CN116478918A - Stem cell exosome extraction kit and extraction method - Google Patents
Stem cell exosome extraction kit and extraction method Download PDFInfo
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- 210000001808 exosome Anatomy 0.000 title claims abstract description 50
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 41
- 238000000605 extraction Methods 0.000 title claims abstract description 21
- 239000004005 microsphere Substances 0.000 claims abstract description 45
- 239000007853 buffer solution Substances 0.000 claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 26
- 229920000371 poly(diallyldimethylammonium chloride) polymer Polymers 0.000 claims abstract description 25
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 10
- 238000004062 sedimentation Methods 0.000 claims abstract description 3
- GFLJTEHFZZNCTR-UHFFFAOYSA-N 3-prop-2-enoyloxypropyl prop-2-enoate Chemical compound C=CC(=O)OCCCOC(=O)C=C GFLJTEHFZZNCTR-UHFFFAOYSA-N 0.000 claims abstract 6
- 239000002244 precipitate Substances 0.000 claims description 52
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 22
- 239000002245 particle Substances 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 14
- 239000003153 chemical reaction reagent Substances 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 11
- 238000001556 precipitation Methods 0.000 claims description 11
- 239000011780 sodium chloride Substances 0.000 claims description 11
- 239000012488 sample solution Substances 0.000 claims description 10
- 239000000725 suspension Substances 0.000 claims description 10
- 238000004113 cell culture Methods 0.000 claims description 8
- 230000001376 precipitating effect Effects 0.000 claims description 8
- 239000006185 dispersion Substances 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000012467 final product Substances 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 238000001132 ultrasonic dispersion Methods 0.000 claims description 5
- 238000000464 low-speed centrifugation Methods 0.000 claims description 4
- 239000000872 buffer Substances 0.000 description 15
- 239000012535 impurity Substances 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000000710 polymer precipitation Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- 239000011324 bead Substances 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000012716 precipitator Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000005199 ultracentrifugation Methods 0.000 description 2
- 210000003954 umbilical cord Anatomy 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
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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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Abstract
The invention discloses a stem cell exosome extraction kit, which comprises: the sedimentation agent PEG, PDDA coated PS microsphere, a first buffer solution, a second buffer solution and a third buffer solution. The invention also discloses a method for extracting stem cell exosomes by using the kit. The method for extracting stem cell exosomes has the characteristics of low cost, simplicity in operation and high purity, and is suitable for large-scale application.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a stem cell exosome extraction kit and an extraction method.
Background
The stem cell exosomes are extracellular vesicles secreted by stem cells and have complete membrane structures, and comprise a plurality of bioactive factors such as lipid, protein, RNA and the like, and the diameters of the extracellular vesicles are about 30-150 nm. The stem cell exosomes have similar biological performance as stem cells, but compared with stem cells, the exosomes have lower immunogenicity, are safer, more stable and more efficient, and have high development and application potential in the fields of modern biology and medicine.
Currently, stem cell exosomes are mainly extracted from culture fluids harvested from culturing stem cells. The extraction method of stem cell exosomes mainly comprises an ultracentrifugation method, a magnetic bead antibody capturing method, a polymer precipitation method, a chromatography exclusion method, an ultrafiltration method and the like. The methods have certain defects, the ultracentrifugation method and the chromatographic exclusion method have high requirements on equipment, the magnetic bead antibody capture method has high reagent price, the method is not suitable for processing a large number of samples, the polymer precipitation method has low purity, the ultrafiltration method has reduced service life of an ultrafiltration membrane, and the exosome yield is low. Therefore, there is a need to develop an exosome extraction method that is low in cost, simple to operate, and high in purity.
Disclosure of Invention
Based on the technical problems in the background technology, the invention provides a stem cell exosome extraction kit and an extraction method.
