CN105132366A - Three-dimensional dynamic induction culture method for mesenchymal stem cells - Google Patents
Three-dimensional dynamic induction culture method for mesenchymal stem cells Download PDFInfo
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Abstract
The invention relates to a three-dimensional dynamic induction culture method for mesenchymal stem cells. The three-dimensional dynamic induction culture method includes the steps of acquiring mesenchymal stem cells, mixing the mesenchymal stem cells with soluble alginate containing magnetic oxide particles to obtain a mixture, and then mixing the mixture with slat solution of divalent metal ions except magnesium ion to form gel beads; arranging the gel beads in a magnetic field, and then subjecting the mesenchymal stem cells in the gel beads to induction culture. The three-dimension space structure of alginate gel provides a microenvironment simulating growth in vivo for the mesenchymal stem cells and expands growth space thereof; meanwhile, under the action of a dynamic culture environment built by the magnetic field, substance signal exchange between cells and culture solution and among cells is enhanced, growth and differentiation of the mesenchymal stem cells are improved, conversion rate is increased and cell viability is improved. Besides, a process of separating two types of cells is omitted from the culture method as compared with the co-culture method of the prior art.
Description
Technical field
The present invention relates to cell engineering field, particularly a kind of Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell.
Background technology
Articular cartilage tissue defect is a kind of common disease in the present age, the damage caused by tumour, the pathology caused by the age, and various genetic diseases and cartilaginous tissue misgrowth and wound can cause damage and the disappearance of joint cartilage.Joint cartilage self-repairing capability is lower, mainly contains the reason of two aspects: one is that chondrocyte belongs to terminally differentiated cells, and metabolic activity is low, self duplication ability; Two is that cartilaginous tissue does not have blood vessel and nerve.What the most often apply clinically is a kind of therapeutic method of surgery of so-called micro-fracture, the method a fine needle is penetrated damaged cartilage tissue to go directly marrow, lure into medullary cell (containing mescenchymal stem cell) along with blood flows to damaged part, then through original position inducing mesenchymal stem cell formed chondrocyte.But what this restorative procedure was finally formed is a kind of fibrous cartilage, compare with normal hyaline cartilage tissue, the content of II Collagen Type VI that fibrous cartilage contains and cartilage aggrecan is lower, and can not substitute normal cartilage function completely, long-term result for the treatment of is undesirable.
When traditional method effectively can not solve articular cartilage defect disease, and along with the development of biotechnology, be cartilage tissue engineeredly hopeful the difficult problem that can solve regenerative agent of cartilaginous tissue.In cartilage tissue engineered, chondrocyte has two large defects, is first that cell derived is restricted, and is secondly the video picture that chondrocyte can dedifferente when cultivating in vitro, loses chondrocyte phenotype and secretes the ability of specific extracellular matrix.Three-dimensional stent material can maintain chondrocyte's form effectively, but the multiplication rate of chondrocyte on timbering material is cultivated still lower relative to plane, is difficult to the cell obtaining q.s.
And mescenchymal stem cell is a kind of cell with multi-lineage potential, it can directional induction be the various kinds of cell such as insulin-like cell, chondrocyte.And mesenchymal cell is present in human multiple tissue; especially the mescenchymal stem cell of umbilical cord, placenta, marrow, adipose tissue-derived; there are wide material sources, be easy to gather, without advantages such as ethics problems; mass-producing can be induced to differentiate into chondrocyte, for clinical treatment articular cartilage damage provides new technical scheme.But the static inducing mesenchymal stem cell of many employings is converted into chondrocyte at present, and this induction mode not only induction duration is long, and Induction Transformation rate is low, and cytoactive is lower, and chondrocyte is less.
Summary of the invention
Technical problem to be solved by this invention is, it is long to there is induction duration in the mode being converted into chondrocyte for inducing mesenchymal stem cell static in prior art, inductivity is low, cytoactive is poor, the defects such as the chondrocyte's quantity obtained is few, there is provided a kind of Induction Transformation rate that can improve mescenchymal stem cell, the Three-Dimensional Dynamic method for inducing and cultivating of the mescenchymal stem cell of cell activity enhancing.
The technical solution adopted for the present invention to solve the technical problems is: the Three-Dimensional Dynamic method for inducing and cultivating providing a kind of mescenchymal stem cell, comprises the following steps:
Obtain mescenchymal stem cell;
After being mixed with the alginates of the solubility containing magnetic oxide particle by mescenchymal stem cell, then be mixed to formation gel ball with the divalent-metal ion salts solution except magnesium ion;
Described gel ball is placed in magnetic field, and with inductor, dynamic inducing culture is carried out to the mescenchymal stem cell in described gel ball;
Wherein, the diameter of magnetic oxide particle is 1-100nm.
