CN111321115A - Preparation method and application of umbilical cord and placenta mesenchymal stem cells - Google Patents
Preparation method and application of umbilical cord and placenta mesenchymal stem cells Download PDFInfo
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Abstract
The invention discloses a preparation method of umbilical cord and placenta mesenchymal stem cells and application thereof, belonging to the field of preparation of mesenchymal stem cells. The method has simple and convenient operation and low economic cost, the obtained cells have complete shape and high purity, and the obtained umbilical cord and placenta mesenchymal stem cells do not express hematopoietic stem cells and the like, so the vitality of the cells can be well maintained. The prepared mesenchymal stem cells can be applied to skin diseases, blood system diseases, nervous system diseases, solid organ injuries or lesions, immune system diseases, cardiovascular system diseases, metabolic diseases, bone joint diseases, reproductive system diseases and the like of human bodies.
Description
Technical Field
The invention belongs to the field of mesenchymal stem cell culture, and particularly relates to a preparation method and application of umbilical cord and placenta mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSCs) are important members of the stem cell family, are derived from early-developing mesoderm, belong to pluripotent stem cells, and are originally found in bone marrow, so that MSCs are increasingly concerned by people due to the characteristics of multidirectional differentiation potential, hematopoietic support, stem cell implantation promotion, immune regulation, self-replication and the like. For example, under in vivo or in vitro specific induction conditions, mesenchymal stem cells can be differentiated into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium and the like, still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue and organ injuries caused by aging and pathological changes.
The mesenchymal stem cells are mainly derived from bone marrow and are obtained by adopting a density gradient centrifugation method. Although the separation method is simple, the bone marrow extraction operation is painful, and the material can be highly infected during and after the material is obtained. The mesenchymal stem cells from the placenta and the umbilical cord have the characteristics of large differentiation potential, strong proliferation capacity, low immunogenicity, convenient material taking, no limitation of moral ethical problems, easy industrial preparation and the like, have larger application potential, and can become an ideal substitute of the mesenchymal stem cells.
At present, the existing methods for separating stem cells from placenta and umbilical cord and establishing a placental stem cell bank have the defects of insufficient purity, low quantity and incapability of meeting the use requirement.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of umbilical cord and placenta mesenchymal Stem cells and application thereof, the method of the invention utilizes a differentiation mode of forming embryoid bodies to differentiate into Stem cells, and adds Stem Cell Growth Factor (STGF) which specifically regulates and controls just-developed Stem Cell factors, including bFGF, HGF and OSM to promote the Growth and differentiation of the Stem cells, so that the expression of related Stem genes is similar to the development state, a key for activating human body functions by the Stem cells is successfully opened, the Stem cells can be activated in a large scale, and the purity and the quantity are improved. The specific technical scheme is as follows:
a preparation method of umbilical cord and placenta mesenchymal stem cells comprises the following steps:
step 1:
cleaning umbilical cord blood stain with DPBS under aseptic condition, cutting human umbilical cord and placenta tissue into 3-4 cm segments, removing 3 blood vessels and blood stasis parts, further separating tissue, and cutting into 2-3 mm segments3A size tissue mass;
step 2:
dropwise adding a small amount of mesenchymal stem cell serum-free culture medium into the culture plate, sucking tissue blocks by using a suction pipe, uniformly spreading the tissue blocks in the culture plate to ensure that the distance between the tissue blocks is 0.5-1 cm, then placing at 37 ℃ and 5% CO2Incubating in an incubator for 30-40 min;
and step 3:
taking out the culture plate, slowly adding 1-2 ml of mesenchymal stem cell serum-free culture medium containing additives into each hole of the culture plate, and then putting the culture plate back into the incubator for culture;
and 4, step 4:
