CN109593706A - A kind of cultural method of culture medium and Endometrial stem cell - Google Patents
A kind of cultural method of culture medium and Endometrial stem cell Download PDFInfo
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Abstract
The invention belongs to field of biotechnology more particularly to the cultural methods of a kind of culture medium and Endometrial stem cell.The present invention provides a kind of culture mediums, comprising: serum free medium, platelet cracking content, N-acetylcystein, galacturonic acid and antibiotic.The present invention provides the cultural methods of a kind of culture medium and Endometrial stem cell, will lead to Endometrial stem cell increment low efficiency, cell quality difference and the technological deficiency for being easy differentiation for solving existing culture medium culture.
Description
Technical field
The invention belongs to field of biotechnology more particularly to the cultural methods of a kind of culture medium and Endometrial stem cell.
Background technique
Stem cell refers to that a kind of early stage with hyperproliferation potential, self-renewal capacity and height differentiation capability does not divide
Change cell.Mescenchymal stem cell is stem cell member of greatest concern at present, has height self-renewal capacity and Multidirectional Differentiation
Potential.Human uterine is the tissue with highly efficient regeneration and differentiation capability, and containing in endometrium has hyperproliferation energy
The stem cell of power, scientist separate these stem cells from healthy women menses and come out, and are named as Endometrial stem cell,
Also known as menses derived stem cell.
Endometrial stem cell (endometrial derived stem cells, EDSCs) refer to self-renewing,
The undifferentiated endometrial cell of Multidirectional Differentiation and unlimited multiplication capacity, including minimal amount of endometrial epithelial cell, uterus
Interior theca-titerstitial cells and most blood vessels of endometrium endothelial cell.Since Endometrial stem cell has materials convenient and not
It is limited by morals and legal issue, it is made to have a wide range of applications in terms of experimental study and clinical application.Intrauterine
The preclinical study of film stem-cell therapy disease and clinical test, oneself through largely being studied, such as treat myocardial infarction, apoplexy,
Diabetes, liver fibrosis, pulmonary fibrosis, premature ovarian failure, chronic ischemic pain, muscular atrophy, osteoarthritis.
Endometrial stem cell main separation method Ficoll partition method and cell natural subsidence adherent method at present,
It is mononuclearcell that Ficoll is separated from menses, which contains a plurality of types of cells: human peripheral it is single
Nucleus, vascular endothelial cell, mescenchymal stem cell, intimal epithelium cell etc., thus can by Ficoll density gradient from
Heart enamel method obtains Endometrial stem cell.Using cell nature adherent method, this method cell purity obtained is low, is unfavorable for son
The subsequent purification of endometrial stem cells.It is this to be directly inoculated in addition, i.e. directly inoculation after the separation of existing endometrial stem cells
Mode will affect the adherent of Endometrial stem cell；And cultivating system is common cultivating system, causes endometrium dry thin
Born of the same parents' proliferation is slow, and too long incubation time breaks up Endometrial stem cell.
In conclusion traditional stem cell media is not suitable for the culture of Endometrial stem cell, traditional stem cell
Culture medium will lead to the increment low efficiency of Endometrial stem cell, and cell quality is poor；Endometrial stem cell rises in value in limited generation
Afterwards, just break up；It is not able to satisfy clinical cytology dosage demand.
Summary of the invention
In view of this, purifying suitable for Endometrial stem cell and expanding on a large scale the object of the present invention is to provide a kind of
New culture medium.
It is a further object to provide a kind of separation suitable for Endometrial stem cell and cultural methods.
The present invention provides a kind of culture mediums, comprising: serum free medium, platelet cracking content, N-acetylcystein,
Galacturonic acid and antibiotic.
Preferably, the content of the platelet cracking content in the medium is 10-20wt.%.
Preferably, the platelet cracking content is to obtain blood platelet after ultrasonic treatment.
Preferably, the content of the N-acetylcystein in the medium is 2~5wt.%.
