CN112553138A - Stem cell exosome patch and preparation method thereof - Google Patents
Stem cell exosome patch and preparation method thereof Download PDFInfo
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- CN112553138A CN112553138A CN201910908543.6A CN201910908543A CN112553138A CN 112553138 A CN112553138 A CN 112553138A CN 201910908543 A CN201910908543 A CN 201910908543A CN 112553138 A CN112553138 A CN 112553138A
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- 210000001808 exosome Anatomy 0.000 title claims abstract description 70
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 69
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 238000002955 isolation Methods 0.000 claims abstract description 29
- 239000000758 substrate Substances 0.000 claims abstract description 22
- 239000007788 liquid Substances 0.000 claims abstract description 20
- 238000001523 electrospinning Methods 0.000 claims abstract description 15
- 239000003960 organic solvent Substances 0.000 claims abstract description 15
- 238000009987 spinning Methods 0.000 claims abstract description 15
- VSKXVGWORBZZDY-WNQIDUERSA-N (2s)-2-hydroxypropanoic acid;oxepan-2-one Chemical compound C[C@H](O)C(O)=O.O=C1CCCCCO1 VSKXVGWORBZZDY-WNQIDUERSA-N 0.000 claims abstract description 11
- 229920002635 polyurethane Polymers 0.000 claims abstract description 11
- 239000004814 polyurethane Substances 0.000 claims abstract description 11
- 239000000463 material Substances 0.000 claims description 87
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 40
- 239000000243 solution Substances 0.000 claims description 20
- 238000005507 spraying Methods 0.000 claims description 20
- 238000003756 stirring Methods 0.000 claims description 16
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 14
- 239000003242 anti bacterial agent Substances 0.000 claims description 14
- 229940088710 antibiotic agent Drugs 0.000 claims description 14
- 239000007853 buffer solution Substances 0.000 claims description 14
- 238000010041 electrostatic spinning Methods 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- 230000010355 oscillation Effects 0.000 claims description 10
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 238000002791 soaking Methods 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 8
- 238000000108 ultra-filtration Methods 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 7
- GUTLYIVDDKVIGB-OUBTZVSYSA-N Cobalt-60 Chemical compound [60Co] GUTLYIVDDKVIGB-OUBTZVSYSA-N 0.000 claims description 6
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 6
- -1 polytetrafluoroethylene Polymers 0.000 claims description 6
- 229920001343 polytetrafluoroethylene Polymers 0.000 claims description 6
- 239000004810 polytetrafluoroethylene Substances 0.000 claims description 6
- BYEAHWXPCBROCE-UHFFFAOYSA-N 1,1,1,3,3,3-hexafluoropropan-2-ol Chemical group FC(F)(F)C(O)C(F)(F)F BYEAHWXPCBROCE-UHFFFAOYSA-N 0.000 claims description 4
- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 claims description 4
- KZDCMKVLEYCGQX-UDPGNSCCSA-N 2-(diethylamino)ethyl 4-aminobenzoate;(2s,5r,6r)-3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid;hydrate Chemical compound O.CCN(CC)CCOC(=O)C1=CC=C(N)C=C1.N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 KZDCMKVLEYCGQX-UDPGNSCCSA-N 0.000 claims description 4
- 239000012228 culture supernatant Substances 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 4
- 230000001678 irradiating effect Effects 0.000 claims description 4
- 229940056360 penicillin g Drugs 0.000 claims description 4
- 239000008055 phosphate buffer solution Substances 0.000 claims description 4
- 239000002504 physiological saline solution Substances 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 4
- 239000003755 preservative agent Substances 0.000 claims description 4
- 230000002335 preservative effect Effects 0.000 claims description 4
- 239000012266 salt solution Substances 0.000 claims description 4
- 238000005201 scrubbing Methods 0.000 claims description 4
- 238000007789 sealing Methods 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 238000003860 storage Methods 0.000 claims description 4
- 229960002385 streptomycin sulfate Drugs 0.000 claims description 4
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 claims description 3
- 239000005977 Ethylene Substances 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 230000002175 menstrual effect Effects 0.000 claims description 3
- 239000002033 PVDF binder Substances 0.000 claims description 2
- 210000004504 adult stem cell Anatomy 0.000 claims description 2
- 210000003525 amniotic membrane stem cell Anatomy 0.000 claims description 2
- 210000001671 embryonic stem cell Anatomy 0.000 claims description 2
