CN101423820B - Osseous tissue sublevel perfusion culture method based on medulla ossium mesenchymal stem cell - Google Patents

Osseous tissue sublevel perfusion culture method based on medulla ossium mesenchymal stem cell Download PDF

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CN101423820B
CN101423820B CN2008101626962A CN200810162696A CN101423820B CN 101423820 B CN101423820 B CN 101423820B CN 2008101626962 A CN2008101626962 A CN 2008101626962A CN 200810162696 A CN200810162696 A CN 200810162696A CN 101423820 B CN101423820 B CN 101423820B
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cell
perfusion
truss
mesenchymal stem
support
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CN101423820A (en
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王金福
杨金凤
曹琛
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浙江大学
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Abstract

The invention provides a method for culturing a bone tissue through perfusion by stages based on bone mesenchymal stem cells. An inoculation method of syringe low-speed extraction is adopted, and in particular an adjusting method with different perfusion speeds is performed by before and after two stages of low flow speed perfusion culture and high flow speed perfusion culture to improve the inoculation efficiency of seed cells in a PLGA stent. The method not only increases the adhesive force and the uniform distribution of cells in the PLGA stent, but also effectively improves the proliferation efficiency of the seed cells in the PLGA stent; and through the shear force of a fluid flow to the seed cells, the oriented differentiation ability of the seed cells into osteoblast is increased. The method is designed reasonably and achieves two aims of proliferation of the seed cells in the PLGA stent and osteoblast orientated differentiation.

Description

Based on the osseous tissue of mesenchymal stem cells MSCs perfusion culture method stage by stage

Affiliated technical field

The invention belongs to biotechnology, relate to a kind of osseous tissue based on mesenchymal stem cells MSCs perfusion culture method stage by stage.This method can improve mesenchymal stem cells MSCs plantation in polylactic acid-glycollic acid (PLGA) support, amplification and the osteoblastic ability of directed differentiation effectively.

Background technology

Bone tissue engineer is that large-area bone injury provides a kind of alternative medicine.The external structure tissue engineered bone is one of research content of bone tissue engineer; Its objective is through the amplification in vitro seed cell to some amount; Be compound to and carry out common cultivation on the timbering material, form and come repairing bone defect with the osseous tissue morphology cambium similar with biological character.

Mesenchymal stem cells MSCs is a kind of ideal bone tissue engineer seed cell.The mesenchymal stem cells MSCs of vitro culture has powerful multiplication capacity, can increase 10 9Doubly; Mesenchymal stem cells MSCs is not differentiation certainly in amplification procedure, and has multidirectional differentiation potential, can be divided into bone under certain conditions, ligament, fat, cartilage and muscle etc.In view of it is drawn materials easily, the amplification in vitro ability is strong, have multidirectional differentiation potential, the no immunological rejection afterwards that implants, mesenchymal stem cells MSCs is widely used in coming repairing bone defect with the compound structure tissue engineered bone of timbering material.

Support is that the cell growth provides three-dimensional structure stand and specific Growth and Differentiation signal, makes iuntercellular form suitable spatial distribution and is connected with cell, forms the growth microenvironment similar with in-vivo tissue.POLYACTIC ACID and Sodium bromoacetate homopolymer, SRU and multipolymer thereof have favorable tissue consistency, degradability, and material surface do not influence the growth and the differentiation of cell, in cartilage and bone tissue engineer are used, demonstrate preferable performance and are widely accepted.Polylactic acid-glycollic acid (PLGA) timbering material has obtained widespread use in biomedical and organizational project.

A suitable bioreactor system also is that bone tissue engineer institute is essential.At present, there are three kinds of main bio-reactors to be applied to bone tissue engineer substantially: rolling bottle, rotating wall vessel bioreactor, perfusion bioreactor system.In the rolling bottle culture systems, stirring nutrient solution can increase mass transfer effect, and shearing stress that rack surface produces is uneven can to cause adverse influence by pair cell but be stirred in; Rotating wall vessel bioreactor provides environment, low shear stress and the high matter of dynamic cultivation to pass speed; But when being used for cultivating than bulk tissue; Because oxygen transmits through the mode of molecular diffusion; Therefore can only enough oxygen be provided at rack surface 100 μ m thickness places, the cell of internal stent just can't obtain enough oxygen; The perfusion type bio-reactor utilizes the flow through hole of timbering material of nutrient solution to cell enough mass transport to be provided, and certain mechanical shear stress stimulation effect is provided simultaneously, relatively is fit to the external structure of bulk tissue.Research shows, in contrast to static cultivation and other bio-reactor, and the perfusion reaction system can promote the vigor and the function of cell, strengthens Osteoblast Differentiation, and makes cell being more evenly distributed in support.Therefore, this type of reactor drum is usually used in cultivating bone tissue.

