CN110592005A - Method for separating mesenchymal stem cells - Google Patents
Method for separating mesenchymal stem cells Download PDFInfo
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- CN110592005A CN110592005A CN201910749522.4A CN201910749522A CN110592005A CN 110592005 A CN110592005 A CN 110592005A CN 201910749522 A CN201910749522 A CN 201910749522A CN 110592005 A CN110592005 A CN 110592005A
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- 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
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The application provides a method for separating mesenchymal stem cells, which comprises the following steps: taking complete human-derived placenta tissues, and infusing cleaning perfusate from umbilical veins of the placenta tissues until the cleaning perfusate flowing out of the placenta tissues is colorless; sealing the placenta tissue to form an impervious perfusion space inside the placenta tissue, and infusing enzyme perfusate from the umbilical vein of the placenta tissue for digestion; collecting and culturing a first target cell comprising mesenchymal stem cells in the enzymatic perfusate; culturing the first target cell to logarithmic growth phase, leaving 1/3 free cells of the first target cell for subculture to expand the mesenchymal stem cell. The application can greatly improve the separation yield of the mesenchymal stem cells.
Description
Technical Field
The application relates to the field of stem cells, in particular to a mesenchymal stem cell separation method.
Background
The mesenchymal stem cells exist in various tissues, have the potential of differentiating into adipogenic, osteogenic, chondrogenic, adipogenic, neurogenic, bone marrow stroma, even liver cells and the like, and are important seed cells and gene therapy vectors in cell therapy and tissue engineering research. The clinical transformation application of the mesenchymal stem cells becomes a research hotspot, and how to obtain the primary mesenchymal stem cells in large quantity is the key of each research.
In the prior art, various methods are available for the isolation, culture and purification of mesenchymal stem cells, however, the existing methods have various defects, and the methods which are suitable for large-scale cultivation of clinically applicable mesenchymal stem cells are not available so far.
Disclosure of Invention
The purpose of the application is to provide a mesenchymal stem cell separation method with high yield.
In order to achieve the above object, the present application provides the following technical solutions:
a method for separating mesenchymal stem cells comprises the following steps:
taking complete human-derived placenta tissues, and infusing cleaning perfusate from umbilical veins of the placenta tissues until the cleaning perfusate flowing out of the placenta tissues is colorless;
sealing the placenta tissue to form an impervious perfusion space inside the placenta tissue, and infusing enzyme perfusate from the umbilical vein of the placenta tissue for digestion;
collecting and culturing a first target cell comprising mesenchymal stem cells in the enzymatic perfusate;
culturing the first target cell to logarithmic growth phase, leaving 1/3 free cells of the first target cell for subculture to expand the mesenchymal stem cell.
Preferably, before the perfusion fluid is filled, the method further comprises the following steps: the placental tissue was washed well to remove surface blood stains.
Preferably, the perfusion speed of the cleaning perfusate is 100-150ml/min, and the volume of the cleaning perfusate is 3-5L.
Preferably, the washing perfusate is at least one of D-Hank's, Phosphate Buffered Saline (PBS) and physiological saline.
Preferably, the step of sealing the placenta tissue comprises: and (3) wrapping the placenta tissue by adopting a semipermeable membrane for sealing.
Preferably, the step of sealing the placenta tissue comprises: and closing the outlet of the amnion in the placenta tissue.
Preferably, the perfusion speed of the enzyme perfusate is 80-100ml/min, the perfusion time of the enzyme perfusate is 1-2h, and the process of infusing the enzyme perfusate is carried out under the constant temperature condition.
Preferably, the enzyme perfusate comprises collagenase in an amount of 0.5-5.0mg/ml and trypsin in an amount of 0.5-5.0%.
Preferably, the step of collecting and culturing the first target cells comprising mesenchymal stem cells in the enzyme perfusate comprises:
adjusting the density of the first target cells to 8 × 10 upon seeding7～2×108And (4) performing static culture.
Preferably, after the step of infusing the enzyme perfusate from the umbilical vein of the placenta tissue for digestion, the method further comprises the following steps:
and performing static culture on the digested placenta tissue to obtain a second target cell containing the mesenchymal stem cell.
Preferably, said step of subjecting said digested placental tissue to stationary culture comprises:
respectively taking the following tissue blocks from the placenta tissues for static culture: umbilical cord, amniotic membrane, chorionic smooth muscle layer, chorionic trophoblast, and decidual tissue.
