24 h and cultivate, carry out Alizarin red staining after 14 days, observe the formational situation of mineralising tubercle.
2) with P1 for the conventional osteogenic induction of one group of row of ASCs, other one group adds the FGF2 of 20 ng/mL and the LIF of 1000 units/mL simultaneously in osteogenic induction liquid.Extracted total RNA at the 4th, 7,14 and 21 day respectively, according to preceding method Bone formation-related gene Runx2 and Ocn are detected.
3) according to 1) same grouping, extract respectively total RNA of the ASCs in the 1st, 3,6,11 and 16 generations that have or cultivate without FGF2 and LIF, RT-PCR detects the mrna expression of the early stage genes involved Runx2 of skeletonization, judges whether it has osteogenic potential.
The result shows that under the culture condition without FGF-2 and LIF, ASCs cultivates P16 generation, has no the mineralising tubercle behind the osteogenic induction and forms, and have under the culture condition of FGF-2 and LIF, and ASCs cultivates P16 and forms for still seeing the mineralising tubercle.In the osteogenic induction substratum without FGF-2 and LIF, skeletonization early gene Runx2 expression amount in the time of 7～14 days is higher for ASCs for P1, expresses in the time of 21 days to disappear, and expressing appearred in the time of the 7th day in skeletonization significant gene Ocn in late period, continued to 21 days.And contain in the osteogenic induction substratum of FGF-2 and LIF, the skeletonization early gene Runx2 among the ASCs still had expression in the time of 21 days, and skeletonization late gene Ocn never expresses in Induction Process.In the basic medium that FGF-2 and LIF are arranged, ASCs P16 for the time still can detect the expression of skeletonization early gene Runx2; And in the basic medium without FGF-2 and LIF, ASCs P16 for the time fail to detect the expression of Runx2.
6, the obtaining and cultivating of GFP fat stem cell (with the C57 mouse), this test is the fat stem cell that is marked with GFP in order to obtain, and further carries out the in vivo test checking.
1) adopts method enrichment Lin from GFP transgenosis C57 mouse fatty tissue same as described above
-: CD271
+: Sca-1
+GFP fat stem cell group;
2) with the cell that obtains by 1 * 10
5The cell density of/mL is inoculated in the culture dish of diameter 10cm and cultivates, and 10%FBS (v/ v)+α-MEM substratum (containing FGF-2 and LIF) is at saturated humidity, 37 ℃, 5%CO
2Incubator in cultivate per 3 days replaced mediums.
3) Lin
-: CD271
+: Sca-1
+GFP fat stem cell group's external one-tenth fat differentiation
With the P3 that obtains for the GFP fat stem cell with 1 * 10
5Cells is seeded in the culture dish of diameter 3.5cm, begin into fat behind 24 h and induce, fat comprises α-MEM basic medium, 10% FBS, 1 μ M dexamethasone, 10 μ M Regular Insulin, 0.5mM 3-isobutyl-1-methylxanthine and 0.2mM indomethacin and 100u/mL penicillin, 100 μ g/mL Streptomycin sulphates to inducing culture.Amount was changed the substratum that comprises α-MEM basic medium and 10% FBS (v/ v) in per 3 days half.
RT-PCR is detected as the mrna expression situation that becomes fat genes involved PPAR γ 2 and C/EBP-α in the fat Induction Process.Fat drips formational situation to inducing GFP fat stem cell behind the 10d to detect fat with oil red O stain.
4) Lin
-: CD271
+: Sca-1
+GFP fat stem cell group's external Osteoblast Differentiation
P3 is inoculated in six orifice plates for the GFP fat stem cell, changes the skeletonization induced liquid behind 24 h and cultivate, the osteogenic induction substratum comprises α-MEM basic medium, 10% FBS, 10mM β-phospho-glycerol sodium, 10
-8Mol/L dexamethasone, 50 μ M xitix and 0.01 μ M 1,25-dihydroxyvitamin D3 and 100u/mL penicillin, 100 μ g/mL Streptomycin sulphates.
The expression of Bone formation-related gene Runx2 and Ocn in the RT-PCR detection skeletonization culturing process.Osteogenic induction is cultivated the formation of the capable Alizarin red staining observation of the cell climbing sheet mineralising tubercle of 21d.