- 230000003511 endothelial effect Effects 0.000 claims description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 claims description 2
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims description 2
- 210000001178 neural stem cell Anatomy 0.000 claims description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 claims description 2
- 230000002792 vascular Effects 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 4
- 239000007924 injection Substances 0.000 abstract description 3
- 238000002347 injection Methods 0.000 abstract description 3
- 239000000725 suspension Substances 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000008929 regeneration Effects 0.000 description 4
- 238000011069 regeneration method Methods 0.000 description 4
- 230000001502 supplementing effect Effects 0.000 description 4
- 238000010276 construction Methods 0.000 description 3
- 210000004379 membrane Anatomy 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 206010011732 Cyst Diseases 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000747 cardiac effect Effects 0.000 description 2
- 208000031513 cyst Diseases 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000002861 polymer material Substances 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 208000005422 Foreign-Body reaction Diseases 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000009168 stem cell therapy Methods 0.000 description 1
- 238000009580 stem-cell therapy Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229920001059 synthetic polymer Polymers 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
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Abstract
The invention discloses a stem cell exosome patch and a preparation method thereof, wherein the stem cell exosome patch comprises a patch substrate, an isolation layer is arranged on the upper part of the patch substrate, exosomes of stem cells are arranged on the upper part of the isolation layer, and the exosomes of the stem cells are subcellular double-layer vesicle bubbles secreted by the stem cells and having the molecular diameter of 30-100nm, the patch substrate is prepared by arranging the isolation layer on the upper part of the patch substrate and adopting electrospinning liquid prepared from poly (L-lactic acid-caprolactone), polyurethane and an organic solvent in a liquid spinning mode, the patch substrate is sterilized in various modes, the incompatibility of the patch substrate and human body biology is prevented, the rejection and complications are prevented, compared with the direct injection of an exosome suspension, the patch is used as a good carrier to fix the exosomes at a certain position, the fixed-point directional effect of the implanted exosome is ensured, and the possibility of exosome loss is greatly reduced.
Description
Technical Field
The invention relates to the technical field of stem cell exosome patches, in particular to a stem cell exosome patch and a preparation method thereof.
Background
Compared with the traditional stem cell therapy of directly injecting cell suspension, the stem cell sheet transplantation has the advantages that: firstly, the stem cells form a two-dimensional or three-dimensional tissue structure through in vitro construction, and can still keep the connection between cells and cytoplasm after the cell sheet is transplanted into a body, thereby being beneficial to the maintenance of the biological activity of the stem cells; secondly, the stem cells can be directionally implanted into a target area, the stem cells are not easy to lose, and the cell source, the planting density and the construction method are manually controlled in the construction process, so that the cell treatment process is greatly promoted; and the transplantation treatment of the cell sheet can obviously promote the regeneration of peripheral tissues and cells, and has more obvious effect of repairing the damage of tissues and organs.
The biological patch is a regeneration system of human in the new century, can promote the human body repair function to complete the regeneration of new tissues, has a good supporting function after being implanted into the human body, fills up the missing tissues of the damaged part, and can gradually grow new tissues in the original position by the human body repair function under the induction of the material to replace the biological material so as to complete the regeneration process of organ tissues.
At present, the artificial synthetic polymer material patches are generally used clinically, however, most of the polymer material patches are non-absorbable materials, are incompatible with human organisms, have permanent foreign body reaction and are accompanied with a series of complications.
Therefore, a stem cell exosome patch and a preparation method thereof are provided.
