CN110643570A - Preparation method of ESO (ethylene-vinyl acetate copolymer) multi-factor - Google Patents
Preparation method of ESO (ethylene-vinyl acetate copolymer) multi-factor Download PDFInfo
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- CN110643570A CN110643570A CN201910933942.8A CN201910933942A CN110643570A CN 110643570 A CN110643570 A CN 110643570A CN 201910933942 A CN201910933942 A CN 201910933942A CN 110643570 A CN110643570 A CN 110643570A
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Abstract
The invention discloses a preparation method of ESO (Enteromorpha prolifera) multi-factor, which comprises the steps of taking out umbilical cord mesenchymal stem cells from a low-temperature storage container, and quickly thawing the umbilical cord mesenchymal stem cells; transferring the thawed umbilical cord mesenchymal stem cells into a cell culture dish for culture; uniformly dividing cell colonies in a culture dish into six blocks by using an inoculating needle, then placing the inoculating needle on an alcohol lamp to burn, quickly inserting the inoculating needle into one block of cell colonies, and quickly taking out, wherein each block of colonies is operated for three times as above until each divided block of colonies is burnt completely; then carrying out a second burning infection culture; and use
Description
Technical Field
The invention relates to the technical field of ESO (ethylene-vinyl acetate) multi-factor extraction, in particular to a preparation method of ESO multi-factor.
Background
The ESO is also called stem cell exosome, which is an active substance extracted from stem cell culture solution containing a large amount of growth factors and abundant active substances, wherein the ESO mainly comprises VEGF, oligopeptide-2, oligopeptide-9, oligopeptide-5 and other proteins, and also comprises amino acids and other active substances required by human bodies. It has effects of improving skin cell activity, promoting collagen regeneration, and reconstructing skin defense system, and has excellent antiaging and repairing effects.
At present, the preparation process of ESO is complicated, and the prepared product has more impurities and low purity.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a preparation method of an ESO (ethylene-vinyl acetate copolymer) multi-factor, which solves the problems of complicated preparation process, more impurities in the prepared product and low purity of the existing ESO multi-factor.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: a preparation method of ESO (Enteromorpha prolifera) multi-factor comprises the following steps:
step one, taking out umbilical cord mesenchymal stem cells from a low-temperature storage container, quickly placing the umbilical cord mesenchymal stem cells into a water tank at 36.5 ℃ to shake, and quickly unfreezing the umbilical cord mesenchymal stem cells;
step two, taking a sterile 10ml culture dish, preparing a special MDF (medium for serum free) culture medium which has FDA (food and drug administration) in the culture dish, transferring the thawed umbilical cord mesenchymal stem cells into a cell culture dish for culturing for 3 days, and keeping the temperature of the culture dish at 36.5 ℃;
uniformly dividing the cell colonies in the culture dish into six blocks by using an inoculating needle, then placing the inoculating needle on an alcohol lamp for flame burning until a needle head of the inoculating needle is burned for 10-15s, quickly inserting the inoculating needle into one block of cell colonies, and quickly taking out the cell colonies, wherein each block of colonies is operated for three times as above until each divided block of colonies is pre-burned;
step four, continuously culturing the umbilical cord mesenchymal stem cell culture dish after the burn is finished for 30 hours at the temperature of 36.5 ℃, and executing secondary burning infection culture;
transferring the umbilical cord mesenchymal stem cells after the second culture to a centrifugal separation vessel and using
Removing the conductive medium in the water by using a Reference ultrapure water system;
step six, adding the umbilical cord mesenchymal stem cells and culture solution thereof treated in the centrifugal separation vessel into a centrifugal machine, and carrying out centrifugal treatment at the rotating speeds of 300G, 2000G and 10000G in sequence, wherein the supernatant of the centrifugal separation vessel is ESO (Enteromorpha prolifera);
and seventhly, detecting and centrifuging to obtain the content of impurities in the ESO.
Preferably, the amount of umbilical cord mesenchymal stem cells taken out from the cryogenic storage container at a time in the first step is 3-4 ml.
Preferably, the time for rapidly thawing the mesenchymal stem cells in the first step is 0.9min-1.2 min.
Preferably, during the culture of the umbilical cord mesenchymal stem cells in the second step, 1ml of MDF serum-free of FDA is added into the culture dish every 1 day.
Preferably, in the third step, the inoculating needle draws lines in the culture dish uniformly and is divided into six areas, human eyes can recognize the six areas, each cell colony can be connected together or separated, and the temperature of the needle after burning is 260-280 ℃.
Preferably, the umbilical cord mesenchymal stem cells in the sixth step are rapidly cooled to 4 ℃ before centrifugation, and are centrifuged in an environment of 4 ℃.
Preferably, in the sixth step, the rotation speed of 300G is 15-20min, the bottom cells are removed, and the supernatant is taken for next centrifugation.
Preferably, in the sixth step, the rotating speed of 2000G is used for centrifugation for 18-25min, and cell debris at the bottom is removed to obtain supernatant for next centrifugation.
