Mesenchymal stem cell injection and preparation method thereof

Abstract

The invention relates to a mesenchymal stem cell injection and a preparation method thereof. The injection takes 18AA-V-SF compound amino acid injection as a mother solution, and contains 0.375 to 6 [mu] g / mL of coenzyme Q10, 0.25 to 4 ng / mL of vitamin B2 and 0.5 to 8 Um / 10 ml of low molecular weight heparin sodium. Compared with the prior art, the injection obviously enhances the stability and activity of stem cells in in-vitro culture and clinical transplantation. Tests prove that the high-concentration, medium-concentration and relatively low-concentration injection combined with human umbilical cord mesenchymal stem cells has an anti-inflammatory effect on H9C2 cell LPS damage, and the anti-inflammatory effect of a medium-concentration group is obviously superior to that of other groups. The injection is an efficient and safe umbilical cord mesenchymal stem cell injection for treating myocardial infarction, and has great clinical application potential.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a mesenchymal stem cell injection and a preparation method thereof. Background Art

[0002] In today's medical field, stem cell therapy has become one of the hotspots in medical research due to its unique therapeutic potential and broad application prospects. As pluripotent tissue stem cells, mesenchymal stem cells (MSCs) are widely used in the treatment of various refractory diseases due to their excellent self-renewal ability, significant immunomodulatory effect, and multi-directional differentiation potential. For example, the application of MSCs in the treatment of diseases such as avascular necrosis of the femoral head, myocardial infarction, brain trauma, and vascular diseases has achieved certain therapeutic effects and significantly improved the quality of life of patients. In the treatment of avascular necrosis of the femoral head, the transplantation of MSCs can promote the regeneration and repair of bone tissue, delay the progression of the disease, and reduce the need for joint replacement surgery. In the myocardial repair after myocardial infarction, MSCs can promote myocardial regeneration and angiogenesis, improve cardiac function, and reduce the sequelae of myocardial infarction. In the treatment of brain trauma, the transplantation of MSCs can promote neuron regeneration and axon regeneration, improve nerve function, and reduce nerve dysfunction. In the treatment of vascular diseases, MSCs can promote angiogenesis and vascular regeneration, improve blood circulation, and reduce ischemia-hypoxia injury. However, the stability and safety issues of stem cells in clinical applications remain the key factors restricting their development. For example, there are phenomena such as low survival rate, weakened cell activity, and cell aggregation in the clinical use of stem cells, which seriously affect the clinical application of stem cells and are problems that the industry urgently needs to solve. Summary of the Invention

[0003] To overcome the above technical defects, the present invention provides a mesenchymal stem cell injection and a preparation method thereof. The mesenchymal stem cell injection of the present invention is used for diluting or formulating a mesenchymal stem cell pharmaceutical solution for clinical use.

[0004] The specific technical solutions are as follows:

[0005] The injection uses 18AA-V-SF compound amino acid injection as the mother liquor, which contains 0.375 - 6 μg / mL coenzyme Q10, 0.25 - 4 ng / mL vitamin B2, and 0.5 - 8 Um / 10 ml low molecular weight heparin sodium.

[0006] As one of the preferred solutions, the preferred concentrations are: using 18AA-V-SF compound amino acid injection as the mother liquor, containing 1.5 g / mL coenzyme Q10, 1 ng / mL vitamin B2, and 2 Um / 10 ml low molecular weight heparin sodium.

[0007] Another preferred concentration is: containing 0.375 μg / mL coenzyme Q10, 0.25 ng / mL vitamin B2 and 0.5 Um / 10 ml low molecular weight heparin sodium.

[0008] The present invention further provides a preparation method of the injection, and the preparation steps are as follows:

[0009] 1. Using 18AA-V-SF compound amino acid injection as the mother liquor, adding coenzyme Q10, vitamin B2 and low molecular weight heparin sodium to the mother liquor according to the required concentration, and mixing evenly to obtain a mixed solution;

[0010] 2. Adjusting the pH value of the mixed solution to 5.5 to 7.5;

[0011] 3. Adjusting the osmotic pressure of the mixed solution to be between 260 mOsm and 320 mOsm;

[0012] 4. Filtering, filtering the mixed solution through a filter with a pore size of 0.22 μm to obtain the target injection.

