CN115212162A - Extraction method of stem cell exosomes and application of essence of exosomes - Google Patents
Extraction method of stem cell exosomes and application of essence of exosomes Download PDFInfo
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- A61K8/00—Cosmetics or similar toiletry preparations
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- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The invention provides a method for extracting a stem cell exosome and application of essence of the exosome. The method for extracting the stem cell exosomes comprises the following steps: s1, taking biological stem cells, washing the biological stem cells with precooled PBS buffer solution for 2-4 times, and then shearing the biological stem cells into fragments-tissue blocks, wherein the size of the tissue blocks is about 1mm 3 (ii) a S2, washing the tissue block processed in the step S1 again through a precooled PBS buffer solution until the liquid is clear; s3, placing the tissue blocks processed in the step S2 into a centrifuge tube, adding type II collagenase with the content of 0.1%, and then performing shock digestion in a water bath kettle with the temperature of 37 ℃ for 1 hour. The stem cell exosome extraction method and the application of the essence of the exosome provided by the invention can save cost and simultaneouslyThe production yield of exosomes can be increased, and the advantage of improving facial wrinkles can be enhanced.
Description
Technical Field
The invention belongs to the technical field of exosome combined preparations, and particularly relates to a stem cell exosome extraction method and application of an essence of exosome.
Background
Exosomes are vesicles of several tens to several hundreds nanometers in size, and are composed of a phospholipid bilayer membrane having the same structure as a cell membrane, and proteins, nucleic acids, and the like that are included in the exosomes are loaded. It is known that exosome carrier proteins comprise a wide range of signaling factors, and that these signaling factors are specific to cell types and are regulated differently depending on the environment of the exosome secreting cell. In addition, since exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted therethrough modulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. The exosome can reflect the characteristics of cells from which the exosome is derived, the biological functions of exosomes derived from different stem cells are not complete, skin is the largest organ of a human body and is positioned on the outermost side of the human body structure, the exosome has the main functions of playing an immune barrier role, protecting the human body from invasion of external harmful substances, pathogens and the like, and has the functions of regulating the metabolism, feeling and the like of the human body. The method comprises the following steps: culturing the biological source stem cells in a culture medium to a logarithmic phase, and collecting cell culture supernatant; centrifuging 300g of the supernatant for 8-11min, and then keeping the supernatant; centrifuging at 2000g for 13-16min, and collecting supernatant; centrifuging at 10000g for 28-31min, and collecting supernatant; centrifuging 100000g for 69-72min, discarding the supernatant, re-suspending with a re-suspension solution, centrifuging 100000g for 69-72min, discarding the supernatant, re-suspending with the re-suspension solution, and concentrating the re-suspension solution in an ultrafiltration centrifugal tube to obtain exosome; the essence is prepared by dissolving and mixing the following raw materials in percentage by weight: 5-10% of nicotinamide; 4-8% of allantoin; 4-6% of tranexamic acid; 3-6% of sodium polyglutamate; 3-6% of sodium hyaluronate; 2-5% of exosomes; the balance of deionized water. The essence of the application can be used for facial application, and has the advantage of obviously improving facial wrinkles.
However, the above-mentioned structure has disadvantages that although the above-mentioned structure can achieve the effect of preparing exosome, the above-mentioned structure uses human umbilical cord stem cell as raw material, so the price is expensive.
Therefore, there is a need to provide a new method for extracting exosomes from stem cells and application of the essence of exosomes to solve the above technical problems.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a stem cell exosome extraction method and application of an essence of exosome, wherein the stem cell exosome extraction method can save cost, increase the preparation yield of exosome and strengthen and improve facial wrinkles.
In order to solve the technical problem, the method for extracting the stem cell exosomes provided by the invention comprises the following steps: s1, taking biological stem cells, washing the biological stem cells with precooled PBS buffer solution for 2-4 times, and then shearing the biological stem cells into fragments-tissue blocks, wherein the size of the tissue blocks is about 1mm 3 ；
S2, washing the tissue block processed in the step S1 again through a precooled PBS buffer solution until the liquid is clear;
s3, placing the tissue blocks processed in the step S2 into a centrifuge tube, adding type II collagenase with the content of 0.1%, and then performing shock digestion in a water bath kettle with the temperature of 37 ℃ for 1 hour;
s4, lightly blowing and beating the digested product processed in the step S3 through a dropper to separate cells from loose tissue blocks and generate cell supernatant, then standing in an ice block for 10min to enable undigested tissue blocks to be deposited at the bottom of the centrifuge tube, and enabling the cell supernatant to move to the upper part of the centrifuge tube;
s5, taking out the cell supernatant obtained in the step S4, then dripping the cell supernatant into a centrifuge tube, carrying out centrifugation, centrifuging for 10min at 400g, sucking the supernatant, centrifuging for 25min at 600g, sucking the supernatant, centrifuging for 40min at 800g, sucking the supernatant, centrifuging for 55min at 1000g, discarding the supernatant, centrifuging for 70min at 1200g, and discarding the supernatant;
s6, carrying out heavy suspension work on the product processed in the step S5 through a heavy suspension solution, and finally placing the heavy suspension solution into a centrifuge tube for precipitation to obtain the exosome.