Yet; With the mesenchymal stem cells MSCs is seed cell; In the PLGA support, make up osseous tissue under the perfusion culture condition, relating to seed cell variation of culture condition in the serial processes such as uniform distribution, adhesion, propagation and directed differentiation scleroblast in support.Therefore; The perfusion culture method stage by stage of the plantation of design support inner cell, propagation and directional induction differentiation; Improve uniform distribution, proliferate efficiency and the directed differentiation effect of seed cell in support, the external osseous tissue that makes up is effectively had important practical application meaning.

Summary of the invention

The present invention is in order effectively to improve plantation, distribution, propagation and the scleroblast directed differentiation efficient of mesenchymal stem cells MSCs in the PLGA support; A kind of osseous tissue based on mesenchymal stem cells MSCs perfusion culture method stage by stage is provided, is based on the external bone tissue engineer technology of mesenchymal stem cells MSCs.

Realize through following steps:

I. make up the perfusion culture apparatus: connect peristaltic pump, gas-filtering device, reservoir and sample cell through silica gel hose and make up the perfusion culture apparatus; The exit end of reservoir links to each other through truss entrance end on silica gel hose and the sample cell, and the reservoir entrance end passes through that the truss exit end links to each other under silica gel hose and the sample cell, forms an airtight circulation passage; Reservoir also is provided with the filtration unit that is used to carry out gaseous interchange; Sample cell is provided with and can unloads the last truss and following truss that connects through screw socket, and last truss top has the nutrient solution import, and following truss bottom has the nutrient solution outlet; Following truss intracavity bottom is lined with network of micropores; Be placed in the sample cell, be positioned at the network of micropores top, O type packing ring 9 places top of chamber in the sample cell.

Sample cell internal diameter in the said perfusion culture apparatus of step I is 10mm, is processed by the Vestolen PP 7052 pressing mold, and stent diameter is fitted with the sample cell internal diameter mutually.

Timbering material in the said perfusion culture apparatus of step I is a polylactic acid-glycollic acid, diameter 10mm, thickness 3mm, porosity 94.5%-98.0%, aperture 280-450 μ m.

The filter membrane of the built-in 0.22 μ m of the described filtration unit of step I.

II. cell/scaffold complex preparation:

1) collects the people's mesenchymal stem cells MSCs of former generation (MSCs) that is cultured to the third generation; With containing 10% foetal calf serum (Hangzhou Sijiqing Biological Engineering Material Co., Ltd.; Hangzhou) and 2mM L-glutaminate (Sigma company; Shanghai) α-MEM nutrient solution (α-Modified Eagle Medium, the safe Bioisystech Co., Ltd of Hangzhou fluorescence, Hangzhou) regulates cell density to 2 * 10 6Individual/ml, with the preparation cell suspension, subsequent use;

2) the learn from else's experience PLGA support of 24 hours 75% alcohol disinfectings; After blotting with sterile gauze, blower fan dries up at a slow speed in ultra quiet, truss under the sample thief groove; The PLGA support that dries up is put into truss under the sample cell; The lower end that connects truss under the sample cell with silica gel hose exports and asepsis injector, draws the cell suspension of above-mentioned about 250 μ l, evenly drips to rack upper surface; Extracting syringe gently with the flow velocity of 0.05ml/min then makes suspension evenly get into internal stent to carry out the mesenchymal stem cells MSCs inoculation, improve mesenchymal stem cells MSCs is planted efficient in support purpose to reach;

3) inoculation finishes and takes out support and be put in Tissue Culture Plate six orifice plates and left standstill 2 hours; Every then hole adds α-MEM nutrient solution that 5ml contains 10% (v/v) foetal calf serum, 100U/ml penicillium mould and 100U/ml Streptomycin sulphate; Static cultivation 24 hours obtains cell/scaffold complex.

