CN106975105A - The preparation method of the autologous bone marrow mesenchymal stem cells compound rest of matrix induction - Google Patents
The preparation method of the autologous bone marrow mesenchymal stem cells compound rest of matrix induction Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of the autologous bone marrow mesenchymal stem cells compound rest of matrix induction, concretely comprise the following steps：Prepare bone marrow suspension；Peripheral blood in patients is taken, is placed in blood bag；Prepare autoserum；Obtain aim cell；Take 15ml DMEM high glucose mediums to be added in aim cell, be inoculated in T25 Tissue Culture Flasks, be placed in incubator and cultivate；Cell is rinsed with PBS liquid twice；DMEM in high glucose culture medium is instilled in blake bottle and terminates digestion；Precipitation after being cleaned；Cell is inoculated in Tissue Culture Flask and continues to cultivate；Passage 3 times；Trypsin-EDTA solution is added into blake bottle；Cell is washed with PBS 3 times；Cell is resuspended；A sterile Collagen Type VI film of pig source I/III is taken, is positioned in aseptic plastic culture dish, with DMEM in high glucose culture medium soaked overnight；Cell suspension is instilled on the Collagen Type VI film of pig source I/III, cultivated 3 days.The present invention has the advantages that treatment accuracy is high.
Description
Technical field
The present invention relates to organizational project articular cartilage damage repairing and treating field, specifically a kind of matrix induction from
The preparation method of body mesenchymal stem cells MSCs compound rest.
Background technology
Articular cartilage damage is clinically a kind of common disease, and clinical manifestation is arthralgia, is born a heavy burden, motion then adds
Weight, limits the activity in affected part joint, frequently-occurring disease in sportsman, elderly, because articular cartilage is hyaline cartilage, lack blood
Pipe, lymph, neurotrophy etc., therefore be difficult self-regeneration, once morbidity, then often develop into Osteoarthritis, severe one needs
Prosthetic replacement.
There are a variety for the treatment of means, including debridement, marrow stimulation before the treatment of this disease（Such as microfrature,
Subchondral bone Drilling etc.）, autologous or homogenous cartilage film and periosteum transplantation, bone cartilage grafting, articular cartilage transplantation etc.,
But these technology repair tissues are more based on fibrocartilage, the mechanical property and durability of shortage normal hyaline, while
Undue growth, periosteal proliferation, cartilage hypertrophy, damage normal structure, disease propagation after usually occurring some complication as repaired
Deng.
With Autologous Chondrocyte implantation technique（Autologous chondrocyte implantation, ACI）'s
Maturation, cartilage damage repairing and treating progresses into organizational project and repairs field, and Self periosteum valve is used first generation ACI technologies more,
Easily form periosteum loose, periosteal proliferation stretches into articular cavity and generally requires second operation excision, the periosteum of transplanting also makes subchondral bone
Density increase, causes neocartilage tissue deterioration, the biomechanical property of the hyaline cartilage sample tissue of the periosteum generation of transplanting, resistance to
Grind persistence not good, easily change in quality, whole surgical procedure complex operation, high cost.
Second generation cell therapy substitutes Self periosteum using a kind of absorbable support, reduces the damage to health tissues
Evil, but it still needs to two operations, still needs to suture, and technology is relative complex, and cell distribution is unbalanced, the risk that there is cell loss.
The cell therapy of the third generation then further optimizes to above technology, Autologous Chondrocyte is implanted into a kind of absorbable
Membrane support, and continue cultivate some days, cell can be made to be uniformly distributed in collagem membrane, then by this cell collagen film be implanted into
Repaired at cartilage damage, be referred to as the Autologous Chondrocyte implantation technique of matrix induction（Matrix-induced
autologous chondrocyte implantation (MACI)）, this technology post-operative recovery time is short, easy to operate,
Wound is small, repaired area reaches, up to 20cm2, postoperative to produce more hyaline cartilages, therapeutic effect is ideal, and this is also avoided
Before complication caused by technology.But MACI technologies still need collection autologous patient cartilage cell, damage normal soft
Bone tissue, still needs to second operation, adds patient suffering, while medical expense is expensive.
On the other hand, with the development of tissue engineering technique, stem cells technology is increasingly mature, and it is medically applied and also got over
To be more concerned.Because stem cell has tissue totipotency, various histocytes can be divided into, it is thin to be especially divided into cartilage
Born of the same parents, this makes it possible that it is clinically applied to repair cartilage damage.
The content of the invention
The invention aims to solve it is of the prior art detection poor accuracy, under-sensitive defect there is provided
The preparation method of the autologous bone marrow mesenchymal stem cells compound rest of matrix induction a kind of solves the above problems.
