Preparation method of umbilical cord mesenchymal stem cell local injection

Abstract

The invention relates to the technical field of stem cell medicines, and discloses a preparation method of an umbilical cord mesenchymal stem cell local injection, which is characterized by comprising the following steps: detecting infectious diseases; detecting four infectious diseases by using a colloidal gold method and couple reaction, and preparing and culturing umbilical cord mesenchymal stem cells; preparing autologous platelet rich plasma (PRP); preparing an umbilical cord mesenchymal stem cell local injection; after the structure is adopted, the umbilical cord mesenchymal stem cell local injection has the following advantages that the umbilical cord mesenchymal stem cells are cultured, and the umbilical cord mesenchymal stem cell local injection with the motility rate of more than 95% is prepared. The problems can be specifically repaired by local injection of the umbilical cord mesenchymal stem cells, the long-term and efficient whitening and anti-aging effects of the skin are achieved, the renewal of the skin cell level is efficiently and rapidly completed, and the skin is finer and more glossy.

Description

Technical Field

[0001] The invention relates to the technical field of stem cell medicines, and in particular to a method for preparing a local injection of umbilical cord mesenchymal stem cells. Background Art

[0002] The unbalanced pace of life in today's society, staying up late and irregular diet lead to a series of problems such as dryness, dull skin, inflammation, and skin aging caused by cell damage. Studies have proven the anti-aging and tissue regeneration capabilities of umbilical cord mesenchymal stem cells. At present, most of the skin care products used in the market for whitening, removing acne marks, and filling facial depressions contain chemical ingredients. There will be certain side effects after use, such as facial deformation caused by growth factors and facial stiffness caused by hyaluronic acid. Summary of the invention

[0003] The problem to be solved by the present invention is that most skin care products used in the market contain chemical ingredients, which may cause certain side effects after use, such as facial deformation caused by growth factors and facial stiffness caused by hyaluronic acid.

[0004] In order to solve the above technical problems, the technical solution provided by the present invention is: a method for preparing a local injection of umbilical cord mesenchymal stem cells, characterized in that it comprises:

[0005] (1) Detection of infectious diseases;

[0006] Use colloidal gold method and enzyme-linked method to conduct 4 infectious disease tests.

[0007] (2) Preparation and culture of umbilical cord mesenchymal stem cells;

[0008] 2.1. Preparation of complete culture medium for primary culture: add 25 mL of factor and one bottle of gentamicin to every 500 mL of primary culture medium;

[0009] 2.1. Cleaning of the umbilical cord;

[0010] 2.3, separation of Wharton's jelly;

[0011] 2.4. Umbilical cord tissue culture;

[0012] 2.5. Harvest cells;

[0013] (3) Preparation of autologous platelet-rich plasma (PRP);

[0014] 3.1. Collect 8mL of venous blood from the user using a blood collection tube;

[0015] 3.2. Let the collected venous blood stand at 4°C for 20 minutes. After the standing time, centrifuge it and draw the upper platelet-rich plasma (PRP) into a sterile storage tube and store it at 4°C for later use.

[0016] (4) Preparation of umbilical cord mesenchymal stem cell local injection solution;

[0017] 4.1. After 3 to 5 days of cell culture, observe the cells;

[0018] 4.2. Perform sterility and endotoxin tests 48 hours in advance;

[0019] 4.3. Examination under microscope;

[0020] 4.4. Collect the culture supernatant;

[0021] 4.5. Digestion and collection of cells;

[0022] 4.6. Gently pipette and mix the 50mL centrifuge tube containing the cell suspension, make up the volume to 40mL, and centrifuge at 2000rpm for 5min.

[0023] 4.7. After centrifugation, discard the supernatant, resuspend with 10 mL of physiological saline, pipette and beat evenly, and finally adjust the volume to 40 mL. Centrifuge at 2000 rpm for 5 min, and take an appropriate amount for cell counting and activity determination;

[0024] 4.8. After centrifugation, remove the supernatant, resuspend with 4 mL platelet-rich plasma (PRP), pipette evenly, take a new 50 mL centrifuge tube, and pass the cell suspension through a 70 μm mesh to form a cell suspension;

[0025] 4.9. Put the prepared cell suspension into a syringe and store it for later use.

[0026] After adopting the above structure, the present invention has the following advantages: the present invention cultivates umbilical cord mesenchymal stem cells and prepares 1×107 / mL umbilical cord mesenchymal stem cell local injection solution with an activity rate of >95%. The local injection of umbilical cord mesenchymal stem cells can specifically repair the above problem, achieve long-term and efficient whitening and anti-aging effects on the skin, and efficiently and quickly complete the renewal of the skin cell level, making the skin more delicate and lustrous.