III. low flow velocity perfusion is cultivated:

Get cell/scaffold complex that above-mentioned static cultivation obtained after 24 hours; Move under the sample cell of perfusion culture apparatus in the truss; Upper and lower then truss rotation is connected the sealed sample groove; Add α-MEM nutrient solution that 50ml contains 10% (v/v) foetal calf serum, 100U/ml penicillium mould and 100U/ml Streptomycin sulphate in the reservoir, set flow velocity 0.2ml/min and carry out perfusion cultivation 11 days, whenever change liquid at a distance from three and half amounts.Selecting the nutrient solution flow velocity for use is 0.2ml/min, for reaching low flow velocity feeding to guarantee the purpose of internal stent mesenchymal stem cells MSCs good adhesion, uniform distribution and high proliferation efficient.

IV. the high flow rate perfusion is cultivated:

Above-mentioned perfusion was cultivated after 11 days, increased the perfusion flow velocity and carried out perfusion cultivation 21 days to 3.0ml/min, and every measuring at a distance from three and half changed liquid.Selecting the nutrient solution flow velocity for use is 3.0ml/min, for reaching the purpose that the high flow rate feeding promotes the bone mesenchymal stem cell to osteoblast directed differentiation.

The invention has the beneficial effects as follows:

(1) adopts syringe low speed to extract inoculation method in the technology provided by the invention, improved the inoculation efficient of mesenchymal stem cells MSCs in the PLGA support.

(2) adopt the perfusion culture method that hangs down flow velocity, not only increased adhesion and the uniform distribution of mesenchymal stem cells MSCs in the PLGA support, and improved the proliferate efficiency of mesenchymal stem cells MSCs in the PLGA support effectively.

(3) perfusion culture method of employing high flow rate to the shearing force that mesenchymal stem cells MSCs produces, has increased the ability of bone mesenchymal stem cell to osteoblast directed differentiation through flow liquid.

(4) under perfused culture system of the present invention, the adjusting through the different perfusion rate of front and back phase in has reached the dp of mesenchymal stem cells MSCs in internal breeding of PLGA support and scleroblast directed differentiation.

Description of drawings

Fig. 1 is a perfusion culture apparatus synoptic diagram.

Fig. 2 is the sample cell structural representation.

Fig. 3 is the distribution of fluorescence microscope mesenchymal stem cells MSCs three aspects in the PLGA support.

Fig. 4 is the alkaline phosphatase staining of calcium cobalt method.

Fig. 5 is the alkaline phosphatase activities detected result.

Embodiment

The present invention combines specific embodiment and accompanying drawing to be described further.Should be understood that these embodiment only are used for illustration purpose, and be not used in the restriction scope of the invention.

Embodiment 1

Referring to Fig. 1, Fig. 2, the present invention makes up the perfusion culture apparatus of being made up of reservoir 3, sample cell 4 and the silica gel hose 5 of peristaltic pump 1, gas-filtering device 2, splendid attire nutrient solution, connects gas-filtering device 2, reservoir 3 and sample cell 4 through silica gel hose 5; Peristaltic pump 1 adopts 7524-55 type (Cole-Parmer company, Shanghai), to produce perfusion power; Silica gel hose 5 is as nutrient solution perfusion passage, its internal diameter 0.16cm (Cole-Parmer company, Shanghai); Sample cell 4 (bio-reactor National Key Laboratory of East China University of Science, Shanghai) is processed by the Vestolen PP 7052 pressing mold, in order to placing rack 8; Sample cell 4 internal diameters are 10mm, are provided with through screw thread 12 and unload the last truss 6 and following truss 7 that connects, and the nutrient solution import is arranged at last truss 6 tops; The nutrient solution outlet is arranged at following truss 7 bottoms, and following truss 7 inner bottom parts are lined with network of micropores 9 (200 order), and support 8 places down truss 7; Be positioned at network of micropores 9 tops, support 8 diameters are fitted with sample cell 4 internal diameters mutually, and packing ring 10 is the O type; And be positioned at truss 6 tops, be used for truss 6 and the following truss 7 usefulness screw threads 12 sealed sample groove inner chamber 11 afterwards that spins.The exit end of reservoir 3 links to each other through truss 6 entrance ends on silica gel hose 5 and the sample cell, and reservoir 3 entrance ends pass through that truss 7 exit end link to each other under silica gel hose 5 and the sample cell, form an airtight circulation passage.Reservoir also is provided with the filtration unit 2 that is used to carry out gaseous interchange for 3 bottles, the filter membrane of filtration unit 2 built-in 0.22 μ m.

Confession cell stereoscopic culture that the present invention is used and the support of differentiation 8 are polylactic acid-glycollic acid (PLGA) support, the support parameter: diameter 10mm, thickness 3mm, porosity 94.5%-98.0%, aperture 280-450 μ m.

