CN107513518A - A kind of umbilical cord mesenchymal stem cells extracorporeal culturing method - Google Patents
A kind of umbilical cord mesenchymal stem cells extracorporeal culturing method Download PDFInfo
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Abstract
The invention discloses a kind of umbilical cord mesenchymal stem cells extracorporeal culturing method, its step are as follows：Take the nearly tire section 5cm of umbilical cord；Umbilical cord tissue is put into the blue wide-mouth bottle for the PBS liquid for being pre-loaded with content 90U/ml penicillin and 90U/ml streptomysins；Tissue wet is kept by DMEM/F12 nutrient solutions are added dropwise in tissue shear history；Washed 3 times using 90U/ml penicillin and 90U/ml streptomysins, the centrifuge tube for taking 15ml is standby by umbilical cord tissue centrifugation 5min；The preparation of digestive ferment；1h, 2h, 3h terminate digestion with the DMEM/F12 culture mediums containing 10%FBS, 90U/ml penicillin and 0.1mg/ml streptomysins after digestion respectively, obtain umbilical cord mesenchymal stem cells；By the umbilical cord mesenchymal stem cells of separation with 1 × 106/ ml cell densities are inoculated into the culture dish coated with chitosan film；Culture dish is placed on 37 DEG C, volume fraction 5%CO248h is cultivated in incubator, old nutrient solution is carefully drawn along culture dish wall using sample loading gun, new nutrient solution is reheated, cultivates to the 5th day, the present invention improves stem cell separation and the efficiency prepared.
Description
Technical field
The invention belongs to technical field of stem cell culture, more specifically, more particularly to a kind of umbilical cord mesenchymal stem cells
Extracorporeal culturing method.
Background technology
Stem cell is cells of origin, is the cell with propagation and differentiation potential, has the ability that self-renewing replicates
(Self-renewing) well differentiated functioning cell can, be produced.Briefly, it is that one kind has multi-lineage potential
With the original neoblast of the of self-replication capacity, be each histoorgan to form mammal initial cell.It is dry thin
Born of the same parents morphologically have general character, are generally circular in shape or oval, and cell volume is small, and core is relatively large, and nucleus is mostly often dyeing
Matter, and there is higher telomerase activation.Stem cell is self-replacation or differentiation function cell, mainly due to cell itself
State and microenvironment factor are determined.Various cycles plain (Cyclin) and cycle element dependent kinase including adjusting the cell cycle
(Cyclin-DependentKinase), gene transcription factor, the cytoplasmic factor of influence asymmetric cell division.Microenvironment
Factor, including stem cell and peripheral cell, stem cell and epimatrix and stem cell and the interaction of various soluble factors.
Stage of development according to residing for stem cell is divided into embryonic stem cell (embryonicstemcell, ES cell) and adult stem cell
(somaticstemcell).It is divided into three classes according to the potentiality of development of stem cell：Myeloid-lymphoid stem cell (totipotentstemcell,
TSC), multipotential stem cell (pluripotentstemcell) and unipotent stem cell (unipotentstemcell).Stem cell
(StemCell) it is a kind of inabundant differentiation, still jejune cell, there is the potential work(for regenerating various histoorgans and human body
Can, medical field is referred to as " general-purpose cell ".The mankind place hope on the separation and in vitro culture using stem cell, breed out group in vitro
Knit or organ, and eventually through tissue or organ transplant, realize the treatment to clinical disease, in existing stem cell incubation
Aging is cured, activity reduces.
The content of the invention
The invention aims to solve shortcoming present in prior art, and a kind of umbilical cord mesenchyma proposed is dry thin
Born of the same parents' extracorporeal culturing method.