Preferably, in the sixth step, the rotation speed of 10000G is used for centrifugation for 25-30min, bottom impurities are removed, and supernate is obtained, so that the ESO multi-factor is obtained.
Preferably, the umbilical cord mesenchymal stem cells are high-expression umbilical cord mesenchymal stem cells of miR-525-3 p.
(III) advantageous effects
The invention provides a preparation method of ESO (ethylene-vinyl acetate copolymer) multi-factor. The method has the following beneficial effects:
according to the preparation method of the ESO multi-factor, umbilical cord mesenchymal stem cells are taken out from a low-temperature storage container and quickly placed in a water tank at 36.5 ℃ to shake, so that the umbilical cord mesenchymal stem cells are quickly thawed; in culturePreparing a special MDF (medium for serum free) culture medium with FDA (food and drug administration), transferring the thawed umbilical cord mesenchymal stem cells into a cell culture dish for culturing for 3 days, and keeping the temperature of the culture dish at 36.5 ℃; uniformly dividing cell colonies in a culture dish into six blocks by an inoculation needle, then placing the inoculation needle on an alcohol lamp to burn by outer flame until a needle head of the inoculation needle is burnt for 10-15s, quickly inserting the inoculation needle into one cell colony, and quickly taking out the cell colony, wherein each colony is operated for three times as above until each divided colony is burnt completely; then the temperature is kept at 36.5 ℃ for continuous culture for 30h, and the second burning infection culture is executed; transferring the umbilical cord mesenchymal stem cells after the second culture to a centrifugal separation vessel, and using
Removing the conductive medium in the water by using a Reference ultrapure water system; and centrifuging at the rotating speeds of 300G, 2000G and 10000G in sequence, and finally centrifuging the supernatant of the vessel to obtain the ESO multi-factor, so that the preparation procedures of the ESO multi-factor are reduced, and the preparation purity of the ESO multi-factor is improved.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Examples
A preparation method of ESO (Enteromorpha prolifera) multi-factor comprises the following steps:
step one, taking out umbilical cord mesenchymal stem cells from a low-temperature storage container, quickly placing the umbilical cord mesenchymal stem cells into a water tank at 36.5 ℃ to shake, and quickly unfreezing the umbilical cord mesenchymal stem cells;
step two, taking a sterile 10ml culture dish, preparing a special MDF (medium for serum free) culture medium which has FDA (food and drug administration) in the culture dish, transferring the thawed umbilical cord mesenchymal stem cells into a cell culture dish for culturing for 3 days, and keeping the temperature of the culture dish at 36.5 ℃;
uniformly dividing the cell colonies in the culture dish into six blocks by using an inoculating needle, then placing the inoculating needle on an alcohol lamp for flame burning until a needle head of the inoculating needle is burned for 10-15s, quickly inserting the inoculating needle into one block of cell colonies, and quickly taking out the cell colonies, wherein each block of colonies is operated for three times as above until each divided block of colonies is pre-burned;
step four, continuously culturing the umbilical cord mesenchymal stem cell culture dish after the burn is finished for 30 hours at the temperature of 36.5 ℃, and executing secondary burning infection culture;
transferring the umbilical cord mesenchymal stem cells after the second culture to a centrifugal separation vessel and usingRemoving the conductive medium in the water by using a Reference ultrapure water system;
step six, adding the umbilical cord mesenchymal stem cells and culture solution thereof treated in the centrifugal separation vessel into a centrifugal machine, and carrying out centrifugal treatment at the rotating speeds of 300G, 2000G and 10000G in sequence, wherein the supernatant of the centrifugal separation vessel is ESO (Enteromorpha prolifera);
and seventhly, detecting and centrifuging to obtain the content of impurities in the ESO.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation. The use of the phrase "comprising one of the elements does not exclude the presence of other like elements in the process, method, article, or apparatus that comprises the element.
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 (10)
1. A preparation method of ESO (Enteromorpha prolifera) is characterized by comprising the following steps:
step one, taking out umbilical cord mesenchymal stem cells from a low-temperature storage container, quickly placing the umbilical cord mesenchymal stem cells into a water tank at 36.5 ℃ to shake, and quickly unfreezing the umbilical cord mesenchymal stem cells;
step two, taking a sterile 10ml culture dish, preparing a special MDF (medium for serum free) culture medium which has FDA (food and drug administration) in the culture dish, transferring the thawed umbilical cord mesenchymal stem cells into a cell culture dish for culturing for 3 days, and keeping the temperature of the culture dish at 36.5 ℃;
uniformly dividing the cell colonies in the culture dish into six blocks by using an inoculating needle, then placing the inoculating needle on an alcohol lamp for flame burning until a needle head of the inoculating needle is burned for 10-15s, quickly inserting the inoculating needle into one block of cell colonies, and quickly taking out the cell colonies, wherein each block of colonies is operated for three times as above until each divided block of colonies is pre-burned;
step four, continuously culturing the umbilical cord mesenchymal stem cell culture dish after the burn is finished for 30 hours at the temperature of 36.5 ℃, and executing secondary burning infection culture;
transferring the umbilical cord mesenchymal stem cells after the second culture to a centrifugal separation vessel and using
Removing the conductive medium in the water by using a Reference ultrapure water system;
step six, adding the umbilical cord mesenchymal stem cells and culture solution thereof treated in the centrifugal separation vessel into a centrifugal machine, and carrying out centrifugal treatment at the rotating speeds of 300G, 2000G and 10000G in sequence, wherein the supernatant of the centrifugal separation vessel is ESO (Enteromorpha prolifera);
and seventhly, detecting and centrifuging to obtain the content of impurities in the ESO.
