CN110157665A - A kind of separation of human umbilical cord mesenchymal stem cells and cultural method - Google Patents
A kind of separation of human umbilical cord mesenchymal stem cells and cultural method Download PDFInfo
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Abstract
The invention discloses a kind of separation of human umbilical cord mesenchymal stem cells and cultural methods, which is characterized in that the cultural method is the following steps are included: sample collection reception and cell separation, primitive cell culture, secondary culture, cell detection.The beneficial effects of the present invention are: a kind of human umbilical cord mesenchymal stem cells separation of the invention and cultural method substantially reduce cell culture period, have been quickly obtained the mescenchymal stem cell that a large amount of purity are higher, in good condition；Separation and cultural method of the invention has also removed the outer membrane of two arteries, the hUC-MSCs growth conditions of acquisition are good, and the P1 of acquisition is for cell number up to 3~12.5 × 10 in addition to having separated huatong plastic7, flow cytometer detection surface antibody CD44+CD105+Up to (99.6 ± 0.2) %, and have good at rouge, Osteoblast Differentiation ability.
Description
Technical field
The invention belongs to field of biotechnology, are related to technical field of stem cell culture, and in particular to fill between a kind of people's umbilical cord
The separation of matter stem cell and cultural method.
Background technique
Mescenchymal stem cell (Mesenchymal Stem Cells, MSCs) stock is latent with Multidirectional Differentiation in mesoderm
Can, it is distributed in various organization and organ relatively broad.Since MSCs preferably breaks up and self-renewing potential, in tissue repair
It is used as one of main seed cell in the research of regeneration field.In recent years, the clinical value of stem cell was highly visible, wherein
Human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells, hUC-MSCs) are because of it
Source is wide, easy acquisition, dispute of ethic is less, amplification in vitro ability is strong, the high and low oncogenicity of differentiation potential, immunogenicity are low
The advantages that, clinically there is boundless application.
At this stage, MSCs cell therapy is in neurodegenerative disease, cardiovascular and cerebrovascular disease, diabetes, spinal cord injury, heart
Interim progress is achieved in the treatment of sick chronic diseases.The cultural method for preparing of hUC-MSCs is Romanov et al. in 2003
Year obtains from the magnificent Tong Shi glue of removing for the first time and is established.Currently, the preparative separation method of hUC-MSCs is mainly tissue block patch
Wall approach, although the advantage that the method has production cost low, it is long that there is also cell culture periods, is unsuitable for larger scale clinical
The shortcomings that research and application demand.
Summary of the invention
In order to make up for the deficiencies of the prior art, the present invention provides a kind of separation of human umbilical cord mesenchymal stem cells and culture sides
Method, the method prepare cell using enzyme digestion, substantially reduce cell culture period, can be quickly obtained a large amount of purity compared with
Mescenchymal stem cell high, in good condition, and then ideal seed cell is provided for tissue repair and regenerative medicine field.
A kind of separation of human umbilical cord mesenchymal stem cells and cultural method, comprising the following steps:
(1) sample collection reception and cell separation:
(1.1) newborn fetal cord and female blood are acquired: newborn fetal cord is acquired, at away from two end of umbilical cord about 2-3cm
Then the newborn fetal cord of acquisition is placed in the umbilical cord bottle containing amphotericin B and gentamicin storage and transportation liquid by ligation,
It is put into 4-25 DEG C of incubator and saves together together with female blood；
(1.2) the newborn fetal cord of acquisition is transported into laboratory in 22h；
(1.3) specification is received, is registered, and signs informed consent form, carries out HBV, HCV, CMV, TP, HIV and ALT to female blood
Detection；
(1.4) the new life fetal cord described in PBS repeated flushing removes the exocuticle trace of blood, washes out the residual blood in vein；
(1.5) one end of the newborn fetal cord is fixed on autopsy table, is using scalpel at bearing 1cm
The annular mouth of standardized depth appropriateness tears the exocuticle of umbilical cord with dissecting forceps, obtains China's Tong Shi glue, and peel the new green tire
The outer membrane of two arteries in youngster's umbilical cord；
(1.6) digestion reaction is being carried out by digestion enzyme solutions are added in step (1.5) treated newborn fetal cord,
Complete medium is added after the reaction was completed and terminates digestion, obtains cell suspension；
(1.7) cell suspension is sieved with 100 mesh sieve into net, after 200 mesh screens, 1700rpm, is centrifuged 7min later, abandons
Supernatant, precipitating are added complete medium and suspend；
(2) primitive cell culture: by cell inoculation in the culture dish equipped with primitive cell culture liquid, in 37 DEG C, 5%
CO2It is cultivated in incubator, obtains primary cell；The primitive cell culture liquid is made of basal medium and additive, described
Basal medium is DMEM/F12, the additive are as follows: Ultroser G serum substitute 6-8% (v/v), vitamin 80-100
μ g/mL, stem cell factor 30-80ng/mL, amino acid 5-15mmol/mL, microelement 100-200mmol/L；
(3) secondary culture: when the degrees of fusion of primary cell reaches 80% or more, primary cell is inoculated in equipped with passage
In the culture dish of cell culture fluid, in 37 DEG C, 5%CO2It is cultivated in incubator, obtains passage cell；
(4) when the degrees of fusion of passage cell reaches 80% or more, secondary culture cell detection: can be continued；It selects later
Well-grown third generation cell is taken to carry out cytoactive detection, viable count and the inspection of cell streaming after antibody incubation washs
It surveys.