The result shows
The GFP fat stem cellShow as the inoblast sample form of fusiformis, reach 80% ~ 90% cytogamy about 6 days, the egfp expression of all cells is all positive.After the GFP fat stem cell became fat to induce, the lipid that forms in its endochylema identified that with oil red O stain lipid is dyed to orange red, and control group dyeing is negative; Detect by RT-PCR, become to become in the fat Induction Process mrna expression of fat specific gene PPAR γ 2 and C/EBP-α obviously to strengthen; Confirm that the GFP fat stem cell successfully is divided into adipocyte after becoming fat to induce.The capable Alizarin red staining of cell climbing sheet of 21d can be observed the formation of red mineralized material behind the GFP fat stem cell osteogenic induction; The expression of skeletonization specific gene Runx2 and Ocn in the RT-PCR detection skeletonization culturing process; Confirm that GFP fat stem cell process bone is to inducing backward skeletonization direction differentiation.
7, structure and the body of cell-scleroproein mixture are implanted into, and the effect of this test is the interior one-tenth of the body fat differentiation of checking fat stem cell.
1) adopt P3 for GFP
+Lin
-: CD271
+: Sca-1
+Fat stem cell is with 2 * 10
7The density of cells/mL mixes with the 100 μ l fibrinogen solutions that contain 1 μ g FGF2.With Lin
-: CD271
-And Lin
-: CD271
+: Sca-1
-Groups of cells in contrast.
2) under the aseptic condition, the chloral hydrate anesthesia nude mice with 3.5%.
3) after the anesthesia produce effects, with 1) in celliferous fibrinogen solution and 100 μ L thrombin solutions to inject simultaneously the nude mice back subcutaneous.
8, structure and the body of cell-BCP timbering material mixture are implanted into, and the effect of this test is the interior Osteoblast Differentiation of the body of checking fat stem cell.
8.1 the structure of the characteristics of BCP timbering material and cell-BCP timbering material mixture
(ratio of β-TCP) is 30:70, long 5mm, diameter 1.5mm for hydroxyapatite (HA) and bata-tricalcium phosphate in porous biophasic calcium phosphate ceramic (Biphasic calcium phosphate ceramics, the BCP) timbering material.Adopt H
2O
2The drilling of microwave foaming method, macropore pore size 200
~500 μ m, connectivity is good between the hole, and tens to tens microns a large amount of micropores that mutually connect are arranged on the macropore hole wall.For subsequent use behind ultrasonic cleaning, high-temp steam sterilizing.
With 5 * 10
6The density of cells/mL is seeded in BCP timbering material surface with the GFP fat stem cell.After 5 days with the cell-scaffold material composite with PBS flushing 2 times, 4 ℃ of 2.5% glutaraldehyde are fixedly spent the night; 30%, 50%, 75%, 85%, 95%, 100% volume fraction alcohol gradient dehydration; Be immersed in displacement 1 h in the isoamyl acetate after the dehydration; Critical point drying, metal spraying; Scanning electron microscope detects.
8.2 the body of cell-BCP timbering material mixture is implanted into
1) adopt above-mentioned same method to make up GFP fat stem cell-BCP timbering material mixture, totally 4 samples.With Lin
-: CD271
-And Lin
-: CD271
+: Sca-1
-Groups of cells in contrast.
2) under the aseptic condition, the chloral hydrate anesthesia male common C57 mouse in 8 age in week with 3.5%.
3) after the anesthesia produce effects, do otch in the femur district under the aseptic condition, blunt separation muscle exposes femur.Femur defect with the long 5mm of low speed electric drill preparation.
4) GFP fat stem cell-BCP timbering material mixture implantable bone defective region, silk thread are fixed.The layering apposition suture.
9, the detection of repopulating cell-timbering material mixture.
9.1 the detection of cell-scleroproein mixture
1) mixture is implanted 6w, and laboratory animal is put to death in excessive anesthesia, the cell that taking-up is implanted-scleroproein mixture, routine paraffin wax embedding, HE section statining.
