CN116926003A - Preparation method and application of amniotic mesenchymal stem cells - Google Patents
Preparation method and application of amniotic mesenchymal stem cells Download PDFInfo
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- 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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- 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
- C12N5/0653—Adipocytes; Adipose tissue
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- 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
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
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Abstract
The invention provides a preparation method of amniotic mesenchymal stem cells, which comprises the steps of treating pancreatin and dispase II, and then treating with collagenase, DNAase and neutral protease. The buffer solution is used for pretreatment of the amniotic membrane, so that the mixed cells on the surface of the amniotic membrane can be effectively removed, the type of enzyme to be treated is increased, and impurities such as mucus and the like on the surface of the amniotic membrane can be effectively removed. The mesenchymal stem cells prepared by the method have higher proliferation capacity, and can keep higher growth proliferation level in a serum culture medium and a serum-free culture medium. The cells of the secondary culture have obvious markers of mesenchymal stem cells, and can normally further perform osteoblast differentiation and adipogenic differentiation. The mesenchymal stem cells prepared by the method can be subjected to proliferation culture under various conditions, have strong proliferation capacity, and can meet the requirement of preparing the mesenchymal stem cells on a large scale.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method and application of amniotic mesenchymal stem cells.
Background
Mesenchymal stem cells (mesenchymal stem cells, MSCs) have the ability to differentiate into different cell lines, including adipose connective tissue, bone tissue and myocardial tissue. In clinical use, human bone marrow derived MSCs are one of the most common stem cells. However, adult bone marrow contains less MSCs, because both bone marrow MSCs content and differentiation capacity decrease with age and bone marrow derived MSCs must be obtained by invasive routes, which increases the chance of infection in the donor. At present, fetal tissues have become a hotspot for MSCs research, such as amniotic membrane, chorion, placenta and the like in bone marrow, liver, umbilical cord, cord blood, amniotic fluid and placenta tissues of fetuses all contain MSCs. Since the application of fetal tissues in MSCs treatment is greatly limited by ethics, the clinical application and development of the fetal tissues are affected, so that the research of searching for MSCs with rich sources, high content, convenient material taking, no invasiveness and no ethics limitation has become a research hot spot nowadays.
Human placental tissue is readily available as post-gestation reject and its use is essentially non-ethical controversial. Placenta is a transitional organ of exchange substance between mother and son which is formed by combining embryo membrane of embryo and mother endometrium, and contains a large amount of blood vessel and mesenchymal components besides trophoblast; the placental mesenchymal stem cells are a recently newly discovered type of stem cells obtained from placenta, and compared with stem cells extracted from umbilical cord blood or bone marrow, the stem cells are not only large in number but also can differentiate into various tissue cells. And the placenta tissue is derived from postpartum, and the collection process does not cause any damage to the mother and the neonate, and is not limited by ethics and clinical medicine.
The placenta mainly comprises 3 layers, namely amniotic membrane, chorion and decidua, wherein the chorion and the decidua are rich in blood vessels and erythrocytes and are not easy to separate; the amniotic membrane is a tissue which is easy to separate and has a simple structure. If the mesenchymal stem cells can be separated from the amniotic membrane, the amniotic membrane can be a good donor of the mesenchymal stem cells, and a simple way for obtaining the mesenchymal stem cells is provided for researchers.
MSCs derived from amniotic membrane in placental tissue have now been demonstrated to differentiate into multiple cell lines, into endodermal layers, such as hepatocytes; differentiation into mesoderm such as bone cells, cartilage cells, fat cells, cardiac muscle cells; differentiation to the ectoderm, such as neural cells. In addition, researchers have found from cell surface antigen analysis that placental mesenchymal stem cells are stem cells intermediate between embryonic stem cells and adult stem cells, which are not easily rejected by the recipient's immune system due to their low immunogenicity, and are ideal resources for stem cell preservation and utilization.
In the prior art, mesenchymal stem cells are separated based on an amniotic membrane, and are mainly treated based on pancreatin and collagenase, and the enzyme types are single, so that impurities on amniotic membrane components such as red blood cells or fragments, mucus and the like cannot be effectively separated and removed, and the proliferation capacity, the subsequent differentiation capacity and the subsequent functions of the mesenchymal stem cells are seriously influenced along with the mesenchymal stem cells obtained by preparation. And the prepared mesenchymal stem cells have single culture method, and severely restrict the large-scale preparation and subsequent application of the mesenchymal stem cells separated from the amniotic membrane. Therefore, it is necessary to find a simple, convenient and effective method for extracting human amniotic mesenchymal stem cells (human amniotic mesenchymal stem cells, HAMSCs).