Preferably, the step of stationary culture comprises:
and pretreating the tissue block, placing the pretreated tissue block on the bottom surface of a culture vessel for tiling, and adding a culture medium along the edge of the culture vessel until the tissue block is submerged.
Preferably, the step of performing static culture on the digested placenta tissue further comprises:
when the cells in the culture vessel grow to logarithmic growth phase, digesting with pancreatin to obtain a second target cell comprising the mesenchymal stem cells.
Compared with the prior art, the scheme of the application has the following advantages:
1. this application forms the digestion space of the inside non-seepage of placenta tissue through the mode of closing the processing to placenta tissue, makes placenta tissue be soaked by the perfusate completely to improve the digestion degree, and improve mesenchymal stem cell's separation output greatly.
2. The enzyme perfusate adopted by the application comprises collagenase and trypsin, the placenta scaffold can be better digested by combining collagenase with trypsin, the structure becomes loose, cells are easier to separate, and compared with the digestion by adopting single collagenase or trypsin, the enzyme perfusate can be beneficial to greatly improving the digestion degree of placenta tissues.
3. According to the method, after the step of perfusion, the digested placenta tissue is further taken for culture, so that the target cells containing the mesenchymal stem cells can be separated from loose cells in the perfused placenta tissue, specifically, on one hand, the activity of the cells can be effectively recovered, on the other hand, the amplification of the target cells can be realized, and the yield is greatly improved.
4. The method is simple in steps and high in operation efficiency, is suitable for large-scale culture of the mesenchymal stem cells, and provides a foundation for establishment of the seed cell bank.
Additional aspects and advantages of the present application will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the present application.
Drawings
FIG. 1 is a flow chart of a method according to an exemplary embodiment of a method for isolating mesenchymal stem cells of the present application;
FIG. 2 is a flow chart of a method according to another exemplary embodiment of a method of isolating mesenchymal stem cells according to the present application;
FIG. 3 is a diagram of the growth state of mesenchymal stem cells obtained by the method for separating mesenchymal stem cells according to the present application;
FIG. 4 is a diagram of the result of the identification of the cellular immunophenotype of the mesenchymal stem cells obtained by the method for separating mesenchymal stem cells according to the present application;
FIG. 5 is a diagram illustrating the results of adipogenic differentiation of mesenchymal stem cells obtained by the method for isolating mesenchymal stem cells according to the present application;
fig. 6 is a diagram illustrating osteogenic differentiation results of mesenchymal stem cells obtained by the mesenchymal stem cell isolation method of the present application;
fig. 7 is a diagram illustrating a neural differentiation result of mesenchymal stem cells obtained by the mesenchymal stem cell separation method of the present application.
Detailed Description
The following description of the embodiments is merely exemplary in nature and is in no way intended to limit the present disclosure. In addition, if a detailed description of the known art is not necessary to show the features of the present application, it is omitted.
In the present example, maternal placenta (with 25-35cm umbilical cord remaining) was obtained as human placental tissue by obstetrical and gynecological informed consent of the maternal and family members approved and certified by the ethical committee of the medical center of hospital, cantonese, guangzhou, who was within 35 years of age, negative for infectious disease, more than 35 weeks of gestational age, no more than 18 hours of rupture of fetal membranes, and regular birth tests, and was free of abnormalities in birth, infection, or other diseases that might interfere with placental viability.
Referring to fig. 1, the method for isolating mesenchymal stem cells provided by the present application includes the following steps:
s11, taking the complete human placenta tissue, and infusing a cleaning perfusate from the umbilical vein of the placenta tissue until the cleaning perfusate flowing out of the placenta tissue is colorless;
s12, sealing the placenta tissue to form an impervious perfusion space inside the placenta tissue, and infusing enzyme perfusate from the umbilical vein of the placenta tissue for digestion;
s13, collecting and culturing a first target cell containing mesenchymal stem cells in the enzyme perfusate;
s14, culturing the first target cell to a logarithmic growth phase, and subculturing the first target cell which retains 1/3 to expand the mesenchymal stem cell.
The placental tissue is washed thoroughly to remove surface blood contaminants prior to perfusion with a wash perfusate. Specifically, the method comprises the steps of preliminarily cleaning blood stains on the surfaces of the umbilical cord and the placenta by using physiological saline containing 1% of streptomycin, and transporting the blood stains to a laboratory within 3 hours at the temperature of 2-8 ℃ for treatment by a professional researcher. Placenta tissue is taken and placed in an ultraclean workbench, and blood stain on the surface of the placenta is sufficiently cleaned by PBS (phosphate buffered saline) until the flowing PBS is colorless.