Preferably, the content of the galacturonic acid in the medium is 5~10wt.%.
Preferably, the antibiotic is specially mycillin；The content of the antibiotic in the medium be 1 ×
~2 ×.Blueness-streptomysin working concentration is 1 ×~2 × (blueness-streptomysin of the culture medium addition 1-2mL of every 100mL).
Preferably, the serum free medium is specially human mesenchymal stem cell serum free medium.
It should be noted that the antibiotic in culture medium is that can have inhibiting effect to the microorganism in menses.
The present invention also provides a kind of cultural methods of Endometrial stem cell, heavy in culture dish with the culture medium
It is cultivated after outstanding Endometrial stem cell, every the culture medium that replacement in 2-4 days is fresh.
More preferably, the Endometrial stem cell is used by second day of collector and the menses in third day
After FFicoll density-gradient centrifugation method isolates tunica albuginea layer, with erythrocyte cracked liquid (Red Blood Cell Lysis
Buffer it is obtained after) cracking the red blood cell of menses.The present invention uses erythrocyte cracked liquid, and effectively red blood cell can be avoided to intrauterine
The adherent influence of film stem cell.
Wherein, first day menses can wash away the thin fungi of intravaginal, and the menses quality of 2~3 day period is preferable, simultaneously
Pollution can also be reduced.
Preferably, the culture dish first passes through the infiltration and incubation processing that human mesenchymal stem cell promotees adherent reagent in advance.
It should be noted that promoting adherent reagent using human mesenchymal stem cell handles culture dish, promote on the inner membrance of menses
The rush wall growth of chrotoplast, vascular endothelial cell, avoids the pollution of the intimal epithelium cell, vascular endothelial cell of menses, together
When play the role of cell purification.
Preferably, the density that Endometrial stem cell is resuspended is 8 × 104Cells/mL~1 × 105cells/mL。
Compared with prior art, culture medium provided by the invention uses platelet cracking content, and platelet cracking content contains
Natural nutritional ingredient, and platelet cracking content is blood platelet by ultrasound cracking, so that the internal substance of blood platelet is split into
Basic ingredient, it is easier to be utilized by the cells, platelet cracking content of the invention substitutes fetal calf serum, and it is dirty to avoid ox borne virus
Dye.The present invention selects being used in combination for platelet cracking content, N-acetylcystein and galacturonic acid, on the one hand, the present invention
Culture medium be conducive to the growth of mescenchymal stem cell, nutrition needed for supplement growth of mesenchymal stem cells that can be comprehensive at
Point, enable mescenchymal stem cell quickly to divide increment in vitro；On the other hand, culture medium of the invention it is possible to prevente effectively from
Mesenchymal stem cells are broken up in incubation in vitro, moreover it is possible to so that filling between this type of Endometrial stem cell is more mature
Matter stem cell is able to maintain original stemness, and division increment, cell purity are high rapidly, and quality is uniform, and quantity is sufficient, overcomes point
The higher mescenchymal stem cell of change degree is cultivated in vitro is easy to the shortcomings that breaking up.
In addition, cultural method of the invention uses the present invention also provides a kind of cultural method of Endometrial stem cell
Human mesenchymal stem cell is first passed through in advance to promote the infiltration of adherent reagent and be incubated for the culture dish of processing, can further limit menses
Endothelial cell and epithelial cell it is adherent, play the role of cell purification.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 shows the surface antigen testing result for the Endometrial stem cell that the group 4 that the embodiment of the present invention 3 provides is cultivated；
Fig. 2 shows the cell proliferation trend of the Endometrial stem cell for the group 1-4 culture that the embodiment of the present invention 4 provides；
Fig. 3 shows the cell for the Endometrial stem cell that group 4, patent 1 and patent 2 that the embodiment of the present invention 4 provides are cultivated
Increment trend；
Fig. 4 show the group 4 that the embodiment of the present invention 5 provides, the Endometrial stem cell induced osteogenesis of patent 1 and patent 2 at
Rouge result figure；
Fig. 5 shows the Endometrial stem cell induced osteogenesis for the culture of group 1 that the embodiment of the present invention 5 provides at rouge result figure；
Fig. 6 shows the Endometrial stem cell induced osteogenesis for the culture of group 2 that the embodiment of the present invention 5 provides at rouge result figure；
Fig. 7 shows the Endometrial stem cell induced osteogenesis for the culture of group 3 that the embodiment of the present invention 5 provides at rouge result figure；
Fig. 8 shows the Endometrial stem cell induced osteogenesis for the culture of group 4 that the embodiment of the present invention 5 provides at rouge result figure.