2) frozen section oil red O stain, step is as follows:
The true tumor that A takes out is fixedly spent the night with 10% neutral formalin.
Frozen section in the B-25 ℃ cryostat, slice thickness 10 μ m.
Slightly wash in C 60% Virahol.
D oil red O dye liquor infects 10 min.
E flowing water washes 5 min.
F Hematorylin liquid is contaminated 2 min.
G flowing water washes 5 min, the differentiation of 1% (v/ v) hydrochloride alcohol.
10min is to the nucleus oil blackeite in the H washing.
I sucks excessive moisture on every side, the glycogelatin mounting.
3) immunohistochemistry of anti-GFP detects, and step is as follows:
A makes the thick paraffin section of 5 μ m, 24 h in 60 ℃ of baking ovens.
C dehydrated alcohol 5 min, each 5 min of 95%, 85%, 75% (v/ v) ethanol.
D flowing water washes 5 min.
E 3% (v/ v) H
2O
2Incubated at room 15 min, the activity of blocking-up endogenous peroxydase.
F ddH
2O soaks 3 min * 3 time, jolting on the low speed shaking table in the immersion process.
G adds antigen retrieval liquid, 95 ℃ of water-bath 45 min.
Cooling 30 min under the H room temperature.Outwell antigen retrieval liquid, ddH2O soaks 1 min.
I 1% (v/ v) hydrochloride alcohol breaks up 3 sec.
The sealing of J 10% (v/ v) normal goats serum, incubated at room 10 min.Serum deprivation is not washed, and drips the primary antibodie of the anti-GFP of mouse of 1:500, and 4 ℃ are spent the night.
37 ℃ of K are hatched 45 min, PBS flushing, 5 min * 3 time.
It is anti-that L drips the anti-mouse of the rabbit of 1:100 two, hatches 30 min for 37 ℃, PBS flushing, 5 min * 3 time
M drips the 1:100 horseradish peroxidase complex, hatches 30 min for 37 ℃, PBS flushing, 5 min * 3 time.
N DAB colour developing is grasped dye levels at microscopically, and flowing water washes 10 min.
O haematoxylin redyeing 2min, the differentiation of 1% (v/ v) hydrochloride alcohol, flowing water flushing 10min.
P dehydration, transparent, the neutral gum mounting, microscopically is observed.
9.2 the detection of cell-BCP timbering material mixture
1) mixture is implanted 6w, and laboratory animal is put to death in excessive anesthesia, the GFP fat stem cell that taking-up is implanted-BCP timbering material mixture, 12% EDTA decalcification 2w in 37 ℃ of water-baths.Make the thick paraffin section of 5 μ m, conventional H E dyeing, Masson dyeing.
2) make the thick paraffin section of 5 μ m, the immunohistochemistry of the anti-GFP of row detects.
The result shows 6w in the implantation nude mouse, and the true tumor of taking-up is yellow-white, obviously reduces when volume is implanted.Through HE, oil red O stain, confirm the fatty tissue of true tumor.Fat is obviously many than not inducing group to the adipocyte amount of inducing group.The anti-GFP immunohistochemistry of the new adipocyte that forms detects positive, and the GFP fat stem cell that is indicated as implantation is transformed.
GFP fat stem cell-BCP timbering material mixture is implanted C57 mouse femur defective region 6w, and the true tumor of taking-up confirms to have new bone forming through HE, Masson dyeing.The capable anti-GFP immunohistochemistry of tissue slice is detected, and as seen its part scleroblast is positive, and other scleroblast is negative, shows that the GFP fat stem cell Partial Conversion of implantation is scleroblast, participates in the formation of new bone.
In the present invention, proved that from aspects such as morphology, immunocytochemistry, mRNA and protein levels the ASCs that obtains has the potential of self-renewal capacity and Multidirectional Differentiation, clonality is stronger, and confirms that it can be converted into adipocyte/scleroblast under the microenvironment that becomes fat/skeletonization.The method has been set up a kind of novel method that obtains efficiently, fast high purity ASCs, for external obtain a large amount of seed cells with dryness and be applied to tissue regeneration new way is provided.