In the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell provided by the invention, the forming process of described gel ball is: at the uniform velocity add after being mixed with the alginates of the solubility containing magnetic oxide particle by described mescenchymal stem cell in the divalent-metal ion salts solution except magnesium ion.
In the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell provided by the invention, described magnetic oxide is Fe
30
4or Co
3o
4.
In the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell provided by the invention, described magnetic oxide particle is Fe
30
4particle, and in described alginates, Fe
30
4concentration be 2-10mg/ml.
In the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell provided by the invention, described alginates is alginate calcium or sodium alginate.
In the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell provided by the invention, described alginates is sodium alginate, and after mixing with described mescenchymal stem cell, the concentration of sodium alginate is 2%-10%W/V.
In the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell provided by the invention, described divalent-metal ion salts solution is calcium chloride solution, and the concentration of calcium chloride is 1%-5%W/V.
In the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell provided by the invention, before described gel ball is placed in magnetic field, also comprise the step adopting substratum to clean described gel ball.
In the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell provided by the invention, the maximum magnetic energy product in described magnetic field is 35MGOe.
In the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell provided by the invention, described inductor comprises the ITS+Premix of TGF-β, 7-10mmol/l dexamethasone of 5-15ng/ml, 50-100ug/ml xitix, 5-15mmol/l Sodium Glycerophosphate and 50-150ng/ml.
Implement the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell provided by the invention, following beneficial effect can be reached: magnetic oxide and mescenchymal stem cell are bundled together by Lalgine colloid, and force mescenchymal stem cell to move with magnetic oxide by externally-applied magnetic field; Lalgine colloid provides a three dimensional growth space for mescenchymal stem cell, in conjunction with magnetic movement, microenvironment is grown dynamically for mescenchymal stem cell provides in an analogue body, expand the growing space of mescenchymal stem cell, and exchange with nutrient solution and intercellular PM signals, promote the Growth and Differentiation of mescenchymal stem cell, improve transformation efficiency, cell activity enhancing; Meanwhile, the inducing culture process of mescenchymal stem cell can also be shortened.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described, in accompanying drawing:
Fig. 1 is in test experience three provided by the invention, according to the canonical plotting made by the concentration of Chs standard model working fluid and OD value thereof.
Embodiment
In order to solve static inducing culture mescenchymal stem cell is converted into chondrocyte in prior art mode, to there is induction duration long, low conversion rate, cytoactive is poor, the defects such as chondrocyte's comparatively small amt, innovative point of the present invention is the Three-Dimensional Dynamic cultural method providing a kind of mescenchymal stem cell to change into chondrocyte, by utilizing the three-dimensional structure of Lalgine gel, magnetic oxide and mescenchymal stem cell are wrapped up wherein, and at the move under influence of externally-applied magnetic field, with analog cell dynamic living environment in vivo, and for mescenchymal stem cell provides sufficient growing space, increase the contact area of mescenchymal stem cell and nutrient solution, thus achieve the transformation efficiency improving mescenchymal stem cell, cell activity enhancing, increase the quantity of chondrocyte, and shorten induction duration.
The Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell provided by the invention, comprises the following steps:
One, mescenchymal stem cell is obtained;
Two, after mescenchymal stem cell being mixed with the alginates of the solubility containing magnetic oxide particle, add in the divalent-metal ion salts solution except magnesium ion, to forming gel ball;
Three, the mescenchymal stem cell in gel ball is placed in magnetic field, and adopts inductor to carry out dynamic inducing culture.
In step one, mescenchymal stem cell can derive from umbilical cord, placenta, marrow etc., preferably, from marrow, obtains mescenchymal stem cell.Preferably, the density of mescenchymal stem cell is 5-15 × 10
6individual/ml.
In step 2, crosslinked and after losing rheological under the effect of the divalent-metal ion of alginates polymer beyond demagging, the flowing of water molecules receives suppression, and then it is high and have the gelinite of through hole to create water content, and the gelinite of alginates is not thermal reversion, there is good stability and biocompatibility, therefore, stable three dimensional growth space can be provided for mescenchymal stem cell, analog cell growing environment in vivo, expand the growing space of mescenchymal stem cell, and increase the contact area of mescenchymal stem cell and nutrient solution, be conducive to the attaching of cell, growth and differ entiation.