The used cell of the present invention former generation mesenchymal stem cells MSCs of behaving.

Whole device (removing peristaltic pump) all can carry out high pressure steam sterilization.

Embodiment 2

Get the perfusion culture apparatus that embodiment 1 makes up, when perfusion is cultivated, support 8 is placed sample cell; Silica gel hose (Cole-Parmer company, the Shanghai) stage casing that will connect the outlet of reservoir nutrient solution with dop (Cole-Parmer company, Shanghai) is fixed to peristaltic pump pump head (Cole-Parmer company; Shanghai) on, lean on the rotary action of peristaltic pump (Cole-Parmer company, Shanghai) pump head; Nutrient solution in the reservoir is pumped from the nutrient solution outlet of reservoir; Behind silica gel hose entering sample cell inner chamber, pass the cell/scaffold complex in the sample cell, flow back to reservoir via the silica gel pipeline that connects the sample cell outlet again.The sample cell detailed structure is referring to Fig. 2.Because the diameter of support 8 is fitted with sample cell 4 internal diameters mutually, makes cell/scaffold complex airtight all right in sample cell, so nutrient solution is unlikely to flow through all around from support, and from cell/scaffold complex, passes.The filtration unit 2 that reservoir 3 bottlenecks of dress nutrient solution are equipped with built-in 0.22 μ m filter membrane is used for carrying out gaseous interchange.The PLGA support of preparation cell/scaffold complex is provided by Zhejiang University's material and bio-medical macromole institute of chemical engineering institute (Hangzhou).

The confession cell stereoscopic culture that the present invention is used and the support of differentiation are polylactic acid-glycollic acid (PLGA) support, support parameter: diameter 10mm, thickness 3mm, porosity 94.5%-98.0%; Aperture 280-450 μ m.

Concrete through following steps realization the present invention:

1. cell/scaffold complex preparation:

1) collects the people's mesenchymal stem cells MSCs of former generation that is cultured to the third generation; With containing 10% foetal calf serum (Hangzhou Sijiqing Biological Engineering Material Co., Ltd.; Hangzhou) and 2mM L-glutaminate (Sigma company; Shanghai) α-MEM nutrient solution (α-Modified Eagle Medium, the safe Bioisystech Co., Ltd of Hangzhou fluorescence, Hangzhou) regulates cell density to 2 * 10 6Individual/ml, with the preparation cell suspension, subsequent use;

2) the learn from else's experience PLGA support of 24 hours 75% alcohol disinfectings; After blotting with sterile gauze, blower fan dries up at a slow speed in ultra quiet, truss under the sample thief groove; The PLGA support that dries up is put into truss under the sample cell; The lower end that connects truss under the sample cell with silica gel hose exports and asepsis injector, draws the cell suspension of above-mentioned about 250 μ l, evenly drips to rack upper surface; Extracting syringe gently with the flow velocity of 0.05ml/min then makes suspension evenly get into internal stent to carry out the mesenchymal stem cells MSCs inoculation, improve mesenchymal stem cells MSCs is planted efficient in support purpose to reach;

3) inoculation finishes and takes out support and be put in Tissue Culture Plate six orifice plates and left standstill 2 hours; Every then hole adds α-MEM nutrient solution that 5ml contains 10% (v/v) foetal calf serum, 100U/ml penicillium mould and 100U/ml Streptomycin sulphate; Static cultivation 24 hours prepares cell/scaffold complex.

2. low flow velocity perfusion is cultivated:

Get cell/scaffold complex that above-mentioned static cultivation obtained after 24 hours; Move under the sample cell of perfusion culture apparatus in the truss; Upper and lower then truss rotation is connected the sealed sample groove; Add α-MEM nutrient solution that 50ml contains 10% (v/v) foetal calf serum, 100U/ml penicillium mould and 100U/ml Streptomycin sulphate in the reservoir, set flow velocity 0.2ml/min and carry out perfusion cultivation 11 days, whenever change liquid at a distance from three and half amounts.Selecting the nutrient solution flow velocity for use is 0.2ml/min, for reaching low flow velocity feeding to guarantee the purpose of internal stent mesenchymal stem cells MSCs good adhesion, uniform distribution and high proliferation efficient.

