CN109182263A - A method of palace film mescenchymal stem cell is separated with Tea Saponin dissolution menstruation erythrocyte - Google Patents
A method of palace film mescenchymal stem cell is separated with Tea Saponin dissolution menstruation erythrocyte Download PDFInfo
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- CN109182263A CN109182263A CN201811117472.XA CN201811117472A CN109182263A CN 109182263 A CN109182263 A CN 109182263A CN 201811117472 A CN201811117472 A CN 201811117472A CN 109182263 A CN109182263 A CN 109182263A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The present invention relates to a kind of methods with Tea Saponin dissolution menstruation erythrocyte separation palace film mescenchymal stem cell, include the following steps: (1) preparation of reagents: configuration Tea Saponin solution；(2) it obtains cell precipitation: by menstrual blood sample filtering, being centrifuged off upper plasma, obtain cell precipitation A；Tea Saponin solution is added into cell precipitation A, is sufficiently mixed uniformly, centrifugation removal supernatant, obtains cell precipitation B after standing；(3) cell culture: cell precipitation B is suspended in adhere-wall culture in incubator with cell culture fluid, collects the 3rd generation cell, as palace film mescenchymal stem cell.The method of the present invention uses Tea Saponin, do not cause damage that the palace membrane tissue in addition to the single core that conventional method obtains can be obtained aim cell, can rapidly and efficiently separation and Extraction mononuclearcell and the induction of palace membrane tissue obtain palace film mescenchymal stem cell from menstrual blood, and quantity is the several times of traditional tunica albuginea layer.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to it is a kind of with Tea Saponin dissolution menstruation erythrocyte separation palace film between
The method of mesenchymal stem cells.
Background technique
Stem cell is a kind of cell with self-renewing and Proliferation, Differentiation ability, in cell and tissue repair, Yi Jizuo
There is huge application value for the carrier etc. of gene therapy.Mescenchymal stem cell (MesenchymAlstemcells,
MSCs) from a wealth of sources, plasticity is strong, while possessing and being easily isolated culture, a variety of cytokine secretion functions and immunoloregulation function
Many advantages, such as, it is the research hotspot of current stem cell field.It is many research shows that MSCs is in tissue damage reparation and various diseases
Disease treatment aspect has good application prospect, including participates in the Repair of tissue defect such as bone, cartilage and tendon, improves myocardial infarction mould
The cardiac function of type, reduce acute lung injury degree, promote the glucose in diabetes model adjust restore, reduce it is drug-induced
Level of Hepatic Fibrosis and promote neuron regeneration, skin regeneration and wound healing etc..
Research finds to organize in addition to marrow, umbilical cord, fat etc., and it is dry that there is also the membrane derived mesenchymas in palace abundant in menstrual blood
Cell (palace film mescenchymal stem cell) passes on 50 times, differentiation potential is big, exempts from since its in-vitro multiplication ability can be proliferated by force 390 times
The advantages that epidemic focus is low, and growth factor secreting rate is high and the concern by regenerative medicine research field.Palace film stem cell at present
Separation mainly removes red blood cell in menstrual blood using lymphocyte separation medium, hydroxyethyl starch etc., obtains karyocyte, goes forward side by side one
Step is obtained by adhere-wall culture method, and separation process takes a long time, agents useful for same complicated component.Therefore, it further studies and opens
It sends out palace film stem cell isolation techniques efficient, shortens disengaging time, reduces and be exogenously introduced agent formulations, reduce separation costs, it is right
It is of great significance in the industrialization and raising palace film stem cell application security for promoting the preparation of palace film stem cell.
The Chinese patent that number of patent application is 201810065994.3 mainly makes in disclosed palace film stem cell acquisition methods
The method associated with density gradient centrifugation and hydroxyethyl starch sedimentation, traditional density-gradient centrifugation method and hydroxyethyl starch sedimentation
What is harvested is only the mononuclearcell of tunica albuginea layer, and the tissue block to fall off can then be deposited in bottom and discard, this method harvest
The cell quantity arrived is less.