As a further scheme of the invention, the exosomes obtained in the step S6 are washed, precipitated and resuspended by PBS buffer solution, the centrifugation is carried out for 90min at 1200g, finally, the resuspension precipitation is carried out by the PBS buffer solution, and then the exosomes are frozen for standby use in an environment with the temperature of-90 ℃, and when the exosomes are used, the exosomes are placed on ice for unfreezing and then can be added into essence for use.
As a further scheme of the invention, all the centrifuge tubes used in the method are sterile centrifuge tubes.
As a further embodiment of the present invention, the biological stem cell in S1 is an animal umbilical cord stem cell.
In addition, the invention also provides application of the stem cell exosome extraction essence.
In one embodiment of the invention, the external secretion comprises essence and a frozen standby exosome;
the essence is formed by mixing the first mixture and the second mixture with the thawed exosomes.
As a further aspect of the present invention, the first mixture is a mixture of polysorbate, oxidized lecithin and tocopherol;
as a further aspect of the present invention, the second mixture is formed by mixing glycerin, ethylene glycol, xanthan gum, hyaluronic acid, EDTA, and distilled water.
As a further scheme of the invention, the essence obtained by mixing the components can be applied to the face, and has the advantage of improving facial wrinkles.
Compared with the related technology, the extraction method of the stem cell exosomes and the application of the essence of the exosomes provided by the invention have the following beneficial effects:
1. the invention adopts the animal umbilical cord stem cells as the raw material to prepare the exosome, so that the cost can be saved, the preparation yield of the exosome can be increased, and the advantage of improving the facial wrinkles can be enhanced.
Drawings
In order to facilitate understanding for those skilled in the art, the present invention will be further described with reference to the accompanying drawings.
FIG. 1 is a graph of a data record of the present invention before testing;
FIG. 2 is a graph of data records after testing in accordance with the present invention.
Detailed Description
Please refer to fig. 1 and fig. 2 in combination, wherein fig. 1 is a data record chart before testing according to the present invention; FIG. 2 is a graph of data records after testing of the present invention. The extraction method of the stem cell exosomes and the application of the essence of the exosomes comprise the following steps:
s1, taking biological stem cells, washing the biological stem cells with precooled PBS buffer solution for 2-4 times, and then shearing the biological stem cells into fragments-tissue blocks, wherein the size of the tissue blocks is about 1mm 3 ；
S2, washing the tissue block processed in the step S1 again through a precooled PBS buffer solution until the liquid is clear;
s3, placing the tissue blocks processed in the step S2 into a centrifuge tube, adding type II collagenase with the content of 0.1%, and then performing shock digestion in a water bath kettle with the temperature of 37 ℃ for 1 hour;
s4, lightly blowing and beating the digested product processed in the step S3 through a dropper to separate cells from loose tissue blocks and generate cell supernatant, then standing in an ice block for 10min to enable undigested tissue blocks to be deposited at the bottom of the centrifuge tube, and enabling the cell supernatant to move to the upper part of the centrifuge tube;
s5, taking out the cell supernatant obtained in the step S4, then dripping the cell supernatant into a centrifuge tube, carrying out centrifugation, centrifuging for 10min at 400g, sucking the supernatant, centrifuging for 25min at 600g, sucking the supernatant, centrifuging for 40min at 800g, sucking the supernatant, centrifuging for 55min at 1000g, discarding the supernatant, centrifuging for 70min at 1200g, and discarding the supernatant;
s6, carrying out heavy suspension work on the product processed in the step S5 through a heavy suspension solution, and finally placing the heavy suspension solution into a centrifuge tube for precipitation to obtain the exosome.
Washing, precipitating and re-suspending the exosome obtained in the step S6 by using PBS buffer solution, centrifuging for 90min at 1200g, finally performing re-suspending precipitation by using the PBS buffer solution, then freezing at-90 ℃ for later use, and when in use, placing the exosome on ice for unfreezing and adding the exosome into essence for use.