A kind of preparation method of the autologous bone marrow mesenchymal stem cells compound rest induced the invention discloses matrix, specifically
Step is：
（1）, aseptically, using 20ml syringe, 1ml liquaemin physiological saline is pumped into syringe, then
It is continuing with syringe and is pumped into marrow 9ml, remove after syringe needle, it is high that liquaemin physiological saline and marrow are added into DMEM containing 10ml
In the blake bottle of sugar culture-medium, after being well mixed, bone marrow suspension is obtained；
（2）, aseptically, take peripheral blood in patients 100-200ml, be placed in blood bag, seal；
（3）, the peripheral blood of patient is sub-packed in 50ml centrifuge tubes, every pipe 45ml, under conditions of 3000rpm, centrifugation
30min, takes supernatant, under conditions of 3000rpm, and supernatant is centrifuged into 30min, goes precipitation, degerming through 0.22 μm of bore filter, obtains
To autoserum, dispense stand-by；
（4）, bone marrow suspension is slowly added to along tube wall in the centrifuge tube of the liquid of cell separation containing 20ml, under conditions of 2000rpm,
20min is centrifuged, middle tunica albuginea layer is then drawn, 50ml PBS is then added into tunica albuginea layer, and blow and beat uniformly, in 1500rpm
Under conditions of, 5min is centrifuged, supernatant is abandoned, 50ml PBS piping and druming is added uniformly, under conditions of 1500rpm, 5min is centrifuged, abandons
Supernatant, obtained precipitation as aim cell；
（5）, autologous blood from DMEM high glucose mediums to DMEM high glucose mediums that add in autoserum and gentamicin sulphate,
Clear volumetric concentration is that the concentration of the gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, then takes 15ml to contain
The DMEM high glucose mediums of autoserum and gentamicin sulphate are added in aim cell, are then blown and beaten cell uniformly, and
Count, with 5 × 105/ cm2It is inoculated in T25 Tissue Culture Flasks, is placed in 37 DEG C, 5% CO2Cultivated in incubator, later every three
It changes a DMEM high glucose medium, and the DMEM high glucose mediums also contain 10% autoserum and 40U/ml gentamicin sulphates；
（6）, after cell attachment reaches more than 80%, blot nutrient solution, cell rinsed twice with PBS liquid；
（7）, trypsase-EDTA solution is added into blake bottle, the matter of the trypsase in trypsase-EDTA solution
It is 0.25% to measure concentration, and the mass concentration of the EDTA in trypsase-EDTA solution is 0.01%, until covering all cells
Untill, it is placed in 37 DEG C, 5% CO2After incubator 2-3min, it is inverted under the microscope, it was observed that cell retraction, space between cells increase
When, then terminate digestion to instillation 5ml DMEM in high glucose culture medium in blake bottle；
（8）, with suction pipe blow and beat bottom of bottle repeatedly, cell is come off from bottle wall, then cell suspension is transferred in 50ml centrifuge tubes,
Add 45ml DMEM in high glucose culture mediums, centrifuge 7min, go after supernatant, repeat the above steps twice, that is, it is heavy after being cleaned
Form sediment；
（9）, autologous blood from DMEM high glucose mediums to DMEM high glucose mediums that add in autoserum and gentamicin sulphate,
Clear volumetric concentration is that the concentration of the gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, is then taken containing autologous blood
Clearly, the DMEM in high glucose culture medium regulation cell concentration of gentamicin sulphate is 1 × 108/ L, 75cm is inoculated in by cell2Cell is trained
Support and continue to cultivate in bottle；
（10）, after cell culture 3-4 weeks, passage 3 times；
（11）, blot nutrient solution, trypsase-EDTA solution is added into blake bottle, in trypsase-EDTA solution
The mass concentration of trypsase be 0.25%, the mass concentration of the EDTA in trypsase-EDTA solution is 0.01%, directly
Untill all cells are covered, it is placed in after incubator 2-3min, cell retraction, space between cells increase is observed under inverted microscope
When, then terminate digestion to instillation DMEM in high glucose culture medium in blake bottle；
（12）, cell suspension centrifuged into 7min, abandon supernatant, then wash with PBS cell 3 times；
（13）, cell count, autoserum and gentamicin sulphate, DMEM high glucose mediums are added into DMEM high glucose mediums
In the volumetric concentration of autoserum be that the concentration of gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, then
Cell is resuspended with the DMEM in high glucose culture medium containing autoserum, gentamicin sulphate, adjustment cell concentration is 5 × 106/ml；
（14）, add into DMEM high glucose mediums it is autologous in autoserum and gentamicin sulphate, DMEM high glucose mediums
The volumetric concentration of serum is that the concentration of the gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, takes a sterile pig
The Collagen Type VI film of source I/III, upward, smooth surface is positioned in aseptic plastic culture dish matte down, then with containing autoserum, sulfuric acid
The DMEM in high glucose culture medium soaked overnight of gentamicin；
（15）, by step（13）The cell suspension prepared is instilled on the ready Collagen Type VI film of pig source I/III, cell number about 1
×106/cm2, continue to cultivate 3 days, you can.
