CN117305235A - Method for separating and extracting decidua stem cells from placenta - Google Patents
Method for separating and extracting decidua stem cells from placenta Download PDFInfo
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- CN117305235A CN117305235A CN202311078348.8A CN202311078348A CN117305235A CN 117305235 A CN117305235 A CN 117305235A CN 202311078348 A CN202311078348 A CN 202311078348A CN 117305235 A CN117305235 A CN 117305235A
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- 210000003785 decidua Anatomy 0.000 title claims abstract description 42
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 33
- 210000002826 placenta Anatomy 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000005119 centrifugation Methods 0.000 claims abstract description 22
- 230000029087 digestion Effects 0.000 claims abstract description 17
- 238000004140 cleaning Methods 0.000 claims abstract description 15
- 238000012258 culturing Methods 0.000 claims abstract description 10
- 238000000605 extraction Methods 0.000 claims abstract description 10
- 230000000694 effects Effects 0.000 claims abstract description 9
- 102000029816 Collagenase Human genes 0.000 claims abstract description 7
- 108060005980 Collagenase Proteins 0.000 claims abstract description 7
- 229960002424 collagenase Drugs 0.000 claims abstract description 7
- 102000004142 Trypsin Human genes 0.000 claims abstract description 6
- 108090000631 Trypsin Proteins 0.000 claims abstract description 6
- 239000012588 trypsin Substances 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 21
- 239000001963 growth medium Substances 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 7
- 239000003242 anti bacterial agent Substances 0.000 claims description 6
- 229940088710 antibiotic agent Drugs 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 239000003761 preservation solution Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 5
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 claims description 3
- 239000001045 blue dye Substances 0.000 claims description 3
- 239000006285 cell suspension Substances 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 238000004659 sterilization and disinfection Methods 0.000 claims description 2
- 238000010186 staining Methods 0.000 claims 1
- 230000003169 placental effect Effects 0.000 abstract description 7
- 239000008055 phosphate buffer solution Substances 0.000 description 5
- 239000012634 fragment Substances 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 230000003394 haemopoietic effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 208000009527 Refractory anemia Diseases 0.000 description 1
- 206010072684 Refractory cytopenia with unilineage dysplasia Diseases 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
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- Bioinformatics & Cheminformatics (AREA)
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- Organic Chemistry (AREA)
- Developmental Biology & Embryology (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
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- Rheumatology (AREA)
- Microbiology (AREA)
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- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A method for separating and extracting decidua stem cells from placenta comprises the steps of firstly, placenta collection pretreatment; step two, stripping decidua and cleaning; step three, primary enzymolysis digestion; step four, primary centrifugation; step five, secondary enzymolysis digestion; step six, secondary centrifugation; step seven, inoculating and culturing; in the fourth step, the running speed of the centrifugal machine is set to be 1000r/min, and the duration of centrifugation is 5min; in the fifth step, 1g/L collagenase is used for digestion in the secondary enzymolysis, and the enzymolysis digestion time is 60min; according to the invention, the extraction efficiency of the placental decidua stem cells is greatly improved by a secondary enzymolysis scheme of trypsin and collagenase, a more proper enzymolysis environment is provided by adding the culture solution in the enzymolysis process, so that the extracted stem cells have higher activity, and in addition, the decidua stem cells are prevented from being damaged in the multiple centrifugation process by a secondary centrifugation scheme.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for separating and extracting decidua stem cells from placenta.
Background
The placental decidua stem cells are important components of human microenvironment, the mesenchymal stem cells can be transplanted to change the hematopoietic microenvironment, reconstruct the immune system, promote the recovery of hematopoietic function, and the treatment effects of leukemia, refractory anemia and the like can be obviously improved by being transplanted together with the hematopoietic stem cells, the genotype of the placental decidua stem cells is completely the same as that of a mother, and the placental decidua stem cells are the exclusive stem cells of the mother and the maternal relatives, and have obvious effects in aspects of beautifying, anti-aging, postpartum recovery, female diseases and the like.