The cleaning perfusate is at least one of D-Hank's, PBS and physiological saline. In one embodiment of the present application, the perfusion rate is 100-150ml/min, and the volume of the cleaning perfusate is 3-5L. When the washing perfusate flowing out of the placenta tissue is colorless, the red blood cells of the placenta tissue are basically washed.
In one embodiment of the present application, the step of sealing the placenta tissue is specifically: and in the final stage of filling the enzyme perfusate, closing the outlet of the amnion in the placenta tissue. Specifically, in the final stage of filling the enzyme perfusate, the last 15-40min, preferably the last 30min of perfusion is used in circulating the enzyme perfusate. The outlet of the amniotic membrane may be tied with a surgical thread. Due to the delivered placenta, the inner umbilical cord and amniotic membrane are turned to the outer side, so that the amniotic membrane at the outermost layer is exposed. The amnion is a semi-permeable membrane, tough and non-vascular, so that the amnion is wrapped on the outermost layer of the placenta tissue, the non-penetration of the placenta tissue in the perfusion process can be ensured, and the full digestion is ensured. In another embodiment of the present application, the placental tissue may also be encapsulated and encapsulated with a semi-permeable medical membrane for perfusion. The placenta tissue is subjected to sealing treatment, so that an impervious digestion space in the placenta tissue can be formed, and the placenta tissue is completely soaked by the perfusate, thereby greatly improving the digestion degree and obtaining a large amount of mesenchymal stem cells.
The enzyme perfusate comprises collagenase and trypsin, wherein the dosage of the collagenase is 0.5-5.0mg/ml, and the dosage of the trypsin is 0.5-5.0%. In one embodiment of the present application, the collagenase is used in an amount of 1.0-2.0mg/ml, the trypsin is used in an amount of 1.0-2.5%, and the process of infusing the enzyme perfusate is performed under a constant temperature condition, the temperature is adjusted to 37.5 ℃, and the infusion speed is controlled to be in a range of
80-100ml/min, perfusion time 1-2h, dosage 400 ml. The collagenase and trypsin are pre-incubated for use before performing the experiment, and specifically, the collagenase and trypsin are pre-heated in a shaker at 37 ℃. The placenta scaffold can be better digested by combining collagenase digestion with trypsin digestion, so that the structure becomes loose, cells are easier to separate, and the digestion degree of placenta tissues is improved.
When complete digestion of placental chorion was observed, all enzyme perfusate was collected, digestion was terminated, and target cells, first target cells, were collected after centrifugation. In one embodiment of the present application, 400ml of the perfusate is collected, neutralized with soybean trypsin stop solution, centrifuged at 1500 rpm for 5min, and the precipitate is collected to obtain the first target cell of the target cells.
In one embodiment of the present application, the step of collecting and culturing the first target cells comprising mesenchymal stem cells in the enzyme perfusate comprises:
adjusting the density of the first target cells to 8 × 10 upon seeding7～2×108And (4) performing static culture.
In one embodiment of the present application, the density of the first target cells is adjusted to 1 × 108After inoculation at 75cm2The culture flask of (4) is added with a proper amount of culture medium. CO at 37 deg.C and 5% volume fraction2And (5) standing and culturing in an incubator.
Referring to fig. 2, in another embodiment of the present application, a method of isolating mesenchymal stem cells includes the steps of:
s01, pouring enzyme perfusate into the intact human placenta tissue for digestion;
s010, collecting the enzyme perfusate containing the mesenchymal stem cells;
s011, obtaining a first target cultured cell containing mesenchymal stem cells;
s020, performing static culture on the digested placenta tissue;
and S021, obtaining a second target cell containing the mesenchymal stem cell.
Specifically, after the complete human placenta tissue is poured into the enzyme perfusate for digestion, the following steps are respectively carried out: collecting the enzyme perfusate containing the mesenchymal stem cells to obtain first target cultured cells containing the mesenchymal stem cells; and performing static culture on the digested placenta tissue to obtain a second target cell containing the mesenchymal stem cell.