2. The method for preparing an ESO-gene according to claim 1, wherein: the amount of umbilical cord mesenchymal stem cells taken out from the cryogenic storage container in the first step is 3-4ml each time.
3. The method for preparing an ESO-gene according to claim 1, wherein: in the first step, the time for rapidly thawing the mesenchymal stem cells is 0.9min-1.2 min.
4. The method for preparing an ESO-gene according to claim 1, wherein: and in the second step, 1ml of MDF serum-free of FDA is added into the culture dish every 1 day during the culture of the umbilical cord mesenchymal stem cells.
5. The method for preparing an ESO-gene according to claim 1, wherein: in the third step, the inoculating needle draws lines in the culture dish uniformly and is divided into six areas, human eyes can recognize the six areas, each cell colony can be connected together or separated, and the temperature of the needle head after firing is 260-280 ℃.
6. The method for preparing an ESO-gene according to claim 1, wherein: and in the sixth step, the umbilical cord mesenchymal stem cells are quickly cooled to 4 ℃ before centrifugation, and are centrifuged at the temperature of 4 ℃.
7. The method for preparing ESO multi-factor according to claim 6, wherein: and in the sixth step, the rotating speed of 300G is used for centrifuging for 15-20min, bottom cells are removed, and supernate is obtained and is used for next centrifugation.
8. The method for preparing an ESO-gene according to claim 7, wherein: and in the sixth step, the centrifugal time at the rotating speed of 2000G is 18-25min, cell debris at the bottom is removed, and supernatant is taken for next centrifugal use.
9. The method for preparing an ESO-gene according to claim 8, wherein: and in the sixth step, the rotation speed of 10000G is used for centrifugation for 25-30min, bottom impurities are removed, and supernate is obtained to obtain the ESO multi-factor.
10. The method for preparing an ESO-gene according to claim 1, wherein: the umbilical cord mesenchymal stem cells adopt the high-expression umbilical cord mesenchymal stem cells of miR-525-3 p.
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| CN201910933942.8A CN110643570A (en) | 2019-09-29 | 2019-09-29 | Preparation method of ESO (ethylene-vinyl acetate copolymer) multi-factor |
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| CN201910933942.8A CN110643570A (en) | 2019-09-29 | 2019-09-29 | Preparation method of ESO (ethylene-vinyl acetate copolymer) multi-factor |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105769916A (en) * | 2016-04-29 | 2016-07-20 | 南京大学 | Application of mesenchymal stem cell-derived exosome in preparing drug or preparation for treating preeclampsia |
| CN106399247A (en) * | 2016-09-30 | 2017-02-15 | 广州赛莱拉干细胞科技股份有限公司 | Purpose of umbilical cord mesenchymal stem cell exosome |
| CN107245472A (en) * | 2017-06-08 | 2017-10-13 | 黄兵 | A kind of preparation method, the application method of human mesenchymal stem cell excretion body freeze-dried powder |
| CN109880797A (en) * | 2019-04-08 | 2019-06-14 | 济南磐升生物技术有限公司 | A method of preparing human umbilical cord mesenchymal stem cells excretion body |
-
2019
- 2019-09-29 CN CN201910933942.8A patent/CN110643570A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105769916A (en) * | 2016-04-29 | 2016-07-20 | 南京大学 | Application of mesenchymal stem cell-derived exosome in preparing drug or preparation for treating preeclampsia |
| CN106399247A (en) * | 2016-09-30 | 2017-02-15 | 广州赛莱拉干细胞科技股份有限公司 | Purpose of umbilical cord mesenchymal stem cell exosome |
| CN107245472A (en) * | 2017-06-08 | 2017-10-13 | 黄兵 | A kind of preparation method, the application method of human mesenchymal stem cell excretion body freeze-dried powder |
| CN109880797A (en) * | 2019-04-08 | 2019-06-14 | 济南磐升生物技术有限公司 | A method of preparing human umbilical cord mesenchymal stem cells excretion body |
Non-Patent Citations (1)
| Title |
|---|
| 郭莹等: "人脐带间充质干细胞来源外泌体提取方法的比较"", 《中国组织工程研究》 * |
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