Preferably, described step（1）In, the concentration of described liquaemin physiological saline is 1200U/ml.
Preferably, described step（1）In, the sulfuric acid celebrating containing 40U/ml is big mould in described DMEM high glucose mediums
Element.
Preferably, described step（8）And step（12）In, centrifugal rotational speed is 1200rpm.
Described step（7）、（11）In, trypsase-EDTA solution is purchased from Beijing Lei Gen Bioisystech Co., Ltd.
The present invention has advantages below compared with prior art：
1st, the transplanting same day, with physiological saline to step（15）The autologous bone marrow mesenchymal stem cells composite support of obtained matrix induction
Frame is rinsed 3 times, is trimmed the Collagen Type VI film of pig source I/III according to the shape at cartilage damage, is then made matte towards articular cartilage defect
Place, smooth surface is fixed at cartilage defect towards articular cavity, to repair cartilage damage, and present invention employs a boar peritonaeum source
I/III Collagen Type VI bilayer membrane structure, one face has relatively highdensity collagenous fibres, and mantle friction is relatively low, and cell is not
It is penetrating, cell can be prevented to be spread to articular cavity, another side be coarse surface, above space it is larger, be conducive to cartilage cell attached
Wherein, this film has persistence, tear-resistant, the operation such as it can bear to cut, punch, sutures, its is flexible, can accomplish
Different shape, will not shrink over time.It has absorbability, and 2 Zhou Houke of transplanting are degraded and absorbed, can conduct
Splendid tissue engineering bracket material；
2nd, using autologous bone marrow mesenchymal stem cells（Bone marrow–derived mesenchymal
Stem cells, BMSC) as seed cell, its abundance of originating can be drawn materials by simple bone marrow aspiration, not deposited
In tissue matching and immunological rejection, in vitro culture performance is stable, it is easy to passage amplification, can overcome internal cartilage cell's conduct
Seed cell limited source, amplification in vitro are easily caused the shortcoming of seed cell aging and biological function decline, while also reducing
Corrective surgery number of times, reduces medical expense；
3rd, using autoserum, it is to avoid using hyclone may caused by immunogenic response and disease infect；
4th, this technology is using gentamicin sulphate as bacteriostatic agent, and its concentration is 40U/ml, to a variety of gram-negative bacterias and the positive
Bacterium all has antibacterial and bactericidal action, the alternative penicillin and streptomysin used at present；
5th, the present invention can be used for treatment 3-20cm2Cartilage damage area, repair surface is big, and accuracy is high, and sensitivity is good.
Embodiment
Embodiments of the invention are elaborated below, the present embodiment is carried out lower premised on technical solution of the present invention
Implement, give detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following implementations
Example.