The Chinese patent that number of patent application is 201711371272.2, it is disclosed a kind of to fill between Endometrium from being separated in menses
The method of matter stem cell, cell separation process handle cell using sodium chloride solution, can cause to damage to cell, thus
Influence the quantity of subsequent cell culture.
Summary of the invention
Technical problem to be solved by the present invention lies in provide a kind of Tea Saponin dissolution menstruation erythrocyte separation palace film
The method of mescenchymal stem cell does not cause to damage to aim cell and tissue, can from menstrual blood rapidly and efficiently separation and Extraction list
A nucleus and the induction of palace membrane tissue obtain palace film mescenchymal stem cell, and quantity is the several times of traditional tunica albuginea layer.
In order to solve the above technical problems, between a kind of dissolution menstruation erythrocyte separation palace film with Tea Saponin provided by the invention
The method of mesenchymal stem cells, includes the following steps:
(1) preparation of reagents: configuration Tea Saponin solution, it is 375-1500mg/L that the Tea Saponin solution, which is Tea Saponin concentration,
PBS solution is saved through filtration sterilization；(2) it obtains cell precipitation: by menstrual blood sample filtering, being centrifuged off upper plasma, obtain
Cell precipitation A；The Tea Saponin solution is added into the cell precipitation A, after mixing centrifugation removal supernatant, obtains thin
Born of the same parents' precipitate B；(3) cell culture: being suspended in adhere-wall culture in incubator with cell culture fluid for the cell precipitation B, for 24 hours-
Topple over after 48h and remove non-attached cell, changes fresh cell medium, change cell culture fluid within then every 2-3 days, it is long extremely to cell
When 85%-90% converges, had digestive transfer culture collects the 3rd generation cell, as palace film mescenchymal stem cell.
Further, the menstruation blood sample is menstrual cycle of female second day or the menstrual blood in third day, the bodies such as addition
The long-pending buffer containing at least one antibacterial material.
Further, the antibacterial material be Cefotaxime Sodium, amphotericin B, vancomycin hydrochloride, Ciprofloxacin,
One or more of kanamycins and quadracycline.
Further, the volume ratio of Tea Saponin solution and cell precipitation A are 9:1 in the step (2).
Further, cell culture fluid is the basis culture for adding 5% serum substitute of volumetric concentration in the step (3)
Base.
Further, incubator is that temperature is 37 DEG C, saturated humidity, contains 5%CO in the step (3)2Environment.
Further, in the step (3), after the cell precipitation B is suspended with cell culture fluid, with 2*105/cm2Density
It is seeded in incubator.
Further, in the step (3), the adhere-wall culture includes originally culture and passage amplification.
The invention has the beneficial effects that:
1, it is had an effect using on the saponin E ring of Tea Saponin 22 rouge key and ferroheme, permeability of erythrocyte membrane is caused to be sent out
It is raw to change, make erythrocytorrhexis, only red blood cell is acted on, on other cells and tissue without influence, damage is not caused i.e. to cell
It can get purpose karyocyte, the palace film mescenchymal stem cell of purifying obtained after secondary culture；It can obtain removing conventional method
Palace membrane tissue other than obtained single core, the palace film mescenchymal stem cell quantity of harvest are the several times of traditional tunica albuginea layer, and tea
Saponin is natural bacteriostatic object, effectively controls sample contamination situation, and preparation time is shorter, and gained cell activity is kept preferably, identical
Cell concentration obtained by generation is obviously higher than obtained by conventional method.
2, separation agent used is Tea Saponin solution, removes red blood cell in menstrual blood by Tea Saponin solution, ingredient is simple
Controllably, easy to operate, preparation cost is substantially reduced, and improve safety.
3, the completeness that antibacterial material guarantees degerming, the palace film mescenchymal stem cell made are added in menstruation blood sample
Product is not contaminated.