As a preferred solution, enzyme solutions clostridiopetidase A containing 1-2mg/mL, 4-6 μ g/mL are digested described in step (1.6)
Hyaluronidase, digestion process are 37 DEG C of digestive ferment digestion to be added, and shake 1 time every 10min, after digesting 2h, are added complete
Culture medium terminates digestion, obtains digestive juice；And the preparation method of the digestion enzyme solutions is: first using Collagenase I described in 60mg
The PBS dissolution of 60mL obtains mixed liquor, then takes mixed liquor described in 40mL that 200 μ L hyaluronidases are added, later with 0.22 μm
Filter filtering, obtains the digestion enzyme solutions.
It is further preferred that step (1.6) can be using the mixed of 1-2mg/mL clostridiopetidase A and 4-6 μ g/mL hyaluronidase
1,37 DEG C of digestion 60min of liquid are closed, the mode of 0.25% pancreatin and 2,37 DEG C of 0.1%DNaseI mixed liquor digestion 20min, is obtained later
Obtain cell suspension.
It is further preferred that step (1.6) can use 37 DEG C of digestion 1.5h of 1-2mg/mL clostridiopetidase A, later 0.25% pancreas
The mode of 37 DEG C of enzyme digestion 30min, obtains cell suspension.
It is further preferred that the clostridiopetidase A in step (1.6) the digestion enzyme solutions can be Collagenase I, clostridiopetidase A II
With clostridiopetidase A IV.
It is further preferred that vitamin described in step (2) includes vitamin C, vitamin B2, vitamin B12, vitamin
D, the stem cell factor include epidermal growth factor, fibroblast growth factor, platelet derived growth factor, blood
Endothelial tube growth factor, the amino acid include glutamine, arginine, valine, isoleucine, leucine, phenylalanine,
The microelement includes calcium ion and magnesium ion.
It is further preferred that labelled antibody used in flow cytometer detection described in step (4) is CD34-PE, CD44- respectively
FITC、CD45-FITC、CD105-PE。
It is further preferred that the stem cell of the cultural method preparation is with good at rouge, Osteoblast Differentiation potential.
The beneficial effects of the present invention are: a kind of human umbilical cord mesenchymal stem cells separation of the invention and cultural method have
Following advantage:
1. a kind of human umbilical cord mesenchymal stem cells separation of the invention and cultural method substantially reduce cell culture week
Phase has been quickly obtained the mescenchymal stem cell that a large amount of purity are higher, in good condition.
2. a kind of human umbilical cord mesenchymal stem cells separation of the invention and cultural method are also removed in addition to having separated huatong plastic
The outer membrane of two arteries, the hUC-MSCs growth conditions of acquisition are good.
3. the P1 that a kind of human umbilical cord mesenchymal stem cells separation of the invention and cultural method obtain for cell number up to 3~
12.5×107, flow cytometer detection surface antibody CD44+CD105+Up to (99.6 ± 0.2) %, and have good at rouge skeletonization point
Change ability.