Sequence table
＜110〉Sichuan University
＜120 〉
A kind of isolation cultivation method of fat stem cell
<160>?16
<210>?1
<211>?22
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: PPAR-γ 2 upstream primers
<400>?1
agaccactcg?cattcctttg?ac?22
<210>?2
<211>?21
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: PPAR-γ 2 downstream primers
<400>?2
tgctttatcc?ccacagactc?g?21
<210>?3
<211>?22
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: C/EBP-α upstream primer
<400>?3
ggatggtttc?gggtcgctgg?at?22
<210>?4
<211>?23
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: C/EBP-α downstream primer
<400>?4
cacggcctga?ctccctcatc?tta?23
<210>?5
<211>?18
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: Lp1 upstream primer
<400>?5
gaggatggca?agcaacac?18
<210>?6
<211>?18
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: Lp1 downstream primer
<400>?6
tgggttagcc?accgttta?18
<210>?7
<211>?23
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: Runx2 upstream primer
<400>?7
actacccagc?cacctttacc?tac?23
<210>?8
<211>?22
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: Runx2 downstream primer
<400>?8
gtcagcgtca?acaccatcat?tc?22
<210>?9
<211>?18
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: Opn upstream primer
<400>?9
tggtgcctga?cccatctc?18
<210>?10
<211>?18
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: Opn downstream primer
<400>?10
gctgcccttt?ccgttgtt?18
<210>?11
<211>?20
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: Sox9 upstream primer
<400>?11
gctctactcc?accttcactt?20
<210>?12
<211>?18
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: Sox9 downstream primer
<400>?12
ttctccaatc?gtcctcca?18
<210>?13
<211>?22
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: Acan upstream primer
<400>?13
atggaaacca?gcacggagac?ac?22
<210>?14
<211>?22
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: Acan downstream primer
<400>?14
ccagcctcag?ggtaagcaga?ca?22
<210>?15
<211>?18
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: GAPDH upstream primer
<400>?15
tcaacggcac?agtcaagg?18
<210>?16
<211>?18
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: GAPDH downstream primer
<400>?16
accagtggat?gcagggat?18
Claims (9)
1. the isolation cultivation method of a fat stem cell comprises separating from fatty tissue and obtains, cultivates the SVF cell, characterized by further comprising following steps: remove Lin in the SVF cell with the immunological magnetic bead sorting method
+Cell obtains Lin
-Cell mass; With the Lin of fluidic cell separating method from obtaining
-Enrichment CD271 in the cell mass
+Sca-1
+Cell obtains fat stem cell; With the fat stem cell culture medium culturing that contains LIF, FGF2 that obtains; The marker that adopts in the described immunological magnetic bead sorting method is CD5, CD45R, CD11b, Anti-Gr-1 and Ter-119.
2. the isolation cultivation method of fat stem cell according to claim 1, it is characterized in that: the marker that uses in the fluidic cell separating method is Lin, CD271 and Sca-1.
3. the isolation cultivation method of fat stem cell according to claim 1 is characterized in that: cultivating the used substratum of fat stem cell that obtains is to contain 10% volume ratio foetal calf serum, 10
3-10
5The α of U/mL LIF, 10-100 ng/mL FGF2-MEM substratum.
4. the isolation cultivation method of fat stem cell according to claim 3 is characterized in that: cultivating the used substratum of fat stem cell that obtains is to contain 10% volume ratio foetal calf serum, 10
3The α of U/mL LIF, 20 ng/mL FGF2-MEM substratum.
5. the isolation cultivation method of fat stem cell according to claim 1 is characterized in that: also comprise the fat stem cell that the obtains cultivation of going down to posterity.
6. the isolation cultivation method of fat stem cell according to claim 5 is characterized in that: describedly go down to posterity that to cultivate be adherent culture.
7. the isolation cultivation method of fat stem cell according to claim 6 is characterized in that: when the stand density of the fat stem cell that obtains in the culture dish bottom reaches 70-90%, and the cultivation of going down to posterity.
8. according to claim 1-7 application of the fat stem cell of the isolation cultivation method of any one fat stem cell institute separation and Culture, it is characterized in that purifying that it is applied to non-therapeutic purpose or clone stem cell, non-therapeutic purpose the transgenosis stem cell line production or be used for the fat/scleroblast that produces of non-therapeutic purpose.