To achieve the above object, the present invention provides following technical scheme：
A kind of umbilical cord mesenchymal stem cells extracorporeal culturing method, the umbilical cord mesenchymal stem cells extracorporeal culturing method specifically walk
It is rapid as follows：
S1：The nearly tire section 5cm of umbilical cord of normal mature caesarean birth is taken under germ-free condition, umbilical cord tissue is divided into 5 deciles；
S2：The good umbilical cord tissue of S1 deciles is put into and is pre-loaded with content 90U/ml penicillin and 90U/ml streptomysins
In the blue wide-mouth bottle of PBS liquid, preserve at 4 DEG C, and entered in 2 hours using 90U/ml penicillin and 90U/ml streptomysins
Bloodstain and vein tissue remaining in tissue are rejected in row washing；
S3：Umbilical cord tissue is cut into the broken lamellar structure of 2mm × 2mm × 2mm sizes, DMEM/ is added dropwise in shear history
F12 nutrient solutions keep tissue wet；
S4：Washed 3 times using 90U/ml penicillin and 90U/ml streptomysins, take 15ml centrifuge tube by umbilical cord tissue from
Heart 5min is standby；
S5：The preparation of digestive ferment, II Collagenase Type 0.1-0.3w/v% of configuration, pancreatin 0.1-0.3w/v%, EDTA
0.03w/v%, hyaluronidase 0.2w/v% and DNA enzymatic 0-0.2w/v% are blended in sterilized culture medium, will be located in S4
The umbilical cord tissue managed is blended in digestive ferment culture medium, 37.5 DEG C of concussion digestion；
S6：Respectively after digestion 1h, 2h, 3h with containing 10%FBS, 90U/ml penicillin and 0.1mg/ml streptomysins
DMEM/F12 culture mediums terminate digestion, and 1200r/min is centrifuged 5 minutes, abandoned supernatant, obtain umbilical cord mesenchymal stem cells；
S7：By the umbilical cord mesenchymal stem cells of separation with 1 × 106/ ml cell densities are inoculated into coated with chitosan film
Culture dish in；
S8：By culture dish be placed on 37 DEG C, volume fraction be 5% food-grade carbon-dioxide incubator in cultivate 48h, make
Old nutrient solution is carefully drawn along culture dish wall with sample loading gun, new nutrient solution is reheated, cultivates to the 5th day.
Preferably, the surface coated on culture dish after dissolving the chitosan in acetic acid, placement 12h blows in super-clean bench
It is dry, form chitosan film.
Preferably, described people source specific virus include HIV, HBV, HCV, TP and CMV.
Preferably, the umbilical cord is received by collection specification, archives are established in registration, enters pedestrian source virus to the Cord blood of collection
Detection, ensure umbilical cord securely and reliably and trace to the source.
The technique effect and advantage of the present invention：A kind of umbilical cord mesenchymal stem cells extracorporeal culturing method provided by the invention,
Compared with conventional art, the activity that the present invention significantly improves mescenchymal stem cell has certain anti-aging, improves mesenchyma
The efficiency of the culture of stem cell, more mescenchymal stem cells can be obtained in the shorter time, facilitate clinical research.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with specific embodiment, to this
Invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, not
For limiting the present invention.Based on the embodiment in the present invention, those of ordinary skill in the art are not before creative work is made
The every other embodiment obtained is put, belongs to the scope of protection of the invention.
