Preparation method of mesenchymal stem cell-derived secretory preparation

Abstract

The invention provides a preparation method of a mesenchymal stem cell-derived secretory preparation, and belongs to the technical field of biological preparation development. The preparation method of the mesenchymal stem cell-derived secretory preparation provided by the invention can solve the problems that the existing exosome preparation is low in product purity, limited in quantity and easy to inactivate. According to the preparation method provided by the invention, impurities in the production process can be effectively removed, and the purity of the obtained secretory body is improved; according to the preparation method, the extraction rate and purity of the secretory body can be improved, the activity of the preparation is kept after the liquid preparation is finally obtained, and an excellent curative effect is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological preparation development, and in particular to a method for preparing a mesenchymal stem cell-derived secretory body preparation. Background Art

[0002] A secretome is a tiny membrane vesicle that can be secreted by most cells in the body. It has a lipid bilayer membrane and is about 30-150nm in diameter. Secretomes contain a large amount of repair factors, proteins, lipids and other substances, and these substances often need to be carefully cared for. If they are not strictly stored at low temperatures after a period of fresh preparation, they will quickly become ineffective. In clinical trials, freshly prepared secretomes are often not able to be used quickly; during transportation, it is also difficult to ensure that the transportation environment meets the preservation standards for secretomes, which greatly reduces the efficacy of secretome preparations. In the application process of secretomes, either they are constrained by geographical requirements, and there are clear requirements for the distance between the preparation laboratory and the application department; otherwise, the secretome can only be frozen and stored before transportation, which increases transportation costs and increases the risk of product contamination; or freeze-drying technology is used, which greatly increases the preparation cost, places too high demands on the laboratory, and is not suitable for promotion.

[0003] Chinese patent CN112011498A discloses a method for purifying and concentrating stem cell exosomes, comprising the following steps: S1, quickly filtering the cell culture fluid with a 0.22 μm filter screen to separate intact cells and residues, and ultracentrifuging at a speed of 10,000-11,500 g for 2 hours using a centrifuge; S2, resuspending and washing the vesicles with 1 mL of room temperature phosphate buffered saline solution, and ultracentrifuging again at a speed of 10,000-11,500 g for 2 hours using a centrifuge; S3, resuspending with 100 μL of room temperature phosphate buffered saline solution and transferring to a low-adhesion tube to obtain a high-concentration exosome. The invention purifies the exosomes by multiple ultracentrifugation, is simple to operate, and obtains a large number of vesicles.

[0004] However, the exosome preparations obtained by the above scheme still fail to solve the problems of low product purity, limited quantity, and easy inactivation. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing a mesenchymal stem cell-derived secretory body preparation, in which the secretory bodies in the product preparation have higher purity, greater quantity and can maintain activity and therapeutic effect for a long time.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for preparing a mesenchymal stem cell-derived secretory body preparation, comprising the following steps: S1. culturing mesenchymal stem cells to obtain a culture supernatant; S2, centrifuging the culture supernatant to obtain a supernatant; S3, filtering the supernatant using a filter membrane to obtain a filtrate; S4, centrifuging the filtrate to obtain precipitate 1; S5, resuspending the precipitate 1 to obtain a suspension; S6, mixing the suspension with the purified solution, and performing purification treatment to obtain a purified solution; S7, centrifuging the purified solution to obtain precipitate 2; S8, resuspending the precipitate 2 to obtain a secretion suspension; S9. Mix the secretory body suspension with mesenchymal stem cells to obtain a mesenchymal stem cell-derived secretory body preparation.

[0007] Preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0008] Preferably, the mesenchymal stem cells are P5 progeny cells.

[0009] Preferably, the degree of fusion of the mesenchymal stem cells is 80-90%.

[0010] Preferably, in S2, the rotation speed of the centrifugal treatment is 1000~1500g; And / or, the centrifugal treatment time is 20 to 40 minutes; And / or, the temperature of the centrifugal treatment is 5-15°C; And / or, the number of centrifugal treatments is 1 to 3 times.

[0011] Preferably, in S4, the rotation speed of the centrifugal treatment is 15000~20000g; The centrifugal treatment time is 60 to 120 minutes; The temperature of the centrifugal treatment is 5-15°C; The reagent used for the resuspension is PBS.

[0012] Preferably, in S6, the volume ratio of the suspension to the purified solution is 1:1-3.

[0013] Preferably, the purified solution contains polyethylene glycol and sodium chloride; The final concentration of polyethylene glycol in the purified solution is 200-400 g / L; The final concentration of sodium chloride in the purified solution is 50-150 g / L; The purification time is 12 to 36 hours; The temperature of the purification treatment is 5-15°C.

[0014] Preferably, in S7, the rotation speed of the centrifugal treatment is 8000~12000g; The centrifugal treatment time is 1.5h~3h; The temperature of the centrifugal treatment is 5-15°C.