Disclosure of Invention
The invention aims to provide a stem cell exosome patch and a preparation method thereof, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: the utility model provides a stem cell exosome patch, includes the patch substrate, the upper portion of patch substrate is equipped with the isolation layer, the upper portion of isolation layer is equipped with the exosome of stem cell, the exosome of stem cell is the subcellular bilayer capsule membrane bubble that the molecule diameter that stem cell excretes is 30-100 nm.
Meanwhile, the invention also provides a preparation method of the stem cell exosome patch, which comprises the following steps:
s1: preparing an electrospinning solution: adding poly (L-lactic acid-caprolactone) and polyurethane into an organic solvent, wherein the mass ratio of the poly (L-lactic acid-caprolactone) to the polyurethane to the organic solvent is 6:2:92, stirring and dissolving for 1-3 days, sealing the opening of a beaker by using a preservative film, placing the beaker in a constant-temperature multi-magnetic-head stirrer, stirring and dissolving at the stirring speed of 200-2000rpm, and stirring for 1-3 days to obtain an electrospinning solution;
s2: liquid spinning: and (4) performing electrostatic spinning on the electrospinning liquid prepared in the step (S1) in a high-voltage electrostatic dynamic spinning device, wherein the high-voltage electrostatic dynamic spinning device comprises: the device comprises a solution storage device, a spraying device, a water bath device and a receiving scroll, wherein the temperature of electrostatic spinning is 37-42 ℃, the voltage of the electrostatic spinning is 1-3kv, the speed of the solution conveyed by the spraying device is 0.5-3ml/h, the type of a required needle in the spraying device is No. 8, the distance between the needle and the water bath device is 10-20cm, the depth of water in the water bath device is 5-40cm, the bottom aperture of the water bath device is 0.1-1.5cm, and the distance between the water bath device and the receiving scroll is 5-20cm, so that a patch base material is obtained;
s3: spraying an isolation layer: taking the patch base material prepared in the step S3 down from the receiving reel, and spraying an isolation layer on the upper side and the lower side of the patch base material, wherein the isolation layer is one or more of polytetrafluoroethylene, polyvinylidene fluoride and ethylene polytetrafluoroethylene, so that the patch base material sprayed with the isolation layer is obtained;
s4: post-treatment of the patch substrate: placing the patch base material obtained in the step S3 in a refrigerator at-80 ℃ for 2-12 hours, taking out, placing in a freeze dryer for 24-48 hours at the temperature of- (20-80) DEG C and under the pressure of 0.6-1.0MPa, and cutting the patch after freeze drying into pieces with the length of 10cm and the width of 10 cm;
s5: preparing a filtering exosome: collecting 10mL of culture supernatant of purified stem cells under aseptic condition, centrifuging at 300r/min at 4 ℃ for 10 minutes, sucking the supernatant into another clean and aseptic 15mL centrifuge tube, centrifuging at 2000r/min for 20 min at 4 deg.C, sucking the supernatant into another clean and sterile 15mL centrifuge tube, filtering the collected liquid with 0.22 μm filter membrane in a clean bench, collecting the filtered liquid, centrifuging at 4 deg.C at 1000r/min for 70 min to obtain white precipitate as exosome, removing supernatant, washing with sterile PBS phosphate buffer solution, centrifuging at 1000r/min for 60 min at 4 deg.C, resuspending exosome with 1mL sterile PBS, centrifuging at 300r/min for 30 min at 4 deg.C with 100nm ultrafiltration centrifuge tube, and centrifuging at 300r/min for 30 min with 0.22 μm ultrafiltration centrifuge tube at 4 deg.C to filter exosome for use;
s6: sterilizing a patch substrate: scrubbing the patch base material obtained in the step S4 with sterile physiological saline for 10 times, placing the patch base material in a sterile square plate, soaking the patch base material in carbonate buffer solution containing antibiotics for 2 times, 5 minutes each time, placing the patch base material in sterile pure water, soaking the patch base material in water bath at 37 ℃ for 30 minutes, placing the soaked patch base material in 10mL of sterile PBS salt solution (0.01mol/L, pH-6-8) containing 0.3% (w/v) of sodium dodecyl sulfate, performing oscillation treatment in the water bath at 37 ℃, wherein the oscillation rotation speed is 185rpm, performing oscillation for 10-12 hours, placing the soaked patch base material in 50mL of carbonate buffer solution containing antibiotics, performing oscillation washing in the water bath at 25 ℃ for 6 times, 60 minutes each time, obtaining the cleaned patch base material, irradiating and sterilizing the cleaned patch base material with cobalt 60, and storing the cleaned patch base material at 4 ℃ for later use;
s7: preparing a stem cell exosome patch: and (4) taking out the patch base material sterilized in the step S6, dripping the filtered exosome prepared in the step S5, and standing in an incubator at 37 ℃ for 5-10min to solidify, thus obtaining the stem cell exosome patch.