Disclosure of Invention
The invention aims at solving the problems of the prior art that the mesenchymal stem cells are separated based on the amniotic membrane, and provides a method for separating and preparing the mesenchymal stem cells from the amniotic membrane.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention discloses a preparation method of amniotic mesenchymal stem cells, which comprises the following steps:
1) Separating the amniotic membrane from the chorion layer, shearing into small pieces, and repeatedly cleaning with PBS buffer solution;
2) Treating the washed amniotic fragment in Hank's balanced salt solution containing pancreatin and dispase II, centrifuging, collecting amniotic fragment precipitate, and repeatedly washing with PBS;
3) Treating the cleaned amniotic membrane fragments in Hank's balanced salt solution containing collagenase, DNAase and neutral protease until the amniotic membrane fragments disappear;
4) Filtering the digestion solution obtained in the step 3) through a cell filter screen, and centrifugally collecting to obtain the amniotic mesenchymal cells.
5) And carrying out subculture on the obtained amniotic mesenchymal stem cells.
Preferably, after washing with PBS buffer in step 1), the procedure of room temperature treatment with buffer A is further employed.
Preferably, the buffer solution A has the formula: NH (NH) 4 Cl、NaHCO 3 、EDTA-Na 2 Salt and sterilized deionized water.
Preferably, the treatment conditions of buffer A are room temperature for 8-15 minutes.
Preferably, the concentration of pancreatin and dispase II is 100-500U/mL and 100-300U/mL, respectively.
Preferably, the concentration of collagenase, DNAase, neutral protease is 0.5-2mg/ml, 0.05% -0.2% and 0.05-0.2%, respectively.
Preferably, in step 4) the digestion solution is passed through a 100 μm sieve and a 40 μm sieve, respectively.
Preferably, the size of the small pieces in step 1) is 0.5mm x0.5 mm.
Preferably, the subculture in step 5) comprises a serum culture and a serum-free culture.
Preferably, the method further comprises the step of inducing the prepared mesenchymal stem cells to differentiate other cell types.
Preferably, the induced differentiation includes cell adipogenic differentiation and cell osteogenic differentiation.
According to the invention, the buffer solution is used for pretreatment of the amniotic membrane, so that the mixed cells on the surface of the amniotic membrane can be effectively removed, and the impurities such as mucus and the like on the surface of the amniotic membrane can be effectively removed by increasing the type of enzyme to be treated. The mesenchymal stem cells prepared by the method have higher proliferation capacity, and can keep better growth proliferation level in serum culture medium and serum-free culture medium. The cells of the secondary culture have obvious markers of mesenchymal stem cells, and can normally further perform osteoblast differentiation and adipogenic differentiation. The mesenchymal stem cells prepared by the method can be subjected to proliferation culture under various conditions, have strong proliferation capacity, and can meet the requirement of preparing the mesenchymal stem cells on a large scale.
Drawings
FIG. 1 microscopic observation of the primary cells and the P1-substituted cells prepared by separation, wherein FIG. 1a is the primary cells isolated and FIG. 1b is the P1-substituted cells cultured;
FIG. 2 shows the results of serum culture and serum-free culture of P5-generation cells observed by an inverted microscope, wherein FIG. 2a shows the results of serum culture and FIG. 2b shows the results of serum-free culture;
FIG. 3 proliferation curves of mesenchymal stem cells of each generation (P3, P4, P5) cultured in serum medium;
FIG. 4 proliferation curves of mesenchymal stem cells of each generation (P3, P4, P5) cultured in serum-free medium;
FIG. 5 identification of surface markers of serum-cultured amniotic mesenchymal stem cells;
FIG. 6. Surface marker identification of serum-free cultured amniotic mesenchymal stem cells;
FIG. 7. Results of adipogenic identification of serum-cultured amniotic mesenchymal stem cells;
FIG. 8. Results of adipogenic identification of serum-free cultured amniotic mesenchymal stem cells;
FIG. 9. Results of osteogenic identification of serum-cultured amniotic mesenchymal stem cells;
FIG. 10 results of osteoblast identification of serum free cultured amniotic mesenchymal stem cells.