adding 1ml of complete culture medium into each hole of the culture plate on day 1, changing the culture medium in half on day 3, changing the culture medium in full on day 6, changing the culture medium 2 times per week, observing and recording the cell morphology of tissue adherent growth by using an inverted microscope every day, observing under an inverted phase contrast microscope after the earliest 5 days, and finding that fine fusiform cells climb out from the edge;
and 5:
after the cells are confluent and grow for more than 80 percent, absorbing and removing tissue blocks and culture media in the culture plate, washing for 1 time by using a DPBS solution, then adding 1ml of recombinant pancreatin for digestion, tapping the culture plate to completely separate the cells, adding 5-6 ml of soybean trypsin inhibitor diluted by using the DPBS solution to stop digestion, centrifuging to remove supernatant, suspending the cells by using a small amount of complete culture medium, and counting the cells;
step 6:
adjusting cell density to 5000 cells/cm2Inoculated in a T25 flask at 37 ℃ with 5% CO2Culturing in an incubator; observing and recording the growth condition of the cells by using an inverted microscope, and carrying out subculture when the cultured cells reach 80% fusion;
and 7:
centrifugally collecting cells, suspending the cells in the mesenchymal stem cell frozen stock solution, and adjusting the cell density to (0.5-1) × 106Putting the seeds/ml into a freezing storage tube, putting the freezing storage tube into a program cooling box, standing the freezing storage tube in a refrigerator at the temperature of minus 80 ℃ for 24 hours, and then transferring the freezing storage tube into liquid nitrogen for storage for later use;
in the step 3, the additive is a B27 serum-free additive of 1-10 ppmbFGF;
in the step 5, the Recombinant pancreatin is Recombinant Trypsin EDTA Solution Recombinant pancreatin;
in the step 6, subculturing is carried out for 4-5 days;
in the step 7, the specification of the freezing storage tube is 1 ml/tube;
the mesenchymal stem cell serum-free culture medium is
MSC XF basic Medium mesenchymal stem cell serum-free culture Medium;
the culture plate is a 6-hole culture plate;
the application of the mesenchymal stem cell comprises the application to skin diseases, blood system diseases, nervous system diseases, solid organ injuries or lesions, immune system diseases, cardiovascular system diseases, metabolic diseases, bone joint diseases and reproductive system diseases of a human body.
Compared with the prior art, the preparation method and the application of the umbilical cord and placenta mesenchymal stem cells have the beneficial effects that:
the differentiation mode of forming embryoid bodies is utilized to differentiate into Stem cells, Stem Cell Growth Factor (STGF) which specifically regulates the just-developed Stem Cell is added, and the STGF comprises bFGF, HGF and OSM to promote the Growth and differentiation of the Stem cells, so that the expression of related Stem genes is similar to the development state, a key for activating human body functions by the Stem cells is successfully opened, the Stem cells can be activated in a large scale, the purity and the quantity are improved, and the use requirement is met.
The STGF stem cell factor mainly comprises bFGF basic fibroblast cell factor which is transmitted by a nano-secretion technology, and is a universal factor aiming at activating MSC mesenchymal stem cells, wherein the bFGF basic fibroblast growth factor is a key component of culture media of human embryonic stem cells and iPS cells, 146 amino acids of ReProCell recombinant human FGF-2(bFGF) can maintain the normal growth of the pluripotent stem cells no matter the culture condition of the feeding cells exists or not, the cooperation of the STGF and the MSC can improve the autoimmunity of a human body, repair 8 major systems of the human body and resist aging.
And the serum-free culture system is different from the traditional serum-containing culture system in that the use of a culture medium, fetal calf serum and a double antibody is reduced, the culture cost is reduced, the tissue block adherence rate can be improved, and the cell migration rate is increased. The method shortens the time for the cells to migrate from the tissue block, reduces the influence of antibiotics and the like on the cell growth as much as possible, eliminates the risk caused by unidentified pathogens, and ensures the purity and the integrity of the cells.
Human umbilical cord mesenchymal stem cells (HUC-MSCs) are adult stem cells which are derived from umbilical cord tissues of newborns and have self-renewal and multidirectional differentiation potential, and have the potential of differentiating towards various functional cells such as nerves, blood vessels, muscles, bones, cartilages, fats and the like. The mesenchymal stem cells prepared by the method have high purity and strong repairability, can greatly improve the body immunity and the anti-aging repair of a human body system, slow down the aging of self-aging cells, and improve injured visceral tissues and an immune system.