The invention provides a stem cell exosome extraction kit, which comprises:
the sedimentation agent PEG, PDDA coated PS microsphere, a first buffer solution, a second buffer solution and a third buffer solution;
the first buffer solution contains 0.1-0.5M NaCl and 20-100 mM PBS, and the pH value is 7.4;
the second buffer solution is 10-20 mM PBS buffer solution, and the pH value is 7.4;
the third buffer solution contains 0.3-0.5M NaCl and 50-100mM PBS, and the pH value is 6.5.
Preferably, the preparation method of the PDDA coated PS microsphere comprises the following steps: adding PDDA into aqueous dispersion of carboxylated PS microspheres, performing ultrasonic dispersion for 10-15 min, stirring for 1-2 h, centrifuging, and freeze-drying the obtained precipitate to obtain the final product.
Wherein, the carboxylated PS microspheres can be obtained by commercial purchase or prepared by a conventional preparation method.
Preferably, the mass ratio of the PDDA to the carboxylated PS microspheres is (2-5): 1, a step of; the particle size of the carboxylated PS microspheres is 1-25 mu m.
Preferably, the aqueous dispersion of carboxylated PS microspheres has a solids content of 1 to 5%.
Preferably, the precipitator PEG is PEG with a molecular weight of 6000-15000.
A method for extracting stem cell exosomes, using the kit; the extraction method comprises the following steps:
s1, performing low-speed centrifugation on a stem cell culture solution to obtain a supernatant;
s2, adding a precipitating agent PEG into the first buffer solution to obtain a precipitating agent; uniformly mixing 3-5 mL of supernatant obtained by S1 with 2-3 mL of the precipitation reagent, incubating for 6-12 h at 0-4 ℃, and centrifuging for 10-15 min at 15000-18000 g to obtain a precipitate;
s3, re-suspending the precipitate obtained in the step S2 in 50-200 mu L of a second buffer solution to obtain a sample solution; re-suspending 10-15 mg PDDA coated PS microsphere in 10-20 mL second buffer solution to obtain microsphere suspension; after uniformly mixing the sample solution and microsphere suspension, incubating for 2-3 hours at 0-4 ℃, and centrifuging for 20-30 min at 10000-15000 g to obtain a precipitate;
s4, re-suspending the precipitate obtained in the S3 in a third buffer solution, incubating for 0.5-1 h at 0-4 ℃, centrifuging for 20-30 min at 10000-15000 g, and discarding the precipitate to obtain an exosome solution.
Preferably, in S1, the specific steps of the low-speed centrifugation process include: centrifuging for 10-15 min at 300-500 g, removing precipitate, centrifuging for 10-15 min at 2000-3000 g, removing precipitate, centrifuging for 30-40 min at 8000-10000 g, and removing precipitate.
Preferably, in the precipitating reagent, the concentration of the precipitating agent PEG is 80-150 mg/mL.
The beneficial effects of the invention are as follows:
the invention firstly uses a precipitator PEG to carry out precipitation treatment on the supernatant fluid of stem cells to obtain precipitate containing exosomes, then adopts PDDA (polydiallyl dimethyl ammonium chloride) with positive charges on the surface to coat PS microspheres, enables exosomes to be adsorbed on the surfaces of the microspheres through electrostatic adsorption in buffer solution with proper pH value, removes a part of impurities and impurity proteins through centrifugal separation, and then enables exosomes to fall off from the surfaces of the microspheres through adjustment of pH value and ionic strength, and be dispersed in the buffer solution, and a part of impurity proteins are still adsorbed on the surfaces of the microspheres, so that impurities are further removed through centrifugal separation, and a purified exosome solution is obtained. The exosome extraction method provided by the invention can be used for better removing impurities and protein fragments in the exosome by combining a polymer precipitation method with an electrostatic adsorption method, has the characteristics of low cost, simplicity in operation and high purity of the harvested exosome, and is suitable for large-scale application.
Detailed Description
The technical scheme of the invention is described in detail through specific embodiments.