Specific embodiment
The present invention provides the cultural methods of a kind of culture medium and Endometrial stem cell, for solving existing culture medium
Culture will lead to Endometrial stem cell increment low efficiency, cell quality difference and the technological deficiency for being easy differentiation.
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Wherein, raw material used in following embodiment is commercially available or self-control.
Embodiment 1
The embodiment of the invention provides portion of reagent preparation methods:
1, human mesenchymal stem cell promotees the preparation method of adherent reagent: by human mesenchymal stem cells promote adherent reagent with
1640 basal mediums are to obtain human mesenchymal stem cell after 1:99 is mixed to promote adherent reagent according to volume ratio (v:v), in 4 DEG C of rings
It is spare in border.
2, the preparation method of platelet cracking content: under 4 DEG C of environment, taking 100mL human peripheral, is centrifuged in 2000rpm
15min is transferred to upper plasma in new centrifuge tube；Blood plasma is centrifuged 20min, the blood of remaining bottom 20mL in 3000rpm
Slurry and blood platelet, the blood plasma on upper layer, which is inhaled, to be abandoned.After pasteur pipet piping and druming uniformly, as ultrasound cracking 10min, cracking in Ultrasound Instrument
After obtain platelet cracking content, be transferred to spare in 4 DEG C of environment.
3, the preparation method of the culture medium of group 1: MSCXF Medium (it buys in BI company, article No.:
It 05-200-1A) is mixed with 2 × blueness-streptomysin, obtains group 1.
4, the preparation method of the culture medium of group 2: MSCXF Medium (it buys in BI company, article No.:
05-200-1A), 10wt.% platelet cracking content and mycillin mix, and obtain group 2.
5, the preparation method of the culture medium of group 3: MSCXF Medium (it buys in BI company, article No.:
05-200-1A), 5wt.%N- acetylcysteine, 5wt.% galacturonic acid and mycillin mix, and obtain group 3.
6, the preparation method of the culture medium of group 4: MSCXF Medium (it buys in BI company, article No.:
05-200-1A), 10wt.% platelet cracking content, 5wt.%N- acetylcysteine, 5wt.% galacturonic acid and green strepto-
Element mixes, and obtains group 4.
Embodiment 2
The embodiment of the present invention provides the method for the culture of Endometrial stem cell:
1, the human mesenchymal stem cell that embodiment 1 is prepared is added in culture dish and promotees adherent reagent, makes human mesenchyme
Stem cell promotees adherent reagent and infiltrates ware bottom, is placed at 37 DEG C and is incubated for, and after 1h, inhales and abandons the adherent examination of human mesenchymal stem cell rush
Agent.
2, the menses in the second day~third day of collector.
3, reference literature (Zhou Yunfan, Yang Bo, Hu Xiang, etc.；Separation, the training of menses source temper Endometrium mescenchymal stem cell
It supports and identification [J], Chinese Tissue Engineering Study and clinical rehabilitation；2010,14 (32): 5952~5956.), by the menses of step 2
After isolating tunica albuginea layer with Ficoll density-gradient centrifugation method, with erythrocyte cracked liquid (Red Blood Cell Lysis
Buffer after the red blood cell for) cracking menses, 1500rpm is centrifuged 5min, after discarding erythrocyte cracked liquid, with 1640 basal mediums
It is resuspended, abandons supernatant after 1500rpm is centrifuged 5min, obtain cell precipitation.