[0013] Among them, sodium chloride or hydrochloric acid is used to adjust the pH value of the injection; sodium chloride adjusts the osmotic pressure.

[0014] Compared with the prior art, this injection uses compound amino acid injection (18AA-V-SF) as the mother liquor, and adds vitamin B2, coenzyme Q10 and heparin sodium in the best ratio, significantly enhancing the stability and activity of stem cells in in vitro culture and clinical transplantation. It has been proved by experiments that the high-concentration, medium-concentration and low-concentration injections of the present invention combined with human umbilical cord mesenchymal stem cells have an anti-inflammatory effect on LPS-induced injury of H9C2 cells, and the anti-inflammatory effect of the medium-concentration group is significantly better than that of other groups. Therefore, this injection is a highly efficient and safe umbilical cord mesenchymal stem cell injection for the treatment of myocardial infarction, and has great potential for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a result diagram of the test on the influence of the mesenchymal stem cell injection of the example on the survival rate of human umbilical cord mesenchymal stem cells;

[0016] Figure 2 It is a result diagram of the test on the optimal modeling concentration of LPS inflammatory factors in the example;

[0017] Figure 3 It is a result diagram of the test on the anti-inflammatory effect of the mesenchymal stem cell injection combined with human umbilical cord mesenchymal stem cells on LPS-induced injury of H9C2 cells in the example. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be further described below with reference to specific embodiments.

[0019] The present invention aims to develop an efficient and safe umbilical cord mesenchymal stem cell injection for the treatment of myocardial infarction, particularly to enhance the stability and activity of mesenchymal stem cells (MSCs) during in vitro culture and transplantation. By using the ratio and concentration of compound amino acid injection (18AA-V-SF), vitamin B2 injection, coenzyme Q10 injection, and sodium heparin, as well as their ratios with different cell concentration suspensions, the optimal combination conditions are explored to improve the survival rate of MSCs, maintain cell activity, reduce cell aggregation, and optimize cell surface markers and solution properties, thereby enhancing the efficacy and safety in clinical treatment.

[0020] I. Experiment on the effect of the stem cell injection of the present invention on the survival rate of human umbilical cord mesenchymal stem cells

[0021] (I) Experimental subjects: Rat H9C2 cardiomyocytes, purchased from the Cell Bank of the Chinese Academy of Sciences. Human umbilical cord mesenchymal stem cells, donated by Guangdong New Southern Group Co., Ltd.

[0022] (II) Preparation of MSCs

[0023] 1. Sample preparation: Wash the blood stains on the surface of the umbilical cord tissue with pre-cooled washing solution, cut it into small sections of 2-3 cm, wash several times, and strip the Wharton's jelly tissue of the umbilical cord and place it in pre-cooled tissue washing solution for standby.

[0024] 2. Isolation, culture and phenotypic identification of human umbilical cord mesenchymal stem cells.

[0025] 2.1 MSCs culture steps:

[0026] Primary culture: Take the Wharton's jelly tissue of the umbilical cord and culture it in a serum-free medium under hypoxic conditions;

[0027] Subculture: Collect the above-mentioned P1 primary cells, prepare a single-cell suspension, and centrifuge to obtain a cell pellet; Add a serum-free medium to the cell pellet and culture it under hypoxic conditions until passage, and continuously culture to P2-P3 generations; Add ligustrazine hydrochloride each time during subculture, and when the cells grow to the predetermined confluence, digest and collect the cells at P6 generation;

[0028] The MSCs preparation cultured by this method reduces the characteristic that stem cells are prone to aggregate into clusters, thus avoiding the situations of cell adhesion, rouleaux formation of red blood cells, and cell mass embolism after intravenous infusion into the human body, enabling the better application of MSCs stem cell technology in clinical practice.

[0029] 2.2 Phenotypic detection: Detect the phenotypes of the above-mentioned collected cells for future use.