The method has simple and convenient operation and low economic cost, the obtained cells have complete shape and high purity, and the obtained umbilical cord mesenchymal stem cells do not express hematopoietic stem cells and the like, can better keep the activity of the cells and have good industrial realizability.
Drawings
FIG. 1 shows the density of HUC-MSCs primary cells, wherein the left image is the electron micrograph of cells just after they have crawled out of the tissue mass after 5 days (10 × 4) of culture, and the right image is the electron micrograph of cells dispersed after 10 days (10 × 10) of culture;
FIG. 2 is the first generation cell density of HUC-MSC, wherein the left image is the electron micrograph of 24h (10 × 4) in culture, and the right image is the electron micrograph of 24h (10 × 10) in culture;
FIG. 3 shows the first generation cell density of HUC-MSC, wherein the left image is the electron micrograph of cultured cells cultured for 72h (10 × 4), and the right image is the electron micrograph of cultured cells cultured for 72h (10 × 10);
FIG. 4 shows the first generation cell density of HUC-MSC, wherein the left image is the electron micrograph of 96h (10 × 4) in culture, and the right image is the electron micrograph of 96h (10 × 10) in culture;
FIG. 5 is an electron micrograph of a long, flat, spindle-shaped, vortex-shaped fibroblast;
FIG. 6 is a chart of immunophenotypes of flow cytometric identification of hUC-MSCs;
FIG. 7 shows the differentiation and staining patterns of HUC-MSCs induced in vitro, wherein the 1-osteogenic staining is 4 ×, the 2-adipogenic staining is 20 ×, the 3-chondrogenic staining is 10 ×, and the 4-chondrogenic staining is 20 ×.
Detailed description of the invention
The present invention will be further described with reference to specific examples, but the present invention is not limited to these examples.
Example 1
A preparation method of umbilical cord and placenta mesenchymal stem cells comprises the following steps:
step 1:
cleaning umbilical cord blood stain with DPBS under aseptic condition, cutting human umbilical cord and placenta tissue into 3cm segments, removing 3 blood vessels and blood stasis parts, further separating tissue, and cutting into 3mm pieces3A size tissue mass;
step 2:
dripping a small amount of mesenchymal stem cell serum-free culture medium into a 6-hole culture plate, sucking tissue blocks with a suction pipe, uniformly spreading the tissue blocks in the culture plate to make the distance between the tissue blocks be 1cm, and then placing at 37 ℃ and 5% CO2Incubating in incubator for 30 min;
and step 3:
taking out the culture plate, slightly adding 1ml of mesenchymal stem cell serum-free culture medium containing 10ppmbFGF B27 serum-free additive into each hole of the culture plate, and then putting the culture plate back to the incubator for culture;
and 4, step 4:
adding 1ml of complete culture medium into each hole of the culture plate on day 1, changing the culture medium in half on day 3, changing the culture medium in full on day 6, changing the culture medium 2 times per week, observing and recording the cell morphology of tissue adherent growth by using an inverted microscope every day, observing under an inverted phase contrast microscope after the earliest 5 days, and finding that fine fusiform cells climb out from the edge, as shown in figure 1;
and 5:
when the cells are confluent and grow 85%, removing tissue blocks and culture media in the culture plate, soaking and washing for 1 time by using a DPBS Solution, then adding 1ml of recombined Trypsin EDTA Solution for digestion, tapping the culture plate to completely separate the cells, adding 6ml of soybean Trypsin inhibitor diluted by the DPBS Solution to stop digestion, centrifuging to remove supernatant, suspending the cells by using a small amount of complete culture medium, and counting the cells;
step 6:
adjusting cell density to 5000 cells/cm2Inoculated in a T25 flask at 37 ℃ with 5% CO2Culturing in an incubator; observing the cell growth condition with an inverted microscope, recording, and performing subculture for 5 days when the cultured cells reach 80% confluency, as shown in FIGS. 2-4;
and 7:
centrifuging to collect cells, suspending the cells in the mesenchymal stem cell frozen stock solution, and adjusting the cell density to 1 × 106Loading into 1 ml/ml freezing tube, placing into a programmed cooling box, standing in a refrigerator at-80 deg.C for 24 hr, and storing in liquid nitrogen.