Detailed description of the invention
The present invention is described in further detail with embodiment with reference to the accompanying drawing.
Fig. 1 is the palace film stem cell morphology figure that present invention separation obtains.
Fig. 2 is the palace film stem cell morphology figure that conventional method separation obtains.
It is differentiating cartilage-forming cell figure that Fig. 3 present invention, which separates the palace film stem cell three obtained,.
Fig. 4 present invention separates three system of palace film stem cell obtained and breaks up osteoblast figure.
Fig. 5 present invention separates three system of the palace film stem cell differentiation lipoblast figure obtained.
Specific embodiment
Embodiment 1
A method of palace film mescenchymal stem cell being separated with Tea Saponin dissolution menstruation erythrocyte, is included the following steps:
(1) preparation of reagents: configuration Tea Saponin solution, the Tea Saponin solution is that the PBS that Tea Saponin concentration is 750mg/L is molten
Liquid, through filtration sterilization；Wherein Tea Saponin pulvis can be by commercially available acquisition.
(2) obtain cell precipitation: by 20ml menstrual blood sample filtering, 300g is centrifuged 3min and removes upper plasma, obtains thin
Born of the same parents' precipitate A；45ml Tea Saponin solution is added into 5ml cell precipitation A, is sufficiently mixed uniformly, stands 0.5-2.5min, mixing is equal
300g centrifugation 5min removes supernatant after even, obtains cell precipitation B.
Wherein menstruation blood sample is menstrual cycle of female second day or the menstrual blood in third day, adds isometric contain at least
A kind of buffer of antibacterial material, the antibacterial material are Cefotaxime Sodium, amphotericin B, vancomycin hydrochloride, cyclopropyl sand
One or more of star, kanamycins and quadracycline.
(3) cell culture: it (is biological tech stock purchased from section is reached that cell precipitation B, which is used containing 5% volumetric concentration serum substitute,
Part Co., Ltd) basal medium (purchased from being Biotechnology Ltd. up to section) suspend, while taking a small amount of suspension logical
Automatic blood analyzer is crossed to be counted, finally by karyocyte adjust density after with 2*105/cm2Density is seeded to 75cm2
In plastic cell culture bottle, it is placed in 37 DEG C, saturated humidity, contains 5%CO2It is cultivated in incubator, topples over removing after -48h for 24 hours and do not paste
Parietal cell changes fresh medium, changes liquid within then every 2-3 days, and when cell length is converged to 85%-90%, had digestive transfer culture, P3 generation is carefully
Born of the same parents collect identification, as palace film mescenchymal stem cell.
Embodiment 2
A method of palace film mescenchymal stem cell being separated with Tea Saponin dissolution menstruation erythrocyte, is included the following steps:
(1) preparation of reagents: configuration Tea Saponin solution, the Tea Saponin solution is that the PBS that Tea Saponin concentration is 375mg/L is molten
Liquid, through filtration sterilization；Wherein Tea Saponin pulvis can be by commercially available acquisition.
(2) obtain cell precipitation: by 20ml menstrual blood sample filtering, 300g is centrifuged 3min and removes upper plasma, obtains thin
Born of the same parents' precipitate A；45ml Tea Saponin solution is added into 5ml cell precipitation A, is sufficiently mixed uniformly, stands 0.5-2.5min, mixing is equal
300g centrifugation 5min removes supernatant after even, obtains cell precipitation B.
Wherein menstruation blood sample is menstrual cycle of female second day or the menstrual blood in third day, adds isometric contain at least
A kind of buffer of antibacterial material, the antibacterial material are Cefotaxime Sodium, amphotericin B, vancomycin hydrochloride, cyclopropyl sand
One or more of star, kanamycins and quadracycline.