3. the high flow rate perfusion is cultivated:

Above-mentioned perfusion was cultivated after 11 days, increased the perfusion flow velocity and carried out perfusion cultivation 21 days to 3.0ml/min, and every measuring at a distance from three and half changed liquid.Selecting the nutrient solution flow velocity for use is 3.0ml/min, for reaching the purpose that the high flow rate feeding promotes the bone mesenchymal stem cell to osteoblast directed differentiation.

Embodiment 3

The concrete grammar that the inventive method is taked is: getting the adult's marrow that exsomatizes carry out carrying out after the separation of mononuclearcell mesenchymal stem cells MSCs purifying and former generation amplification cultivation, preparation is based on the seed cell of the bone tissue engineer of mesenchymal stem cells MSCs.

1, get adult's marrow 3-5ml that exsomatizes, behind the phosphoric acid buffer (PBS) of the not calcic magnesium of adding equivalent, centrifugal 10 minutes of 900 * g discards lipid layer and supernatant.Deposition is with the α-MEM nutrient solution that contains 10% foetal calf serum (FBS, Hangzhou SIJIQING biotech company, Hangzhou) mixing again; Slowly join density and be (Pharmacia company on the Ficoll parting liquid of 1.077g/ml; Shanghai), centrifugal 20 minutes of 900 * g collects the mononuclearcell layer; After PBS washing 2-3 time, again with α-MEM nutrient solution re-suspended cell that contains 10% foetal calf serum and numeration.Regulate cell density to 5 * 10 6Behind/the ml, being inoculated in floorage is 25cm 2Culturing bottle in, be positioned over 37 ℃, 5%CO 2Cultivate in the incubator, later half amount was changed liquid in 48 hours, and every later on separated 3-4 days half amount is changed liquid once.

2, about 90% time at the bottom of former generation, cultured cells grew into culturing bottle; With digestion under 0.25% pancreatin/1mMEDTA room temperature 5 minutes; Then with the ratio of 1:1 add contain 10% foetal calf serum α-MEM substratum with the tryptic activity that neutralizes; And blow and beat cell gently with suction pipe, suspend up to attached cell.The centrifuge washing cell once, α-MEM nutrient solution of using 10% foetal calf serum again is suspension cell again, by the 1:3 cultivation of going down to posterity.Change a nutrient solution next day of in the culturing process that goes down to posterity, carry out amplification cultivation.The mesenchymal stem cells MSCs that reaches the third generation is ready for use on the inoculation support.

Embodiment 4 seed cells inoculation efficient

Alcohol with 75% imports PLGA porous support inside and carried out disinfection one day.Alcohol is wherein removed in aseptic PBS displacement.Support is soaked in the α-MEM nutrient solution of 10% foetal calf serum, subsequent use.

PLGA support behind the cancellation poison blots with sterile gauze, and blower fan dries up at a slow speed in ultra quiet.Truss under the sample thief groove, with silica gel hose with its outlet at bottom with after asepsis injector links to each other, the PLGA support is put into down in the inner chamber of truss.Get the mesenchymal stem cells MSCs of the above-mentioned third generation, regulate cell density to 2 * 10 with the α-MEM nutrient solution of 10% foetal calf serum 6Behind individual/ml, draw 250 μ l cell suspensions and evenly drip to support, extract syringe gently and make cell suspension get into internal stent (flow velocity: 0.05ml/min).Directly put the inoculation method of injection PLGA support and the inoculation method of 250 μ l cell suspension naturally osmotics compares with 250 μ l cell suspensions.

After inoculation finishes, take out support and be put in Tissue Culture Plate six orifice plates, behind the 2h, add α-MEM nutrient solution that 5ml contains 10% foetal calf serum, 100U/ml penicillium mould, 100U/ml Streptomycin sulphate, in static cultivation preparation in 24 hours cell/scaffold complex.Then, take out cell/scaffold complex, wash 1 time with PBS after, put in the distilled water and be stored in-20 ℃, DNA total amount to be checked.During detection, take out cell/scaffold complex, after room temperature is melted, add lysis liquid cracking 30 minutes.Get lysate 50 μ l and be added on 96 orifice plates, mix, add 100 μ l Hoechst33258 staining fluid (Sigma companies again with 50 μ l TNE damping fluids; Shanghai, 2 *, 2 μ g/ml); After the incubated at room 5 minutes; (Infinite M200, Tecan Austrilia) survey fluorescence intensity (Ex350nm/Em450nm) with multi-functional ELIASA.Calf thymus DNA (Sigma company, Shanghai) is as standard substance drawing standard curve.According to the linear relationship of cell density and DNA total amount, calculate the cell inoculation density in the support at last.