The centrifugal tubes used in the method are all sterile centrifugal tubes.
The biological stem cells in the S1 are animal umbilical cord stem cells.
The working principle of the stem cell exosome extraction method provided by the invention is as follows:
by adopting the animal umbilical cord stem cells as raw materials to prepare the exosomes, the cost can be saved, and the preparation yield of the exosomes can be increased.
In addition, the invention also provides application of the stem cell exosome extraction essence.
In one embodiment of the invention, the external secretion comprises essence and a frozen standby exosome;
the essence is formed by mixing the first mixture and the second mixture with the thawed exosomes.
The first mixture is formed by mixing polysorbate, oxidized lecithin and tocopherol;
the second mixture is prepared by mixing glycerol, ethylene glycol, xanthan gum, hyaluronic acid, EDTA and distillation.
The essence obtained by mixing can be applied to face, and has the advantage of improving facial wrinkles.
Performance test
The essences of examples 1-2 and comparative example 1 were used for human face measurement for wrinkle removal ability, and 40 volunteers were randomly selected, wherein the ratio of men to women was 1:1, cleaning facial skin once in the morning and evening, uniformly smearing the face with essence in a penicillin bottle, lightly beating and absorbing, wherein after the essence in the figure 1 is applied to the face of a volunteer, the wrinkle depth, the wrinkle number, the wrinkle length and the wrinkle volume data of the face of the volunteer are shown, and figure 2 is a data table obtained after the essence used by the volunteer is calculated.
Claims (8)
1. A method for extracting exosomes of stem cells is characterized by comprising the following steps:
s1, taking biological stem cells, washing the biological stem cells with precooled PBS buffer solution for 2-4 times, and then shearing the biological stem cells into fragments-tissue blocks, wherein the size of the tissue blocks is about 1mm 3 ；
S2, washing the tissue block processed in the step S1 again through a precooled PBS buffer solution until the liquid is clear;
s3, placing the tissue blocks processed in the step S2 into a centrifuge tube, adding type II collagenase with the content of 0.1%, and then performing shock digestion in a water bath kettle with the temperature of 37 ℃ for 1 hour;
s4, lightly blowing and beating the digested product processed in the step S3 through a dropper to separate cells from loose tissue blocks and generate cell supernatant, then standing in an ice block for 10min to enable undigested tissue blocks to be deposited at the bottom of the centrifuge tube, and enabling the cell supernatant to move to the upper part of the centrifuge tube;
s5, taking out the cell supernatant obtained in the step S4, then dripping the cell supernatant into a centrifuge tube, carrying out centrifugation, centrifuging for 10min at 400g, sucking the supernatant, centrifuging for 25min at 600g, sucking the supernatant, centrifuging for 40min at 800g, sucking the supernatant, centrifuging for 55min at 1000g, discarding the supernatant, centrifuging for 70min at 1200g, and discarding the supernatant;
s6, carrying out heavy suspension work on the product processed in the step S5 through heavy suspension solution, and finally placing the heavy suspension solution into a centrifuge tube for precipitation to obtain the exosome.
2. The method for extracting exosomes from stem cells according to claim 1, characterized in that: washing, precipitating and re-suspending the exosome obtained in the step S6 by using PBS buffer solution, centrifuging for 90min at 1200g, finally performing re-suspending precipitation by using the PBS buffer solution, then freezing at-90 ℃ for later use, and when in use, placing the exosome on ice for unfreezing and adding the exosome into essence for use.
3. The method for extracting a stem cell exosome according to claim 1, characterized in that: the centrifuge tubes used in the method are all aseptic centrifuge tubes.
4. The method for extracting exosomes from stem cells according to claim 2, characterized in that: the biological stem cell in the S1 is an animal umbilical cord stem cell.
5. The application of the stem cell exosome extraction essence is characterized in that: comprises essence and frozen standby exosome for the stem cell exosome extraction method according to any one of claims 1-4;
the essence is formed by mixing the first mixture and the second mixture with the thawed exosomes.
6. The use of the essence for the extraction of stem cell exosomes according to claim 5, characterized in that: the first mixture is formed by mixing polysorbate, oxidized lecithin and tocopherol.
7. The use of the essence for the extraction of stem cell exosomes according to claim 5, characterized in that: the second mixture is prepared by mixing glycerol, glycol, xanthan gum, hyaluronic acid, EDTA and distillation.
8. The use of the essence for the extraction of stem cell exosomes according to claim 5, characterized in that: the essence obtained by mixing can be applied to face, and has the advantage of improving facial wrinkles.
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