(3) cell culture: it (is biological tech stock purchased from section is reached that cell precipitation B, which is used containing 5% volumetric concentration serum substitute,
Part Co., Ltd) basal medium (purchased from being Biotechnology Ltd. up to section) suspend, while taking a small amount of suspension logical
Automatic blood analyzer is crossed to be counted, finally by karyocyte adjust density after with 2*105/cm2Density is seeded to 75cm2
In plastic cell culture bottle, it is placed in 37 DEG C, saturated humidity, contains 5%CO2It is cultivated in incubator, topples over removing after -48h for 24 hours and do not paste
Parietal cell changes fresh medium, changes liquid within then every 2-3 days, and when cell length is converged to 85%-90%, had digestive transfer culture, P3 generation is carefully
Born of the same parents collect identification, as palace film mescenchymal stem cell.
Embodiment 3
A method of palace film mescenchymal stem cell being separated with Tea Saponin dissolution menstruation erythrocyte, is included the following steps:
(1) preparation of reagents: configuration Tea Saponin solution, the Tea Saponin solution is the PBS that Tea Saponin concentration is 1500mg/L
Solution, through filtration sterilization；Wherein Tea Saponin pulvis can be by commercially available acquisition.
(2) obtain cell precipitation: by 20ml menstrual blood sample filtering, 300g is centrifuged 3min and removes upper plasma, obtains thin
Born of the same parents' precipitate A；45ml Tea Saponin solution is added into 5ml cell precipitation A, is sufficiently mixed uniformly, stands 0.5-2.5min, mixing is equal
300g centrifugation 5min removes supernatant after even, obtains cell precipitation B.
Wherein menstruation blood sample is menstrual cycle of female second day or the menstrual blood in third day, adds isometric contain at least
A kind of buffer of antibacterial material, the antibacterial material are Cefotaxime Sodium, amphotericin B, vancomycin hydrochloride, cyclopropyl sand
One or more of star, kanamycins and quadracycline.
(3) cell culture: it (is biological tech stock purchased from section is reached that cell precipitation B, which is used containing 5% volumetric concentration serum substitute,
Part Co., Ltd) basal medium (purchased from being Biotechnology Ltd. up to section) suspend, while taking a small amount of suspension logical
Automatic blood analyzer is crossed to be counted, finally by karyocyte adjust density after with 2*105/cm2Density is seeded to 75cm2
In plastic cell culture bottle, it is placed in 37 DEG C, saturated humidity, contains 5%CO2It is cultivated in incubator, topples over removing after -48h for 24 hours and do not paste
Parietal cell changes fresh medium, changes liquid within then every 2-3 days, and when cell length is converged to 85%-90%, had digestive transfer culture, P3 generation is carefully
Born of the same parents collect identification, as palace film mescenchymal stem cell.
Embodiment 4
The present embodiment is check experiment, separates palace film stem cell for conventional method, comprising the following steps:
(1) 20ml menstrual blood 300g is centrifuged 3min, after removing upper plasma, obtains cell precipitation A；To 5ml cell precipitation A
Middle addition 15ml physiological saline simultaneously mixes well, and obtains dilution blood sample.
(2) clean centrifuge tube is taken, transfer 10ml human lymphocyte separating liquid (is purchased from the limited public affairs of Hangzhou connection section biotechnology
Department) into 50ml centrifuge tube.
(3) laying is loaded: the dilution blood sample in 20ml step 1 is added to the liquid level of human lymphocyte separating liquid in step 2
On, 400g is centrifuged 20min；Extract mononuclearcell: occurring apparent layering after centrifugation, in centrifuge tube, from bottom to top according to
Secondary is red blood cell granulocyte layer, separation liquid layer, mononuclearcell layer and plasma layer.
(4) mist mononuclearcell layer obtained in careful aspiration step (3), and physiological saline is added and is centrifuged with 300g
5min abandons supernatant, obtains cell precipitation.