Detailed Description
The following describes specific embodiments of the present invention in detail with reference to the drawings. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
EXAMPLE 1 isolation of human amniotic mesenchymal Stem cells
Placenta obtained from healthy women after delivery (informed consent obtained during pregnancy), the amniotic membrane was peeled off with forceps and separated from the chorion layer, the amniotic membrane was cut into small pieces (about 0.5cm x0.5 cm) with surgical scissors, and repeatedly washed with Phosphate Buffer (PBS) buffer to remove blood stains.
The amniotic membrane fragments are treated with buffer A for about 8-15 minutes at room temperature to remove as much as possible the surface-adherent foreign cells from the amniotic membrane, and the amniotic membrane fragments are continuously stirred during the treatment and then washed with PBS buffer. The formula of the adopted buffer solution A is as follows: NH (NH) 4 Cl、NaHCO 3 、EDTA-Na 2 Salt and sterilized deionized water, the concentration of NH4 Cl is 100mM, the NaHCO 3 Is 5mM, the EDTA-Na 2 Is 0.1mM.
The washed amniotic membrane pieces were treated in Hank's balanced salt solution (pH 7.4) containing 240U/mL pancreatin and 200U/mL dispase II at 37℃for 45 minutes. Centrifugation, collection of amniotic fragment pellet, and repeated washing with PBS. The washed amniotic membrane fragments were digested in Hank's balanced salt solution containing 1mg/ml collagenase (Sigma) +0.08% DNAase (Sigma) +0.1% neutral protease at 37℃until the amniotic membrane fragments were substantially disappeared, with gentle stirring with a stirring bar without interruption.
Wherein, the formula of the adopted Hank's balanced salt solution is as follows: 8.0g NaCl, 0.4g KCl, 0.06g KH 2 PO 4 Na of 0.08g 2 HPO 4 .12H 2 O, 0.35g NaHCO 3 Water to 1000ml, and adjusting the pH value to 7.4.
The resulting solutions were passed through a 100 μm sieve and a 40 μm sieve, respectively, and collected by centrifugation at 2000Xg for 10 minutes to isolate amniotic mesenchymal cells (primary cells).
After the amniotic mesenchymal stem cells (primary cells) collected by centrifugation are suspended in a DMEM medium containing 10% FBS+100U/mL penicillin+100 ug/mL streptomycin, the cells are inoculated in a cell culture dish and inoculated with CO at 37 ℃ with a volume fraction of 5% 2 The culture was performed in a saturated moderate incubator with medium changes every 2-3 days.
After about 7 days, when the confluence of the primary cultured mesenchymal stem cells reached more than 85%, the primary cultured mesenchymal stem cells were treated with 0.05% trypsin and 0.2mM EDTA. Mesenchymal stem cells were then collected from the dishes and scored as P1 generation cells. The primary cells and the P1 generation cells are taken respectively for morphological inversion microscopic observation, and the detection results are shown in figure 1. Wherein, FIG. 1a shows isolated primary cells, FIG. 1b shows cultured P1 generation cells, and from microscopic images, the primary (P0) generation cells show a shuttle shape, but the cell morphology is slightly short and thick, but after the culture, small cell clusters are formed in the P1 generation, and the cells further show a long shuttle shape.
EXAMPLE 2 proliferation culture of human amniotic mesenchymal Stem cells
The isolated and prepared P1 generation cells are respectively subjected to serum culture and serum-free culture to detect the proliferation capacity of the prepared human amniotic mesenchymal stem cells, and specific culture conditions are as follows.
2.1 serum culture of human amniotic mesenchymal Stem cells
1) The P1 generation cells prepared by separation are replaced by 4 multiplied by 10 3 Cells/cm 2 Is cultured on a culture plate in a serum medium added with 1ng/ml bFGF at 37℃under 5% CO 2 The serum medium used was DMEM medium containing 10% fbs.
2) The culture broth was refreshed every 3 days, and when the cells were nearly confluent, they were subcultured and so on repeatedly until passage 10.
3) After each generation of incubation, the plates were treated with 0.05% trypsin+0.2 mM EDTA. Cells were collected from the plates and stored frozen for later use.
4) The P5-generation cultured cells are taken under an inverted microscope to observe the growth condition of the cells.