The existing extraction method of the placental decidua stem cells has the following defects that firstly, the activity of the extracted stem cells is low, the requirement on the subsequent separation and purification is high, secondly, the number of the extracted stem cells is small, the extraction efficiency is low, thirdly, the existing extraction method needs to perform centrifugal separation for more than three times in the extraction process, and the stem cells are easy to damage in the centrifugal process.
Disclosure of Invention
The invention aims to provide a method for separating and extracting decidua stem cells from placenta, which aims to solve the problems in the background technology.
In order to achieve the above purpose, the present invention provides the following technical solutions: a method for separating and extracting decidua stem cells from placenta comprises the steps of firstly, placenta collection pretreatment; step two, stripping decidua and cleaning; step three, primary enzymolysis digestion; step four, primary centrifugation; step five, secondary enzymolysis digestion; step six, secondary centrifugation; step seven, inoculating and culturing;
in the first step, placenta is collected and stored in corresponding preservation solution, and pretreatment before extraction is carried out;
in the second step, the decidua on the placenta is peeled off, and is cleaned by using a cleaning solution, and the decidua is sheared;
in the third step, adding a culture solution, and performing primary enzymolysis digestion on decidua tissues to obtain a cell sap;
transferring the cell sap into a centrifuge tube, adding a cleaning solution, opening a low-temperature centrifuge, balancing, centrifuging the cell sap once, precipitating, and discarding the supernatant;
in the fifth step, adding a culture solution into the cell sap in the fourth step for secondary enzymolysis;
in the sixth step, cleaning liquid is added for secondary centrifugation after enzymolysis, supernatant is discarded, and the mixture is filtered by a cell screen;
in the seventh step, the cell fluid is transferred to a medium, and the medium is added for culturing.
Preferably, in the first step, the preservation solution is a tissue protection solution containing 99% low sugar DMEM and 1% diab.
Preferably, in the second step, the washing solution used is 0-4deg.C calcium-magnesium-free PBS, washing is performed twice to three times, the decidua tissue is sheared into pieces of about 1mm, and the washing solution is rinsed again with calcium-magnesium-free PBS until colorless.
Preferably, in the third step, 2g/L trypsin is used for one enzymolysis, and the enzymolysis time is 30min.
Preferably, in the fourth step, the running speed of the centrifuge is set to 1000r/min, and the duration of centrifugation is 5min.
Preferably, in the fifth step, the secondary enzymolysis is performed by using 1g/L collagenase, and the enzymolysis and digestion time is 60min.
Preferably, in the sixth step, the rotation speed of the centrifuge is set to 1200r/min, the centrifugation duration is 5min, a 300 mesh cell screen is used for filtering, 10mL of cleaning solution is added into the pipette after filtering, about 50 μL of cell liquid is transferred to the pipette for resuspension, 50 μL of cell suspension is taken and added into a sterile test tube, 50 μL of trypan blue dye with concentration of 0.4% is added into the sterile test tube, and the activity and the quantity of cells are measured after dyeing for 2-3 min.
Preferably, in the seventh step, the preparation method of the culture solution comprises adding 55mL of FBS serum into 500mL of DMEM high-sugar culture medium until the concentration is 10%, adding double antibiotics to ensure that the unit of the antibiotics reaches 100Units, placing the culture medium into a pure water bath at 37 ℃ for warm bath before use, sterilizing by using an ultraviolet lamp, taking the culture medium out of a water bath kettle before use, and placing the inoculated culture medium into CO at 37 DEG C 2 Culturing in an incubator.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, the extraction efficiency of the placental decidua stem cells is greatly improved by a secondary enzymolysis scheme of trypsin and collagenase, a more proper enzymolysis environment is provided by adding the culture solution in the enzymolysis process, so that the extracted stem cells have higher activity, and in addition, the decidua stem cells are prevented from being damaged in the multiple centrifugation process by a secondary centrifugation scheme.