4, the cell precipitation of step 3 is resuspended in the culture medium of the group 1- group 4 prepared respectively with embodiment 1, adjusts density
It is 105Then cells/mL is seeded in the culture dish of step 1 being incubated for.
5, the culture dish of step 4 is transferred to 37 DEG C, 5%CO2, 95% humidity environment in cultivate, changed afterwards for 24 hours
Liquid.
6, a not good liquor (culture medium for replacing fresh corresponding group 1- group 4) is changed within every 3 days, is reached to cell confluency
80% or so, carry out cell secondary culture.
Embodiment 3
The embodiment of the present invention provides the surface antigen detection of Endometrial stem cell, the specific steps are as follows:
Endometrial stem cells are taken, are made 1 × 106The cell suspension of cells/mL, take respectively anti-human CD45, CD14, CD29,
Each 5 μ L of the monoclonal antibody of CD90, CD105, HLA-DR is added the 500 μ L of cell suspension that group 4 is cultivated, is protected from light incubates at room temperature
20min is educated, while setting up blank Isotype control, 1500r/min is centrifuged 5min, abandons supernatant, washs 2 with the PBS containing 10%FBS
Time, upper machine testing after being resuspended with 500 μ L PBS, according to domestic patent CN105586308A, (stem cell media and culture are in utero
The method of film stem cell, is denoted as patent 1) and CN106801034A (a kind of Endometrial stem cell large-scale preparation method and its
Using being denoted as patent 2) method respectively obtain the Endometrial stem cell of two patents, and detected using above-mentioned detection method
The surface antigen of patent 1 and patent 2, as a result as shown in table 1 and Fig. 1.
Table 1
| Surface antigen title | CD29 | CD90 | CD105 | CD14 | CD45 | HLA-DR |
| Group 4 | 97.5% | 98.6% | 95.4% | 1.1% | 0.8% | 0.3% |
| Patent 1 | 98.4% | 94.7% | 91.7% | 1.3% | 2.8% | 2.6% |
| Patent 2 | 96.9% | 97.2% | 93.8% | 1.5% | 5.9% | 4.8% |
| Group 1 | 47.5% | 57.6% | 72.8% | 0.8% | 5.4% | 0.1% |
| Group 2 | 95.2% | 93.8% | 96.7% | 1.6% | 1.3% | 0.0% |
| Group 3 | 96.8% | 95.3% | 98.1% | 2.5% | 5.7% | 3.3% |
From the result of table 1 and Fig. 1 it is found that comparing the culture medium of patent 1 and patent 2, what the culture medium culture of group 4 obtained
Cell is Endometrial stem cell, and the purity of Endometrial stem cell is very high.The culture medium of group 1 do not have effective nutrition at
Point, when Endometrial stem cell is cultivated in vitro, there is automatic differentiating phenomenon；The cell proliferation rate of the culture medium of group 2 compared with
Slowly, part cell occur volume become larger, model deformation；The cell proliferation rate of the culture medium of group 3 is slower, and part cell occurs
Volume becomes larger, model deformation, illustrates that there is collaboration to promote uterus for platelet cracking content, N-acetylcystein and galacturonic acid
The increment of inner membrance stem cell acts on.
Embodiment 4
The embodiment of the invention provides the methods of the growth curve of detection Endometrial stem cell:
The patent 1 of the group 1- group 4 and step 3 that take step 2 culture to obtain and P2~P3 of patent 2 are dry for endometrium
Cell resuspension is seeded in 6 orifice plates, and cell-seeding-density is 5 × 104The hole cells/mL, 2mL/.A hole cell was taken every 2 days
Calculate total number of cells, the growth curve of the cell of the group 1- group 4 and patent 1 and patent 2 of record, as a result such as table 2 and Fig. 2-3
It is shown.