2.2 serum-free culture of human amniotic mesenchymal Stem cells
1) The P1 generation cells prepared by separation are replaced by 4 multiplied by 10 3 Cells/cm 2 Is cultured on a culture plate in a serum-free medium at 37 ℃ under 5% CO 2 The formula of the serum-free medium used was: DMEM/F12 serum-free medium containing 0.5% human serum albumin, 10ng/mL bFGF, 0.2g/L human transferrin, 1g/L human insulin, 0.00067g/L sodium selenite, wherein DMEM/F12 medium was used as DMEM medium and F12 medium according to 1:1, and the like.
2) The culture broth was refreshed every 3 days, and when the cells were nearly confluent, they were subcultured and so on repeatedly until passage 10.
3) After each generation of incubation, the plates were treated with 0.05% trypsin+0.2 mM EDTA. Cells were collected from the plates and stored frozen for later use.
4) The P5-generation cultured cells are taken under an inverted microscope to observe the growth condition of the cells.
Results: the photographs of the P5-generation cells of the serum culture and the serum-free culture observed by the inverted microscope are shown in FIG. 2, wherein FIG. 2a shows the serum culture results and FIG. 2b shows the serum-free culture results. The observation results show that: the living state of the cells is good, the cells can grow on the wall, the cell morphology is normal, and the cells are long fusiform. The P1 generation cells prepared by separation have strong proliferation capability, can grow in a serum culture medium and can be cultured in a serum-free culture medium, and the cells cultured until the P5 generation grow and proliferate normally.
2.3 proliferation potency detection of each generation of cells under different culture medium culture conditions was performed using WST-1 (cell proliferation and cytotoxicity detection kit).
1) Taking P3, P4 and P5 generation cultured cells, and preparing single cell suspension by using a corresponding culture medium;
2) 96-well plates were inoculated at a density of about 1500/well and tested at the same time point every day from day 2.
The detection method comprises the following steps: discarding the culture solution, replacing 100uL of fresh culture solution after PBS washing, adding 10uL of WST-1 into each hole, and incubating for 2 hours in a cell incubator; after the cells were removed, absorbance (A) was read by selecting a wavelength of 450 nm. Then, the cell proliferation curve for continuous detection within 7 days was plotted on the abscissa of the cell culture time and on the ordinate of the absorbance value at a wavelength of 450 nm.
Results: the proliferation curve of each generation of mesenchymal stem cells cultured in the serum medium is shown in fig. 3, and the proliferation curve of each generation of mesenchymal stem cells cultured in the serum-free medium is shown in fig. 4. From the results of cell proliferation measured in fig. 3 and 4, the trend of cell proliferation growth in different media is similar: on days 1-2, cells grow slowly and proliferate unobvious, starting from days 2-4, cells enter the fast growth phase and proliferate obviously, and starting from day 5, growth enters the slow state and enters the proliferation plateau phase.
Example 3 identification of surface markers of human amniotic mesenchymal Stem cells
And (3) selecting corresponding fluorescent marked antibodies from the cultured cells by adopting a flow cytometer for specific binding reaction, detecting CD73, CD90 and CD105 by positive indexes, detecting HLA-DR by negative indexes, and then carrying out classified counting by the flow cytometer.
1) Preparing a cell sample to be tested: sampling the mesenchymal stem cell suspension P3 generation which is recovered in advance according to different culture types, and taking the cells to be detected respectively to be not less than 6 multiplied by 10 6 Centrifuging 500g in a flow cell tube for 6min, discarding supernatant, and adding 800ul PBS to resuspend cells for later use;
2) Antibody addition: taking 6 branch cell tubes from each sample, and adding corresponding fluorescent-labeled specific antibodies and negative and positive controls;
3) Sample addition: 100ul of pre-resuscitated stem cell suspension was added to each of the flow tubes 1-6.
4) And (3) light shielding and standing: and after fully and uniformly mixing, putting the mixture into a refrigerator for refrigeration for half an hour.
5) Washing: the sample was removed from the refrigerator and centrifuged for 2 times at 300g for 5min to remove unbound antibody.
6) 300ul of cell suspension was added to each tube and the tube was checked on-machine.