A kind of preparation method of the autologous bone marrow mesenchymal stem cells compound rest induced the invention discloses matrix, specifically
Step is：
（1）, aseptically, using 20ml syringe, 1ml liquaemin physiological saline is pumped into syringe, then
It is continuing with syringe and is pumped into marrow 9ml, remove after syringe needle, it is high that liquaemin physiological saline and marrow are added into DMEM containing 10ml
In the blake bottle of sugar culture-medium, after being well mixed, bone marrow suspension is obtained；
（2）, aseptically, take peripheral blood in patients 100-200ml, be placed in blood bag, seal；
（3）, the peripheral blood of patient is sub-packed in 50ml centrifuge tubes, every pipe 45ml, under conditions of 3000rpm, centrifugation
30min, takes supernatant, under conditions of 3000rpm, and supernatant is centrifuged into 30min, goes precipitation, degerming through 0.22 μm of bore filter, obtains
To autoserum, dispense stand-by；
（4）, bone marrow suspension is slowly added to along tube wall in the centrifuge tube of the liquid of cell separation containing 20ml, under conditions of 2000rpm,
20min is centrifuged, middle tunica albuginea layer is then drawn, 50ml PBS is then added into tunica albuginea layer, and blow and beat uniformly, in 1500rpm
Under conditions of, 5min is centrifuged, supernatant is abandoned, 50ml PBS piping and druming is added uniformly, under conditions of 1500rpm, 5min is centrifuged, abandons
Supernatant, obtained precipitation as aim cell；
（5）, autologous blood from DMEM high glucose mediums to DMEM high glucose mediums that add in autoserum and gentamicin sulphate,
Clear volumetric concentration is that the concentration of the gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, then takes 15ml to contain
The DMEM high glucose mediums of autoserum and gentamicin sulphate are added in aim cell, are then blown and beaten cell uniformly, and
Count, with 5 × 105/ cm2It is inoculated in T25 Tissue Culture Flasks, is placed in 37 DEG C, 5% CO2Cultivated in incubator, later every three
It changes a DMEM high glucose medium, and the DMEM high glucose mediums also contain 10% autoserum and 40U/ml gentamicin sulphates；
（6）, after cell attachment reaches more than 80%, blot nutrient solution, cell rinsed twice with PBS liquid；
（7）, trypsase-EDTA solution is added into blake bottle, the matter of the trypsase in trypsase-EDTA solution
It is 0.25% to measure concentration, and the mass concentration of the EDTA in trypsase-EDTA solution is 0.01%, until covering all cells
Untill, it is placed in 37 DEG C, 5% CO2After incubator 2-3min, it is inverted under the microscope, it was observed that cell retraction, space between cells increase
When, then terminate digestion to instillation 5ml DMEM in high glucose culture medium in blake bottle；
（8）, with suction pipe blow and beat bottom of bottle repeatedly, cell is come off from bottle wall, then cell suspension is transferred in 50ml centrifuge tubes,
Add 45ml DMEM in high glucose culture mediums, centrifuge 7min, go after supernatant, repeat the above steps twice, that is, it is heavy after being cleaned
Form sediment；
（9）, autologous blood from DMEM high glucose mediums to DMEM high glucose mediums that add in autoserum and gentamicin sulphate,
Clear volumetric concentration is that the concentration of the gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, is then taken containing autologous blood
Clearly, the DMEM in high glucose culture medium regulation cell concentration of gentamicin sulphate is 1 × 108/ L, 75cm is inoculated in by cell2Cell is trained
Support and continue to cultivate in bottle；
（10）, after cell culture 3-4 weeks, passage 3 times；
（11）, blot nutrient solution, trypsase-EDTA solution is added into blake bottle, in trypsase-EDTA solution
The mass concentration of trypsase be 0.25%, the mass concentration of the EDTA in trypsase-EDTA solution is 0.01%, directly
Untill all cells are covered, it is placed in after incubator 2-3min, cell retraction, space between cells increase is observed under inverted microscope
When, then terminate digestion to instillation DMEM in high glucose culture medium in blake bottle；
（12）, cell suspension centrifuged into 7min, abandon supernatant, then wash with PBS cell 3 times；
（13）, cell count, autoserum and gentamicin sulphate, DMEM high glucose mediums are added into DMEM high glucose mediums
In the volumetric concentration of autoserum be that the concentration of gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, then
Cell is resuspended with the DMEM in high glucose culture medium containing autoserum, gentamicin sulphate, adjustment cell concentration is 5 × 106/ml；
（14）, add into DMEM high glucose mediums it is autologous in autoserum and gentamicin sulphate, DMEM high glucose mediums
The volumetric concentration of serum is that the concentration of the gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, takes a sterile pig
The Collagen Type VI film of source I/III, upward, smooth surface is positioned in aseptic plastic culture dish matte down, then with containing autoserum, sulfuric acid
The DMEM in high glucose culture medium soaked overnight of gentamicin；
（15）, by step（13）The cell suspension prepared is instilled on the ready Collagen Type VI film of pig source I/III, cell number about 1
×106/cm2, continue to cultivate 3 days, you can.
Preferably, described step（1）In, the concentration of described liquaemin physiological saline is 1200U/ml.
Preferably, described step（1）In, the sulfuric acid celebrating containing 40U/ml is big mould in described DMEM high glucose mediums
Element.
Preferably, described step（8）And step（12）In, centrifugal rotational speed is 1200rpm, can so prevent cell
It is damaged.
Described step（7）、（11）In, trypsase-EDTA solution is purchased from Beijing Lei Gen Bioisystech Co., Ltd.