Detailed description of the invention
Fig. 1 is human umbilical cord mesenchymal stem cells primitive cell culture effect picture (× 40) in the embodiment of the present invention；
Fig. 2 is human umbilical cord mesenchymal stem cells third generation cell culture effect picture (× 400) in the embodiment of the present invention；
Streaming of the human umbilical cord mesenchymal stem cells third generation cell after antibody incubation washs is thin in Fig. 3 embodiment of the present invention
Born of the same parents' testing result；
Human umbilical cord mesenchymal stem cells adipogenic induction differentiation qualification result figure (× 400) in Fig. 4 inventive embodiments；
Human umbilical cord mesenchymal stem cells Osteoinductive differentiation qualification result figure (× 400) in Fig. 5 inventive embodiments.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is
A part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art
Every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
A kind of separation of human umbilical cord mesenchymal stem cells and cultural method, the cultural method the following steps are included:
(1) sample collection reception and cell are separately cultured:
(1.1) newborn fetal cord and female blood are acquired: newborn fetal cord is acquired, at away from two end of umbilical cord about 2-3cm
Then the newborn fetal cord of acquisition is placed in the umbilical cord bottle containing amphotericin B and gentamicin storage and transportation liquid by ligation,
It is put into 4-25 DEG C of incubator and saves together together with female blood；
(1.2) the newborn fetal cord of acquisition is transported into laboratory in 22h；
(1.3) specification is received, is registered, and signs informed consent form, carries out HBV, HCV, CMV, TP, HIV and ALT to female blood
Detection；
(1.4) digestive ferment is prepared: the PBS dissolution of 60mL of 60mg clostridiopetidase A being obtained mixed liquor, is then taken described in 40mL
200 μ L hyaluronidases are added in mixed liquor, are filtered later with 0.22 μm of filter, obtain digestion enzyme solutions, spare；
(1.5) the new life fetal cord described in PBS repeated flushing removes the exocuticle trace of blood, washes out the residual blood in vein；
(1.6) one end of the newborn fetal cord is fixed on autopsy table, is using scalpel at bearing 1cm
The annular mouth of standardized depth appropriateness tears the exocuticle of umbilical cord with dissecting forceps, obtains China's Tong Shi glue, and peel the new green tire
The outer membrane of two arteries in youngster's umbilical cord；
(1.7) by being added digestion enzyme solutions in step (1.6) treated newborn fetal cord, 37 DEG C of digestion, and
It is shaken 1 time every 10min, after digesting 2h, complete medium is added and terminates digestion, obtains cell suspension；
(1.8) cell suspension is sieved with 100 mesh sieve into net, after 200 mesh screens, 1700rpm, is centrifuged 7min later, abandons
Supernatant, precipitating are added complete medium and suspend；
(2) cell culture: by cell inoculation in the culture dish equipped with primitive cell culture liquid, in 37 DEG C, 5%CO2 is trained
It supports and is cultivated in case, obtain primary cell, specific cultivation results are shown in Fig. 1；The primitive cell culture liquid is by basal medium and adds
Object is added to form, the basal medium is DMEM/F12, the additive are as follows: Ultroser G serum substitute 8% (v/v),
80 μ g/mL of vitamin, stem cell factor 60ng/mL, amino acid 1 2mmol/mL, microelement 130mmol/L；
(3) it secondary culture: when the degrees of fusion of primary cell reaches 80% or more, inhales and abandons primitive cell culture liquid, with life
It manages salt water to clean cell surface 2 times, every ware adds 0.25% pancreatin 1mL, the adherent face of homogeneous immersion cell, 37 DEG C of incubation 1min or room
Temperature is incubated for 3min；Every ware adds complete medium to terminate digestion after cell rounding, quickly shakes, and blows and beats the adherent face of cell repeatedly,
Cell suspension is sucked out into 2 15mL centrifuge tubes, every ware adds appropriate physiological saline, and purging is primary, imports in 15mL centrifuge tube；
1200rpm is centrifuged 6min, abandons supernatant, and precipitating is resuspended with passage cell culture solution, is filtered through cell sieve, counts, makes cell density
For 1-2 × 104/ mL or so sets 37 DEG C, 5%CO2Secondary culture is carried out in incubator, obtains passage cell；The passage cell
Culture solution is made of primitive cell culture liquid and passage additive, the passage additive are as follows: serine 20-30 μ g/mL, sweet ammonia
Sour 20-40 μ g/mL, biotin 3-5mmol/mL, folic acid 2-5mmol/mL, pyridoxol 2-3mmol/mL, thiamine 1-2mmol/
ML, B family vitamin 3-5mmol/mL；
(4) when the degrees of fusion of passage cell reaches 80% or more, secondary culture cell detection: can be continued；It selects later
Well-grown third generation cell (specific cultivation results are shown in Fig. 2) is taken to carry out cytoactive detection, work after antibody incubation washs
Cell count and cell flow cytometer detection, specifically:
(4.1) cell activity and counting: total number of cells measurement is carried out using Countstar cell counter；Utilize blood cell
Tally detects cell activity, and dead cell number is calculated as N, cell activity=[100-10N/C] × 100%；
(4.2) cell flow cytometer detection: cell surface antibodies detection include CD34-PE, CD44-FITC, CD45-FITC,
CD105-PE collects growth conditions good 3rd generation hUC-MSCs, 1500rpm, discards supernatant after being centrifuged 5min, adjustment is thin
Born of the same parents' concentration is to 4 × 106/mL；Later respectively mark IgG-FITC/IgG-PE (Isotype control), CD44-FITC/CD34-PE,
5~10 μ L fluorescence antibodies and 100 μ L cell suspensions to be measured are added in CD45-FITC/CD105-PE, 3 streaming loading pipes, every pipe,
After abundant shaken well, it is protected from light and is incubated for 20min, every pipe adds 2mL physiological saline, centrifuge washing later, and 1200rpm is centrifuged 5min,
Supernatant is abandoned, is repeated aforesaid operations 1 time.Every pipe adds 2mL PBS, 1500rpm to be centrifuged 5min, abandons supernatant, adds 500 μ l PBS, mixes
Upper machine testing afterwards.Flow cytometer detection result: CD44+, CD105+ are above 95%, and CD34+, CD45+ are below 2%, concrete outcome
See Fig. 3.