[0030] P6 hUC-MSCs were collected for pathogen detection to exclude pathogenic contamination. After washing with saline, the cell concentration was adjusted to 2×106 cells / mL, and 5 μL of CD29, CD31, CD34, CD44, CD45, CD73, CD90, CD105, CD166, and human leukocyte antigen (HLA-DR) monoclonal antibodies were added to each tube at 100 μL, mixed, and incubated at 4°C in the dark for 30 minutes; after washing with saline and resuspending, the expression of hUC-MSC cell surface markers was detected by flow cytometry.

[0031] Record the flow cytometry phenotype test results of umbilical cord mesenchymal stem cells to ensure that they are umbilical cord mesenchymal stem cell populations.

[0032] Before the experiment, the prepared and preserved stem cells were revived and cultured, and when the cell density reached 70%-80%, they were passaged and centrifuged for cell passage; the serum-free non-programmed freezing solution was taken out, the supernatant was discarded, and 2 ml of the freezing solution was taken to resuspend the cells, with 1 ml of cell suspension in each cryotube, which was placed in a -80°C refrigerator overnight and transferred to a liquid nitrogen tank after 24 hours for storage.

[0033] (III) Preparation of stem cell injection solution

[0034] Five groups of preparation samples and a control group were prepared using compound amino acid injection (18AA-V-SF) as the mother solution and adding vitamin B2, coenzyme Q10 and heparin sodium according to the concentration requirements.

[0035] High concentration group: Coenzyme Q10, 6μg / mL; Vitamin B2, 4ng / mL; Low molecular weight heparin sodium, 8Um / 10ml;

[0036] Higher concentration group: Coenzyme Q10, 3μg / mL; Vitamin B2, 2ng / mL; Low molecular weight heparin sodium, 4Um / 10ml;

[0037] Medium concentration group: Coenzyme Q10, 1.5μg / mL; Vitamin B2, 1ng / mL; Low molecular weight heparin sodium, 2Um /

[0038] 10ml;

[0039] Low concentration group: Coenzyme Q10, 0.75μg / mL; Vitamin B2, 0.5ng / mL; Low molecular weight heparin sodium 1Um / 10ml;

[0040] Lower concentration group: Coenzyme Q10, 0.375μg / mL; Vitamin B2, 0.25ng / mL; Low molecular weight heparin sodium, 0.5Um / 10ml;

[0041] Normal control group: normal saline group and compound amino acid injection (18AA-V-SF) group.

[0042] Adjust the pH value of the preparation sample: Use sodium chloride and hydrochloric acid to adjust the pH value of the solution to 5.5 to 7.5.

[0043] Adjust the osmotic pressure of the preparation sample: Adjust the concentration of sodium chloride to make the osmotic pressure between 260 mOsm and 320 mOsm.

[0044] Filter the preparation sample through a 0.22 μm pore size filter and store it at 2°C to 8°C for future use.

[0045] (IV) Experiment

[0046] 1. Centrifuge and resuspend the cells in the logarithmic growth phase, count them, inoculate 1x104 cells per well into a 96-well plate, and incubate in a constant temperature incubator for 24 h. Discard the old complete medium, wash twice with normal saline, and add stem cell injection preparation, normal saline, and compound amino acid injection to seven groups respectively. Place it in a three-gas incubator with 94% N2, 5% CO2, and 5% O2 for 72 h, and perform cck-8 detection after completion. Determine whether the combined drug is safe according to the experimental results.

[0047] 2. CCK-8 detection of cell viability

[0048] 2.1 Inoculate human umbilical cord mesenchymal cells into a 96-well plate and place it in a 37°C three-gas constant temperature incubator;

[0049] 2.2 After the cell treatment is completed, discard the old medium, wash twice with normal saline, turn off the light on the experimental bench, and add a mixture of 10 μL CCK-8 and 100 μL serum-free medium for mesenchymal stem cells to each well;

[0050] 2.3 Wrap the 96-well plate with tin foil to avoid light, place it in a 37°C constant temperature incubator for incubation for 1.5 h, and then perform detection. Measure the OD value of each well at a wavelength of 450 nm with an enzyme-labeled instrument;

[0051] 2.4 Calculate the cell viability according to the formula; Cell viability (%) = [A((drug added)-A(blank)] / [A(0 drug added)-A(blank)] x 100

[0052] 3 Data statistical analysis

[0053] The numerical data were tested for normality. If they followed normal distribution, the mean ± standard deviation was used for description. When the numerical data followed normal distribution, variance analysis was used for multiple group comparisons. LSD test was used for homogeneous variances. Dunnett'T3 test was used for unequal variances. If they did not meet normal distribution, quartiles M (P25-P75) were used for representation, and then nonparametric Krusckal--Wallis rank sum test was used. SPSS 26.0 software was used for statistical analysis. At the a=0.05 level, P<0.05 was considered statistically significant.