Embodiment 1
The invention discloses a kind of preparation method of the autologous bone marrow mesenchymal stem cells compound rest of matrix induction, specific steps
For：
（1）, aseptically, using 20ml syringe, 1ml liquaemin physiological saline is pumped into syringe, then
It is continuing with syringe and is pumped into marrow 9ml, remove after syringe needle, it is high that liquaemin physiological saline and marrow are added into DMEM containing 10ml
In the blake bottle of sugar culture-medium, after being well mixed, bone marrow suspension is obtained；
（2）, aseptically, take peripheral blood in patients 150ml, be placed in blood bag, seal；
（3）, the peripheral blood of patient is sub-packed in 50ml centrifuge tubes, every pipe 45ml, under conditions of 3000rpm, centrifugation
30min, takes supernatant, under conditions of 3000rpm, and supernatant is centrifuged into 30min, goes precipitation, degerming through 0.22 μm of bore filter, obtains
To autoserum, dispense stand-by；
（4）, bone marrow suspension is slowly added to along tube wall in the centrifuge tube of the liquid of cell separation containing 20ml, under conditions of 2000rpm,
20min is centrifuged, middle tunica albuginea layer is then drawn, 50ml PBS is then added into tunica albuginea layer, and blow and beat uniformly, in 1500rpm
Under conditions of, 5min is centrifuged, supernatant is abandoned, 50ml PBS piping and druming is added uniformly, under conditions of 1500rpm, 5min is centrifuged, abandons
Supernatant, obtained precipitation as aim cell；
（5）, autologous blood from DMEM high glucose mediums to DMEM high glucose mediums that add in autoserum and gentamicin sulphate,
Clear volumetric concentration is that the concentration of the gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, then takes 15ml to contain
The DMEM high glucose mediums of autoserum and gentamicin sulphate are added in aim cell, are then blown and beaten cell uniformly, and
Count, with 5 × 105/ cm2It is inoculated in T25 Tissue Culture Flasks, is placed in 37 DEG C, 5% CO2Cultivated in incubator, later every three
It changes a DMEM high glucose medium, and the DMEM high glucose mediums also contain 10% autoserum and 40U/ml gentamicin sulphates；
（6）, after cell attachment reaches more than 80%, blot nutrient solution, cell rinsed twice with PBS liquid；
（7）, trypsase-EDTA solution is added into blake bottle, the matter of the trypsase in trypsase-EDTA solution
It is 0.25% to measure concentration, and the mass concentration of the EDTA in trypsase-EDTA solution is 0.01%, until covering all cells
Untill, it is placed in 37 DEG C, 5% CO2After incubator 2-3min, it is inverted under the microscope, it was observed that cell retraction, space between cells increase
When, then terminate digestion to instillation 5ml DMEM in high glucose culture medium in blake bottle；
（8）, with suction pipe blow and beat bottom of bottle repeatedly, cell is come off from bottle wall, then cell suspension is transferred in 50ml centrifuge tubes,
Add 45ml DMEM in high glucose culture mediums, centrifuge 7min, go after supernatant, repeat the above steps twice, that is, it is heavy after being cleaned
Form sediment；
（9）, autologous blood from DMEM high glucose mediums to DMEM high glucose mediums that add in autoserum and gentamicin sulphate,
Clear volumetric concentration is that the concentration of the gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, is then taken containing autologous blood
Clearly, the DMEM in high glucose culture medium regulation cell concentration of gentamicin sulphate is 1 × 108/ L, 75cm is inoculated in by cell2Cell is trained
Support and continue to cultivate in bottle；
（10）, after cell culture 3 weeks, passage 3 times；
（11）, blot nutrient solution, trypsase-EDTA solution is added into blake bottle, in trypsase-EDTA solution
The mass concentration of trypsase be 0.25%, the mass concentration of the EDTA in trypsase-EDTA solution is 0.01%, directly
Untill all cells are covered, it is placed in after incubator 2min, cell retraction is observed under inverted microscope, when space between cells increases,
Terminate and digest to instillation DMEM in high glucose culture medium in blake bottle again；
（12）, cell suspension centrifuged into 7min, abandon supernatant, then wash with PBS cell 3 times；
（13）, cell count, autoserum and gentamicin sulphate, DMEM high glucose mediums are added into DMEM high glucose mediums
In the volumetric concentration of autoserum be that the concentration of gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, then
Cell is resuspended with the DMEM in high glucose culture medium containing autoserum, gentamicin sulphate, adjustment cell concentration is 5 × 106/ml；
（14）, add into DMEM high glucose mediums it is autologous in autoserum and gentamicin sulphate, DMEM high glucose mediums
The volumetric concentration of serum is that the concentration of the gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, takes suitable size
The sterile Collagen Type VI film of pig source I/III, upward, smooth surface is positioned in aseptic plastic culture dish matte down, then with containing autologous blood
Clearly, the DMEM in high glucose culture medium soaked overnight of gentamicin sulphate；
（15）, by step（13）The cell suspension prepared is instilled on the ready Collagen Type VI film of pig source I/III, cell number about 1
×106/cm2, continue to cultivate 3 days, you can.