Wherein, univalent cation and Mg
2+can not gel be formed with alginates, and Ba
2+and Sr
2+the gel formed compares Ca
2+the gellifying property formed is stronger.Other polyvalent cations, as Pb
2+, Cu
2+, Cd
2+, Co
2+, Ni
2+, Zn
2+and Mn
2+sodium alginate cross-linking gel can be formed Deng also, but limited because having its application of toxicity; Due to Ca
2+source is comparatively wide, and preferably, divalent-metal ion is calcium ion, and divalent-metal ion salts solution is CaCl
2solution, CaCl
2concentration be 1%-5%W/V, calcium ion concn is larger, and gel ball diameter is larger, and the pore texture of formation is more, and the penetrating power of gel ball is also stronger, be more conducive to cell attachment growth, differentiation; Alginates is sodium alginate or alginate calcium etc., and be preferably sodium alginate soln, the concentration of sodium alginate is 2%-10%W/V.In the present invention, unit W/V is as being mg/ml without particularly pointing out.
It should be noted that being swift in response due to divalent-metal ion and alginates, by improving gelling temperature, calcium ion total amount or reduce alginate solution concentration and accelerate gel process; And the alginates that the curing speed that slows down, raising alginate solution concentration, calcium ion total amount and use molecular mass are higher, its mechanical property can be strengthened, be more conducive to the differentiation of mescenchymal stem cell.In addition, the dropping order of alginates and metal ion salt solution and rate of addition can affect the character of colloid, and rate of addition is too fast, and the colloid of generation is the gel structure of strip gel and interruption, mescenchymal stem cell skewness; Preferably, the present invention at the uniform velocity instills in metal ion salt solution after adopting and being mixed with alginates by mescenchymal stem cell, and the gel ball size formed with this is comparatively even, also can not affect mescenchymal stem cell being uniformly distributed in colloid.
In the present invention, first magnetic oxide is mixed with alginates, then add mescenchymal stem cell, after adding divalent-metal ion salts solution, can magnetic oxide and mescenchymal stem cell are evenly wrapped in Lalgine gel ball; Then, Lalgine gel ball carries out certain motion under the effect of externally-applied magnetic field.Lalgine gel ball is that mescenchymal stem cell provides three dimensional growth space, and magnetic oxide by with externally-applied magnetic field coordinate also for mescenchymal stem cell provides a dynamic cultivation environment, to simulate the environment of human inner cell's dynamic growth, thus the proliferation and differentiation of mescenchymal stem cell can be promoted, and improve cytoactive.
Preferably, the diameter of magnetic oxide particle is 1-100nm nano level, and diameter crosses conference affects the coated of Lalgine gel ball, and diameter is too little also can affect magnetic exercise intensity; Magnetic oxide can be Fe
30
4or Co
3o
4.Due to Fe
30
4source is comparatively extensive, therefore preferably selects Fe
30
4, and Fe
30
4concentration be 2-10mg/ml.
In step 3, after treating that gel ball is formed, remove unnecessary divalent-metal ion salts solution, and repeatedly clean by NaCl solution, then with basic medium cleaning, avoid metal ion to produce toxic action in cell cultivation process; Then, then the gel ball containing mescenchymal stem cell and magnetic oxide is placed in externally-applied magnetic field carries out dynamic inducing culture, preferably, adopt strong magnets as externally-applied magnetic field, and the maximum magnetic energy product of strong magnets is 35MGOe.Dynamic cultivation condition shear-stress can promote that mescenchymal stem cell forms chondrocyte to a certain extent, improves Induction Transformation rate.
Further, in inducing culture process, inductor of the present invention comprises TGF-β, dexamethasone, xitix, Sodium Glycerophosphate and ITS+Premix.
Wherein, TGF-β (transforminggrowthfactor-β, TGF-β transforming growth factor-beta) be that gang extensively exists, structure relevant, intimate multifunction activity peptide, there is the polypeptide growth factor of several functions, the reproduction restraint of mescenchymal stem cell can be promoted, promote the synthesis of extracellular matrix, promote that collagen produces, stimulate synthesis and the extracellular matrix secretion albumen of chondrocyte, promote the propagation of osteocyte.TGF-β has three kinds of isomer, be respectively TGF-β 1, β 2 and β 3, TGF-β 2, β 3 comparatively β 1 can more fast and effeciently promote that mesenchymal stem cells into chondrocytes breaks up, but between TGF-β 2, β 3, the level of induced dry-cell is substantially identical.Because the biological function of TGF-β in osteogenesis is dual regulation, can the proliferation and differentiation effect of irritation cell, also can carry out restraining effect, in general, lower concentration TGF-β acts as a spur, and promotes the proliferation and differentiation of cell; High density plays restraining effect, and therefore, preferably, the concentration of TGF-β is 5-15ng/ml.