Drawings
FIG. 1 is a flow chart of the method of the present invention.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and all other embodiments obtained by those skilled in the art without making creative efforts based on the embodiments of the present invention are included in the protection scope of the present invention.
Referring to fig. 1, an embodiment of the present invention is provided: a method for separating and extracting decidua stem cells from placenta comprises the steps of firstly, placenta collection pretreatment; step two, stripping decidua and cleaning; step three, primary enzymolysis digestion; step four, primary centrifugation; step five, secondary enzymolysis digestion; step six, secondary centrifugation; step seven, inoculating and culturing;
in the first step, placenta is collected and stored in corresponding preservation solution, pretreatment is carried out before extraction, and the preservation solution is tissue protection solution containing 99% of low-sugar DMEM and 1% of diab;
in the second step, the decidua on the placenta is peeled off, the decidua is washed by using a washing liquid, the decidua is sheared, the washing liquid is 0-4 ℃ calcium-magnesium-free PBS (phosphate buffer solution), the washing is carried out for two to three times, the decidua tissue is sheared into fragments with the diameter of about 1mm, and the fragments are rinsed again by using the calcium-magnesium-free PBS until the fragments are colorless;
in the third step, adding a culture solution, and performing primary enzymolysis digestion on decidua tissues to obtain a cell fluid, wherein 2g/L trypsin is used for primary enzymolysis, and the enzymolysis time is 30min;
transferring the cell sap into a centrifuge tube, adding a cleaning solution, opening a low-temperature centrifuge, balancing, centrifuging the cell sap once, setting the running speed of the centrifuge to 1000r/min, centrifuging for 5min, and discarding the supernatant after precipitation;
in the fifth step, adding a culture solution into the cell sap in the fourth step, and performing secondary enzymolysis, wherein the secondary enzymolysis uses 1g/L collagenase for digestion, and the enzymolysis digestion time is 60min;
in the sixth step, cleaning liquid is added for secondary centrifugation after enzymolysis, the rotation speed of the centrifuge is set to 1200r/min, the centrifugation duration is 5min, supernatant is discarded, a 300-mesh cell screen is used for filtering, 10mL of cleaning liquid is added into a pipette after filtering, about 50 mu L of cell liquid is transferred to the pipette for resuspension, 50 mu L of cell suspension is taken and added into a sterile test tube, 50 mu L of trypan blue dye liquid with the concentration of 0.4% is added into the sterile test tube, and the activity and the number of cells are measured after dyeing for 2-3 min;
in the seventh step, the cell sap is transferred to a culture medium, the culture medium is added for culturing, the preparation method of the culture medium is that 55mL of FBS serum is added to 500mL of DMEM high-sugar culture medium until the concentration is 10%, then double antibiotics are added to ensure that the unit of the antibiotics reaches 100Units, the culture medium is put into a pure water bath with the temperature of 37 ℃ for medium temperature bath before use, an ultraviolet lamp is used for sterilization, the culture medium is taken out from a water bath kettle before use, and the inoculated culture medium is put into CO with the temperature of 37 DEG C 2 Culturing in an incubator.