Preferably, described step（1）In, the concentration of described liquaemin physiological saline is 1200U/ml.
Preferably, described step（1）In, the sulfuric acid celebrating containing 40U/ml is big mould in described DMEM high glucose mediums
Element.
Preferably, described step（8）And step（12）In, centrifugal rotational speed is 1200rpm.
Described step（7）、（11）In, trypsase-EDTA solution is purchased from Beijing Lei Gen Bioisystech Co., Ltd.
Embodiment 2
The invention discloses a kind of preparation method of the autologous bone marrow mesenchymal stem cells compound rest of matrix induction, specific steps
For：
（1）, aseptically, using 20ml syringe, 1ml liquaemin physiological saline is pumped into syringe, then
It is continuing with syringe and is pumped into marrow 9ml, remove after syringe needle, it is high that liquaemin physiological saline and marrow are added into DMEM containing 10ml
In the blake bottle of sugar culture-medium, after being well mixed, bone marrow suspension is obtained；
（2）, aseptically, take peripheral blood in patients 150ml, be placed in blood bag, seal；
（3）, the peripheral blood of patient is sub-packed in 50ml centrifuge tubes, every pipe 45ml, under conditions of 3000rpm, centrifugation
30min, takes supernatant, under conditions of 3000rpm, and supernatant is centrifuged into 30min, goes precipitation, degerming through 0.22 μm of bore filter, obtains
To autoserum, dispense stand-by；
（4）, bone marrow suspension is slowly added to along tube wall in the centrifuge tube of the liquid of cell separation containing 20ml, under conditions of 2000rpm,
20min is centrifuged, middle tunica albuginea layer is then drawn, 50ml PBS is then added into tunica albuginea layer, and blow and beat uniformly, in 1500rpm
Under conditions of, 5min is centrifuged, supernatant is abandoned, 50ml PBS piping and druming is added uniformly, under conditions of 1500rpm, 5min is centrifuged, abandons
Supernatant, obtained precipitation as aim cell；
（5）, autologous blood from DMEM high glucose mediums to DMEM high glucose mediums that add in autoserum and gentamicin sulphate,
Clear volumetric concentration is that the concentration of the gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, then takes 15ml to contain
The DMEM high glucose mediums of autoserum and gentamicin sulphate are added in aim cell, are then blown and beaten cell uniformly, and
Count, with 5 × 105/ cm2It is inoculated in T25 Tissue Culture Flasks, is placed in 37 DEG C, 5% CO2Cultivated in incubator, later every three
It changes a DMEM high glucose medium, and the DMEM high glucose mediums also contain 10% autoserum and 40U/ml gentamicin sulphates；
（6）, after cell attachment reaches more than 80%, blot nutrient solution, cell rinsed twice with PBS liquid；
（7）, trypsase-EDTA solution is added into blake bottle, the matter of the trypsase in trypsase-EDTA solution
It is 0.25% to measure concentration, and the mass concentration of the EDTA in trypsase-EDTA solution is 0.01%, until covering all cells
Untill, it is placed in 37 DEG C, 5% CO2After incubator 3min, it is inverted under the microscope, it was observed that when cell retraction, space between cells increase,
Terminate and digest to instillation 5ml DMEM in high glucose culture medium in blake bottle again；
（8）, with suction pipe blow and beat bottom of bottle repeatedly, cell is come off from bottle wall, then cell suspension is transferred in 50ml centrifuge tubes,
Add 45ml DMEM in high glucose culture mediums, centrifuge 7min, go after supernatant, repeat the above steps twice, that is, it is heavy after being cleaned
Form sediment；
（9）, autologous blood from DMEM high glucose mediums to DMEM high glucose mediums that add in autoserum and gentamicin sulphate,
Clear volumetric concentration is that the concentration of the gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, is then taken containing autologous blood
Clearly, the DMEM in high glucose culture medium regulation cell concentration of gentamicin sulphate is 1 × 108/ L, 75cm is inoculated in by cell2Cell is trained
Support and continue to cultivate in bottle；
（10）, after cell culture 4 weeks, passage 3 times；
（11）, blot nutrient solution, added in blake bottle in trypsase-EDTA solution, trypsase-EDTA solution
The mass concentration of trypsase is 0.25%, and the mass concentration of the EDTA in trypsase-EDTA solution is 0.01%, until
Untill covering all cells, it is placed in after incubator 3min, cell retraction is observed under inverted microscope, when space between cells increases, then
DMEM in high glucose culture medium is instilled in blake bottle and terminates digestion；
（12）, cell suspension centrifuged into 7min, abandon supernatant, then wash with PBS cell 3 times；
（13）, cell count, autoserum and gentamicin sulphate, DMEM high glucose mediums are added into DMEM high glucose mediums
In the volumetric concentration of autoserum be that the concentration of gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, then
Cell is resuspended with the DMEM in high glucose culture medium containing autoserum, gentamicin sulphate, adjustment cell concentration is 5 × 106/ml；
（14）, add into DMEM high glucose mediums it is autologous in autoserum and gentamicin sulphate, DMEM high glucose mediums
The volumetric concentration of serum is that the concentration of the gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, takes suitable size
The sterile Collagen Type VI film of pig source I/III, upward, smooth surface is positioned in aseptic plastic culture dish matte down, then with containing autologous blood
Clearly, the DMEM in high glucose culture medium soaked overnight of gentamicin sulphate；
（15）, by step（13）The cell suspension prepared is instilled on the ready Collagen Type VI film of pig source I/III, cell number about 1
×106/cm2, continue to cultivate 3 days, you can.