Dexamethasone, be purchased from Sigma company, the expression of alkaline phosphatase, Bone Gla protein and NTx etc. can be stimulated, increase the activity of alkaline phosphatase significantly, alkaline phosphatase is enzyme necessary in bone forming process, and the expression of alkaline phosphatase represents osteoplastic situation, the glucocorticoid receptor being positioned at mescenchymal stem cell surface can be activated, promote mesenchymal stem cells into chondrocytes differentiation, meanwhile, improve the differentiation rate of mesenchymal stem cells into chondrocytes; Preferably, the concentration of dexamethasone is 7-10mmol/ml.
Xitix is also known as vitamins C, it is the one of water-soluble vitamins, participate in redox processes, play an important role in the oxidation of biology and reduction process and cellular respiration, simultaneously, xitix is that collage synthesis is necessary, also the synthesis of adjustable ATP enzyme and ALP activity and non-collagen stroma protein; Xitix, to the promoter action of Chondrocyte Differentiation, mainly by increasing the accumulation of collagen, then increases the expression of alkaline phosphatase in chondrocyte; In addition, also can suppress or reduce apoptotic generation in Induction Process as a kind of antioxidant.Preferably, the concentration of xitix is 50-100ug/ml.
Sodium Glycerophosphate can provide phosphate ion for chondrocyte, promotes deposition and the calcification of physiological calcium salt simultaneously, is the prerequisite that bone marrow matrix mescenchymal stem cell produces Mineral nodules; Preferably, the concentration of Sodium Glycerophosphate is 5-15mmol/ml.
ITS+Premix adds basic medium as a kind of somatomedin, is purchased from BD company, and its composition includes bovine serum albumin and linolic acid etc., can promote the proliferate of cell; Preferably, the concentration of ITS+Premix is 50-150ng/ml.
Further, induced liquid provided by the invention also comprises Regular Insulin and Transferrins,iron complexes; Regular Insulin can reduce protein degradation, increase the absorption of amino acid and glucose, for Growth of Cells and differentiation provide energy, mescenchymal stem cell is impelled to be in low sugar environment, keep cytoactive, be conducive to the differentiation of mesenchymal stem cells into chondrocytes, preferably, the concentration range of Regular Insulin is 5-10mg/ml; And Transferrins,iron complexes can intervene ion metabolism in chondrocyte, promote the generation of chondrocyte, preferably, the concentration range of Transferrins,iron complexes is 5-10mg/ml.
Further, induced liquid provided by the invention also comprises glutamine, and glutamine participates in synthesis and the nucleic acid metabolism of protein, can be used as the energy derive in induced liquid, in addition, glutamine, by cell compatibilization, also can promote the growth and differ entiation of cell; Preferably, the concentration range of glutamine is 1-10mmol/l.
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1
The Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell provided by the invention, comprises the following steps:
1, the separation of mescenchymal stem cell;
By air Injection new zealand white rabbit auricular vein, make it dead.The four limbs of White Rabbit smeared by alcohol with 75%, sterilize and reduce the pollution of the rabbit hair.Cut femur and the spinal joints of rabbit four limbs, removing skin histology retains the muscle reticular tissue of joint, and the muscle tissue at all the other positions cuts off.The tissue processed is immersed in the alcohol of 75%, takes out after 30min, clean with aseptic PBS, be placed in super clean bench.Cut away the muscle of joint with aseptic operation cutter, joint is exposed.Cut off joint with bone shears, bone chamber is exposed.Use disposable syringe to draw aseptic PBS, repeatedly rinse bone chamber, the marrow in bone chamber is all flushed in disposable plate.Liquid rotating is moved on 200 aseptic object screen clothes, cross and filter large tissue, obtain suspension.Suspension is slowly joined the 50ml centrifuge tube of the Ficoll lymph parting liquid that 15ml is housed in advance, the centrifugal 30min of 2790rpm.After centrifugal end, with sharp suction pipe imbitition middle white nepheloid layer cell, after cell being transferred to the large square vase of T150, adding 50ml growth of mesenchymal stem cells substratum, after 3 days, change liquid.Go down to posterity after cytogamy degree reaches 90%, trysinization 3-5min, absorption suspension is transferred in new square vase to be cultivated, and this cell is designated as P2 cell, and partial freeze is preserved, and rest part continues to go down to posterity.
2, the Secondary Culture of mescenchymal stem cell, obtains P3 for mescenchymal stem cell;
Cytogamy degree gets final product had digestive transfer culture when reaching about 90%, sucking-off substratum, cleans cell surface, wash away residual substratum with aseptic PBS.Add 5ml pancreatin, leave standstill 5min, the most cell of microscopic examination becomes spherical, and when can float, the substratum added containing FBS stops digestion.Sucking-off cell suspension, blood counting chamber counts, then with 1.0 × 10
4individual/cm
2density be inoculated in T150 square vase.Square vase is placed in 37 DEG C, 5%CO
2cultivate in animal cell culture case, treating that cytogamy degree reaches 90% can had digestive transfer culture again; Adopt P3 for mescenchymal stem cell in the present embodiment.