[0027] As an improvement, the umbilical cord described in step 2.1 is cleaned by soaking and disinfecting it in 75% alcohol for 1 to 2 minutes, then washing it with saline for more than 2 times, cutting the umbilical cord into small segments of 2 to 3 cm with surgical scissors, squeezing out the blood from each segment, and washing it with saline for 3 times to clean the blood.

[0028] As an improvement, the umbilical cord tissue culture and observation described in step 2.4:

[0029] On day 0 of culture, the cut umbilical cord tissue was placed in a 10 mm culture dish containing 7 mL of complete culture medium, and then placed in a 5% CO2, 37°C incubator for culture;

[0030] After culturing for 5 days, observe the tissue block to see if a small amount of cells have crawled out, then add 5 mL of complete culture medium, shake to evenly distribute the culture medium, and place in a 5% CO2, 37°C incubator for culture;

[0031] After 8 days of culture, observe the tissue block. A small number of cells should crawl out. Then observe the cell crawling situation every day.

[0032] After 10 days of culture, observe the cells crawling out, then add 5 mL of complete medium, shake to make the medium evenly distributed, and culture in a 5% CO2, 37°C incubator;

[0033] After 15 days of culture, observe the cells and see if they have crawled out. If there are too few cells, add 5 mL of complete culture medium, shake to make the culture medium evenly distributed, and culture in a 5% CO2, 37°C incubator.

[0034] As an improvement, the cells harvested in step 2.5 were subcultured using a 150 mm culture dish and umbilical cord mesenchymal stem cell culture medium. When the number of cells reached 1 × 10 7 At the same time, the umbilical cord mesenchymal stem cell local injection solution was prepared.

[0035] As an improvement, the venous blood after standing in step 3.2 is placed in a centrifuge at 3500 rpm and centrifuged for 10 minutes.

[0036] As an improvement, the microscopic examination described in step 4.3 is performed under a 40x (high-power) microscope to observe whether the cells have uniformly moving black particles and whether they are contaminated.

[0037] As an improvement, collect the culture supernatant as described in step 4.4 in an ultra-clean workbench, and then neutralize and digest with about 10 mL of saline and gently wash the dish once.

[0038] As an improvement, in step 4.5, add 3 mL of trypsin to a 150 mm dish, add culture supernatant after digestion is complete, neutralize and digest, collect the digested cell suspension into a 50 mL centrifuge tube, and then wash the dish with about 10 mL of physiological saline to make the cell collection more complete.

[0039] As an improvement, the cell suspension described in step 4.9 is loaded into a 5 mL syringe and stored at 4-8°C. DETAILED DESCRIPTION

[0040] A method for preparing a local injection of umbilical cord mesenchymal stem cells, characterized in that it comprises:

[0041] (1) Detection of infectious diseases;

[0042] Use colloidal gold method and enzyme-linked method to conduct 4 infectious disease tests.

[0043] (2) Preparation and culture of umbilical cord mesenchymal stem cells;

[0044] 2.1. Preparation of complete culture medium for primary culture: add 25 mL of factor and one bottle of gentamicin to every 500 mL of primary culture medium;

[0045] 2.1. Cleaning of the umbilical cord;

[0046] 2.3, separation of Wharton's jelly;

[0047] 2.4. Umbilical cord tissue culture;

[0048] 2.5. Harvest cells;

[0049] (3) Preparation of autologous platelet-rich plasma (PRP);

[0050] 3.1. Collect 8mL of venous blood from the user using a blood collection tube;

[0051] 3.2. Let the collected venous blood stand at 4°C for 20 minutes. After the standing time, centrifuge it and draw the upper platelet-rich plasma (PRP) into a sterile storage tube and store it at 4°C for later use.

[0052] (4) Preparation of umbilical cord mesenchymal stem cell local injection solution;

[0053] 4.1. After 3 to 5 days of cell culture, observe the cells;

[0054] 4.2. Perform sterility and endotoxin tests 48 hours in advance;

[0055] 4.3. Examination under microscope;

[0056] 4.4. Collect the culture supernatant;

[0057] 4.5. Digestion and collection of cells;

[0058] 4.6. Gently pipette and mix the 50mL centrifuge tube containing the cell suspension, make up the volume to 40mL, and centrifuge at 2000rpm for 5min.