9. the application of fat stem cell according to claim 8 is characterized in that it is applied to produce fat/osteoblastic method and is:
(1), the classification of fluorescence-activation fluidic cell combines to separate with Magnetic activated cell sorting and obtains ASCs;
(2), with ASCs respectively conjugated fibre albumen and BCP timbering material, be inoculated in the damaged place of nude mice back or mouse femur.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010568873 CN102002478B (en) | 2010-12-01 | 2010-12-01 | Adipose-derived stem cell separation culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010568873 CN102002478B (en) | 2010-12-01 | 2010-12-01 | Adipose-derived stem cell separation culture method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102002478A CN102002478A (en) | 2011-04-06 |
| CN102002478B true CN102002478B (en) | 2013-01-02 |
Family
ID=43810180
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010568873 Active CN102002478B (en) | 2010-12-01 | 2010-12-01 | Adipose-derived stem cell separation culture method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102002478B (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102485885A (en) * | 2011-06-09 | 2012-06-06 | 臻景生物技术（上海）有限公司 | Separating method and application of fat stem cells |
| TWI654983B (en) * | 2012-11-21 | 2019-04-01 | 瑟弗維股份有限公司 | Adipose tissue-derived stem cells for veterinary use |
| US9580678B2 (en) | 2013-06-21 | 2017-02-28 | The Regents Of The University Of California | Microfluidic tumor tissue dissociation device |
| CN104630140A (en) * | 2013-11-06 | 2015-05-20 | 吉林济惠生物科技有限公司 | Isolation and culture method of placenta mesenchyma precursor stem cells |
| US10722540B1 (en) | 2016-02-01 | 2020-07-28 | The Regents Of The University Of California | Microfluidic device and method for shear stress-induced transformation of cells |
| CN107345897A (en) * | 2016-05-04 | 2017-11-14 | 上海米洋杨生物科技有限公司 | A kind of screening technique of retinal photoreceptor cells specific surfaces albumen |
| JP7072228B2 (en) | 2016-06-08 | 2022-05-20 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | Methods and equipment for treating tissues and cells |
| CN106337036A (en) * | 2016-08-26 | 2017-01-18 | 杭州易文赛生物技术有限公司 | Method for increasing extraction rate of CD34 positive stromal vascular fraction (SVF) in high-fat tissue |
| CN106399234A (en) * | 2016-08-31 | 2017-02-15 | 肖锷 | Processing method of adipose tissue, mesenchymal stem cell, single processed adipose cell, and extracellular matrix |
| CN107254435A (en) * | 2017-06-08 | 2017-10-17 | 黎洪棉 | A kind of internal construction method of study of vascularized tissue engineering fat flap |
| CN109266601A (en) * | 2018-07-12 | 2019-01-25 | 江苏瑞思坦生物科技有限公司 | The method for constructing clinical fat stem cell bank |
| CN111040994A (en) * | 2019-11-20 | 2020-04-21 | 中国人民解放军陆军军医大学第一附属医院 | Method for efficiently separating adipose-derived mesenchymal stem cells |
| CN113215092B (en) * | 2021-05-26 | 2022-04-05 | 广州峰缘生物科技有限公司 | Rapid adipose-derived mesenchymal stem cell adipogenic differentiation method |
-
2010
- 2010-12-01 CN CN 201010568873 patent/CN102002478B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| 管晓光.脂肪基质细胞骨向诱导分化的研究进展.《国际口腔医学杂志》.2008,第35卷(第5期), * |
| 陈希哲等.人体脂肪基质细胞分离培养及其成骨潜能.《实用口腔医学杂志》.2004,第20卷(第1期), * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102002478A (en) | 2011-04-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102002478B (en) | Adipose-derived stem cell separation culture method | |
| Williams et al. | Young developmental age cardiac extracellular matrix promotes the expansion of neonatal cardiomyocytes in vitro | |
| US20180117087A1 (en) | Stem cells from urine and methods for using the same | |
| Greiner et al. | Efficient animal-serum free 3D cultivation method for adult human neural crest-derived stem cell therapeutics | |
| JP6687757B2 (en) | Methods for preparing 3D cartilage organoid blocks | |
| JP5785394B2 (en) | Optimized and defined method for isolating and storing progenitor cells from human umbilical cord | |