The mesenchymal stem cells MSCs inoculum density is following in the PLGA support that above-mentioned three kinds of cell suspension inoculation methods are produced: inoculation back static cultivation one day reaches 2.97 (± 0.69) * 10 with cell quantity in the PLGA support of the low flow velocity abstracting method inoculation of syringe 5Individual cell/support, PLGA support (1.35 (± 0.86) * 10 of significantly inoculating more than the naturally osmotic method 5Individual cell/support) and directly put PLGA support (1.99 (± 0.55) * 10 of injecting method inoculation 5Individual cell/support) (P<0.05), show that with relatively other two kinds of method inoculations of the low flow velocity abstracting method inoculation of syringe better effect of inoculation is arranged.

Mesenchymal stem cells MSCs distributes and proliferate efficiency in the PLGA support that embodiment 5 different cultural methods obtain

Static cultivation, the cultivation of 0.2ml/min flow velocity perfusion and 1ml/min flow velocity perfusion are set cultivate three kinds of cultural methods.Get cell/scaffold complex of one day of said syringe low flow velocity abstracting method inoculation back static cultivation, continued in six orifice plates static cultivation 11 days, be the static cultivation group; Get cell/scaffold complex of one day of said syringe low flow velocity abstracting method inoculation back static cultivation, change in the sample cell of perfusion culture system and cultivated 11 days, be 0.2ml/min flow velocity perfusion culture group with 0.2ml/min flow velocity perfusion; Get cell/scaffold complex of one day of said syringe low flow velocity abstracting method inoculation back static cultivation, change in the sample cell of perfusion culture system and cultivated 11 days, be 1ml/min flow velocity perfusion culture group with 1ml/min flow velocity perfusion.Three kinds of cultural methods are cultivated after 11 days down, and regularly carry out the total flow measurement of DNA of above-mentioned cell/scaffold complex, analyze the proliferation potential of mesenchymal stem cells MSCs in the PLGA support.

Take out above-mentioned cytoskeleton mixture, wash 1 time with PBS after, cut from the centre; Add resorcinolphthalein diethyl ester (FDA, Merck company, Shanghai) staining fluid; Hatched under 37 ℃ 5 minutes; The unnecessary staining fluid of PBS flush away, inverted fluorescence microscope is observed and is taken pictures, and analyzes the distribution of mesenchymal stem cells MSCs in support.

The detection of DNA total amount and TCS conversion interpretation of result in cell/scaffold complex, cultivate 3,6,9 and 11 days in the static cultivation group after, the cell quantity in cell/scaffold complex is respectively 3.54 (± 1.12) * 10 5, 5.72 (± 1.31) * 10 5, 4.34 (± 0.93) * 10 5With 3.85 (± 0.66) * 10 5Individual cell/support; 0.2ml/min after cultivating 3,6,9 and 11 days in the flow velocity perfusion culture group, cell quantity is respectively 3.92 (± 0.94) * 10 in cell/scaffold complex 5, 5.45 (± 1.15) * 10 5, 8.29 (± 1.38) * 10 5With 9.78 (± 1.55) * 10 5Individual cell/support; After cultivating 3,6,9 and 11 days in the 1ml/min flow velocity perfusion culture group, cell quantity is respectively 1.82 (± 0.51) * 10 in cell/scaffold complex 5, 0.95 (± 0.35) * 10 5, 1.45 (± 0.97) * 10 5With 2.58 (± 1.88) * 10 5Individual cell/support.The result shows, static cultivation is cultivated with high flow rate relatively, and the perfusion of low flow velocity (0.2ml/min) is cultivated the adhesion that more helps the interior mesenchymal stem cells MSCs of PLGA support and propagation (P < 0.05).

Through to cultivating the upper and lower surface of cell/scaffold complex and the cell fluorescence colour developing of middle cross-sectional area after 11 days; Analyze the distribution of the interior mesenchymal stem cells MSCs of PLGA support under three kinds of different culture condition and see Fig. 3; Wherein each group of A, B and C is respectively the upper surface of cytoskeleton mixture, middle cross-sectional area and lower surface, 10 * 10 times.In the static cultivation group; The distribution of upper and lower near surface duct inner cell is similar with 0.2ml/min flow velocity perfusion culture group; But the cell distribution result of cross-sectional area relatively, the cell distribution of visible static cultivation group is starkly lower than 0.2ml/min flow velocity perfusion culture group.Explain that static cultivation is unfavorable for the propagation of PLGA internal stent cell.Cell/scaffold complex under the inspection 1ml/min flow velocity perfusion culture condition, all less in the upper and lower near surface of the support duct with the cell distribution of middle cross-sectional area.In conjunction with DNA macroanalysis result, explain that high flow rate has caused the loss of early stage support inner cell, reduced distribution and the cell total amount of cell in support thus.