3, containing Fe
30
4the preparation of the sodium alginate soln of particle;
Cut-off footpath is the magnetic nano-particle Fe of 20nm
30
4mix in 50ml centrifuge tube with sodium alginate, initial Fe
30
4concentration is 4mg/ml, and the solution after mixing is shaken 30min in ultrasonic washing instrument, and 105 DEG C of sterilizing 20min, now with the current afterwards.
4, embed P3 for mescenchymal stem cell, namely mix with solubility marine alga salt;
Get P3 in step 2 for mescenchymal stem cell, and resuspended adjustment cell density is 5 × 10
6individual/ml, uses the isopyknic of preparation in syringe aspiration step 3 to contain Fe
30
4the aseptic 4%W/V sodium alginate of particle, with cell suspension Homogeneous phase mixing, now cell density is 2.5 × 10
6individual/ml, sodium alginate final concentration is 2%W/V, Fe
30
4concentration be 2mg/ml.Syringe is used to draw mescenchymal stem cell and Fe
30
4particle and sodium alginate mixed solution, load onto aseptic syringe needle, the air in Inside Syringe.Slow pushing syringe afterwards, by mescenchymal stem cell and Fe
30
4the 1%W/VCaCl of 100ml is equipped with in particle and the instillation of sodium alginate mixed solution average rate
2in the beaker of solution.Ensure that needle position maintains an equal level with beaker rim of a cup all the time as far as possible, at the uniform velocity instill mescenchymal stem cell and Fe
30
4particle and sodium alginate mixed solution.After question response 10min, containing mescenchymal stem cell and Fe
30
4the sodium alginate of particle is own through being cross-linked to form gel ball.
5, clean gel ball, and inducing culture is carried out to the mescenchymal stem cell in gel ball;
Sucking-off CaCl
2solution, with 10ml aseptic 0.9%NaCl solution washing gel ball, be divided in rolling bottle after not washing twice containing the α-MEM of FBS with 10ml again, 50ml chondrocyte induction substratum is added in rolling bottle, rolling bottle is placed in the powerful magnetic field that maximum magnetic energy product is 35MGOe, full dose changes liquid 3 times weekly, and half amount changes liquid 1 time, be placed in 37 DEG C, 5%CO
2cultivate 28 days in animal cell culture case.
Wherein, containing induced liquid in chondrocyte induction substratum, induced liquid composition is: the ITS+Premix of TGF-β, 10mmol/l dexamethasone of 10ng/ml, 70ug/ml xitix, 10mmol/l Sodium Glycerophosphate and 100ng/ml.
Embodiment 2
Be with the difference of embodiment 1, the step 3 in this embodiment 2 is:
Get magnetic nano-particle Fe
30
4mix in 50ml centrifuge tube with sodium alginate, initial Fe
30
4concentration is 10mg/ml, and the solution after mixing is shaken 30min in ultrasonic washing instrument, and 105 DEG C of sterilizing 20min, now with the current afterwards.
Step 4:
Get P3 in step 2 for mescenchymal stem cell, and resuspended adjustment cell density is 1.5 × 10
7individual/ml, uses the isopyknic of preparation in syringe aspiration step 3 to contain Fe
30
4the aseptic 20%W/V sodium alginate of particle, all hooks with cell suspension and mixes, and now cell density is 0.75 × 10
7individual/ml, sodium alginate final concentration is 10%W/V, Fe
30
4concentration be 5mg/ml.Use syringe to draw mescenchymal stem cell and sodium alginate mixed solution, load onto aseptic syringe needle, the air in Inside Syringe.Slow pushing syringe afterwards, by mescenchymal stem cell and Fe
30
4the 5%CaCl of 100ml is equipped with in particle and the instillation of sodium alginate mixed solution average rate
2in the beaker of solution.Ensure that needle position maintains an equal level with beaker rim of a cup all the time as far as possible, at the uniform velocity instill mescenchymal stem cell and Fe
30
4particle and sodium alginate mixed solution.After question response 10min, containing mescenchymal stem cell and Fe
30
4the sodium alginate of particle is own through being cross-linked to form gel ball.
Embodiment 3
Be with the difference of embodiment 1, in this embodiment, step 3 is:
Cut-off footpath is the magnetic nano-particle Fe of 20nm
30
4mix in 50ml centrifuge tube with sodium alginate, initial Fe
30
4concentration is 4mg/ml, and the solution after mixing is shaken 30min in ultrasonic washing instrument, and 105 DEG C of sterilizing 20min, now with the current afterwards.