Preferably, the organic solvent is hexafluoroisopropanol solvent.
Preferably, the stem cell is one or more of a cardiac stem cell, a skin stem cell, a vascular endothelial progenitor cell, a neural stem cell, a hematopoietic stem cell, an embryonic stem cell, an induced pluripotent differentiation cell, a mesenchymal stem cell, an adipose stem cell, an amniotic membrane stem cell, a limbal stem cell, an adult stem cell and a menstrual stem cell.
Preferably, the antibiotics are 100U/mL penicillin G and 100. mu.g/mL streptomycin sulfate.
Preferably, the concentration of the carbonate buffer solution is 0.05mol/L, and the pH value is 9.0-9.6.
Compared with the prior art, the invention has the beneficial effects that: the invention arranges the isolation layer on the upper part of the patch base material, adopts the electrospinning solution prepared from poly (L-lactic acid-caprolactone), polyurethane and organic solvent, prepares the patch base material by liquid spinning, scrubs the patch base material for 10 times by aseptic normal saline, soaks the patch base material by carbonate buffer solution containing antibiotics, puts the soaked patch base material into aseptic PBS saline solution of sodium dodecyl sulfate, sterilizes the patch base material by cobalt 60 irradiation and other modes, prevents the patch base material from being incompatible with human organism, prevents rejection and complication, compared with the direct injection of exosome suspension, the patch is used as a good carrier to fix exosomes at a certain position, ensures the fixed-point directional effect after the exosomes are implanted, and greatly reduces the possibility of exosome loss.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The utility model provides a stem cell exosome patch, includes the patch substrate, the upper portion of patch substrate is equipped with the isolation layer, the upper portion of isolation layer is equipped with the exosome of stem cell, the exosome of stem cell is the subcellular bilayer cyst membrane bubble that the molecule diameter that stem cell excreted is 30 nm.