In the following examples and comparative examples, the stem cell culture broth used was prepared as follows:
cleaning umbilical cord, removing blood vessel, cutting, adding 0.1% type I collagenase, digesting at 37deg.C for 12 hr, adding DMEM medium containing 10% fetal bovine serum, filtering with a screen to remove tissue mass, centrifuging, removing supernatant, resuspending precipitate with DMEM medium containing 10% fetal bovine serum at 37deg.C, and 5% CO 2 Culturing under the condition, when the cell fusion degree is more than 80%, carrying out passage, when the passage is carried out to the P3 generation, taking the P3 generation stem cells, culturing the P3 generation stem cells by using a DMEM (medium-medium) containing 10% of fetal bovine serum until the cell fusion degree is more than 80%, replacing the P3 generation stem cells by a fresh serum-free DMEM medium, and continuously culturing the P3 generation stem cells for 48 hours to obtain the umbilical cord mesenchymal stem cell culture solution.
Example 1
A method for extracting stem cell exosomes, comprising the steps of:
s1, centrifuging a stem cell culture solution for 10min at 300g, removing precipitates, centrifuging for 10min at 2000g, removing the precipitates, centrifuging for 30min at 8000g, and removing the precipitates to obtain a supernatant;
s2, adding PEG with the molecular weight of 6000 into the first buffer solution to obtain a precipitation reagent with the concentration of 80 mg/mL; uniformly mixing 3mL of supernatant obtained in S1 with 2mL of the precipitation reagent, incubating for 6h at 4 ℃, and centrifuging for 10min at 15000g to obtain a precipitate;
the first buffer contained 0.1M NaCl and 20mM PBS, pH 7.4;
s3, re-suspending the precipitate obtained in the step S2 in 50 mu L of a second buffer solution to obtain a sample solution; re-suspending 10mg of PDDA coated PS microspheres in 10mL of a second buffer solution to obtain microsphere suspension; after uniformly mixing the sample solution and microsphere suspension, incubating for 2 hours at 4 ℃, and centrifuging for 20 minutes at 10000g to obtain a precipitate;
the second buffer was 10mM PBS buffer, pH 7.4;
the preparation method of the PDDA coated PS microsphere comprises the following steps: adding PDDA into aqueous dispersion of carboxylated PS microspheres with solid content of 1%, performing ultrasonic dispersion for 10min, stirring for 1h, centrifuging, and freeze-drying the obtained precipitate to obtain the final product; wherein the mass ratio of PDDA to carboxylated PS microspheres is 2:1, the particle size of the carboxylated PS microspheres is 1 mu m;
s4, re-suspending the precipitate obtained in the step S3 in 100 mu L of a third buffer solution, incubating for 0.5h at 4 ℃, centrifuging for 20min at 10000g, and discarding the precipitate to obtain an exosome solution;
the third buffer contained 0.3M NaCl and 50mM PBS, pH 6.5.
The particle size of the exosomes is analyzed by a Markov particle size analyzer, and the result shows that the average particle size of the exosomes extracted by the method is 74.0nm.