This example was observed by morphology of HUC-MSCs, as shown in FIG. 5: when observed under an inverted microscope, the typical fibroblast morphology is seen, long and flat spindle cells are arranged in order of polarity, colonies are in a vortex shape, and the morphology is uniform.
This example was immunophenotyping with HUC-MSCs, and as shown in FIG. 6, adherent cultures of P3 cells were harvested by digestion, washed 3 times with 0.9% NS, and divided into 7 tubes containing 1 × 10 cells per tube6Adding CD to each cell29-PE、CD44-PE、CD90-PE、CD105-FITC、CD34-FITC、CD4510 μ l of FITC antibody and isotype control, incubating for 30min at 4 ℃ in the dark, washing to remove unbound antibody, and detecting the expression level of the surface antigen marker by flow cytometry, with the following results: CD (compact disc)29、CD44、CD90、CD105Expression is positive; does not express CD34、CD45。
In this example, HUC-MSCs were subjected to in vitro induced differentiation and staining, as shown in FIG. 7: 1) adipogenic induced differentiation, adherent culture of P3 generation cells was collected by digestion, and 3 × 10 generation cells were collected4cells/cm2Was inoculated into 6-well plates and placed in 5% CO2And (2) performing static culture in an incubator at 37 ℃, discarding the culture solution when cell fusion reaches 80-90%, adding a adipogenic differentiation induction solution for culture, changing the solution every 3-4 days, replacing a differentiation culture medium with a maintenance culture medium after adipogenic induction is finished, culturing for 3-6 days, changing the solution every 2-4 days, performing alternate culture, culturing for at least 2 periods, and observing the formationMature adipocytes, fixed with 4% formaldehyde and stained with oil red O, and the cytoplasm is full of lipid droplets under biological microscope, as shown in FIG. 7 (2). 2) osteogenic induced differentiation, adherent culture of P3 generation cells is collected by digestion, using 3 × 104cell/cm2Was inoculated into 6-well plates and placed in 5% CO2Performing static culture in an incubator at 37 ℃ until cell fusion reaches 80%, discarding the culture solution, adding osteogenic differentiation inducing solution for culture, changing the solution every 3 days, fixing at room temperature (30-60) min after culturing for 21 days, adding 2% ARS for staining, and performing red calcium nodule under a biological microscope, as shown in (1) of figure 7, 3) performing chondrogenic induced differentiation, collecting adherent cultured P3 generation cells by digestion, and adopting 1 × 10 generation cells5cells/ml are inoculated into a 6-well plate, statically cultured for 2 hours and placed in 5% CO2After culturing for 24 hours in an incubator at 37 ℃, adding chondrogenic differentiation culture solution for culturing, and changing the culture solution every 3 days. After 14 days of culture, after fixation with 4% formaldehyde, alcian blue staining was added, and a blue-colored cartilage collagen matrix was observed under a biological microscope, as shown in fig. 7(3), 7 (4).
In this example, HUC-MSCs still retain mesenchymal stem cell characteristics after multiple passages and were identified according to the minimum criteria of HUC-MSCs defined by International Society for Cell Therapy (ISCT), which was identified as having CD29、CD44、CD90、CD105Positive expression; CD (compact disc)34、CD45Negative expression, and differentiation towards adipogenic, osteogenic and chondrogenic directions, all in accordance with international cytology.