Meanwhile, the invention also provides a preparation method of the stem cell exosome patch, which comprises the following steps:
s1: preparing an electrospinning solution: adding poly (L-lactic acid-caprolactone) and polyurethane into an organic solvent, wherein the mass ratio of the poly (L-lactic acid-caprolactone) to the polyurethane to the organic solvent is 6:2:92, stirring and dissolving for 1 day, sealing the opening of a beaker by using a preservative film, placing the beaker in a constant-temperature multi-magnetic head stirrer, stirring and dissolving at the stirring speed of 250rpm, and stirring for 3 days to obtain an electrospinning solution;
s2: liquid spinning: and (4) performing electrostatic spinning on the electrospinning liquid prepared in the step (S1) in a high-voltage electrostatic dynamic spinning device, wherein the high-voltage electrostatic dynamic spinning device comprises: the film supplementing device comprises a solution storage device, a spraying device, a water bath device and a receiving scroll, wherein the temperature of electrostatic spinning is 42 ℃, the voltage of the electrostatic spinning is 3kv, the solution conveying speed of the spraying device is 0.5ml/h, the type of a required needle in the spraying device is 8, the distance between the needle and the water bath device is 10cm, the depth of water in the water bath device is 25cm, the aperture at the bottom of the water bath device is 1.5cm, and the distance between the water bath device and the receiving scroll is 12cm, so that a film supplementing substrate is obtained;
s3: spraying an isolation layer: taking the patch base material prepared in the step S3 down from the receiving reel, and spraying isolation layers on the upper side and the lower side of the patch base material, wherein the isolation layers are made of polytetrafluoroethylene, so that the patch base material sprayed with the isolation layers is obtained;
s4: post-treatment of the patch substrate: placing the patch base material obtained in the step S3 in a refrigerator at-80 ℃ for 2 hours, taking out the patch base material, placing the patch base material in a freeze dryer at-80 ℃ for about 48 hours under the pressure of 1.0MPa, and cutting the freeze-dried patch into a length of 10cm and a width of 10 cm;
s5: preparing a filtering exosome: collecting 10mL of culture supernatant of purified stem cells under aseptic condition, centrifuging at 300r/min at 4 ℃ for 10 minutes, sucking the supernatant into another clean and aseptic 15mL centrifuge tube, centrifuging at 2000r/min for 20 min at 4 deg.C, sucking the supernatant into another clean and sterile 15mL centrifuge tube, filtering the collected liquid with 0.22 μm filter membrane in a clean bench, collecting the filtered liquid, centrifuging at 4 deg.C at 1000r/min for 70 min to obtain white precipitate as exosome, removing supernatant, washing with sterile PBS phosphate buffer solution, centrifuging at 1000r/min for 60 min at 4 deg.C, resuspending exosome with 1mL sterile PBS, centrifuging at 300r/min for 30 min at 4 deg.C with 100nm ultrafiltration centrifuge tube, and centrifuging at 300r/min for 30 min with 0.22 μm ultrafiltration centrifuge tube at 4 deg.C to filter exosome for use;
s6: sterilizing a patch substrate: scrubbing the patch base material obtained in the step S4 with sterile physiological saline for 10 times, placing the patch base material in a sterile square plate, soaking the patch base material in carbonate buffer solution containing antibiotics for 2 times, 5 minutes each time, placing the patch base material in sterile pure water, soaking the patch base material in water bath at 37 ℃ for 30 minutes, placing the soaked patch base material in 10mL of sterile PBS salt solution (0.01mol/L, pH-6) containing 0.3% (w/v) of sodium dodecyl sulfate, shaking the patch base material in the water bath at 37 ℃, shaking the patch base material at 185rpm for 12 hours, placing the soaked patch base material in 50mL of carbonate buffer solution containing antibiotics, shaking and washing the patch base material in the water bath at 25 ℃ for 6 times, 60 minutes each time, obtaining a cleaned patch base material, irradiating and sterilizing the cleaned patch base material with cobalt 60, and storing the cleaned patch base material at 4 ℃ for later use;
s7: preparing a stem cell exosome patch: and (4) taking out the patch base material sterilized in the step S6, dripping the filtered exosome prepared in the step S5, and standing in an incubator at 37 ℃ for 5-10min to solidify, thus obtaining the stem cell exosome patch.
Specifically, the organic solvent is hexafluoroisopropanol solvent.
In particular, the stem cells are cardiac stem cells.
Specifically, the antibiotics are 100U/mL penicillin G and 100 mu G/mL streptomycin sulfate.
Specifically, the concentration of the carbonate buffer solution is 0.05mol/L, and the pH value is 9.0.
Example 2
The utility model provides a stem cell exosome patch, includes the patch substrate, the upper portion of patch substrate is equipped with the isolation layer, the upper portion of isolation layer is equipped with the exosome of stem cell, the exosome of stem cell is the subcellular bilayer cyst membrane bubble that the molecule diameter that stem cell excreted is 88 nm.