Step 4:
Get P3 in step 2 for mescenchymal stem cell, and resuspended adjustment cell density is 2 × 10
7individual/ml, uses the isopyknic of preparation in syringe aspiration step 3 to contain Fe
30
4the aseptic 8%W/V sodium alginate of particle, all hooks with cell suspension and mixes, and now cell density is 1 × 10
7individual/ml, sodium alginate final concentration is 4%W/V, Fe
30
4concentration be 2mg/ml.Use syringe to draw mescenchymal stem cell and sodium alginate mixed solution, load onto aseptic syringe needle, the air in Inside Syringe.Slow pushing syringe afterwards, by mescenchymal stem cell and Fe
30
4the 5%W/VCaCl of 100ml is equipped with in particle and the instillation of sodium alginate mixed solution average rate
2in the beaker of solution.Ensure that needle position maintains an equal level with beaker rim of a cup all the time as far as possible, at the uniform velocity instill mescenchymal stem cell and Fe
30
4particle and sodium alginate mixed solution.After question response 10min, containing mescenchymal stem cell and Fe
30
4the sodium alginate of particle is own through being cross-linked to form gel ball.
Embodiment 4
Be with the difference of step 1, the induced liquid composition that step 5 adopts in this embodiment is:
The concentration of TGF-β is 10ng/ml, and the concentration of dexamethasone is 10mmol/l, and the concentration of xitix is 70ug/ml, the concentration of Sodium Glycerophosphate is 10mmol/l, the concentration of ITS+Premix is 100ng/ml, and the concentration of Regular Insulin is 6.25mg/ml, and the concentration of Transferrins,iron complexes is 6.25mg/ml.
Embodiment 5
Be with the difference of step 2, the induced liquid composition that step 5 adopts in this embodiment is:
The concentration of TGF-β is 5ng/ml, the concentration of dexamethasone is 7mmol/l, the concentration of xitix is 50ug/ml, the concentration of Sodium Glycerophosphate is 5mmol/l, the concentration of ITS+Premix is 50ng/ml, the concentration of Regular Insulin is 5mg/ml, and the concentration of Transferrins,iron complexes is 5mg/ml, and the concentration of glutamine is 1mmoL/L.
Embodiment 6
Be with the difference of step 2, the induced liquid composition that step 5 adopts in this embodiment is:
The concentration of TGF-β is 15ng/ml, the concentration of dexamethasone is 10mmol/l, the concentration of xitix is 100ug/ml, the concentration of Sodium Glycerophosphate is 15mmol/l, the concentration of ITS+Premix is 150ng/ml, the concentration of Regular Insulin is 10mg/ml, and the concentration of Transferrins,iron complexes is 10mg/ml, and the concentration of glutamine is 10mmoL/L.
For verifying the unusual effect of the three-dimensional culture method of stem cell provided by the invention further, be specifically described by following experiment and experimental data.
Test experience one, fluorescent dye anyway
The chondrocyte that detected object: embodiment 1-6 obtains
0,35 day time, in rolling bottle, get gel ball, be positioned in EP pipe (centrifuge tube), often pipe 1 gel ball, washs with 0.9%NaCl.Prepare work dye liquor in advance: joined in 1ml0.9%NaCl by 1mg/mlPI and the 1mg/mlCAM fluorescence dye of 2 μ L and mix.Often pipe adds 150-200 μ L working fluid, blows and beats gently, gel ball is suspended in dye liquor.EP pipe is placed in 37 DEG C, 5%CO
2animal cell culture case, lucifuge hatches 30min.Take out EP pipe, sucking-off dye liquor, twice, 0.9%NaCl detergent gel ball.Gel ball is placed on slide glass, fluorescence microscope.Dead cell excites and takes on a red color under green fluorescence irradiates, and viable cell excites in green under blue-fluorescence irradiates, and observation is taken pictures.
Result: in encapsulation process, CaCl
2part necrocytosis can be caused etc. chemical substance.35 days time, under fluorescent microscope, all can be observed the green become clear, and redfree, in the gel ball that the method is described thus, seldom have dead cell.
Test experience two, MTT (tetrazolium bromide) detect
Detected object:
The chondrocyte that test set-embodiment of the present invention 1-6 obtains;
In control group-prior art, the chondrocyte that static inducing culture mescenchymal stem cell obtains.