Example 2
A preparation method of umbilical cord and placenta mesenchymal stem cells comprises the following steps:
step 1:
cleaning umbilical cord blood stain with DPBS under aseptic condition, cutting human umbilical cord and placenta tissue into 4cm segments, removing 3 blood vessels and blood stasis parts, further separating tissue, and cutting into 2mm pieces3A size tissue mass;
step 2:
dripping a small amount of mesenchymal stem cell serum-free culture medium into a 6-hole culture plate, and usingThe tissue blocks were evenly spread in the plate with suction tubes to 0.5cm apart, and then placed at 37 deg.C with 5% CO2Incubating in incubator for 30 min;
and step 3:
taking out the culture plate, slowly adding 1.5ml of the mesenchymal stem cell serum-free culture medium containing the B27 serum-free additive containing 8ppm bFGF into each hole of the culture plate, and then putting the culture plate back to the incubator for culture;
and 4, step 4:
adding 1ml of complete culture medium into each hole of the culture plate on day 1, changing the culture medium in half on day 3, changing the culture medium in full on day 6, changing the culture medium 2 times per week, observing and recording the cell morphology of tissue adherent growth by using an inverted microscope every day, observing under an inverted phase contrast microscope after the earliest 5 days, and finding that fine fusiform cells climb out from the edge;
and 5:
when the cells are confluent and grow 85%, removing tissue blocks and culture media in the culture plate, soaking and washing for 1 time by using a DPBS Solution, then adding 1ml of recombined Trypsin EDTA Solution for digestion, tapping the culture plate to completely separate the cells, adding 5ml of soybean Trypsin inhibitor diluted by the DPBS Solution to stop digestion, centrifuging to remove supernatant, suspending the cells by using a small amount of complete culture medium, and counting the cells;
step 6:
adjusting cell density to 5000 cells/cm2Inoculated in a T25 flask at 37 ℃ with 5% CO2Culturing in an incubator; observing and recording the growth condition of the cells by using an inverted microscope, and carrying out subculture for 6 days when the cultured cells reach 80% fusion;
and 7:
centrifuging to collect cells, suspending the cells in the mesenchymal stem cell frozen stock solution, and adjusting the cell density to 0.5 × 106Loading into 1 ml/ml freezing tube, placing into a programmed cooling box, standing in a refrigerator at-80 deg.C for 24 hr, and storing in liquid nitrogen.
In this example, HUC-MSCs after multiple passages still retain mesenchymal stem cell characteristics, and are identified according to the minimum standards of HUC-MSCs defined by International Society for Cell Therapy (ISCT), and the identification result shows that the HUC-MSCs have the characteristics of mesenchymal stem cellsExamples in which HUC-MSCs have CD29、CD44、CD90、CD105Positive expression; CD (compact disc)34、CD45Negative expression, and differentiation towards adipogenic, osteogenic and chondrogenic directions, all in accordance with international cytology.
Example 3
A preparation method of umbilical cord and placenta mesenchymal stem cells comprises the following steps:
step 1:
cleaning umbilical cord blood stain with DPBS under aseptic condition, cutting human umbilical cord and placenta tissue into 3.5cm segments, removing 3 blood vessels and blood stasis parts, further separating tissue, and cutting into 3mm segments3A size tissue mass;
step 2:
dripping a small amount of mesenchymal stem cell serum-free culture medium into a 6-hole culture plate, sucking tissue blocks with a suction pipe, uniformly spreading the tissue blocks in the culture plate to make the distance between the tissue blocks be 0.8cm, placing at 37 deg.C and 5% CO2Incubating in incubator for 40 min;
and step 3:
taking out the culture plate, slightly adding 2ml of mesenchymal stem cell serum-free culture medium containing 6ppm bFGF B27 serum-free additive into each hole of the culture plate, and then putting the culture plate back into the incubator for culture;
and 4, step 4:
adding 1ml of complete culture medium into each hole of the culture plate on day 1, changing the culture medium in half on day 3, changing the culture medium in full on day 6, changing the culture medium 2 times per week, observing and recording the cell morphology of tissue adherent growth by using an inverted microscope every day, observing under an inverted phase contrast microscope after the earliest 5 days, and finding that fine fusiform cells climb out from the edge;
and 5:
when the cells are confluent and grow 85%, removing tissue blocks and culture media in the culture plate, soaking and washing for 1 time by using a DPBS Solution, then adding 1ml of recombined Trypsin EDTA Solution for digestion, tapping the culture plate to completely separate the cells, adding 5.5ml of soybean Trypsin inhibitor diluted by the DPBS Solution to stop digestion, centrifuging to remove supernatant, suspending the cells by using a small amount of complete culture medium, and counting the cells;
step 6:
adjusting cell density to 5000 cells/cm2Inoculated in a T25 flask at 37 ℃ with 5% CO2Culturing in an incubator; observing and recording the growth condition of the cells by using an inverted microscope, and carrying out subculture for 5 days when the cultured cells reach 80% fusion;
and 7:
centrifuging to collect cells, suspending the cells in the mesenchymal stem cell frozen stock solution, and adjusting the cell density to 0.8 × 106Loading into 1 ml/ml freezing tube, placing into a programmed cooling box, standing in a refrigerator at-80 deg.C for 24 hr, and storing in liquid nitrogen.