Specifically, the method for obtaining the second target cell containing the mesenchymal stem cell by performing static culture on the digested placenta tissue comprises the following steps:
respectively taking the following tissue blocks from the placenta tissues for static culture: umbilical cord, amniotic membrane, chorionic smooth muscle layer, chorionic trophoblast, and decidual tissue.
Specifically, the tissue mass is pre-treated: respectively taking 20g of umbilical cord, amnion, chorion smooth muscle layer, chorion trophoblast and decidual tissue as samples, removing umbilical artery and umbilical vein, and cutting into strips with the length of 1 cm; adding appropriate amount of PBS into the above five samples, processing the samples to granule with diameter of 1-2mm with a hand-held electric homogenizer, centrifuging at 1500r/min for 5min, and removing supernatant to obtain pretreated tissue block.
Placing the pretreated tissue block on the bottom surface of a culture vessel for flat paving, wherein the paving density is 1 block/cm22-4ml of medium was added along the edge of the culture vessel until the tissue mass was submerged. Adjusting CO2Culturing in incubator with CO at 37 deg.C, 95% humidity and 5% volume fraction2So that the tissue mass is in the CO2And (5) standing and culturing in an incubator.
When the cells in the culture vessel grow to the logarithmic growth phase, namely the cell density reaches 80-90% of fusion, the cells are digested by pancreatin to obtain second target cells containing the mesenchymal stem cells, and then the second target cells which are reserved 1/3 are subjected to subculture to expand the mesenchymal stem cells.
This application is after the step of pouring into, still gets the placenta tissue after the digestion to cultivate, cultivates respectively through the different tissues of separation placenta, can improve the utilization ratio of placenta tissue greatly. In addition, the method is also beneficial to separating a second target cell containing the mesenchymal stem cell from loose cells in the placenta tissue after perfusion, and particularly, on one hand, the method can effectively recover the activity of the cells, on the other hand, the target cell can be amplified, and the yield is greatly improved.
In general, the method for separating the mesenchymal stem cells realizes the high-efficiency separation of the mesenchymal stem cells by using the placenta umbilical vein perfusion method, has simple steps and low cost, is suitable for large-scale culture of the mesenchymal stem cells, and can provide sufficient cell sources for the clinical application of the mesenchymal stem cells in the later period.
The biological characteristics of the mesenchymal stem cells obtained by the separation method of the mesenchymal stem cells are detected through morphological observation, cell immunophenotyping and mesenchymal stem cell adipogenic, osteogenic and neurogenic differentiation potential experiments.
Morphological observation of mesenchymal stem cells:
the culture was kept static for 7 days after inoculation, and thereafter the morphology and growth of mesenchymal stem cells were observed under a microscope every day and recorded and photographed. Referring to fig. 3, in fig. 3, a is a graph showing the growth of mesenchymal stem cells at 3 to 4 days of standing after umbilical vein perfusion, and it can be seen that partially adherent cells are generated after 3 to 4 days of standing after umbilical vein perfusion. Panel b shows the presence of small numbers of epithelial cells and flattened stem cells in the culture medium. Panel c shows the spontaneous formation of centers after cell culture. The d picture shows the mesenchymal stem cells obtained after the first target cells obtained by the separation method of the mesenchymal stem cells are cultured for the third generation, and the picture shows that the cells have uniform shape after being attached to the wall, grow in a long prism shape, a parallel shape or a vortex shape, and grow rapidly after passage.
And (3) identifying the cellular immune phenotype:
referring to fig. 4, flow analysis results showed that all cells highly expressed the following proteins: CD73, CD90, CD44 and CD105, low expression CD34, CD19, CD11b, CD45 and HLA-DR, wherein the expression rate of CD73 reaches 99.9 percent, the expression rate of CD105 reaches 99.2 percent, the expression rate of CD44 reaches 100 percent and the expression rate of CD90 reaches 99.4 percent. Therefore, it is demonstrated that the primary cultured cells have surface markers of mesenchymal stem cells, and the cells obtained by isolation are placental mesenchymal stem cells. Meanwhile, the expression rate of the positive molecules is higher than 95%, the expression rate of the negative molecules is lower than 2%, and the expression rate can reach the minimum standard of International Society for Cell Therapy (ISCT), namely, the mesenchymal stem cells which are separated by the method for separating the mesenchymal stem cells are qualified mesenchymal stem cells.