According to The above results; At the osseous tissue that makes up based on mesenchymal stem cells MSCs; When after the mesenchymal stem cells MSCs inoculation, carrying out mesenchymal stem cells MSCs propagation; In order to guarantee the propagation of mesenchymal stem cells MSCs, guarantee not running off of the interior mesenchymal stem cells MSCs of support simultaneously again, should carry out perfusion with low flow velocity and cultivate to appropriate.

Embodiment 6 scleroblast directional induction differentiation capability comparative analysiss

0.2ml/min the flow velocity perfusion is cultivated the cell/scaffold complex after 11 days, continues respectively after perfusion under 0.2ml/min flow velocity and the 3ml/min flow conditions is cultivated 21 days, to take out cell/scaffold complex, carries out paraffin embedding and section, slice thickness 6 μ m.The dyeing of calcium cobalt method is carried out in section, promptly cut into slices, go into Incubating Solution in 37 ℃ of insulations 4 hours, washed with de-ionized water 3 times through the YLENE dewaxing; Added the 20mg/ml cobalt nitrate solution 5 minutes, washed with de-ionized water 3 times, drying; Mounting, om observation is taken pictures, and carries out the detection of alkaline phosphatase active coloring referring to Fig. 4; Wherein A is that 0.2ml/min flow velocity perfusion is cultivated the coloration result after 21 days, and B is that 3ml/min flow velocity perfusion is cultivated the calcium cobalt method coloration result after 21 days, 10 * 10 times.

Simultaneously, the cell/scaffold complex of get above-mentioned cultivation 12 days and 21 days, chopping; Homogenate, behind the multigelation method lysing cell, (bio-engineering research first branch is built up in Nanjing to adopt the alkaline phosphatase assay test kit; Nanjing) carry out the alkaline phosphatase activities quantitative analysis: get lysate 30 μ L and 0.5ml damping fluid and the abundant mixing of 0.5ml matrix liquid, 37 ℃ of water-bath 15min add the 1.5ml developer; Mixing immediately, the 520nm place surveys respectively manages absorbancy.Analyze alkaline phosphatase activity referring to Fig. 5, wherein--expression 0.2ml/min flow velocity perfusion cultivates alkaline phosphatase activity down, alkaline phosphatase activity under-■-expression 3ml/min flow velocity perfusion cultivation.

According to the perfusion cultivation results under Fig. 4 and Fig. 5 comparison 0.2ml/min flow velocity and the 3ml/min flow velocity; The culture condition of high flow rate has significantly promoted the expression activity of cell/scaffold complex neutral and alkali Phosphoric acid esterase, shows that the high flow rate cultivation has the usefulness that strengthens the differentiation of inducing bone mesenchymal stem cell to osteoblast.

Case summary:

(1) adopt the low flow velocity abstracting method of syringe on the PLGA support, to inoculate mesenchymal stem cells MSCs, reached higher seed cell inoculation efficient:

Naturally osmotic method relatively more commonly used and directly some injecting method inoculation; Significantly plant inoculation method with cell quantity in the PLGA support of the low flow velocity abstracting method inoculation of syringe, show with the low flow velocity abstracting method inoculation of syringe to have better effect of inoculation more than the first two.

(2) low flow velocity perfusion culture method helps mesenchymal stem cells MSCs adhesion in the PLGA support, propagation and uniform distribution:

Relatively static cultivation is hanged down the flow velocity perfusion and is cultivated survival and the propagation that helps PLGA internal stent seed cell, makes cell distribution even; Cultivate than high flow velocities perfusion, low flow velocity perfusion is cultivated and is helped the adhesion of cell at internal stent, has reduced the loss of internal stent cell.

(3) the high flow rate perfusion is cultivated the directed differentiation that has promoted bone mesenchymal stem cell to osteoblast:

After mesenchymal stem cells MSCs adhered in the PLGA support and breeds, the high flow rate perfusion was cultivated the directed differentiation that the shearing force that produces has promoted bone mesenchymal stem cell to osteoblast.