Meanwhile, the invention also provides a preparation method of the stem cell exosome patch, which comprises the following steps:
s1: preparing an electrospinning solution: adding poly (L-lactic acid-caprolactone) and polyurethane into an organic solvent, wherein the mass ratio of the poly (L-lactic acid-caprolactone) to the polyurethane to the organic solvent is 6:2:92, stirring and dissolving for 2 days, sealing the opening of a beaker by using a preservative film, placing the beaker in a constant-temperature multi-magnetic head stirrer, stirring and dissolving at the stirring speed of 1800rpm, and stirring for 1 day to obtain an electrospinning solution;
s2: liquid spinning: and (4) performing electrostatic spinning on the electrospinning liquid prepared in the step (S1) in a high-voltage electrostatic dynamic spinning device, wherein the high-voltage electrostatic dynamic spinning device comprises: the film supplementing device comprises a solution storage device, a spraying device, a water bath device and a receiving scroll, wherein the temperature of electrostatic spinning is 39 ℃, the voltage of the electrostatic spinning is 2kv, the solution conveying speed of the spraying device is 2ml/h, the type of a required needle in the spraying device is 8, the distance between the needle and the water bath device is 11cm, the depth of water in the water bath device is 25cm, the aperture of the bottom of the water bath device is 1cm, and the distance between the water bath device and the receiving scroll is 9cm, so that a film supplementing base material is obtained;
s3: spraying an isolation layer: taking the patch base material prepared in the step S3 down from the receiving reel, and spraying isolation layers on the upper side and the lower side of the patch base material, wherein the isolation layers are made of ethylene polytetrafluoroethylene, so that the patch base material sprayed with the isolation layers is obtained;
s4: post-treatment of the patch substrate: placing the patch base material obtained in the step S3 in a refrigerator at-80 ℃ for 7 hours, taking out the patch base material, placing the patch base material in a freeze dryer for about 30 hours at-44 ℃ under the pressure of 0.8MPa, and cutting the patch after freeze drying into a length of 10cm and a width of 10 cm;
s5: preparing a filtering exosome: collecting 10mL of culture supernatant of purified stem cells under aseptic condition, centrifuging at 300r/min at 4 ℃ for 10 minutes, sucking the supernatant into another clean and aseptic 15mL centrifuge tube, centrifuging at 2000r/min for 20 min at 4 deg.C, sucking the supernatant into another clean and sterile 15mL centrifuge tube, filtering the collected liquid with 0.22 μm filter membrane in a clean bench, collecting the filtered liquid, centrifuging at 4 deg.C at 1000r/min for 70 min to obtain white precipitate as exosome, removing supernatant, washing with sterile PBS phosphate buffer solution, centrifuging at 1000r/min for 60 min at 4 deg.C, resuspending exosome with 1mL sterile PBS, centrifuging at 300r/min for 30 min at 4 deg.C with 100nm ultrafiltration centrifuge tube, and centrifuging at 300r/min for 30 min with 0.22 μm ultrafiltration centrifuge tube at 4 deg.C to filter exosome for use;
s6: sterilizing a patch substrate: scrubbing the patch base material obtained in the step S4 with sterile physiological saline for 10 times, placing the patch base material in a sterile square plate, soaking the patch base material in carbonate buffer solution containing antibiotics for 2 times, 5 minutes each time, placing the patch base material in sterile pure water, soaking the patch base material in water bath at 37 ℃ for 30 minutes, placing the soaked patch base material in 10mL of sterile PBS salt solution (0.01mol/L, pH-7) containing 0.3% (w/v) of sodium dodecyl sulfate, performing oscillation treatment in the water bath at 37 ℃, oscillating the solution at 185rpm for 10-12 hours, then placing the soaked patch base material in 50mL of carbonate buffer solution containing antibiotics, performing oscillation washing in the water bath at 25 ℃ for 6 times, 60 minutes each time, obtaining the cleaned patch base material, irradiating and sterilizing the cleaned patch base material with cobalt 60, and storing the cleaned patch base material for later use at 4 ℃;
s7: preparing a stem cell exosome patch: and (4) taking out the patch base material sterilized in the step S6, dripping the filtered exosome prepared in the step S5, and standing in an incubator at 37 ℃ for 6min to solidify, thus obtaining the stem cell exosome patch.