The plastosome of viable cell can produce succinodehydrogenase, and it is water insoluble that this enzyme can make MTT be reduced to, and be but dissolved in the first hairpin of DMSO (dimethyl sulfoxide (DMSO)), dead cell then can not.Respectively following operation is performed to experimental group and control group: 3 gel balls getting 0,7,14,21,28 and 35 day, be divided in 3 EP pipes (n=3) respectively, add the substratum that the MTT of the 5mg/ml of 40 μ L and 200 μ L is fresh, and blow and beat gently, gel ball is made to be suspended in dye liquor, lucifuge in 37 DEG C, 5%CO
2hatch in animal cell culture case 4h reaction terminate after, remove supernatant, gel ball smashed, add the DMSO of 300 μ L, vibration, 10000rpm high speed centrifugation 5min.Draw 200 μ L supernatants, be transferred to 96 orifice plates, use microplate reader to survey light absorption value (OD value) at 490nm place.
Table 1:
| OD value | 0d | 7d | 14d | 21d | 28d | 35d |
| Embodiment 1 | 0.61±0.11 | 0.61±0.10 | 0.61±0.12 | 0.60±0.13 | 0.58±0.19 | 0.57±0.10 |
| Embodiment 2 | 0.83±0.13 | 0.87±0.12 | 0.85±0.11 | 0.84±0.10 | 0.82±0.17 | 0.80±0.10 |
| Embodiment 3 | 0.61±0.11 | 0.61±0.10 | 0.61±0.12 | 0.60±0.13 | 0.58±0.19 | 0.57±0.10 |
| Embodiment 4 | 0.53±0.12 | 0.54±0.11 | 0.53±0.15 | 0.52±0.11 | 0.51±0.17 | 0.50±0.10 |
| Embodiment 5 | 0.77±0.10 | 0.76±0.15 | 0.75±0.15 | 0.76±0.18 | 0.74±0.11 | 0.72±0.10 |
| Embodiment 6 | 0.61±0.11 | 0.61±0.10 | 0.61±0.12 | 0.60±0.13 | 0.58±0.19 | 0.57±0.10 |
| Control group | 0.61±0.11 | 0.56±0.14 | 0.54±0.12 | 0.47±0.15 | 0.42±0.13 | 0.40±0.16 |
Detected result: OD value is larger, the content representing first hairpin in solution is higher, also illustrates simultaneously, impels MTT enzymolysis to be that the succinodehydrogenase that first is worn in one's hair is more, and the quantity of secretion succinodehydrogenase viable cell is more.From data in table 1, in embodiment of the present invention 1-3, OD value is all greater than control group, illustrates thus, and the cytoactive that three-dimensional culture method obtains of stem cell provided by the invention is comparatively strong, and quantity is more.
Test experience three, GAG (glycosaminoglycan) assay
Detected object:
The chondrocyte that experimental group-embodiment 1-6 obtains;
In control group-prior art, the chondrocyte that static inducing mesenchymal stem cell obtains.
Respectively following operation is performed to the chondrocyte in experimental group and control group:
1), sample preparation: each 9 of the gel ball getting 0,7,14,21,28 and 35 day, is divided into 3 groups and is placed in EP pipe (n=3).Add 300 μ L papoids, 60 DEG C of water-baths are spent the night.-20 DEG C of freezen protective.During use, take out sample, room temperature is melted, vibration, and the centrifugal 5min of 4000rpm gets supernatant and is transferred to new EP and manages.
2) Chs (chondroitin sulfate, the covalently bound class glycosaminoglycan forming proteoglycan on protein, is extensively present in animal cartilage tissue) standard model working fluid production standard curve, is got.
Making method:
Precision weighing chondroitin sulfate standard substance are appropriate, make the 0.5mg/ml aqueous solution, get six colorimetric cylinders once to number, No. 0 is blank, add 0.5ml distilled water, liquid 0.10 drawn respectively by 1-5 pipe, 0.20, 0.30, 0.40, 0.50ml is placed in 50ml colorimetric cylinder, mend to 0.5ml with distilled water, add sulfuric acid respectively: each 6ml of sulfuric acid liquid of water (2:1), limit edged shake, then be placed in 95 DEG C of water and heat 60min, after taking-up is chilled to room temperature, measure OD value, as shown in table 2 below with shown in Fig. 1, in Fig. 1, ordinate zou is OD value, X-coordinate is chondroitin sulfate standard concentration.
Table 2:
3), get DMMB (1,9-dimethylated methene base the is blue) solution that 50 μ L samples join 2ml, mixing, prevents bubble.Use ultraviolet spectrophotometer to survey 0D value at 525nm place, substitute into typical curve equation y=0.9930x+0.0041, standard deviation R^2=0.9991, calculate GAG content (μ g/ pearl), GAG content results is as shown in table 3.