(4) based on the bone tissue engineer of mesenchymal stem cells MSCs stage by stage perfusion culture method both guaranteed mesenchymal stem cells MSCs propagation enough in the PLGA support, promoted osteoblastic directed differentiation again:

In perfusion is cultivated; The flow velocity of perfusion stage by stage conversion method of the present invention has guaranteed adhesion and the propagation of mesenchymal stem cells MSCs in early stage in the PLGA support on the one hand, for the directed differentiation of later stage bone mesenchymal stem cell to osteoblast inductive condition is provided again.

Claims (4)

1. one kind based on the osseous tissue of mesenchymal stem cells MSCs perfusion culture method stage by stage, it is characterized in that realizing through following steps:
I. make up the perfusion culture apparatus: connect peristaltic pump (1), gas-filtering device (2), reservoir (3) and sample cell (4) through silica gel hose (5) and make up the perfusion culture apparatus; The exit end of reservoir (3) links to each other through truss (6) entrance end on silica gel hose (5) and the sample cell; Reservoir (3) entrance end links to each other through truss (7) exit end under silica gel hose (5) and the sample cell, and reservoir (3) is provided with the filtration unit (2) that is used to carry out gaseous interchange, and sample cell (4) is provided with through screw thread (12) and unloads last truss (6) and the following truss (7) that connects; Last truss (6) top has the nutrient solution import; Following truss (7) bottom has the nutrient solution outlet, and following truss (7) inner bottom part is lined with network of micropores (9), and support (8) places in the sample cell (4); Be positioned at network of micropores (9) top, packing ring (10) is positioned at sample cell inner chamber (11) top;
II. support/cell complexes preparation:
1) collects the people's mesenchymal stem cells MSCs of former generation that is cultured to the third generation: regulate cell density to 2 * 10 with the α that contains 10% foetal calf serum and 2mM L-glutaminate-MEM nutrient solution 6Individual/ml, with the preparation cell suspension, subsequent use;
2) the learn from else's experience support of 24 hours 75% alcohol disinfectings; Dry up; The support that dries up is put into truss under the sample cell, and the lower end that connects truss under the sample cell with silica gel hose exports and asepsis injector, draws the cell suspension of above-mentioned about 250 μ l; Evenly drip to rack upper surface, extract syringe gently with the flow velocity of 0.05ml/min then and make suspension evenly get into internal stent to carry out the seed cell inoculation;
3) inoculation finishes and takes out support and be put in Tissue Culture Plate six orifice plates and left standstill 2 hours; Every then hole adds α-MEM nutrient solution that 5ml contains 10% (v/v) foetal calf serum, 100U/ml penicillium mould, 100U/ml Streptomycin sulphate; Static cultivation 24 hours obtains support/cell complexes;
Ш. low flow velocity perfusion is cultivated:
Get support/cell complexes that above-mentioned static cultivation obtained after 24 hours; Move under the sample cell in the truss; Upper and lower then truss rotation is connected the sealed sample groove; Add α-MEM nutrient solution that 50ml contains 10% (v/v) foetal calf serum, 100U/ml penicillium mould and 100U/ml Streptomycin sulphate in the reservoir, set flow velocity 0.2ml/min and carry out perfusion cultivation 11 days, whenever change liquid at a distance from three and half amounts;
IV. the high flow rate perfusion is cultivated:
Above-mentioned perfusion was cultivated after 11 days, increased the perfusion flow velocity and carried out perfusion cultivation 21 days to 3.0ml/min, and every measuring at a distance from three and half changed liquid.
2. a kind of osseous tissue based on mesenchymal stem cells MSCs according to claim 1 is perfusion culture method stage by stage; It is characterized in that: sample cell (4) internal diameter in the said perfusion culture apparatus of step I is 10mm; Processed by the Vestolen PP 7052 pressing mold, support (8) diameter is fitted with sample cell (4) internal diameter mutually.
3. a kind of osseous tissue based on mesenchymal stem cells MSCs according to claim 1 is perfusion culture method stage by stage; It is characterized in that: following truss (7) material in the said perfusion culture apparatus of step I is a polylactic acid-glycollic acid; Diameter 10mm; Thickness 3mm, porosity 94.5%-98.0%, aperture 280-450 μ m.
4. a kind of osseous tissue based on mesenchymal stem cells MSCs according to claim 1 is perfusion culture method stage by stage, it is characterized in that: the filter membrane of the built-in 0.22 μ m of the said filtration unit of step I (2).
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