Results: the surface marker identification results of the serum-cultured amniotic mesenchymal stem cells are shown in fig. 5, and the surface marker identification results of the serum-free cultured amniotic mesenchymal stem cells are shown in fig. 6. From the detection results, the positive index detection CD73, CD90 and CD105 occupy most proportion, and the negative index detection HLA-DR signal occupy little, which shows that the cultured cells can express obvious mesenchymal stem cell surface markers, and the prepared mesenchymal stem cells have normal physiological state.
EXAMPLE 4 lipid-induced differentiation Capacity of human amniotic mesenchymal Stem cells
1) Taking the mesenchymal stem cells preserved in the P4 generation under different culture conditions, sucking and discarding the culture medium, and washing the cells with PBS for 3 times;
2) Adding 2mL of adipogenic induced differentiation complete culture medium into each hole, and changing liquid every 3 days;
3) Culturing in incubator for 2-4 weeks until the observed lipid drop under the mirror becomes large and round;
5) Sucking and removing the induced differentiation culture medium, washing the cells for 3 times by using PBS, adding 2mL of cell tissue fixing solution into each hole, and fixing the cells for 30min at room temperature;
6) Absorbing and discarding the fixing solution, washing the cells for 3 times by using PBS, and adding 1mL of oil red O staining solution into each hole for staining for 30min;
7) The oil red O dye solution is sucked and removed, PBS is used for cleaning for 3 times, and the pore plate is placed under a living cell workstation to observe the dyeing effect and take a picture.
Wherein, the formula of the complete culture medium for adipogenic induced differentiation is as follows: high sugar DMEM,10% fetal bovine serum, 2×10 -4 mol/L indomethacin, 10 -6 mo1/L dexamethasone, 10mg/L insulin.
Results: the results of the adipogenic identification of serum-cultured amniotic mesenchymal stem cells are shown in fig. 7, and the results of the adipogenic identification of serum-free cultured amniotic mesenchymal stem cells are shown in fig. 8. After the induction culture medium is added into the p 4-generation cells cultured under different conditions, oil red-O staining is carried out, obvious bright red lipid drops appear in the cells after lipid induction, the cell nucleus is blue-purple, and the matrix is colorless. Illustrating that after adipogenic induction, we tested p4 generation mesenchymal stem cells had differentiated towards adipocytes. The above results indicate that the detected cells are capable of being transformed into adipocytes under certain conditions.
EXAMPLE 5 osteoinductive differentiation Capacity of human amniotic mesenchymal Stem cells
1) Taking the mesenchymal stem cells preserved in the P4 generation under different culture conditions, sucking and removing the culture medium, and washing the cells for 3 times by PBS;
2) Adding 2mL of osteogenesis inducing differentiation complete culture medium into each hole, and changing liquid every 3 days;
3) After 2-4 weeks from the beginning of induction, ending the induction differentiation according to the growth condition and morphological change of the cells;
4) Sucking off the induced differentiation culture medium, washing the cells for 3 times by using PBS, and adding a cell tissue fixing solution into the holes to fix the cells for 30min;
5) The fixative was aspirated and the cells were washed 3 times with PBS. 1mL alizarin red is added into each hole for dyeing for 3-5 min;
6) Alizarin red dye solution was aspirated, cells were washed 3 times with PBS, and staining effect was observed and photographed at a living cell workstation.
Wherein, the formula of the complete culture medium for osteogenesis induced differentiation is as follows: high sugar DMEM,10% fetal bovine serum 10 -2 mol/L beta-glycerophosphate, 50mg/L ascorbic acid, 10 -7 mol/L dexamethasone.
Results: the results of the osteoblast identification of the serum-cultured amniotic mesenchymal stem cells are shown in fig. 9, and the results of the osteoblast identification of the serum-free amniotic mesenchymal stem cells are shown in fig. 10. P4 generation cells cultured under different conditions are subjected to alizarin red staining after being added with an induction medium, and obvious red-stained calcified nodules appear in cells after osteogenesis induction, but no cells are induced. After osteogenic induction, we tested that the P4 generation mesenchymal stem cells had differentiated towards bone cells. The above results indicate that the detected cells are capable of transforming into bone cells under certain conditions.
The process of the present invention is illustrated by the above examples, but the present invention is not limited to the above process steps, i.e., it is not meant that the present invention must be practiced by relying on the above process steps. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.