[0015] Preferably, in the mesenchymal stem cell-derived secretory preparation, the concentration of mesenchymal stem cells is 10 4 ~5×10 5 Pieces / mL.

[0016] Beneficial effects of the present invention: The preparation method provided by the present invention can effectively remove impurities in the production process and improve the purity of the obtained secretion; the preparation method of the present invention can improve the extraction rate and purity of the secretion, and after the liquid preparation is finally obtained, the activity of the preparation is maintained, thereby achieving excellent therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the electron microscopy result diagram; Figure 2 The results of flow cytometry were shown. DETAILED DESCRIPTION

[0018] The present invention provides a method for preparing a mesenchymal stem cell-derived secretory body preparation, comprising the following steps: S1. culturing mesenchymal stem cells to obtain a culture supernatant; S2, centrifuging the culture supernatant to obtain a supernatant; S3, filtering the supernatant using a filter membrane to obtain a filtrate; S4, centrifuging the filtrate to obtain precipitate 1; S5, resuspending the precipitate 1 to obtain a suspension; S6, mixing the suspension with the purified solution, and performing purification treatment to obtain a purified solution; S7, centrifuging the purified solution to obtain precipitate 2; S8, resuspending the precipitate 2 to obtain a secretion suspension; S9. Mix the secretory body suspension with mesenchymal stem cells to obtain a mesenchymal stem cell-derived secretory body preparation.

[0019] In the present invention, preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells; preferably, the mesenchymal stem cells are P5 daughter cells; preferably, the fusion degree of the mesenchymal stem cells is 80-90%; compared with sources such as fat, dental pulp, and hair follicles, the mesenchymal stem cells of the present invention have the advantages of convenient sample source, non-invasive and safe tissue extraction, etc., and are easy to promote and have broad application prospects.

[0020] The umbilical cord mesenchymal stem cells of the P5 daughter cells of the present invention are preferably obtained by the following preparation method: the human umbilical cord of a healthy, full-term primipara who has undergone cesarean section and has been ligated after the fetus is delivered is quickly placed in an umbilical cord preservation solution, wherein the composition of the umbilical cord preservation solution is preferably: based on a serum-free culture medium, double antibodies are added at a volume ratio of 48 to 50:1; the umbilical cord is longitudinally cut, the arteries and veins in the umbilical cord are removed, and the obtained Wharton's jelly is cut into 2 to 5 mm 3 The tissue homogenate was filtered through a 70-900 mesh filter and added to the culture bottle to ensure that the tissue in each culture bottle was within the required range: 0.8 g / bottle for T75 standard and 2 g / bottle for T175 standard; serum-free medium was added to the culture bottle and cultured in a CO2 incubator at 37°C and 3-8% CO2 concentration; after 7 days, the original medium was discarded and replaced with fresh medium; when the fusion degree of adherent cells in the culture bottle reached 80-90%, they were digested with 1 pancreatic enzyme substitute to obtain P0 cells; the P0 cell suspension was cultured at 5000-6500 cells / cm 2 The cells were subcultured at a ratio of , and the obtained cells were recorded as the first generation cells (P1). When the adherent cells grew to 80-90% confluence, the steps were repeated to continue subculture until P5 cells were obtained.

[0021] In the present invention, preferably, in S2, the speed of the centrifugal treatment is 1000-1500g; and / or, the time of the centrifugal treatment is 20-40min; and / or, the temperature of the centrifugal treatment is 5-15°C; and / or, the number of centrifugal treatments is 1-3; preferably, the pore size of the filter membrane is preferably 0.22μm; preferably, in S4, the speed of the centrifugal treatment is 15000-20000g; the time of the centrifugal treatment is 60-120min; the temperature of the centrifugal treatment is 5-15°C; the reagent used for the resuspension is PBS; preferably, in S6, the suspension The volume ratio of the mixture with the purified solution is 1:1~3; preferably, the purified solution contains polyethylene glycol and sodium chloride; the final concentration of polyethylene glycol in the purified solution is 200~400g / L; the final concentration of sodium chloride in the purified solution is 50~150g / L, and the purified solution of the present invention can effectively remove impurities and improve the purity of the final product; the time of the purification treatment is 12~36h; the temperature of the purification treatment is 5~15°C; preferably, in S7, the speed of the centrifugal treatment is 8000~12000g; the time of the centrifugal treatment is 1.5h~3h; the temperature of the centrifugal treatment is 5~15°C.

[0022] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0023] Example 1 Obtain P5 umbilical cord mesenchymal stem cells: The human umbilical cord of a healthy, full-term primipara who underwent cesarean section and had its ligated cord quickly placed in an umbilical cord preservation solution after the fetus was delivered. The composition of the umbilical cord preservation solution is: based on a serum-free culture medium, double antibodies are added at a volume ratio of 49:1.