Detecting the adipogenic potential of the placenta mesenchymal stem cells:
the third generation placenta source mesenchymal stem cells are processed into 3 x104Cell concentration per L was seeded in six-well plates. When the cells grow to the logarithmic phase, removing the growth culture medium, respectively adding the prepared adipogenic induction culture medium, changing the culture medium every 3 days, and inducing for 10-15 days. When lipid droplets were observed in the cytoplasm, the adipogenic induction medium was removed, washed gently with PBS 2 times, fixed with 40g/L paraformaldehyde for 20min, stained with oil red O, observed under a microscope, and photographed. Fig. 5 shows the result of the adipogenic differentiation of the placental mesenchymal stem cells, wherein the morphology of the mesenchymal stem cells cultured by adipogenic induction begins to change from slender fusiform to mast-like cells at day 7, lipid droplets are formed in the cytoplasm of a small part of the mesenchymal stem cells after day 7-8, and a large amount of fat cells are formed after induction for 11 days. Oil red O staining positive, confirmed the induction of intracellular lipid droplet formation.
Detecting the osteogenic potential of the placenta mesenchymal stem cells:
the third generation placenta source mesenchymal stem cells are processed into 3 x104Cell concentration per L was seeded in six-well plates. When the cells grow to the logarithmic growth phase, removing the growth culture medium, respectively adding the prepared osteogenic induction culture medium, and changing the liquid of the differentiation dish every 3 days for induction for 10-15 days. Referring to fig. 6, mesenchymal stem cells induced by osteogenic medium were transformed from long spindle to cubic with a paving stone-like change. Alizarin red staining shows that cells grow in colonies and appear calcium nodules, compact opaque masses are formed among the cells, large flaky red staining areas appear, the staining area is wide, the area is large, and the cells show obvious osteogenic activity.
Detecting the differentiation potential of the placenta mesenchymal stem cells into nerves:
the third generation placenta source mesenchymal stem cells are processed into 3 x104L-1The cell concentration of (a) was seeded in a six-well plate. When the cells grow to the logarithmic growth phase, removing the growth culture medium, respectively adding the prepared neurogenesis induction culture medium, and changing the medium every 2 days in a differentiation dish for induction for 4-6 days. The change of cell morphology is observed under a microscope on the first day, and Nie's staining is identified after at least three days of culture, see figure 7, after 1 day of neuro-induction, the cell morphology becomes round and granular, the Nie's staining shows that the cell is light blue, and the blue-black Nie's corpuscle can be seen in the cell.
The mesenchymal stem cell yield condition obtained by the mesenchymal stem cell separation method of the application is as follows: by adopting the separation method of the mesenchymal stem cells, the number of single cells collected from the enzyme perfusate is 2X10 every time an experiment is carried out7-5X107The number of single cells collected per gram of placenta tissue is 2X10 by subsequently taking the digested placenta tissue for adherent culture6-3X106Compared with the traditional enzyme perfusion method, the separation yield is greatly improved.
In conclusion, by adopting the method for separating the mesenchymal stem cells, the mesenchymal stem cells which have good shapes and meet the international standard can be separated, and the separated mesenchymal stem cells have stronger adipogenic, osteogenic and neurogenic differentiation potentials. More importantly, compared with the traditional method, the separation yield is greatly improved, so that the method for separating the mesenchymal stem cells can be suitable for large-scale culture of the mesenchymal stem cells, and a foundation is provided for establishment of a seed cell bank.
The foregoing is only a partial embodiment of the present application, and it should be noted that, for those skilled in the art, several modifications and decorations can be made without departing from the principle of the present application, and these modifications and decorations should also be regarded as the protection scope of the present application.
Claims (13)
1. A method for separating mesenchymal stem cells is characterized by comprising the following steps:
taking complete human-derived placenta tissues, and infusing cleaning perfusate from umbilical veins of the placenta tissues until the cleaning perfusate flowing out of the placenta tissues is colorless;
sealing the placenta tissue to form an impervious perfusion space inside the placenta tissue, and infusing enzyme perfusate from the umbilical vein of the placenta tissue for digestion;
collecting and culturing a first target cell comprising mesenchymal stem cells in the enzymatic perfusate;
culturing the first target cell to logarithmic growth phase, leaving 1/3 free cells of the first target cell for subculture to expand the mesenchymal stem cell.
2. The method of isolating mesenchymal stem cells according to claim 1, further comprising, prior to the perfusion with a washing perfusate: the placental tissue was washed well to remove surface blood stains.