Described step（1）In, the concentration of described liquaemin physiological saline is 1200U/ml.
Described step（1）In, the gentamicin sulphate containing 40U/ml in described DMEM high glucose mediums.
Preferably, described step（8）And step（12）In, centrifugal rotational speed is 1200rpm.
Described step（7）、（11）In, trypsase-EDTA solution is purchased from Beijing Lei Gen Bioisystech Co., Ltd.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and that described in above-described embodiment and specification is the present invention
Principle, various changes and modifications of the present invention are possible without departing from the spirit and scope of the present invention, these change and
Improvement is both fallen within the range of claimed invention.The protection domain of application claims by appended claims and its
Equivalent is defined.
Claims (4)
1. a kind of preparation method of the autologous bone marrow mesenchymal stem cells compound rest of matrix induction, it is characterised in that：Specific step
Suddenly it is：
（1）, aseptically, using 20ml syringe, 1ml liquaemin physiological saline is pumped into syringe, then
It is continuing with syringe and is pumped into marrow 9ml, remove after syringe needle, it is high that liquaemin physiological saline and marrow are added into DMEM containing 10ml
In the blake bottle of sugar culture-medium, after being well mixed, bone marrow suspension is obtained；
（2）, aseptically, take peripheral blood in patients 100-200ml, be placed in blood bag, seal；
（3）, the peripheral blood of patient is sub-packed in 50ml centrifuge tubes, every pipe 45ml, under conditions of 3000rpm, centrifugation
30min, takes supernatant, under conditions of 3000rpm, and supernatant is centrifuged into 30min, goes precipitation, degerming through 0.22 μm of bore filter, obtains
To autoserum, dispense stand-by；
（4）, bone marrow suspension is slowly added to along tube wall in the centrifuge tube of the liquid of cell separation containing 20ml, under conditions of 2000rpm,
20min is centrifuged, middle tunica albuginea layer is then drawn, 50ml PBS is then added into tunica albuginea layer, and blow and beat uniformly, in 1500rpm
Under conditions of, 5min is centrifuged, supernatant is abandoned, 50ml PBS piping and druming is added uniformly, under conditions of 1500rpm, 5min is centrifuged, abandons
Supernatant, obtained precipitation as aim cell；
（5）, autologous blood from DMEM high glucose mediums to DMEM high glucose mediums that add in autoserum and gentamicin sulphate,
Clear volumetric concentration is that the concentration of the gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, then takes 15ml to contain
The DMEM high glucose mediums of autoserum and gentamicin sulphate are added in aim cell, are then blown and beaten cell uniformly, and
Count, with 5 × 105/ cm2It is inoculated in T25 Tissue Culture Flasks, is placed in 37 DEG C, 5% CO2Cultivated in incubator, later every three
It changes a DMEM high glucose medium, and the DMEM high glucose mediums also contain 10% autoserum and 40U/ml gentamicin sulphates；
（6）, after cell attachment reaches more than 80%, blot nutrient solution, cell rinsed twice with PBS liquid；
（7）, trypsase-EDTA solution is added into blake bottle, the matter of the trypsase in trypsase-EDTA solution
It is 0.25% to measure concentration, and the mass concentration of the EDTA in trypsase-EDTA solution is 0.01%, until covering all cells
Untill, it is placed in 37 DEG C, 5% CO2After incubator 2-3min, it is inverted under the microscope, it was observed that cell retraction, space between cells increase
When, then terminate digestion to instillation 5ml DMEM in high glucose culture medium in blake bottle；
（8）, with suction pipe blow and beat bottom of bottle repeatedly, cell is come off from bottle wall, then cell suspension is transferred in 50ml centrifuge tubes,
Add 45ml DMEM in high glucose culture mediums, centrifuge 7min, go after supernatant, repeat the above steps twice, that is, it is heavy after being cleaned
Form sediment；
（9）, autologous blood from DMEM high glucose mediums to DMEM high glucose mediums that add in autoserum and gentamicin sulphate,