(5) it (is biotechnology purchased from section is reached that the cell precipitation that step (4) obtains, which is used containing 5% volumetric concentration serum substitute,
Limited liability company) basal medium (purchased from being Biotechnology Ltd. up to section) suspend, while taking a small amount of suspension
Counted by automatic blood analyzer, finally by karyocyte adjust density after with 2*105/cm2Density is seeded to
75cm2In plastic cell culture bottle, it is placed in 37 DEG C, saturated humidity, contains 5%CO2It is cultivated in incubator, topples over removing after -48h for 24 hours
Non- attached cell changes fresh medium, changes liquid within then every 2-3 days, when cell length is converged to 85%-90%, had digestive transfer culture, and P3
It collects and identifies for cell, as palace film mescenchymal stem cell.
Cell number is carried out to the cell that the above embodiment of the present invention 1-3 and 4 cell culture of conventional method embodiment are obtained
Amount and cellular morphology analysis, as a result as follows:
1 present invention of table and conventional method gained Cell Culture Cells quantity situation
2 present invention of table and conventional method gained Cell Culture Cells form situation
It takes the embodiment of the present invention 1 and conventional method embodiment 4 to carry out streaming identification: taking the embodiment of the present invention 1 and tradition side
The 3rd generation each 1*10 of palace film stem cell that method embodiment 4 obtains6, it is uniformly distributed into 9 pipes respectively, is resuspended with PBS to 200 μ after centrifugation
L is washed 2 times.1 pipe is stayed to do blank after centrifugation, phenotypic markers are added that (antibody is respectively mouse anti human by one label of every pipe
CD73-PE, CD90-PE, CD105-PE, CD34-PE, CD45-PE, CD19-PE, CD14-FITC and HLA-DR-FITC).It is protected from light
Room temperature is incubated for 30min, washs 2 times after centrifugation, and after being then centrifuged for plus 200 μ lPBS are resuspended, and is measured with flow cytometer, as a result such as
Under:
| Surface marker | Embodiment 1 | Embodiment 4 | Standard (DB33/T 2030-2017) |
| CD73 | 99.62 | 99.54 | > 95% |
| CD90 | 99.66 | 99.90 | > 95% |
| CD105 | 95.74 | 95.78 | > 95% |
| CD34 | 0.34 | 0.44 | < 2% |
| CD45 | 0.63 | 0.48 | < 2% |
| CD19 | 0.45 | 0.26 | < 2% |
| CD14 | 0.55 | 0.47 | < 2% |
| HLA-DR | 0.58 | 0.63 | < 2% |
The differentiation of three systems is carried out to the 1 gained palace film stem cell of embodiment of the method for the present invention, obtains differentiation as in Figure 3-5
Figure, wherein Fig. 3 be palace film stem cell three be differentiating cartilage-forming cell figure, Fig. 4 be three system of palace film stem cell differentiation osteoblast figure,
Fig. 5 is that three system of palace film stem cell breaks up lipoblast figure.
To sum up the result shows that, the method for the present invention be compared with the traditional method compared with: (1) filled using between palace film obtained by the method for the present invention
The purity of matter stem cell and conventional method gained are almost the same, all meet mescenchymal stem cell surface marker standard of perfection
(DB33/T2030-2017)；(2) shorter the time required to cell primary preparation in the method for the present invention, separation process time-consuming is no more than
15min, conventional method separate time-consuming 30min or more；(3) the method for the present invention using Tea Saponin saponin E ring on 22 rouge key and
Ferroheme is had an effect, and permeability of erythrocyte membrane is caused to change, and makes erythrocytorrhexis, is only acted on red blood cell, to other
Cell and tissue do not cause damage to can be obtained purpose karyocyte in cell without influence, and purifying is obtained after secondary culture
Palace film mescenchymal stem cell；The palace membrane tissue in addition to the single core that conventional method obtains can be obtained, is filled between the palace film of harvest
Matter stem cell population is the several times of traditional tunica albuginea layer, and Tea Saponin is natural bacteriostatic object, effectively controls sample contamination situation, preparation
Time is shorter, and gained cell activity is kept preferably, and cell concentration obtained by identical generation is obviously higher than obtained by conventional method；(4) originally
Separation agent used in inventive method is Tea Saponin solution, removes red blood cell in menstrual blood by Tea Saponin solution, ingredient simply may be used
Control, it is easy to operate, preparation cost is substantially reduced, and improve safety；(5) the method for the present invention saves middle addition in liquid in menstrual blood
The multiple means such as antibacterial material ensure that the completeness of degerming, and the palace film stem cell products made are not contaminated.(6) of the invention
Cell obtained by method is able to carry out the differentiation of three systems, meets mescenchymal stem cell characteristic.