3. The method for separating mesenchymal stem cells according to claim 1, wherein the perfusion rate of the washing perfusate is 100-150ml/min, and the volume of the washing perfusate is 3-5L.
4. The method of isolating mesenchymal stem cells according to claim 3, wherein the washing perfusate is at least one of D-Hank's, phosphate buffered saline, and physiological saline.
5. The method for isolating mesenchymal stem cells according to claim 1, wherein the step of sealing the placental tissue comprises: and (3) wrapping the placenta tissue by adopting a semipermeable membrane for sealing.
6. The method for isolating mesenchymal stem cells according to claim 1, wherein the step of sealing the placental tissue comprises: and closing the outlet of the amnion in the placenta tissue.
7. The method for separating mesenchymal stem cells according to claim 1, wherein the perfusion rate of the enzyme perfusate is 80-100ml/min, the perfusion time of the enzyme perfusate is 1-2h, and the process of perfusing the enzyme perfusate is performed under a constant temperature condition.
8. The method for separating mesenchymal stem cells according to claim 1, wherein the enzyme perfusate comprises collagenase in an amount of 0.5-5.0mg/ml and trypsin in an amount of 0.5-5.0%.
9. The method of isolating mesenchymal stem cells of claim 1, wherein the step of collecting and culturing a first target cell comprising mesenchymal stem cells in the enzymatic perfusate comprises:
adjusting the density of the first target cells to 8 × 10 upon seeding7～2×108And (4) performing static culture.
10. The method of isolating mesenchymal stem cells according to claim 1, wherein the step of perfusing an enzymatic perfusate from the umbilical vein of the placental tissue for digestion further comprises:
and performing static culture on the digested placenta tissue to obtain a second target cell containing the mesenchymal stem cell.
11. The method for isolating mesenchymal stem cells according to claim 10, wherein the step of subjecting the digested placental tissue to static culture comprises:
respectively taking the following tissue blocks from the placenta tissues for static culture: umbilical cord, amniotic membrane, chorionic smooth muscle layer, chorionic trophoblast, and decidual tissue.
12. The method of isolating mesenchymal stem cells according to claim 11, wherein the step of static culturing comprises:
and pretreating the tissue block, placing the pretreated tissue block on the bottom surface of a culture vessel for tiling, and adding a culture medium along the edge of the culture vessel until the tissue block is submerged.
13. The method for isolating mesenchymal stem cells according to claim 12, wherein the step of subjecting the digested placental tissue to static culture further comprises:
when the cells in the culture vessel grow to logarithmic growth phase, digesting with pancreatin to obtain a second target cell comprising the mesenchymal stem cells.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116144583A (en) * | 2023-04-19 | 2023-05-23 | 北京雪颜博润生物科技有限公司 | Method for obtaining human placenta embryo-derived stem cells and application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152405A (en) * | 2014-08-15 | 2014-11-19 | 博雅干细胞科技有限公司 | Method for extracting hemopoietic stem cells from placentas |
| CN205602402U (en) * | 2016-01-30 | 2016-09-28 | 张宏玉 | Placenta freshness protection package |
| CN106754680A (en) * | 2015-12-31 | 2017-05-31 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | A kind of separation method of placenta derived stem cells and its application |
| CN108559725A (en) * | 2005-12-29 | 2018-09-21 | 人类起源公司 | placental stem cell populations |
-
2019
- 2019-08-14 CN CN201910749522.4A patent/CN110592005A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108559725A (en) * | 2005-12-29 | 2018-09-21 | 人类起源公司 | placental stem cell populations |
| CN104152405A (en) * | 2014-08-15 | 2014-11-19 | 博雅干细胞科技有限公司 | Method for extracting hemopoietic stem cells from placentas |
| CN106754680A (en) * | 2015-12-31 | 2017-05-31 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | A kind of separation method of placenta derived stem cells and its application |
| CN205602402U (en) * | 2016-01-30 | 2016-09-28 | 张宏玉 | Placenta freshness protection package |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116144583A (en) * | 2023-04-19 | 2023-05-23 | 北京雪颜博润生物科技有限公司 | Method for obtaining human placenta embryo-derived stem cells and application |
| CN116144583B (en) * | 2023-04-19 | 2023-08-25 | 北京雪颜博润生物科技有限公司 | Method for obtaining human placenta embryo-derived stem cells and application |
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