Example 2
A method for extracting stem cell exosomes, comprising the steps of:
s1, centrifuging a stem cell culture solution for 15min at 500g, removing precipitates, centrifuging for 15min at 3000g, removing the precipitates, centrifuging for 40min at 10000g, and removing the precipitates to obtain a supernatant;
s2, adding PEG with molecular weight of 15000 into the first buffer solution to obtain a precipitation reagent with concentration of 150 mg/mL; uniformly mixing 5mL of supernatant obtained in S1 with 3mL of the precipitation reagent, incubating for 12h at 4 ℃, and centrifuging at 18000g for 15min to obtain a precipitate;
the first buffer contained 0.5M NaCl and 100mM PBS, pH 7.4;
s3, re-suspending the precipitate obtained in the step S2 in 200 mu L of a second buffer solution to obtain a sample solution; re-suspending 15mg PDDA coated PS microsphere in 20mL second buffer solution to obtain microsphere suspension; after uniformly mixing the sample solution and microsphere suspension, incubating for 3 hours at 4 ℃, and centrifuging for 30 minutes at 15000g to obtain a precipitate;
the second buffer was 20mM PBS buffer, pH 7.4;
the preparation method of the PDDA coated PS microsphere comprises the following steps: adding PDDA into aqueous dispersion of carboxylated PS microspheres with solid content of 5%, performing ultrasonic dispersion for 15min, stirring for 2h, centrifuging, and freeze-drying the obtained precipitate to obtain the final product; wherein the mass ratio of PDDA to carboxylated PS microspheres is 5:1, the particle size of the carboxylated PS microspheres is 25 mu m;
s4, re-suspending the precipitate obtained in the step S3 in 100 mu L of a third buffer solution, incubating for 1h at 4 ℃, centrifuging for 30min at 15000g, and discarding the precipitate to obtain an exosome solution;
the third buffer contained 0.5M NaCl and 100mM PBS, pH 6.5.
The particle size of the exosomes is analyzed by a Markov particle size analyzer, and the result shows that the average particle size of the exosomes extracted by the method is 72.8nm.
Example 3
A method for extracting stem cell exosomes, comprising the steps of:
s1, centrifuging a stem cell culture solution for 10min at 400g, removing precipitates, centrifuging for 10min at 2000g, removing the precipitates, centrifuging for 30min at 8000g, and removing the precipitates to obtain a supernatant;
s2, adding PEG with molecular weight of 8000 into the first buffer solution to obtain a precipitation reagent with concentration of 100 mg/mL; uniformly mixing 4mL of supernatant obtained by S1 with 2mL of the precipitation reagent, incubating for 10 hours at 4 ℃, and centrifuging for 10 minutes at 16000g to obtain a precipitate;
the first buffer contained 0.2M NaCl and 50mM PBS, pH 7.4;
s3, re-suspending the precipitate obtained in the step S2 in 100 mu L of a second buffer solution to obtain a sample solution; re-suspending 10mg of PDDA coated PS microspheres in 15mL of a second buffer solution to obtain microsphere suspension; after uniformly mixing the sample solution and microsphere suspension, incubating for 2 hours at 4 ℃, and centrifuging for 20 minutes at 12000g to obtain a precipitate;
the second buffer was 10mM PBS buffer, pH 7.4;
the preparation method of the PDDA coated PS microsphere comprises the following steps: adding PDDA into aqueous dispersion of carboxylated PS microspheres with solid content of 2.5%, firstly performing ultrasonic dispersion for 10min, then stirring for 1h, then centrifuging, and freeze-drying the obtained precipitate to obtain the final product; wherein the mass ratio of PDDA to carboxylated PS microspheres is 3:1, carboxylated PS microspheres have a particle size of 5 μm;
s4, re-suspending the precipitate obtained in the S3 in 100 mu L of a third buffer solution, incubating for 0.5h at 4 ℃, centrifuging for 20min at 12000g, and discarding the precipitate to obtain an exosome solution;
the third buffer contained 0.4M NaCl and 60mM PBS, pH 6.5.
The particle size of the exosomes is analyzed by a Markov particle size analyzer, and the result shows that the average particle size of the exosomes extracted by the method is 81.3nm.
Comparative example 1
A method for extracting stem cell exosomes, comprising the steps of:
s1, centrifuging a stem cell culture solution for 10min at 400g, removing precipitates, centrifuging for 10min at 2000g, removing the precipitates, centrifuging for 30min at 8000g, and removing the precipitates to obtain a supernatant;
s2, adding PEG with molecular weight of 8000 into the first buffer solution to obtain a precipitation reagent with concentration of 100 mg/mL; uniformly mixing 4mL of supernatant obtained by S1 with 2mL of the precipitation reagent, incubating for 10 hours at 4 ℃, and centrifuging for 10 minutes at 16000g to obtain a precipitate;
the first buffer contained 0.2M NaCl and 50mM PBS, pH 7.4;
s3, re-suspending the precipitate obtained in the step S2 in 100 mu L of a second buffer solution to obtain an exosome solution; the second buffer was 10mM PBS buffer, pH 7.4.