Above description is exemplary and not limiting.By above description skilled person realizes that originally
Many kinds of change and modification of invention, will also fall within the spirit and scope of the invention.
Claims (8)
1. a kind of method with Tea Saponin dissolution menstruation erythrocyte separation palace film mescenchymal stem cell, which is characterized in that including
Following steps:
(1) preparation of reagents: configuration Tea Saponin solution, the Tea Saponin solution is the PBS that Tea Saponin concentration is 375-1500mg/L
Solution is cooled to room temperature after filtration sterilization；
(2) it obtains cell precipitation: by menstrual blood sample filtering, being centrifuged off upper plasma, obtain cell precipitation A；To described thin
The saponin solution is added in born of the same parents' precipitate A, is sufficiently mixed uniformly, centrifugation removal supernatant, obtains cell precipitation after mixing
B；
(3) cell culture: the cell precipitation B is suspended in adhere-wall culture in incubator with cell culture fluid, after -48h for 24 hours
Topple over and remove non-attached cell, changes fresh cell medium, change cell culture fluid within then every 2-3 days, it is long to 85%- to cell
90% when converging, had digestive transfer culture, collects the 3rd generation cell, as palace film mescenchymal stem cell.
2. a kind of side with Tea Saponin dissolution menstruation erythrocyte separation palace film mescenchymal stem cell according to claim 1
Method, which is characterized in that the menstruation blood sample is menstrual cycle of female second day or the menstrual blood in third day, adds isometric
Buffer containing at least one antibacterial material.
3. a kind of side with Tea Saponin dissolution menstruation erythrocyte separation palace film mescenchymal stem cell according to claim 2
Method, which is characterized in that the antibacterial material is Cefotaxime Sodium, amphotericin B, vancomycin hydrochloride, Ciprofloxacin, blocks that
One or more of mycin and quadracycline.
4. a kind of side with Tea Saponin dissolution menstruation erythrocyte separation palace film mescenchymal stem cell according to claim 1
Method, which is characterized in that the volume ratio of Tea Saponin solution and cell precipitation A are 9:1 in the step (2).
5. a kind of side with Tea Saponin dissolution menstruation erythrocyte separation palace film mescenchymal stem cell according to claim 1
Method, which is characterized in that cell culture fluid is the basal medium for adding 5% serum substitute of volumetric concentration in the step (3).
6. a kind of side with Tea Saponin dissolution menstruation erythrocyte separation palace film mescenchymal stem cell according to claim 1
Method, which is characterized in that incubator is that temperature is 37 DEG C, saturated humidity, contains 5%CO in the step (3)2Environment.
7. a kind of side with Tea Saponin dissolution menstruation erythrocyte separation palace film mescenchymal stem cell according to claim 1
Method, which is characterized in that in the step (3), after the cell precipitation B is suspended with cell culture fluid, with 2*105/cm2Density connects
Kind is into incubator.
8. a kind of side with Tea Saponin dissolution menstruation erythrocyte separation palace film mescenchymal stem cell according to claim 1
Method, which is characterized in that in the step (3), the adhere-wall culture includes originally culture and passage amplification.
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| CN114525248A (en) * | 2022-03-21 | 2022-05-24 | 士泽生物医药(上海)有限公司 | Method for preparing menstrual blood-derived mesenchymal stem cells |
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