[0054] 4 Experimental results

[0055] like Figure 1 As shown, after 72 hours of hypoxia, the cell activity was detected using the CCK-8 method. The results showed that the cell activity in the amino acid group, high concentration group, relatively high concentration group, medium concentration group, low concentration group, and relatively low concentration group increased to varying degrees after 72 hours, indicating that this stem cell injection is beneficial to the survival of umbilical cord mesenchymal stem cells.

[0056] 2. The anti-inflammatory effect of stem cell preparation combined with human umbilical cord mesenchymal stem cells on LPS-induced H9C2 cells.

[0057] (I) H9C2 cell culture: H9C2 cells were revived and subcultured, and 2 ml of cryopreservation solution was taken to resuspend the cells. Each cryopreservation tube contained 1 ml of cell suspension and was placed in a -80°C refrigerator overnight. After 24 hours, the cells were transferred to a liquid nitrogen tank for storage.

[0058] (II) Screening and determination of the concentration of LPS for H9C2 cell modeling

[0059] 1. Extraction of RNA from H9C2 cells

[0060] 1.1 Discard the co-culture dish and culture medium, add 500 μL of saline to each well and wash the cells twice;

[0061] 1. Add 250 μL of lysis buffer to each well of the 2-well plate, place the 6-well plate on a shaker at room temperature for 5 minutes, then blow the cells to fully lyse the cells and transfer the liquid to a new 1.5 ml centrifuge tube;

[0062] 1.3 Add 250 μL of anhydrous ethanol to the centrifuge tube in step (2), shake it up and down evenly, and if white precipitate is seen, blow it with a pipette until the precipitate disappears. Transfer it to the adsorption column, centrifuge it at 4000×g, 4℃, for 1 minute, discard the waste liquid in the centrifuge tube, and absorb it with absorbent paper;

[0063] 1.4 Prepare the required solution according to the ratio of adding 2 μL of DNase (gDNA Remover) to 10 μL of ddH2O for each sample. Mix well with a pipette and add it to the center of the centrifugal column. Let it stand at room temperature for 5 minutes.

[0064] 1.5 Pipette 500 μL of Wash Buffer into the adsorption column, centrifuge at 12,000×g at 4 °C for 1 minute, pour off the waste liquid, blot it dry with absorbent paper, and centrifuge again.

[0065] 1.6 Transfer the adsorption column to a new 1.5 mL centrifuge tube, open the lid, and let it air dry at room temperature for 2 minutes.

[0066] 1.7 Add 20 μL of Elution buffer to the membrane in the center of the adsorption column (note not to touch it), cover the lid, and let it stand at room temperature for 2 minutes.

[0067] 1.8 Transfer it to the centrifuge, centrifuge at 12,000×g at 4 °C for 1 minute. Discard the adsorption column.

[0068] 1.9 Detect the concentration and quality of RNA with a nucleic acid and protein quantitation detector. Store the qualified RNA in an -80 °C refrigerator.

[0069] 2. RNA reverse transcription

[0070] The RNA reverse transcription reaction system is shown in Table 1. The operation of reverse transcribing RNA into cDNA is as follows:

[0071] 2.1 According to the concentration measured by Nanodrop, use a pipette to transfer a total of 1 μg of each RNA sample into an eight-well tube for reverse transcription reaction.

[0072] 2.2 Add 2 μL of reagent 4xRTMasterMix to each sample, and then make up the total volume to 10 μL with reagent Nuclease-free ddH2O.

[0073] 2.3 Start the PCR instrument for cDNA synthesis. The program is shown in Table 2:

[0074] Table 1 RNA reverse transcription system

[0075]

[0076] Table 2 cDNA synthesis and amplification reaction

[0077]

[0078] 2.4 After the program ends, dilute the sample to 200 μL with ddH2O and store it in an -80 °C refrigerator.