The particle size of the exosomes is analyzed by adopting a Markov particle size analyzer, and the result shows that the average particle size of the exosomes extracted by the method is 55.2nm.
From the above results, it can be seen that the extraction method of the present invention has a larger average particle size of the exosomes, more closely approaching the particle size range of the stem cell exosomes, compared with the conventional polymer precipitation method, indicating that the impurities and protein fragments are less and the purity is higher.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (8)
1. A stem cell exosome extraction kit, the kit comprising:
the sedimentation agent PEG, PDDA coated PS microsphere, a first buffer solution, a second buffer solution and a third buffer solution;
the first buffer solution contains 0.1-0.5M NaCl and 20-100 mM PBS, and the pH value is 7.4;
the second buffer solution is 10-20 mM PBS buffer solution, and the pH value is 7.4;
the third buffer solution contains 0.3-0.5M NaCl and 50-100mM PBS, and the pH value is 6.5.
2. The stem cell exosome extraction kit according to claim 1, wherein the preparation method of the PDDA coated PS microsphere comprises: adding PDDA into aqueous dispersion of carboxylated PS microspheres, performing ultrasonic dispersion for 10-15 min, stirring for 1-2 h, centrifuging, and freeze-drying the obtained precipitate to obtain the final product.
3. The stem cell exosome extraction kit according to claim 1, wherein the mass ratio of PDDA to carboxylated PS microspheres is (2-5): 1, a step of; the particle size of the carboxylated PS microspheres is 1-25 mu m.
4. The stem cell exosome extraction kit according to claim 1, wherein the aqueous dispersion of carboxylated PS microspheres has a solids content of 1-5%.
5. The stem cell exosome extraction kit according to claim 1, wherein the precipitant PEG is PEG having a molecular weight of 6000-15000.
6. A method for extracting stem cell exosomes, characterized by using the kit of any one of claims 1 to 5; the extraction method comprises the following steps:
s1, performing low-speed centrifugation on a stem cell culture solution to obtain a supernatant;
s2, adding a precipitating agent PEG into the first buffer solution to obtain a precipitating agent; uniformly mixing 3-5 mL of supernatant obtained by S1 with 2-3 mL of the precipitation reagent, incubating for 6-12 h at 0-4 ℃, and centrifuging for 10-15 min at 15000-18000 g to obtain a precipitate;
s3, re-suspending the precipitate obtained in the step S2 in 50-200 mu L of a second buffer solution to obtain a sample solution; re-suspending 10-15 mg PDDA coated PS microsphere in 10-20 mL second buffer solution to obtain microsphere suspension; after uniformly mixing the sample solution and microsphere suspension, incubating for 2-3 hours at 0-4 ℃, and centrifuging for 20-30 min at 10000-15000 g to obtain a precipitate;
s4, re-suspending the precipitate obtained in the S3 in a third buffer solution, incubating for 0.5-1 h at 0-4 ℃, centrifuging for 20-30 min at 10000-15000 g, and discarding the precipitate to obtain an exosome solution.
7. The method for extracting stem cell exosomes according to claim 6, wherein the specific steps of low-speed centrifugation in S1 comprise: centrifuging for 10-15 min at 300-500 g, removing precipitate, centrifuging for 10-15 min at 2000-3000 g, removing precipitate, centrifuging for 30-40 min at 8000-10000 g, and removing precipitate.
8. The method according to claim 6, wherein the concentration of PEG as a precipitating agent in the precipitating agent is 80-150 mg/mL.
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