[0059] 4.7. After centrifugation, discard the supernatant, resuspend with 10 mL of physiological saline, pipette and beat evenly, and finally adjust the volume to 40 mL. Centrifuge at 2000 rpm for 5 min, and take an appropriate amount for cell counting and activity determination;

[0060] 4.8. After centrifugation, remove the supernatant, resuspend with 4 mL platelet-rich plasma (PRP), pipette evenly, take a new 50 mL centrifuge tube, and pass the cell suspension through a 70 μm mesh to form a cell suspension;

[0061] 4.9. Put the prepared cell suspension into a syringe and store it for later use.

[0062] The umbilical cord described in step 2.1 is cleaned by soaking and disinfecting it in 75% alcohol for 1 to 2 minutes, then washing it with saline for more than 2 times, cutting the umbilical cord into small sections of 2 to 3 cm with surgical scissors, squeezing out the blood from each section, and washing it with saline for 3 times to clean the blood.

[0063] Culture and observe the umbilical cord tissue as described in step 2.4:

[0064] On day 0 of culture, the cut umbilical cord tissue was placed in a 10 mm culture dish containing 7 mL of complete culture medium, and then placed in a 5% CO2, 37°C incubator for culture;

[0065] After culturing for 5 days, observe the tissue block to see if a small amount of cells have crawled out, then add 5 mL of complete culture medium, shake to evenly distribute the culture medium, and place in a 5% CO2, 37°C incubator for culture;

[0066] After 8 days of culture, observe the tissue block. A small number of cells should crawl out. Then observe the cell crawling situation every day.

[0067] After 10 days of culture, observe the cells crawling out, then add 5 mL of complete medium, shake to make the medium evenly distributed, and culture in a 5% CO2, 37°C incubator;

[0068] After 15 days of culture, observe the cells and see if they have crawled out. If there are too few cells, add 5 mL of complete culture medium, shake to make the culture medium evenly distributed, and culture in a 5% CO2, 37°C incubator.

[0069] The cells harvested in step 2.5 were subcultured using a 150 mm culture dish and umbilical cord mesenchymal stem cell culture medium. When the number of cells reached 1 × 10 7 At the same time, the umbilical cord mesenchymal stem cell local injection solution was prepared.

[0070] The venous blood after standing in step 3.2 is placed in a centrifuge at 3500 rpm and centrifuged for 10 minutes.

[0071] The microscopic examination described in step 4.3 is performed under a 40x (high power) microscope to observe whether the cells have uniformly moving black particles and whether they are contaminated.

[0072] Collect the culture supernatant as described in step 4.4 in an ultra-clean workbench, then neutralize and digest, and add about 10 mL of saline to gently wash the dish once.

[0073] In step 4.5, add 3 mL of trypsin to a 150 mm dish. After digestion is complete, add the culture supernatant to neutralize the digestion. Collect the digested cell suspension into a 50 mL centrifuge tube, and then wash the dish with about 10 mL of saline to make the cell collection more complete.

[0074] The cell suspension described in step 4.9 is placed in a 5 mL syringe and stored at 4-8°C.

[0075] The materials used in the present invention only contain umbilical cord mesenchymal stem cells and the user's autologous PRP (platelet-rich plasma). After decades of development, umbilical cord mesenchymal stem cells have not been found to have side effects, and autologous PRP is the user's own platelet-rich plasma and will not cause any problems. Umbilical cord mesenchymal stem cells have a high differentiation potential and can differentiate in multiple directions. It can differentiate into tissues such as muscle, tendon, fat, nerve and endothelium. It can promote angiogenesis, change the local microenvironment, improve local metabolic capacity, remove acne marks, differentiate into adipose tissue to fill acne pits and make the skin fuller and firmer. Due to the improvement of the production and metabolic capacity of blood vessels, it differentiates into damaged cells, making the skin more tender and smooth.

[0076] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0077] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0078] The present invention and its implementation methods are described above, and such description is not restrictive, and the actual structure is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can creatively design a structure and embodiment similar to the technical solution, which should fall within the protection scope of the present invention.

Claims

1. A method for preparing a local injection of umbilical cord mesenchymal stem cells, characterized in that: It includes: (1) Detection of infectious diseases; Use colloidal gold method and enzyme-linked method to conduct 4 infectious disease tests. (2) Preparation and culture of umbilical cord mesenchymal stem cells; 2.

1. Preparation of complete culture medium for primary culture: add 25 mL of factor and one bottle of gentamicin to every 500 mL of primary culture medium; 2.