Embodiment 1
A kind of umbilical cord mesenchymal stem cells extracorporeal culturing method, the umbilical cord mesenchymal stem cells extracorporeal culturing method specifically walk
It is rapid as follows：
S1：The nearly tire section 5cm of umbilical cord of normal mature caesarean birth is taken under germ-free condition, umbilical cord tissue is divided into 5 deciles；
S2：The good umbilical cord tissue of S1 deciles is put into and is pre-loaded with content 90U/ml penicillin and 90U/ml streptomysins
In the blue wide-mouth bottle of PBS liquid, preserve at 4 DEG C, and entered in 2 hours using 90U/ml penicillin and 90U/ml streptomysins
Bloodstain and vein tissue remaining in tissue are rejected in row washing；
S3：Umbilical cord tissue is cut into the broken lamellar structure of 2mm × 2mm × 2mm sizes, DMEM/ is added dropwise in shear history
F12 nutrient solutions keep tissue wet；
S4：Washed 3 times using 90U/ml penicillin and 90U/ml streptomysins, take 15ml centrifuge tube by umbilical cord tissue from
Heart 5min is standby；
S5：The preparation of digestive ferment, configuration II Collagenase Type 0.1w/v%, pancreatin 0.3w/v%, EDTA0.03w/v%, thoroughly
Bright matter acid enzyme 0.2w/v% and DNA enzymatic 0w/v% are blended in sterilized culture medium, and the umbilical cord tissue handled well in S4 is mixed
Close in digestive ferment culture medium, 37.5 DEG C of concussion digestion；
S6：Respectively after digestion 1h, 2h, 3h with containing 10%FBS, 90U/ml penicillin and 0.1mg/ml streptomysins
DMEM/F12 culture mediums terminate digestion, and 1200r/min is centrifuged 5 minutes, abandoned supernatant, obtain umbilical cord mesenchymal stem cells；
S7：By the umbilical cord mesenchymal stem cells of separation with 1 × 106/ ml cell densities are inoculated into coated with chitosan film
Culture dish in；
S8：By culture dish be placed on 37 DEG C, volume fraction be 5% food-grade carbon-dioxide incubator in cultivate 48h, make
Old nutrient solution is carefully drawn along culture dish wall with sample loading gun, new nutrient solution is reheated, cultivates to the 5th day.
Specifically, the surface coated on culture dish after dissolving the chitosan in acetic acid, placement 12h blows in super-clean bench
It is dry, form chitosan film.
Specifically, described people source specific virus include HIV, HBV, HCV, TP and CMV.
Specifically, the umbilical cord is received by collection specification, archives are established in registration, pedestrian source virus is entered to the Cord blood of collection
Detection, ensure umbilical cord securely and reliably and trace to the source.
Embodiment 2
A kind of umbilical cord mesenchymal stem cells extracorporeal culturing method, the umbilical cord mesenchymal stem cells extracorporeal culturing method specifically walk
It is rapid as follows：
S1：The nearly tire section 5cm of umbilical cord of normal mature caesarean birth is taken under germ-free condition, umbilical cord tissue is divided into 5 deciles；
S2：The good umbilical cord tissue of S1 deciles is put into and is pre-loaded with content 90U/ml penicillin and 90U/ml streptomysins
In the blue wide-mouth bottle of PBS liquid, preserve at 4 DEG C, and entered in 2 hours using 90U/ml penicillin and 90U/ml streptomysins
Bloodstain and vein tissue remaining in tissue are rejected in row washing；
S3：Umbilical cord tissue is cut into the broken lamellar structure of 2mm × 2mm × 2mm sizes, DMEM/ is added dropwise in shear history
F12 nutrient solutions keep tissue wet；
S4：Washed 3 times using 90U/ml penicillin and 90U/ml streptomysins, take 15ml centrifuge tube by umbilical cord tissue from
Heart 5min is standby；
S5：The preparation of digestive ferment, configuration II Collagenase Type 0.3w/v%, pancreatin 0.1w/v%, EDTA0.03w/v%, thoroughly
Bright matter acid enzyme 0.2w/v% and DNA enzymatic 0.1w/v% are blended in sterilized culture medium, the umbilical cord tissue that will be handled well in S4
It is blended in digestive ferment culture medium, 37.5 DEG C of concussion digestion；
S6：Respectively after digestion 1h, 2h, 3h with containing 10%FBS, 90U/ml penicillin and 0.1mg/ml streptomysins
DMEM/F12 culture mediums terminate digestion, and 1200r/min is centrifuged 5 minutes, abandoned supernatant, obtain umbilical cord mesenchymal stem cells；
S7：By the umbilical cord mesenchymal stem cells of separation with 1 × 106/ ml cell densities are inoculated into coated with chitosan film
Culture dish in；
S8：By culture dish be placed on 37 DEG C, volume fraction be 5% food-grade carbon-dioxide incubator in cultivate 48h, make
Old nutrient solution is carefully drawn along culture dish wall with sample loading gun, new nutrient solution is reheated, cultivates to the 5th day.