[0079] 3. Real-time fluorescence quantitative detection (RT-qPCR)

[0080] 3.1 The primer sequences were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., and are shown in Table 3 below.

[0081] Table 3 Primer sequences

[0082]

[0083] 3.2 According to the reaction system preparation provided by the RT-qPCR kit of Shanghai Yize Biotechnology Co., Ltd., see Table 4.

[0084] Table 4 RT-qPCR reaction system

[0085]

[0086] 3.3 Add the above components to the 96-well reaction plate in sequence. After covering with a sealing plate film, centrifuge at 2200 rpm / min for 30 seconds using a plate shaker. Set the reaction program according to Table 5.

[0087] Table 5 RT-qPCR reaction program

[0088]

[0089] 3.4 Calculate the expression levels of the relevant mRNAs using the 2-ΔΔCt method and perform statistical analysis.

[0090] 4. Data statistical analysis

[0091] Perform a normal test on the logarithmic data. If it follows a normal distribution, describe it using the mean ± standard deviation: when the numerical data follows a normal distribution, use analysis of variance for multiple group comparisons, use the LSD test when the variances are homogeneous, and use the Dunnett's T3 test when the variances are not homogeneous; if it does not meet the normal distribution, represent it using the quartiles M (P25 - P75), and then use the non-parametric Krusckal-Wallis rank sum test. Perform statistical analysis using SPSS 26.0 software. At the significance level of α = 0.05, a difference is considered statistically significant when P < 0.05.

[0092] 5. Experimental results

[0093] 5.1 To determine the LPS inflammation model, the experiment detected the expression of the inflammation-related index mRNA B. The results are as Figure 2 shown. After H9C2 cells experienced LPS inflammation, compared with the Con group, the activities of IL-1β and IL-18 increased (p < 0.05), showing a statistically significant difference. Moreover, when the LPS concentration in H9C2 cells was 1 μg / ml, the expression of the inflammation-related genes IL-1β and IL-18 was the highest, and this concentration was used for model establishment.

[0094] (III) Anti-inflammatory effect test of the stem cell injection of the present invention combined with human umbilical cord mesenchymal stem cells on LPS-induced injury of H9C2 cells.

[0095] The results are as Figure 3 shown. Compared with the Con group, the stem cell injection of high concentration, medium concentration, and lower concentration combined with human umbilical cord mesenchymal stem cells all have anti-inflammatory effects on LPS-induced injury of H9C2 cells, and the anti-inflammatory effect of the medium concentration group is the best.

Claims

1. A mesenchymal stem cell injection, characterized in that: The injection is based on 18AA-V-SF compound amino acid injection as the mother solution, which contains 0.375-6 μg / mL coenzyme Q10, 0.25-4 ng / mL vitamin B2 and 0.5-8 Um / 10 ml low molecular weight heparin sodium.

2. The injection according to claim 1, characterized in that: The injection contains 1.5g / mL coenzyme Q10, 1ng / mL vitamin B2 and 2Um / 10ml low molecular weight heparin sodium.

3. The injection according to claim 1, characterized in that: The injection contains 0.375 μg / mL coenzyme Q10, 0.25 ng / mL vitamin B2 and 0.5 Um / 10 ml low molecular weight heparin sodium.

4. The method for preparing the injection according to claim 1, characterized in that The following steps are involved: (1) Using 18AA-V-SF compound amino acid injection as a mother solution, adding coenzyme Q10, vitamin B2 and low molecular weight heparin sodium into the mother solution at the required concentration, and mixing evenly to obtain a mixed solution; (2) adjusting the pH value of the mixed solution to 5.5 to 7.5; (3) adjusting the osmotic pressure of the mixed solution to between 260 mOsm and 320 mOsm; (4) Filtration: Filter the mixed solution through a filter with a pore size of 0.22 μm to obtain the target injection solution.

5. The method for preparing the injection according to claim 4, characterized in that: The pH regulator in step (2) is sodium chloride or hydrochloric acid.

6. The method for preparing the injection according to claim 4, characterized in that: The osmotic pressure regulator in step (3) is sodium chloride.

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