Specifically, the organic solvent is hexafluoroisopropanol solvent.
Specifically, the stem cells are menstrual stem cells.
Specifically, the antibiotics are 100U/mL penicillin G and 100 mu G/mL streptomycin sulfate.
Specifically, the concentration of the carbonate buffer solution is 0.05mol/L, and the pH value is 9.5.
The invention arranges the isolation layer on the upper part of the patch base material, adopts the electrospinning solution prepared from poly (L-lactic acid-caprolactone), polyurethane and organic solvent, prepares the patch base material by liquid spinning, scrubs the patch base material for 10 times by aseptic normal saline, soaks the patch base material by carbonate buffer solution containing antibiotics, puts the soaked patch base material into aseptic PBS saline solution of sodium dodecyl sulfate, sterilizes the patch base material by cobalt 60 irradiation and other modes, prevents the patch base material from being incompatible with human organism, prevents rejection and complication, compared with the direct injection of exosome suspension, the patch is used as a good carrier to fix exosomes at a certain position, ensures the fixed-point directional effect after the exosomes are implanted, and greatly reduces the possibility of exosome loss.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A stem cell exosome patch comprising a patch substrate, characterized in that: the patch is characterized in that an isolation layer is arranged on the upper portion of the patch base material, exosomes of stem cells are arranged on the upper portion of the isolation layer, and the exosomes of the stem cells are subcellular double-layer vesicle vesicles secreted by the stem cells and having the molecular diameter of 30-100 nm.
2. A preparation method of a stem cell exosome patch is characterized by comprising the following steps: the method comprises the following steps:
s1: preparing an electrospinning solution: adding poly (L-lactic acid-caprolactone) and polyurethane into an organic solvent, wherein the mass ratio of the poly (L-lactic acid-caprolactone) to the polyurethane to the organic solvent is 6:2:92, stirring and dissolving for 1-3 days, sealing the opening of a beaker by using a preservative film, placing the beaker in a constant-temperature multi-magnetic-head stirrer, stirring and dissolving at the stirring speed of 200-2000rpm, and stirring for 1-3 days to obtain an electrospinning solution;
s2: liquid spinning: and (4) performing electrostatic spinning on the electrospinning liquid prepared in the step (S1) in a high-voltage electrostatic dynamic spinning device, wherein the high-voltage electrostatic dynamic spinning device comprises: the device comprises a solution storage device, a spraying device, a water bath device and a receiving scroll, wherein the temperature of electrostatic spinning is 37-42 ℃, the voltage of the electrostatic spinning is 1-3kv, the speed of the solution conveyed by the spraying device is 0.5-3ml/h, the type of a required needle in the spraying device is No. 8, the distance between the needle and the water bath device is 10-20cm, the depth of water in the water bath device is 5-40cm, the bottom aperture of the water bath device is 0.1-1.5cm, and the distance between the water bath device and the receiving scroll is 5-20cm, so that a patch base material is obtained;
s3: spraying an isolation layer: taking the patch base material prepared in the step S3 down from the receiving reel, and spraying an isolation layer on the upper side and the lower side of the patch base material, wherein the isolation layer is one or more of polytetrafluoroethylene, polyvinylidene fluoride and ethylene polytetrafluoroethylene, so that the patch base material sprayed with the isolation layer is obtained;
s4: post-treatment of the patch substrate: placing the patch base material obtained in the step S3 in a refrigerator at-80 ℃ for 2-12 hours, taking out, placing in a freeze dryer for 24-48 hours at the temperature of- (20-80) DEG C and under the pressure of 0.6-1.0MPa, and cutting the patch after freeze drying into pieces with the length of 10cm and the width of 10 cm;