Table 3:
| Detected object | 0d | 7d | 14d | 21d | 28d | 35d |
| Embodiment 1 | 6.1±0.5 | 7.1±0.3 | 8.4±0.7 | 8.9±0.5 | 10.4±0.5 | 9.8±0.4 |
| Embodiment 2 | 8.7±0.6 | 9.8±0.1 | 10.4±0.7 | 11.6±0.3 | 12.9±0.5 | 11.6±0.3 |
| Embodiment 3 | 7.3±0.7 | 8.9±0.3 | 9.4±0.7 | 9.9±0.5 | 11.4±0.6 | 10.8±0.4 |
| Embodiment 4 | 7.3±0.7 | 8.3±0.3 | 9.1±0.5 | 9.8±0.6 | 11.2±0.5 | 10.1±0.3 |
| Embodiment 5 | 7.3±0.7 | 8.2±0.4 | 8.9±0.7 | 9.3±0.9 | 10.2±0.2 | 9.5±0.6 |
| Embodiment 6 | 7.3±0.8 | 8.5±0.5 | 9.6±0.6 | 10.7±0.9 | 12.2±0.3 | 11.3±0.9 |
| Control group | 7.3±0.7 | 7.6±0.8 | 7.9±0.7 | 8.2±0.5 | 7.8±0.4 | 7.3±0.2 |
As shown in Table 3, the OD value in embodiment 1-6 provided by the invention is all greater than control group, illustrates thus, and GAG contained in chondrocyte in the present invention is more, and chondrocyte's quantity is more, and activity is stronger.
In sum, the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell provided by the invention, by utilizing Lalgine colloid coated magnetic oxide compound and mescenchymal stem cell, and under the effect of externally-applied magnetic field, the gel ball being enclosed with magnetic oxide is impelled to carry mescenchymal stem cell motion, thus provide a Three-Dimensional Dynamic growing space for mescenchymal stem cell, with analog cell dynamic growth microenvironment in vivo, expand the growing space of mescenchymal stem cell, and exchange with nutrient solution and intercellular PM signals, promote the Growth and Differentiation of mescenchymal stem cell, improve transformation efficiency, cell activity enhancing, meanwhile, also relatively shorten the inducing culture time of mescenchymal stem cell, to obtain cartilage cell activity comparatively strong, quantity is more.
Below by reference to the accompanying drawings embodiments of the invention are described; but the present invention is not limited to above-mentioned embodiment; above-mentioned embodiment is only schematic; instead of it is restrictive; those of ordinary skill in the art is under enlightenment of the present invention; do not departing under the ambit that present inventive concept and claim protect, also can make a lot of form, these all belong within protection of the present invention.
Claims (10)
1. a Three-Dimensional Dynamic method for inducing and cultivating for mescenchymal stem cell, is characterized in that, comprise the following steps:
Obtain mescenchymal stem cell;
After being mixed with the alginates of the solubility containing magnetic oxide particle by mescenchymal stem cell, then be mixed to formation gel ball with the divalent-metal ion salts solution except magnesium ion;
Described gel ball is placed in magnetic field, and with inductor, dynamic inducing culture is carried out to the mescenchymal stem cell in described gel ball;
Wherein, the diameter of magnetic oxide particle is 1-100nm.
2. the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell according to claim 1, it is characterized in that, the forming process of described gel ball is: after being mixed with the alginates of the solubility containing magnetic oxide particle by described mescenchymal stem cell, more at the uniform velocity instills in the divalent-metal ion salts solution except magnesium ion.
3. the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell according to claim 1, is characterized in that, described magnetic oxide is Fe
30
4or Co
3o
4.
4. the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell according to claim 3, is characterized in that, described magnetic oxide particle is Fe
30
4particle, and in described alginates, Fe
30
4concentration be 2-10mg/ml.
5. the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell according to claim 1, is characterized in that, described alginates is alginate calcium or sodium alginate.
6. the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell according to claim 5, is characterized in that, described alginates is sodium alginate, and after mixing with described mescenchymal stem cell, the concentration of sodium alginate is 2%-10%W/V.
7. the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell according to claim 1, is characterized in that, described divalent-metal ion salts solution is calcium chloride solution, and the concentration of calcium chloride is 1%-5%W/V.
8. the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell according to claim 1, is characterized in that, before described gel ball is placed in magnetic field, also comprises the step adopting substratum to clean described gel ball.
9. the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell according to claim 1, is characterized in that, the maximum magnetic energy product in described magnetic field is 35MGOe.
10. the Three-Dimensional Dynamic method for inducing and cultivating of mescenchymal stem cell according to claim 1, it is characterized in that, described inductor comprises the ITS+Premix of TGF-β, 7-10mmol/l dexamethasone of 5-15ng/ml, 50-100ug/ml xitix, 5-15mmol/l Sodium Glycerophosphate and 50-150ng/ml.
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