Specifically, the surface coated on culture dish after dissolving the chitosan in acetic acid, placement 12h blows in super-clean bench
It is dry, form chitosan film.
Specifically, described people source specific virus include HIV, HBV, HCV, TP and CMV.
Specifically, the umbilical cord is received by collection specification, archives are established in registration, pedestrian source virus is entered to the Cord blood of collection
Detection, ensure umbilical cord securely and reliably and trace to the source.
Embodiment 3
A kind of umbilical cord mesenchymal stem cells extracorporeal culturing method, the umbilical cord mesenchymal stem cells extracorporeal culturing method specifically walk
It is rapid as follows：
S1：The nearly tire section 5cm of umbilical cord of normal mature caesarean birth is taken under germ-free condition, umbilical cord tissue is divided into 5 deciles；
S2：The good umbilical cord tissue of S1 deciles is put into and is pre-loaded with content 90U/ml penicillin and 90U/ml streptomysins
In the blue wide-mouth bottle of PBS liquid, preserve at 4 DEG C, and entered in 2 hours using 90U/ml penicillin and 90U/ml streptomysins
Bloodstain and vein tissue remaining in tissue are rejected in row washing；
S3：Umbilical cord tissue is cut into the broken lamellar structure of 2mm × 2mm × 2mm sizes, DMEM/ is added dropwise in shear history
F12 nutrient solutions keep tissue wet；
S4：Washed 3 times using 90U/ml penicillin and 90U/ml streptomysins, take 15ml centrifuge tube by umbilical cord tissue from
Heart 5min is standby；
S5：The preparation of digestive ferment, configuration II Collagenase Type 0.2w/v%, pancreatin 0.2w/v%, EDTA0.03w/v%, thoroughly
Bright matter acid enzyme 0.2w/v% and DNA enzymatic 0.2w/v% are blended in sterilized culture medium, the umbilical cord tissue that will be handled well in S4
It is blended in digestive ferment culture medium, 37.5 DEG C of concussion digestion；
S6：Respectively after digestion 1h, 2h, 3h with containing 10%FBS, 90U/ml penicillin and 0.1mg/ml streptomysins
DMEM/F12 culture mediums terminate digestion, and 1200r/min is centrifuged 5 minutes, abandoned supernatant, obtain umbilical cord mesenchymal stem cells；
S7：By the umbilical cord mesenchymal stem cells of separation with 1 × 106/ ml cell densities are inoculated into coated with chitosan film
Culture dish in；
S8：By culture dish be placed on 37 DEG C, volume fraction be 5% food-grade carbon-dioxide incubator in cultivate 48h, make
Old nutrient solution is carefully drawn along culture dish wall with sample loading gun, new nutrient solution is reheated, cultivates to the 5th day.
Specifically, the surface coated on culture dish after dissolving the chitosan in acetic acid, placement 12h blows in super-clean bench
It is dry, form chitosan film.
Specifically, described people source specific virus include HIV, HBV, HCV, TP and CMV.
Specifically, the umbilical cord is received by collection specification, archives are established in registration, pedestrian source virus is entered to the Cord blood of collection
Detection, ensure umbilical cord securely and reliably and trace to the source.
A kind of umbilical cord mesenchymal stem cells extracorporeal culturing method provided by the invention, it is dry thin that the present invention significantly improves mesenchyma
The activity of born of the same parents has certain anti-aging, improves the efficiency of the culture of mescenchymal stem cell, can be obtained in the shorter time
More mescenchymal stem cells, facilitate clinical research.