1. Cleaning of the umbilical cord; 2.3, separation of Wharton's jelly; 2.

4. Umbilical cord tissue culture; 2.

5. Harvest cells; (3) Preparation of autologous platelet-rich plasma (PRP); 3.

1. Collect 8mL of venous blood from the user using a blood collection tube; 3.

2. Let the collected venous blood stand at 4°C for 20 minutes. After the standing time, centrifuge it and draw the upper platelet-rich plasma (PRP) into a sterile storage tube and store it at 4°C for later use. (4) Preparation of umbilical cord mesenchymal stem cell local injection solution; 4.

1. After 3 to 5 days of cell culture, observe the cells; 4.

2. Perform sterility and endotoxin tests 48 hours in advance; 4.

3. Examination under microscope; 4.

4. Collect the culture supernatant; 4.

5. Digestion and collection of cells; 4.

6. Gently pipette and mix the 50mL centrifuge tube containing the cell suspension, make up the volume to 40mL, and centrifuge at 2000rpm for 5min. 4.

7. After centrifugation, discard the supernatant, resuspend with 10 mL of physiological saline, pipette and beat evenly, and finally adjust the volume to 40 mL. Centrifuge at 2000 rpm for 5 min, and take an appropriate amount for cell counting and activity determination; 4.

8. After centrifugation, remove the supernatant, resuspend with 4 mL platelet-rich plasma (PRP), pipette evenly, take a new 50 mL centrifuge tube, and pass the cell suspension through a 70 μm mesh to form a cell suspension; 4.

9. Put the prepared cell suspension into a syringe and store it for later use.

2. The method for preparing a local injection of umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The umbilical cord described in step 2.1 is cleaned by soaking and disinfecting it in 75% alcohol for 1 to 2 minutes, then washing it with saline for more than 2 times, cutting the umbilical cord into small sections of 2 to 3 cm with surgical scissors, squeezing out the blood from each section, and washing it with saline for 3 times to clean the blood.

3. The method for preparing a local injection of umbilical cord mesenchymal stem cells according to claim 1, characterized in that: Culture and observe the umbilical cord tissue as described in step 2.4: On day 0 of culture, the cut umbilical cord tissue was placed in a 10 mm culture dish containing 7 mL of complete culture medium, and then placed in a 5% CO2, 37°C incubator for culture; After culturing for 5 days, observe the tissue block to see if a small amount of cells have crawled out, then add 5 mL of complete culture medium, shake to evenly distribute the culture medium, and place in a 5% CO2, 37°C incubator for culture; After 8 days of culture, observe the tissue block. A small number of cells should crawl out. Then observe the cell crawling situation every day. After 10 days of culture, observe the cells crawling out, then add 5 mL of complete medium, shake to make the medium evenly distributed, and culture in a 5% CO2, 37°C incubator; After 15 days of culture, observe the cells and see if they have crawled out. If there are too few cells, add 5 mL of complete culture medium, shake to make the culture medium evenly distributed, and culture in a 5% CO2, 37°C incubator.

4. The method for preparing a local injection of umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The cells harvested in step 2.5 were subcultured using a 150 mm culture dish and umbilical cord mesenchymal stem cell culture medium. When the number of cells reached 1 × 10 7 At the same time, the umbilical cord mesenchymal stem cell local injection solution was prepared.

5. The method for preparing a local injection of umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The venous blood after standing in step 3.2 is placed in a centrifuge at 3500 rpm and centrifuged for 10 minutes.

6. The method for preparing a local injection of umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The microscopic examination described in step 4.3 is performed under a 40x (high power) microscope to observe whether the cells have uniformly moving black particles and whether they are contaminated.

7. The method for preparing a local injection of umbilical cord mesenchymal stem cells according to claim 1, characterized in that: Collect the culture supernatant as described in step 4.4 in an ultra-clean workbench, then neutralize and digest, and add about 10 mL of saline to gently wash the dish once.

8. The method for preparing a local injection of umbilical cord mesenchymal stem cells according to claim 1, characterized in that: In step 4.5, add 3 mL of trypsin to a 150 mm dish. After digestion is complete, add the culture supernatant to neutralize the digestion. Collect the digested cell suspension into a 50 mL centrifuge tube, and then wash the dish with about 10 mL of saline to make the cell collection more complete.

9. The method for preparing a local injection of umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The cell suspension described in step 4.9 is placed in a 5 mL syringe and stored at 4-8°C.