[0024] The umbilical cord was cut open longitudinally, the arteries and veins in the umbilical cord were removed, and the resulting Wharton's jelly was cut into 3 mm 3 The tissue homogenate block is filtered through an 80-mesh filter and added to the culture bottle to ensure that the tissue in each culture bottle is within the required range: 0.8 g / bottle for T75 standard and 2 g / bottle for T175 standard.

[0025] Add Dako serum-free culture medium into the culture flask and culture it in a CO2 incubator at 37 degrees Celsius and 5% CO2 concentration; after 7 days, discard the original culture medium and replace it with fresh culture medium; when the fusion degree of adherent cells in the culture flask reaches 85%, digest it with 1 trypsin substitute to obtain P0 cells.

[0026] The P0 cell suspension was prepared at 5700 cells / cm 2 The cells were subcultured at a ratio of , and the obtained cells were recorded as the first generation (P1). When the adherent cells grew to 85% of the confluence, the steps were repeated to continue subculture until P5 cells were obtained.

[0027] Preparation of secretome: 1. The p5 generation umbilical cord mesenchymal stem cells were inoculated in a stem cell culture medium (mesenchymal stem cell serum-free culture medium, manufacturer: Dakoway) and cultured. After the degree of confluence reached 85%, the supernatant was collected and centrifuged at 10°C, 1200g for 25 minutes. The supernatant was taken and centrifuged again for 30 minutes under the same conditions. The supernatant was retained to obtain a solution containing secretions. The solution containing secretions was filtered using a 0.22μm sterile filter membrane, and the filtrate was centrifuged at 10°C, 180 00g centrifugation for 90min, discard the supernatant; resuspend with sterile PBS to obtain secretion suspension, add purification solution (add 30g polyethylene glycol and 10g sodium chloride to 80ml ultrapure water, dissolve and mix, and make up to 100ml; the volume ratio of secretion suspension to purification solution is 1:2), invert and mix, let stand at 10℃ for 24h, centrifuge at 10℃, 10000g for 2h, the obtained precipitate is the secretion body, resuspend with sterile PBS to obtain the purified secretion solution (5×10 8 / ml).

[0028] 2. Digest the umbilical cord-derived p5 cells with a confluency of 85% with pancreatic enzyme substitute, centrifuge and fix the volume, calculate the total number of cells, and resuspend the mesenchymal stem cells with the purified secretion solution. The mesenchymal cell content in each secretion solution (10 ml) is 3.0×10 6 , and get the finished product.

[0029] In the above preparation process, sterile PBS and purified solution were pre-cooled at 4°C before use.

[0030] When the finished product is used, the cell activity is strictly required to be above 90%.

[0031] Comparative Example The comparative examples include 2. After the culture supernatant is collected, the difference from Example 1 is that the components of the purified solution are different. The comparative examples are respectively purified with physiological saline and resuspended with PBS only.

[0032] The difference between Comparative Example 1 and Example 1 is that: p5 generation umbilical cord mesenchymal stem cells are inoculated in a stem cell culture medium (mesenchymal stem cell serum-free culture medium, manufacturer: Dakoway) and cultured, and the supernatant is collected after the degree of fusion reaches 85%, centrifuged at 10°C and 1200g for 25min, the supernatant is taken, and centrifuged again for 30min under the same conditions, and the supernatant is retained to obtain a solution containing secretions; the solution containing the secretions is filtered using a 0.22μm sterile filter membrane, and the filtrate is centrifuged at 10°C and 18000g for 90min, and the supernatant is discarded; resuspended with sterile PBS to obtain a secretion suspension, PBS is added, dissolved and mixed, and the volume is adjusted to 100ml; the volume ratio of the secretion suspension to the purified solution is 1:2), inverted and mixed, let stand at 10°C for 24h, and centrifuged at 10°C and 10000g for 2h. The resulting precipitate is the secretion, resuspended with sterile PBS to obtain a purified secretion solution, and the secretion concentration is calculated.

[0033] Comparative Example 2: Umbilical cord-derived p5 cells with a confluency of 85% were digested with pancreatic enzyme substitute, centrifuged and fixed to volume, the total number of cells was calculated, and the mesenchymal stem cells were resuspended in PBS. The content of mesenchymal cells in each secretion solution (10 ml) was 3.0×10 6 The finished product survival rate was calculated and the results are shown in Table 1 below.

[0034] Table 1 Finished product concentration and activity test

[0035] Experimental example The fresh secretion obtained in Example 1 of the invention and the secretion preparation prepared in Comparative Example 1 were subjected to stability tests in the same environment. After being placed in an environment of 4° C. for 12 hours, the reagents were tested.