(4.3) hUC-MSCs is at rouge, Osteoinductive differentiation
Referring to GibcoAdipogenesis/Osteogenesis Differentiation Kit
(Gibco, the U.S.) operating procedure is carried out into rouge, osteogenic induction, respectively with observing after oil red O, Alizarin red staining after 21d, it is seen that
There is formation and the deposition of big and round fat drips and mineralising calcium tubercle in cell peripheral.The result shows that hP-MSCs is with good
At rouge, Osteoblast Differentiation potential, meet the feature of MSCs, concrete outcome is shown in Fig. 4.
HUC-MSCs separation of the invention and cultural method is easy to operate, time-consuming short, at low cost, the cell purity of acquisition
Height, activity is strong, and provides a large amount of seed cell at rouge Osteoblast Differentiation ability with good for clinical research and application.
It should be appreciated that described above, the specific embodiments are only for explaining the present invention, is not intended to limit the present invention.By
The obvious changes or variations that spirit of the invention is extended out are still in the protection scope of this invention.
Claims (8)
1. a kind of human umbilical cord mesenchymal stem cells separation and cultural method, which is characterized in that the cultural method includes following step
It is rapid:
(1) sample collection reception and cell separation:
(1.1) newborn fetal cord and female blood are acquired: acquiring newborn fetal cord, away from the ligation about 2-3cm of two end of umbilical cord at,
Then the newborn fetal cord of acquisition is placed in the umbilical cord bottle containing amphotericin B and gentamicin storage and transportation liquid, together with
Female blood is put into 4-25 DEG C of incubator together and saves；
(1.2) the newborn fetal cord of acquisition is transported into laboratory in 22h；
(1.3) specification is received, is registered, and signs informed consent form, carries out HBV, HCV, CMV, TP, HIV and ALT inspection to female blood
It surveys；
(1.4) the new life fetal cord described in PBS repeated flushing removes the exocuticle trace of blood, washes out the residual blood in vein；
(1.5) one end of the newborn fetal cord is fixed on autopsy table, standardized with scalpel at bearing 1cm
The annular mouth of depth appropriateness tears the exocuticle of umbilical cord with dissecting forceps, obtains China Tong Shi glue, and peels the new life Fetal Umbilical
The outer membrane of two arteries in band；
(1.6) digestion reaction, reaction are being carried out by digestion enzyme solutions are added in step (1.5) treated newborn fetal cord
Complete medium is added after the completion and terminates digestion, obtains cell suspension；
(1.7) cell suspension is first sieved with 100 mesh sieve into net, after 200 mesh screens, 1700rpm later is centrifuged 7min, in abandoning
Clearly, precipitating is added complete medium and suspends；
(2) primitive cell culture: by cell inoculation in the culture dish equipped with primitive cell culture liquid, in 37 DEG C, 5%CO2Culture
It is cultivated in case, obtains primary cell；The primitive cell culture liquid is made of basal medium and additive, the basis culture
Base is DMEM/F12, the additive are as follows: Ultroser G serum substitute 6-8% (v/v), vitamin 80-100 μ g/mL, is done
Porcine HGF 30-80ng/mL, amino acid 5-15mmol/mL, microelement 100-200mmol/L；
(3) secondary culture: when the degrees of fusion of primary cell reaches 80% or more, primary cell is inoculated in equipped with passage cell
In the culture dish of culture solution, in 37 DEG C, 5%CO2It is cultivated in incubator, obtains passage cell；The passage cell culture solution by
Primitive cell culture liquid and passage additive composition, the passage additive are as follows: serine 20-30 μ g/mL, glycine 20-40 μ
G/mL, biotin 3-5mmol/mL, folic acid 2-5mmol/mL, pyridoxol 2-3mmol/mL, thiamine 1-2mmol/mL, B race dimension
Raw element 3-5mmol/mL；
(4) when the degrees of fusion of passage cell reaches 80% or more, secondary culture cell detection: can be continued；Life is chosen later
Long good third generation cell carries out cytoactive detection, viable count and cell flow cytometer detection after antibody incubation washs.