s5: preparing a filtering exosome: collecting 10mL of culture supernatant of purified stem cells under aseptic condition, centrifuging at 300r/min at 4 ℃ for 10 minutes, sucking the supernatant into another clean and aseptic 15mL centrifuge tube, centrifuging at 2000r/min for 20 min at 4 deg.C, sucking the supernatant into another clean and sterile 15mL centrifuge tube, filtering the collected liquid with 0.22 μm filter membrane in a clean bench, collecting the filtered liquid, centrifuging at 4 deg.C at 1000r/min for 70 min to obtain white precipitate as exosome, removing supernatant, washing with sterile PBS phosphate buffer solution, centrifuging at 1000r/min for 60 min at 4 deg.C, resuspending exosome with 1mL sterile PBS, centrifuging at 300r/min for 30 min at 4 deg.C with 100nm ultrafiltration centrifuge tube, and centrifuging at 300r/min for 30 min with 0.22 μm ultrafiltration centrifuge tube at 4 deg.C to filter exosome for use;
s6: sterilizing a patch substrate: scrubbing the patch base material obtained in the step S4 with sterile physiological saline for 10 times, placing the patch base material in a sterile square plate, soaking the patch base material in carbonate buffer solution containing antibiotics for 2 times, 5 minutes each time, placing the patch base material in sterile pure water, soaking the patch base material in water bath at 37 ℃ for 30 minutes, placing the soaked patch base material in 10mL of sterile PBS salt solution (0.01mol/L, pH is 6-8) containing 0.3% (w/v) of sodium dodecyl sulfate, performing oscillation treatment in the water bath at 37 ℃, wherein the oscillation rotation speed is 185rpm, performing oscillation for 10-12 hours, placing the soaked patch base material in 50mL of carbonate buffer solution containing antibiotics, performing oscillation washing in the water bath at 25 ℃ for 6 times, 60 minutes each time, obtaining the cleaned patch base material, irradiating and sterilizing the cleaned patch base material with cobalt 60, and storing the cleaned patch base material for later use at 4 ℃;
s7: preparing a stem cell exosome patch: and (4) taking out the patch base material sterilized in the step S6, dripping the filtered exosome prepared in the step S5, and standing in an incubator at 37 ℃ for 5-10min to solidify, thus obtaining the stem cell exosome patch.
3. A method of preparing a stem cell exosome patch according to claim 2, characterized in that: the organic solvent is hexafluoroisopropanol solvent.
4. A method of preparing a stem cell exosome patch according to claim 2, characterized in that: the stem cells are one or more of heart stem cells, skin stem cells, vascular endothelial progenitor cells, neural stem cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent differentiated cells, mesenchymal stem cells, adipose-derived stem cells, amniotic membrane stem cells, limbal stem cells, adult stem cells and menstrual stem cells.
5. A method of preparing a stem cell exosome patch according to claim 2, characterized in that: the antibiotics are 100U/mL penicillin G and 100 mu G/mL streptomycin sulfate.
6. A method of preparing a stem cell exosome patch according to claim 2, characterized in that: the concentration of the carbonate buffer solution is 0.05mol/L, and the pH value is 9.0-9.6.
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| CN104894062A (en) * | 2015-05-19 | 2015-09-09 | 暨南大学 | Stem cell exosome patch and preparation method and application thereof |
| CN105688275A (en) * | 2014-11-25 | 2016-06-22 | 上海市第六人民医院 | Preparation method of nano-elastic patch material for pelvic floor reconstruction |
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| CN105688275A (en) * | 2014-11-25 | 2016-06-22 | 上海市第六人民医院 | Preparation method of nano-elastic patch material for pelvic floor reconstruction |
| CN104894062A (en) * | 2015-05-19 | 2015-09-09 | 暨南大学 | Stem cell exosome patch and preparation method and application thereof |
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