In this example, HUC-MSCs still retain mesenchymal stem cell characteristics after multiple passages and were identified according to the minimum criteria of HUC-MSCs defined by International Society for Cell Therapy (ISCT), which was identified as having CD29、CD44、CD90、CD105Positive expression; CD (compact disc)34、CD45Negative expression, and differentiation towards adipogenic, osteogenic and chondrogenic directions, all in accordance with international cytology.
Claims (8)
1. A preparation method of umbilical cord and placenta mesenchymal stem cells is characterized by comprising the following steps:
step 1:
cleaning umbilical cord blood stain with DPBS under aseptic condition, cutting human umbilical cord and placenta tissue into 3-4 cm segments, removing 3 blood vessels and blood stasis parts, further separating tissue, and cutting into 2-3 mm segments3A size tissue mass;
step 2:
dropwise adding a small amount of mesenchymal stem cell serum-free culture medium into the culture plate, sucking tissue blocks by using a suction pipe, uniformly spreading the tissue blocks in the culture plate to ensure that the distance between the tissue blocks is 0.5-1 cm, then placing at 37 ℃ and 5% CO2Incubating in an incubator for 30-40 min;
and step 3:
taking out the culture plate, slowly adding 1-2 ml of mesenchymal stem cell serum-free culture medium containing additives into each hole of the culture plate, and then putting the culture plate back into the incubator for culture;
and 4, step 4:
adding 1ml of complete culture medium into each hole of the culture plate on day 1, changing the culture medium in half on day 3, changing the culture medium in full on day 6, changing the culture medium 2 times per week, observing and recording the cell morphology of tissue adherent growth by using an inverted microscope every day, observing under an inverted phase contrast microscope after the earliest 5 days, and finding that fine fusiform cells climb out from the edge;
and 5:
after the cells are confluent and grow for more than 80 percent, absorbing and removing tissue blocks and culture media in the culture plate, washing for 1 time by using a DPBS solution, then adding 1ml of recombinant pancreatin for digestion, tapping the culture plate to completely separate the cells, adding 5-6 ml of soybean trypsin inhibitor diluted by using the DPBS solution to stop digestion, centrifuging to remove supernatant, suspending the cells by using a small amount of complete culture medium, and counting the cells;
step 6:
adjusting cell density to 5000 cells/cm2Inoculated in a T25 flask at 37 ℃ with 5% CO2Culturing in an incubator; observing and recording the growth condition of the cells by using an inverted microscope, and carrying out subculture when the cultured cells reach 80% fusion;
and 7:
centrifugally collecting cells, suspending the cells in MSC frozen stock solution of mesenchymal stem cells, and adjusting the cell density to (0.5-1) × 106And putting the seeds/ml into a freezing storage tube, putting the freezing storage tube into a programmed cooling box, standing the freezing storage tube in a refrigerator at the temperature of minus 80 ℃ for 24 hours, and then transferring the freezing storage tube into liquid nitrogen for storage and standby.
2. The method for preparing umbilical cord and placental mesenchymal stem cells according to claim 1, wherein in step 3, the additive is (1-10) ppmbFGF in B27 serum-free additive.
3. The method for preparing umbilical cord and placental mesenchymal stem cells according to claim 1, wherein in step 5, the Recombinant pancreatin is Recombinant Trypsin EDTA Solution Recombinant pancreatin.
4. The method for preparing umbilical cord and placental mesenchymal stem cells according to claim 1, wherein in step 6, the subculture is performed for 4-5 days.
5. The method of claim 1, wherein in step 7, the frozen tube is 1 ml/tube.
6. The method for preparing mesenchymal stem cells of umbilical cord and placenta according to claim 1, wherein the serum-free culture medium for mesenchymal stem cells is
The MSC XF basic Medium mesenchymal stem cell serum-free culture Medium.