2. a kind of human umbilical cord mesenchymal stem cells separation according to claim 1 and cultural method, which is characterized in that step
(1.6) enzyme solutions clostridiopetidase A containing 1-2mg/mL, 4-6 μ g/mL hyaluronidase are digested described in, digestion process is that digestive ferment is added
37 DEG C of digestion, and shaken 1 time every 10min, after digesting 2h, complete medium is added and terminates digestion, obtains digestive juice；And it is described
The preparation method of digestion enzyme solutions is: the PBS dissolution of clostridiopetidase A 60mL described in 60mg first being obtained mixed liquor, then takes 40mL
200 μ L hyaluronidases are added in the mixed liquor, are filtered later with 0.22 μm of filter, obtain the digestion enzyme solutions.
3. a kind of human umbilical cord mesenchymal stem cells separation according to claim 1 and cultural method, which is characterized in that step
(1.6) 1,37 DEG C of digestion 60min of mixed liquor of 1-2mg/mL clostridiopetidase A and 4-6 μ g/mL hyaluronidase can be used, later
The mode of 0.25% pancreatin and 2,37 DEG C of I mixed liquor of 0.1%DNase digestion 20min, obtains cell suspension.
4. a kind of human umbilical cord mesenchymal stem cells separation according to claim 1 and cultural method, which is characterized in that step
(1.6) 37 DEG C of digestion 1.5h of 1-2mg/mL clostridiopetidase A can be used, the mode of 37 DEG C of 0.25% pancreatin digestion 30min, is obtained later
Obtain cell suspension.
5. a kind of human umbilical cord mesenchymal stem cells separation according to claim 2 and cultural method, which is characterized in that step
(1.6) clostridiopetidase A in the digestion enzyme solutions can be Collagenase I, clostridiopetidase A II and clostridiopetidase A IV.
6. a kind of human umbilical cord mesenchymal stem cells separation according to claim 1 and cultural method, which is characterized in that step
(2) vitamin described in includes vitamin C, vitamin B2, vitamin B12, vitamin D, and the stem cell factor includes
Epidermal growth factor, fibroblast growth factor, platelet derived growth factor, vascular endothelial growth factor, the amino
Acid includes glutamine, arginine, valine, isoleucine, leucine, phenylalanine, and the microelement includes calcium ion
And magnesium ion.
7. a kind of human umbilical cord mesenchymal stem cells separation according to claim 1 and cultural method, which is characterized in that step
(4) labelled antibody used in flow cytometer detection described in is CD34-PE, CD44-FITC, CD45-FITC, CD105-PE respectively.
8. a kind of human umbilical cord mesenchymal stem cells separation according to claim 1 and cultural method, characterized in that the training
The stem cell of the method for supporting preparation has good at rouge, Osteoblast Differentiation potential.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804585A (en) * | 2019-11-19 | 2020-02-18 | 武汉济源高科技有限公司 | Separation method of umbilical cord mesenchymal stem cells |
| CN111690599A (en) * | 2020-07-17 | 2020-09-22 | 天晴干细胞股份有限公司 | Method for promoting mesenchymal stem cells to differentiate into chondroblasts |
| CN112430269A (en) * | 2021-01-28 | 2021-03-02 | 天津汉青生物科技有限公司 | Composition for promoting osteogenic differentiation of umbilical cord blood stem cells and application thereof |
| CN113016781A (en) * | 2021-03-08 | 2021-06-25 | 云南圣衍干细胞技术开发应用股份有限公司 | Mesenchymal stem cell protection solution and preservation method |
| CN114457012A (en) * | 2022-03-23 | 2022-05-10 | 厦门骨本生物科技有限公司 | Separation method of umbilical cord mesenchymal stem cells |
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| CN112430269A (en) * | 2021-01-28 | 2021-03-02 | 天津汉青生物科技有限公司 | Composition for promoting osteogenic differentiation of umbilical cord blood stem cells and application thereof |
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| CN114457012A (en) * | 2022-03-23 | 2022-05-10 | 厦门骨本生物科技有限公司 | Separation method of umbilical cord mesenchymal stem cells |
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