Based on the above, the invention has the advantages that: according to the invention, the target stem cells can be separated more efficiently by adopting a secondary enzymolysis scheme of trypsin and collagenase, so that the extraction efficiency of the placental decidua stem cells is improved, compared with the existing method, the method corresponds to a secondary low-speed centrifugation scheme after secondary enzymolysis, the interference between different enzymolysis stages is avoided while the active cell liquid is purified, the possibility of damage of the stem cells in the mechanical centrifugation process is reduced compared with the existing centrifugation times of three or more times, and a more suitable enzymolysis environment is provided by adding culture liquid in the enzymolysis process, so that the stem cells obtained by enzymolysis have higher activity and are more beneficial to subsequent preservation or use.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (8)
1. A method for separating and extracting decidua stem cells from placenta comprises the steps of firstly, placenta collection pretreatment; step two, stripping decidua and cleaning; step three, primary enzymolysis digestion; step four, primary centrifugation; step five, secondary enzymolysis digestion; step six, secondary centrifugation; step seven, inoculating and culturing; the method is characterized in that:
in the first step, placenta is collected and stored in corresponding preservation solution, and pretreatment before extraction is carried out;
in the second step, the decidua on the placenta is peeled off, and is cleaned by using a cleaning solution, and the decidua is sheared;
in the third step, adding a culture solution, and performing primary enzymolysis digestion on decidua tissues to obtain a cell sap;
transferring the cell sap into a centrifuge tube, adding a cleaning solution, opening a low-temperature centrifuge, balancing, centrifuging the cell sap once, precipitating, and discarding the supernatant;
in the fifth step, adding a culture solution into the cell sap in the fourth step for secondary enzymolysis;
in the sixth step, cleaning liquid is added for secondary centrifugation after enzymolysis, supernatant is discarded, and the mixture is filtered by a cell screen;
in the seventh step, the cell fluid is transferred to a medium, and the medium is added for culturing.
2. The method for separating and extracting decidua stem cells from placenta according to claim 1, wherein: in the first step, the preservation solution is tissue protection solution containing 99% of low-sugar DMEM and 1% of diab.
3. The method for separating and extracting decidua stem cells from placenta according to claim 1, wherein: in the second step, washing the decidua tissue with 0-4deg.C calcium-magnesium-free PBS for two to three times, cutting the decidua tissue into pieces of about 1mm, and washing with the calcium-magnesium-free PBS again to colorless.
4. The method for separating and extracting decidua stem cells from placenta according to claim 1, wherein: in the third step, 2g/L trypsin is used for enzymolysis for 30min.
5. The method for separating and extracting decidua stem cells from placenta according to claim 1, wherein: in the fourth step, the running speed of the centrifugal machine is set to 1000r/min, and the centrifugal duration is 5min.
6. The method for separating and extracting decidua stem cells from placenta according to claim 1, wherein: in the fifth step, the secondary enzymolysis is carried out by using 1g/L collagenase, and the enzymolysis and digestion time is 60min.
7. The method for separating and extracting decidua stem cells from placenta according to claim 1, wherein: in the sixth step, the rotation speed of the centrifuge is set to 1200r/min, the centrifugation duration is 5min, a 300-mesh cell screen is used for filtering, 10mL of cleaning liquid is added into a pipette after the filtration, about 50 mu L of cell liquid is transferred to the pipette for resuspension, 50 mu L of cell suspension is taken and added into a sterile test tube, 50 mu L of trypan blue dye with the concentration of 0.4% is added into the sterile test tube, and the activity and the quantity of cells are measured after the staining is carried out for 2-3 min.
8. The method for separating and extracting decidua stem cells from placenta according to claim 1, wherein: in the seventh step, 55mL of FBS serum is added into 500mL of DMEM high-sugar culture medium until the concentration is 10%, double antibiotics are added into the DMEM high-sugar culture medium, the unit of the antibiotics is ensured to reach 100Units, the culture medium is put into a pure water bath with the temperature of 37 ℃ for warm bath before use, an ultraviolet lamp is used for sterilization, the culture medium is taken out from a water bath kettle before use, and CO with the temperature of 37 ℃ is put into the inoculated culture medium 2 Culturing in an incubator.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311078348.8A CN117305235A (en) | 2023-08-25 | 2023-08-25 | Method for separating and extracting decidua stem cells from placenta |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311078348.8A CN117305235A (en) | 2023-08-25 | 2023-08-25 | Method for separating and extracting decidua stem cells from placenta |
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| Publication Number | Publication Date |
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| CN117305235A true CN117305235A (en) | 2023-12-29 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN202311078348.8A Pending CN117305235A (en) | 2023-08-25 | 2023-08-25 | Method for separating and extracting decidua stem cells from placenta |
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| CN (1) | CN117305235A (en) |
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2023
- 2023-08-25 CN CN202311078348.8A patent/CN117305235A/en active Pending
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