7. The method of claim 1, wherein the culture plate is a 6-well culture plate.
8. The use of umbilical cord and placental mesenchymal stem cells according to claim 1, comprising use in a human subject for a skin disease, a blood system disease, a nervous system disease, a solid organ injury or lesion, an immune system disease, a cardiovascular system disease, a metabolic disease, a bone joint disease, a reproductive system disease.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112410285A (en) * | 2020-11-25 | 2021-02-26 | 四川大学华西医院 | Culture method of human placenta mesenchymal stem cells |
| CN113355279A (en) * | 2021-06-07 | 2021-09-07 | 深圳市中佳生物医疗科技有限公司 | Preparation method of human ligament fibroblast |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101538553A (en) * | 2009-02-06 | 2009-09-23 | 叶尚勉 | Method for separating, purifying, culturing and proliferating totipotent stem cell from tissue of early aborted fetus of human being |
| CN102433301A (en) * | 2011-12-02 | 2012-05-02 | 上海安集协康生物技术有限公司 | Method for extracting and amplifying monoclonal mesenchymal stem cells and culture solution for same |
| CN104630140A (en) * | 2013-11-06 | 2015-05-20 | 吉林济惠生物科技有限公司 | Isolation and culture method of placenta mesenchyma precursor stem cells |
| US20150159137A1 (en) * | 2013-12-11 | 2015-06-11 | Buddhist Tzu Chi Medical Foundation | Adjuvant for rapid proliferation of human mesenchymal stem cells in vitro, method for rapid proliferation of human mesenchymal stem cells in vitro, method for growth factor harvested from rapid proliferation of human mesenchymal stem cells in vitro and use thereof |
| 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 |
| WO2017096618A1 (en) * | 2015-12-11 | 2017-06-15 | 郭镭 | Method for separating and culturing mesenchymal stem cells from outer layer of amniotic membrane tissue of umbilical cord |
-
2020
- 2020-03-17 CN CN202010186010.4A patent/CN111321115A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101538553A (en) * | 2009-02-06 | 2009-09-23 | 叶尚勉 | Method for separating, purifying, culturing and proliferating totipotent stem cell from tissue of early aborted fetus of human being |
| CN102433301A (en) * | 2011-12-02 | 2012-05-02 | 上海安集协康生物技术有限公司 | Method for extracting and amplifying monoclonal mesenchymal stem cells and culture solution for same |
| CN104630140A (en) * | 2013-11-06 | 2015-05-20 | 吉林济惠生物科技有限公司 | Isolation and culture method of placenta mesenchyma precursor stem cells |
| US20150159137A1 (en) * | 2013-12-11 | 2015-06-11 | Buddhist Tzu Chi Medical Foundation | Adjuvant for rapid proliferation of human mesenchymal stem cells in vitro, method for rapid proliferation of human mesenchymal stem cells in vitro, method for growth factor harvested from rapid proliferation of human mesenchymal stem cells in vitro and use thereof |
| 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 |
| WO2017096618A1 (en) * | 2015-12-11 | 2017-06-15 | 郭镭 | Method for separating and culturing mesenchymal stem cells from outer layer of amniotic membrane tissue of umbilical cord |
Non-Patent Citations (4)
| Title |
|---|
| YANXIA LIU等: "A novel chemical-defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
| 任春红等: "无动物源成分培养基用于人脐带间充质干细胞培养的研究", 《延安大学学报(医学科学版)》 * |
| 屈玟等: "人脐带间充质干细胞不同培养方案的比较与优化", 《海南医学院学报》 * |
| 张丽等: "无血清培养体系分离培养人脐带间充质干细胞", 《皮肤病与性病》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112410285A (en) * | 2020-11-25 | 2021-02-26 | 四川大学华西医院 | Culture method of human placenta mesenchymal stem cells |
| CN113355279A (en) * | 2021-06-07 | 2021-09-07 | 深圳市中佳生物医疗科技有限公司 | Preparation method of human ligament fibroblast |
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