Claims (10)
1. The preparation method of the amniotic mesenchymal stem cells is characterized by comprising the following steps:
1) Separating the amniotic membrane from the chorion layer, shearing into small pieces, and repeatedly cleaning with PBS buffer solution;
2) Treating the washed amniotic fragment in Hank's balanced salt solution containing pancreatin and dispase II, centrifuging, collecting amniotic fragment precipitate, and repeatedly washing with PBS;
3) Treating the cleaned amniotic membrane fragments in Hank's balanced salt solution containing collagenase, DNAase and neutral protease until the amniotic membrane fragments disappear;
4) Filtering the digestion solution obtained in the step 3) through a cell filter screen, and centrifugally collecting to obtain the amniotic mesenchymal cells.
5) And carrying out subculture on the obtained amniotic mesenchymal stem cells.
2. The method for preparing amniotic mesenchymal stem cells according to claim 1, wherein after washing with PBS buffer in step 1), the step of further treating with buffer a at room temperature is performed, wherein the buffer a has a formula of: NH (NH) 4 Cl、NaHCO 3 、EDTA-Na 2 Salt and sterilized deionized water.
3. The method for preparing amniotic mesenchymal stem cells according to claim 1, wherein the treatment condition of the buffer a is room temperature treatment for 8-15 minutes.
4. The method for preparing amniotic mesenchymal stem cells according to claim 1, wherein the concentration of pancreatin and dispase II is 100-500U/mL and 100-300U/mL, respectively.
5. The method for preparing amniotic mesenchymal stem cells according to claim 1, wherein the collagenase, dnase and neutral protease are respectively at a concentration of 0.5-2mg/ml, 0.05% -0.2% and 0.05-0.2%.
6. The method for preparing amniotic mesenchymal stem cells according to claim 1, wherein the digestion solution is passed through a 100 μm sieve and a 40 μm sieve in step 4), respectively.
7. The method for preparing amniotic mesenchymal stem cells according to claim 1, wherein the size of the small pieces in step 1) is 0.5mm x0.5 mm.
8. The method for preparing amniotic mesenchymal stem cells according to claim 1, wherein the subculture in step 5) comprises serum culture and serum-free culture.
9. The method for preparing amniotic mesenchymal stem cells according to claim 1, further comprising the step of inducing the prepared mesenchymal stem cells to differentiate other cell types.
10. The method for preparing amniotic mesenchymal stem cells according to claim 9, wherein the induced differentiation comprises cell adipogenic differentiation and cell osteogenic differentiation.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420185A (en) * | 2015-12-11 | 2016-03-23 | 郭镭 | Method for separating and extracting hUC-MSC from umbilical cord outer layer amnion tissue |
| CN105420184A (en) * | 2015-12-11 | 2016-03-23 | 郭镭 | Method for culturing umbilical cord mesenchymal stem cells in separated mode from umbilical cord outer layer amnion tissue |
| US20170029770A1 (en) * | 2014-05-09 | 2017-02-02 | Reelabs Private Limited, a Company Incorporated Under Provisions of The Companies Act 1956 | Foetal polymix of mesenchymal stem cells under hypoxic conditions for the treatment of clinical disorders and diseases |
| CN115322964A (en) * | 2022-08-18 | 2022-11-11 | 宁波希诺赛生物科技有限公司 | Method for constructing 3D culture amniotic mesenchymal stem cell seed bank |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170029770A1 (en) * | 2014-05-09 | 2017-02-02 | Reelabs Private Limited, a Company Incorporated Under Provisions of The Companies Act 1956 | Foetal polymix of mesenchymal stem cells under hypoxic conditions for the treatment of clinical disorders and diseases |
| US11072776B2 (en) * | 2014-05-09 | 2021-07-27 | Reelabs Private Limited, a Company Incorporated Under Provisions of The Companies Act 1956 | Foetal polymix of mesenchymal stem cells under hypoxic conditions for the treatment of clinical disorders and diseases |
| CN105420185A (en) * | 2015-12-11 | 2016-03-23 | 郭镭 | Method for separating and extracting hUC-MSC from umbilical cord outer layer amnion tissue |
| CN105420184A (en) * | 2015-12-11 | 2016-03-23 | 郭镭 | Method for culturing umbilical cord mesenchymal stem cells in separated mode from umbilical cord outer layer amnion tissue |
| CN115322964A (en) * | 2022-08-18 | 2022-11-11 | 宁波希诺赛生物科技有限公司 | Method for constructing 3D culture amniotic mesenchymal stem cell seed bank |
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