Table 2
* P < 0.05, compared with group 1, significant difference are indicated.
* indicates P < 0.001, compared with group 1, extremely significant sex differernce.
From table 2 and Fig. 2 it is found that the cell proliferation curve for the Endometrial stem cell that group 1 is cultivated is gentle, entirely cultivating
In period, increase without there is apparent quantity；The cell incubation period for the Endometrial stem cell that group 2 and group 3 are cultivated
It is longer, started apparent logarithmic growth occur in the 9th day, but cell quantity is still on the low side；The endometrium that group 4 is cultivated is dry thin
Born of the same parents start to occur significantly being proliferated after 6 days latent, enter platform growth period in 15 days.Illustrate platelet cracking content, N- acetyl
Cysteine, 5~10wt.% galacturonic acid three there is apparent collaboration to promote the division increment of Endometrial stem cell
Effect.
As can be seen from Figure 3, the cell proliferation trend for the Endometrial stem cell that group 4 is cultivated is cultivated better than patent 1 and patent 2
Endometrial stem cell cell proliferation trend, illustrate that the culture medium culture of this patent can promote Endometrial stem cell to increase
Value.
Embodiment 5
The embodiment of the invention provides the detections of the osteogenic lipogenesis of Endometrial stem cell:
Bibliography: " the Endometrial stem cell culture of menses source, identification and the research of vitro differentiation potential ", Yan Yan, east
Strong Feng, Sang Yunxia etc., Chinese cell biology journal [J] .2014,36 (7): 892-899, with reference to the document to group 1- group
4, the Endometrial stem cell of patent 1 and patent 2 induces differentiation into rouge skeletonization, as a result as shown in Fig. 4-Fig. 8.
As can be seen from Figure 4, Fig. 4-A is that patent 2 cultivates obtained Endometrial stem cell, does not occur into rouge phenomenon, says
Clear-cells has broken up in incubation in vitro, loses the differentiation capability of cell, when causing to induce into rouge in vitro, not
Form fat drips；Fig. 4-B is that patent 1 turns out the cell come, it is seen that fat drips, but still there is part cell not contain fat drips, explanation
Part cell has already appeared differentiating phenomenon；Fig. 4-C is the cell that group 4 of the invention is cultivated, and " the rouge of large area occurs in cell
When the endometrial stem cells for illustrating that the present invention is cultivated are cultivated in vitro, there is not differentiating phenomenon in drop ", and cell remains good
Stemness.
Fig. 5-A be the Endometrial stem cell skeletonization that the culture medium culture of group 1 obtains as a result, Fig. 5-B be into rouge as a result,
As can be seen from Figure 5, the culture medium of group 1 does not occur osteogenic lipogenesis phenomenon, illustrates that Endometrial stem cell was cultivated in vitro
Break up in journey, has lost the differentiation capability of cell；Fig. 6-A is that the endometrium that the culture medium culture of group 2 obtains is dry
Cell skeletonization is as a result, Fig. 6-B is into rouge as a result, as can be seen from Figure 6, the Endometrial stem cell that group 2 is cultivated is largely without going out
Existing osteogenic lipogenesis phenomenon, small part have the ability of differentiation osteogenic lipogenesis, illustrate the big portion of Endometrial stem cell that group 2 is cultivated
Divide in incubation in vitro and broken up, loses the differentiation capability of cell；Fig. 7-A is that the culture medium culture of group 2 obtains
The Endometrial stem cell skeletonization arrived is as a result, Fig. 7-B is into rouge as a result, as can be seen from Figure 7, the endometrium that group 3 is cultivated is dry thin
Born of the same parents do not occur osteogenic lipogenesis phenomenon largely, and small part has the ability of differentiation osteogenic lipogenesis, illustrate the uterus that group 3 is cultivated
Break up in the most of incubation in vitro of inner membrance stem cell, has lost the differentiation capability of cell；Fig. 8-A is group 4
The obtained Endometrial stem cell skeletonization of culture medium culture as a result, Fig. 8-B is into rouge as a result, as it can be observed in the picture that group 4 is cultivated
Endometrial stem cell largely there is osteogenic lipogenesis phenomenon, illustrate that Endometrial stem cell that group 4 is cultivated is most of and exist
Undifferentiated in Process of in vitro, it is dry thin to illustrate that the culture medium of group 4 is able to maintain endometrium for the differentiation capability with stem cell
The stemness of born of the same parents.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of culture medium characterized by comprising serum free medium, platelet cracking content, N-acetylcystein, half
Lactobionic acid and antibiotic.