| WO2020196986A1 (en) | Method for controlling angiogenesis-related cytokine of stem cell-derived extracellular vesicle by using mechanical strength of hydrogel | |
| CN102085390B (en) | Cartilage cell removal matrix and preparation method and application thereof | |
| Tavakol et al. | Injectable, scalable 3D tissue-engineered model of marrow hematopoiesis | |
| Islam et al. | Mesenchymal stromal cells from human umbilical cords display poor chondrogenic potential in scaffold-free three dimensional cultures | |
| CN105779381A (en) | Clinical treatment grade preparation method used for screening human umbilical cord derived WJ-MSCs (Wharton's jelly mesenchymal stem cells) in large scale by applying extracellular matrices through three-dimensional attachment and for cell treatment | |
| CN107058219A (en) | A kind of method that application stem cell self-characteristic prepares dental pulp stem cell | |
| CN105779384A (en) | Seed cell screening and culturing cryopreservation technical method of human amniotic mesenchymal stem cells for tissue engineering | |
| CN101148656A (en) | Construction method for tissue engineering liver unit and tissue engineering liver unit | |
| CN105331579A (en) | Separation and culture method and application of human testis mesenchymal stem cells | |
| CN105647860A (en) | Serum-free in-vitro extraction and preparation method of clinical treatment-grade placental decidua basalis-mesenchymal stem cells (PDB-MSCs) | |
| Gorkun et al. | The duo of osteogenic and angiogenic differentiation in ADSC-derived spheroids | |
| WO2008049281A1 (en) | Construction method of hepatic tissue engineering construct and the hepatic tissue engineering construct | |
| CN111548990A (en) | Dental pulp stem cell polymer for exfoliating deciduous teeth and preparation method and application thereof | |
| CN105713869A (en) | In-vitro three-dimensional isolated culture and storage method for hCDMSC (human chorion-derived mesenchymal stem cell) for clinical adoptive therapy | |
| JP2013506420A (en) | Breast artery-derived cells and methods of use in tissue repair and regeneration | |
| CN109628388A (en) | With digestive enzyme compositions from placenta blood vessel separating mesenchymal stem cell | |
| WO2009080794A1 (en) | Method for preparing cell-specific extracellular matrices | |
| CN109675109B (en) | Method for directly preparing acellular hypertrophic cartilage matrix by using adipose tissue | |
| CN106676056A (en) | Method for inducing differentiation from umbilical cord mesenchymal stem cells to insulin secretion cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210412 Address after: No.11, 18 / F, building 9, No.399, Xiduan, Fucheng Avenue, Chengdu hi tech Zone, China (Sichuan) pilot Free Trade Zone, Chengdu, Sichuan 610000 Patentee after: Chengdu Lide enterprise management center (L.P.) Address before: 610041 No. three, South Renmin Road, Sichuan, Chengdu Province 14 Patentee before: SICHUAN University |
|
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: No.11, 18th floor, building 9, No.399, Fucheng avenue west section, Chengdu hi tech Zone, 610000 China (Sichuan) pilot Free Trade Zone, Chengdu, Sichuan Province Patentee after: Chengdu Lide enterprise management center (L.P.) Address before: No.11, 18 / F, building 9, No.399, Xiduan, Fucheng Avenue, Chengdu hi tech Zone, China (Sichuan) pilot Free Trade Zone, Chengdu, Sichuan 610000 Patentee before: Chengdu Lide enterprise management center (L.P.) |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210628 Address after: 2-3 / F, building E7, No.16, hemin street, Chengdu hi tech Zone, China (Sichuan) pilot Free Trade Zone, Chengdu, Sichuan 610000 Patentee after: Chengdu worldlink Health Biotechnology Co.,Ltd. Address before: No.11, 18th floor, building 9, No.399, west section of Fucheng Avenue, Chengdu hi tech Zone, China (Sichuan) pilot Free Trade Zone, Chengdu, Sichuan 610000 Patentee before: Chengdu Lide enterprise management center (L.P.) |