Clear volumetric concentration is that the concentration of the gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, is then taken containing autologous blood
Clearly, the DMEM in high glucose culture medium regulation cell concentration of gentamicin sulphate is 1 × 108/ L, 75cm is inoculated in by cell2Cell is trained
Support and continue to cultivate in bottle；
（10）, after cell culture 3-4 weeks, passage 3 times；
（11）, blot nutrient solution, trypsase-EDTA solution is added into blake bottle, in trypsase-EDTA solution
The mass concentration of trypsase be 0.25%, the mass concentration of the EDTA in trypsase-EDTA solution is 0.01%, directly
Untill all cells are covered, it is placed in after incubator 2-3min, cell retraction, space between cells increase is observed under inverted microscope
When, then terminate digestion to instillation DMEM in high glucose culture medium in blake bottle；
（12）, cell suspension centrifuged into 7min, abandon supernatant, then wash with PBS cell 3 times；
（13）, cell count, autoserum and gentamicin sulphate, DMEM high glucose mediums are added into DMEM high glucose mediums
In the volumetric concentration of autoserum be that the concentration of gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, then
Cell is resuspended with the DMEM in high glucose culture medium containing autoserum, gentamicin sulphate, adjustment cell concentration is 5 × 106/ml；
（14）, add into DMEM high glucose mediums it is autologous in autoserum and gentamicin sulphate, DMEM high glucose mediums
The volumetric concentration of serum is that the concentration of the gentamicin sulphate in 10%, DMEM high glucose mediums is 40U/ml, takes a sterile pig
The Collagen Type VI film of source I/III, upward, smooth surface is positioned in aseptic plastic culture dish matte down, then with containing autoserum, sulfuric acid
The DMEM in high glucose culture medium soaked overnight of gentamicin；
（15）, by step（13）The cell suspension prepared is instilled on the ready Collagen Type VI film of pig source I/III, cell number about 1
×106/cm2, continue to cultivate 3 days, you can.
2. a kind of preparation side of the autologous bone marrow mesenchymal stem cells compound rest of matrix induction according to claim 1
Method, it is characterised in that：Described step（1）In, the concentration of described liquaemin physiological saline is 1200U/ml.
3. a kind of preparation of the autologous bone marrow mesenchymal stem cells compound rest of matrix induction according to claim 1 or 2
Method, it is characterised in that：Described step（1）In, the sulfuric acid celebrating containing 40U/ml is big mould in described DMEM high glucose mediums
Element.
4. a kind of preparation side of the autologous bone marrow mesenchymal stem cells compound rest of matrix induction according to claim 1
Method, it is characterised in that：Described step（8）And step（12）In, centrifugal rotational speed is 1200rpm.
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|---|---|---|---|---|
| CN107929817A (en) * | 2017-12-01 | 2018-04-20 | 蒋文明 | A kind of preparation method of degradable blood vessel bracket material |
| CN109289088A (en) * | 2018-10-26 | 2019-02-01 | 福州大学 | A type Ⅰ/type Ⅲ collagen composite scaffold loaded with Scutellaria officinalis |
| CN109675117A (en) * | 2019-02-26 | 2019-04-26 | 百澳瑞派（天津）生物科技有限公司 | A kind of composite bone repairing material and preparation method thereof |
-
2017
- 2017-02-21 CN CN201710094014.8A patent/CN106975105A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107929817A (en) * | 2017-12-01 | 2018-04-20 | 蒋文明 | A kind of preparation method of degradable blood vessel bracket material |
| CN109289088A (en) * | 2018-10-26 | 2019-02-01 | 福州大学 | A type Ⅰ/type Ⅲ collagen composite scaffold loaded with Scutellaria officinalis |
| CN109289088B (en) * | 2018-10-26 | 2021-04-27 | 福州大学 | Type I/III collagen composite bracket loaded with caulis spatholobi |
| CN109675117A (en) * | 2019-02-26 | 2019-04-26 | 百澳瑞派（天津）生物科技有限公司 | A kind of composite bone repairing material and preparation method thereof |
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