2. culture medium according to claim 1, which is characterized in that the platelet cracking content containing in the medium
Amount is 10-20wt.%.
3. culture medium according to claim 2, which is characterized in that the platelet cracking content is by blood platelet by ultrasound
It is obtained after processing.
4. culture medium according to claim 1, which is characterized in that the N-acetylcystein is in the medium
Content is 2~5wt.%.
5. culture medium according to claim 1, which is characterized in that the content of the galacturonic acid in the medium
For 5~10wt.%.
6. culture medium according to claim 1, which is characterized in that the antibiotic is specially blueness-streptomysin；The antibiosis
Element content in the medium is 1 ×~2 ×.
7. culture medium according to claim 1, which is characterized in that the serum free medium is specially that human mesenchyme is dry thin
Born of the same parents' serum free medium.
8. a kind of cultural method of Endometrial stem cell, which is characterized in that with the described in any item culture mediums of claim 1-7
It is cultivated after Endometrial stem cell is resuspended in culture dish, it is described in any item every the 2-4 days fresh claim 1-7 of replacement
Culture medium.
9. cultural method according to claim 8, which is characterized in that the culture dish first passes through human mesenchymal stem cell in advance
Promote the infiltration and incubation processing of adherent reagent.
10. cultural method according to claim 8, which is characterized in that the density that Endometrial stem cell is resuspended is 8
×104Cells/mL~1 × 105cells/mL。
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| CN110592001A (en) * | 2019-09-30 | 2019-12-20 | 广东华夏健康生命科学有限公司 | Purification culture system for oviduct epithelial cells |
| CN110964691A (en) * | 2019-12-31 | 2020-04-07 | 南昌诺汇医药科技有限公司 | Directed adipogenic differentiation culture of endometrial stem cells |
| CN114317421A (en) * | 2021-12-16 | 2022-04-12 | 北京科技大学 | Method, composition and application for enhancing mesenchymal stem cells to promote angiogenesis |
| CN115094025A (en) * | 2022-07-14 | 2022-09-23 | 北京中科细胞控股有限公司 | Preparation method of endometrial stromal stem cells |
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| US20140227336A1 (en) * | 2005-09-09 | 2014-08-14 | Cytex Therapeutics, Inc. | Tissue Engineering Methods and Compositions |
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| CN110592001A (en) * | 2019-09-30 | 2019-12-20 | 广东华夏健康生命科学有限公司 | Purification culture system for oviduct epithelial cells |
| CN110964691A (en) * | 2019-12-31 | 2020-04-07 | 南昌诺汇医药科技有限公司 | Directed adipogenic differentiation culture of endometrial stem cells |
| CN114317421A (en) * | 2021-12-16 | 2022-04-12 | 北京科技大学 | Method, composition and application for enhancing mesenchymal stem cells to promote angiogenesis |
| CN114317421B (en) * | 2021-12-16 | 2024-05-03 | 北京科技大学 | Method, composition and application for strengthening mesenchymal stem cells to promote angiogenesis |
| CN115094025A (en) * | 2022-07-14 | 2022-09-23 | 北京中科细胞控股有限公司 | Preparation method of endometrial stromal stem cells |
| CN115094025B (en) * | 2022-07-14 | 2022-11-08 | 北京中科细胞控股有限公司 | Preparation method of endometrial stromal stem cells |
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