Finally it should be noted that：The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention,
Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, it still may be used
To be modified to the technical scheme described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic,
Within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., it should be included in the present invention's
Within protection domain.
Claims (4)
- A kind of 1. umbilical cord mesenchymal stem cells extracorporeal culturing method, it is characterised in that：The umbilical cord mesenchymal stem cells in vitro culture Method comprises the following steps that：S1：The nearly tire section 5cm of umbilical cord of normal mature caesarean birth is taken under germ-free condition, umbilical cord tissue is divided into 5 deciles；S2：The good umbilical cord tissue of S1 deciles is put into the PBS liquid for being pre-loaded with content 90U/ml penicillin and 90U/ml streptomysins Blue wide-mouth bottle in, preserved at 4 DEG C, and washed in 2 hours using 90U/ml penicillin and 90U/ml streptomysins Wash and reject bloodstain and vein tissue remaining in tissue；S3：Umbilical cord tissue is cut into the broken lamellar structure of 2mm × 2mm × 2mm sizes, DMEM/F12 trainings are added dropwise in shear history Nutrient solution keeps tissue wet；S4：Washed 3 times using 90U/ml penicillin and 90U/ml streptomysins, the centrifuge tube for taking 15ml centrifuges umbilical cord tissue 5min is standby；S5：The preparation of digestive ferment, II Collagenase Type 0.1-0.3w/v% of configuration, pancreatin 0.1-0.3w/v%, EDTA 0.03w/ V%, hyaluronidase 0.2w/v% and DNA enzymatic 0-0.2w/v% are blended in sterilized culture medium, by what is handled well in S4 Umbilical cord tissue is blended in digestive ferment culture medium, 37.5 DEG C of concussion digestion；S6：Distinguish the DMEM/F12 of 1h, 2h, 3h containing 10%FBS, 90U/ml penicillin and 0.1mg/ml streptomysins after digestion Culture medium terminates digestion, and 1200r/min is centrifuged 5 minutes, abandoned supernatant, obtain umbilical cord mesenchymal stem cells；S7：By the umbilical cord mesenchymal stem cells of separation with 1 × 106/ ml cell densities are inoculated into the culture coated with chitosan film In ware；S8：48h will be cultivated in culture dish is placed on 37 DEG C, volume fraction is 5% food-grade carbon-dioxide incubator, using adding Sample rifle carefully draws old nutrient solution along culture dish wall, reheats new nutrient solution, cultivates to the 5th day.
- A kind of 2. umbilical cord mesenchymal stem cells extracorporeal culturing method according to claim 1, it is characterised in that：It is described by shell Glycan is dissolved in the surface coated on culture dish after acetic acid, and 12h dryings are placed in super-clean bench, form chitosan film.
- A kind of 3. umbilical cord mesenchymal stem cells extracorporeal culturing method according to claim 1, it is characterised in that：Described people Source specific virus include HIV, HBV, HCV, TP and CMV.
- A kind of 4. umbilical cord mesenchymal stem cells extracorporeal culturing method according to claim 1, it is characterised in that：The umbilical cord Received by collection specification, register and establish archives, the detection of pedestrian source virus is entered to the Cord blood of collection, ensures that umbilical cord is safe and reliable With trace to the source.
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Cited By (3)
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| CN110157665A (en) * | 2019-05-24 | 2019-08-23 | 吉林省农业科学院 | A kind of separation of human umbilical cord mesenchymal stem cells and cultural method |
| CN112011505A (en) * | 2020-09-09 | 2020-12-01 | 广州同康生物科技有限公司 | Umbilical cord mesenchymal stem cell separation method |
| WO2021036507A1 (en) * | 2019-08-28 | 2021-03-04 | 温州医科大学 | Msc cell thin film integrating gelatin-fibroin composite microspheres, and preparation method therefor |
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