[0036] Experimental results analysis: The fresh secretion prepared in the control group was marked as a, and the fresh secretion prepared in Example 1 was marked as b. 1.5 ml of the final product was taken from both samples for testing. The testing scheme was as follows: (1) Morphological observation of secretory bodies: Take two final products, a and b, and observe them under an electron microscope. The results are as follows: Figure 1 Observation under an electron microscope showed that the secretions obtained by the two preparation methods showed the typical morphology of secretions, which were round or oval vesicle-like structures. The number of vesicles in product b was significantly higher than that in product a.

[0037] (2) Flow cytometry: First, take 1 ml of each of the two samples a and b to be tested and add them to a flow cytometry tube. Centrifuge at 15,000 g for 15 min. Discard the supernatant and resuspend the precipitate in 100 μl PBS (concentration 0.01 M, pH 7.4) buffer to obtain the concentrated secretion sample. Add 2 μL of Alexa Fluor™ 594-labeled Anti-CD63 fluorescent antibody and 2 μL of Alexa Fluor™ 647-labeled Anti-GPC1 fluorescent antibody to the sample. Incubate at 25°C for 30 min to obtain the incubation product. When using a flow cytometer to perform flow cytometric detection on the incubation product, first dilute the standard calibration microspheres of different particle sizes (100 nm / 200 nm / 300 nm / 500 nm) with PBS buffer at a volume ratio of 1:10000 (standard calibration microspheres: PBS buffer) to prepare a calibration microsphere dilution mixture, then take 500 μL of the calibration microsphere dilution mixture and add it to the flow cytometer, adjust the gain and threshold of the detection channel, so that the calibration microspheres of different particle sizes are distributed in different positions, then add 2 ml of PBS buffer to the incubated finished products a and b, centrifuge at 15000 g for 15 min for washing, add 500 μl of PBS buffer after washing twice, mix well, and then detect the microsphere content in the secretion body on the machine to determine the number of particles with inner diameters of 100 nm, 200 nm, 300 nm, and 500 nm in the reagent. The test results are as follows: Figure 2 shown.

[0038] Depend on Figure 2 It can be seen that the content of target vesicles in the secretory body preparation obtained by the preparation method provided by the present invention is relatively high.

[0039] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a mesenchymal stem cell-derived secretory preparation, characterized in that: The following steps are involved: S1. culturing mesenchymal stem cells to obtain a culture supernatant; S2, centrifuging the culture supernatant to obtain a supernatant; S3, filtering the supernatant using a filter membrane to obtain a filtrate; S4, centrifuging the filtrate to obtain precipitate 1; S5, resuspending the precipitate 1 to obtain a suspension; S6, mixing the suspension with the purified solution, and performing purification treatment to obtain a purified solution; S7, centrifuging the purified solution to obtain precipitate 2; S8, resuspending the precipitate 2 to obtain a secretion suspension; S9. Mix the secretory body suspension with mesenchymal stem cells to obtain a mesenchymal stem cell-derived secretory body preparation.

2. The preparation method according to claim 1, characterized in that: The mesenchymal stem cells are umbilical cord mesenchymal stem cells.

3. The preparation method according to claim 1, characterized in that: The mesenchymal stem cells are P5 progeny cells.

4. The preparation method according to claim 3, characterized in that: The fusion degree of the mesenchymal stem cells is 80-90%.

5. The preparation method according to claim 1, characterized in that: In S2, the rotation speed of the centrifugal treatment is 1000~1500g; And / or, the centrifugal treatment time is 20 to 40 minutes; And / or, the temperature of the centrifugal treatment is 5-15°C; And / or, the number of centrifugal treatments is 1 to 3 times.

6. The preparation method according to claim 1, characterized in that: In S4, the rotation speed of the centrifugal treatment is 15000~20000g; The centrifugal treatment time is 60 to 120 minutes; The temperature of the centrifugal treatment is 5-15°C; The reagent used for the resuspension is PBS.

7. The preparation method according to claim 1, characterized in that: In S6, the volume ratio of the suspension to the purified solution is 1:1-3.

8. The preparation method according to claim 1, characterized in that: The purified solution contains polyethylene glycol and sodium chloride; The final concentration of polyethylene glycol in the purified solution is 200-400 g / L; The final concentration of sodium chloride in the purified solution is 50-150 g / L; The purification time is 12 to 36 hours; The temperature of the purification treatment is 5-15°C.

9. The preparation method according to claim 1, characterized in that: In S7, the rotation speed of the centrifugal treatment is 8000~12000g; The centrifugal treatment time is 1.5h~3h; The temperature of the centrifugal treatment is 5-15°C.

10. The preparation method according to claim 1, characterized in that: In the mesenchymal stem cell-derived secretory preparation, the concentration of mesenchymal stem cells is 10 4 ~5×10 5 Pieces / mL.

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