Stem cell preparation for treating premature ovarian failure and preparation method thereof

Abstract

The invention provides a stem cell preparation for treating premature ovarian failure, which is composed of human amniotic stem cells and a matrix solution, and the matrix solution is composed of dextran 40 glucose injection, 1-3% of human albumin, 1-10% of compound amino acid injection (18AA), 1-5% of ginkgo leaf extract injection, 1-5 [mu] M of reduced glutathione and 10-20 [mu] g / ml of coenzyme I, the stem cell preparation prepared from the human amniotic stem cells and the matrix solution can well maintain the activity, dryness and uniformity of the stem cells, the components are clear, heterologous animal serum is not contained, after the stem cell preparation is stored for 96 h at the temperature of 4-8 DEG C, the cell morphology is uniform, the survival rate is still kept to be 90% or above, and the stem cell preparation has a good treatment effect on premature ovarian failure.

Description

Technical Field

[0001] The present invention relates to a stem cell preparation for treating premature ovarian failure and a preparation method thereof, and belongs to the technical field of biomedicine. Background Art

[0002] Premature ovarian failure (POF) refers to amenorrhea in women before the age of 40 due to reduced ovarian follicle reserves or follicle dysfunction. Common symptoms include hot flashes, sweating, facial flushing, decreased libido, amenorrhea, infertility, anxiety, depression, insomnia and other low estrogen symptoms (similar to menopausal symptoms). The incidence of POF is 0.1‰ in women under 30 years old, 1% in women aged 30 to 39, 10-28% in primary amenorrhea, and 4-18% in secondary amenorrhea. In recent years, the incidence has been increasing and younger, seriously affecting the quality of life of patients.

[0003] At present, the treatment of POF is mainly hormone replacement therapy, but it may increase the risk of endometrial cancer, breast cancer, cardiovascular and cerebrovascular diseases. In recent years, with the deepening of clinical research on stem cells, studies have found that stem cells can home to the site of injury to secrete a variety of cytokines and growth factors to promote tissue repair, improve the local tissue inflammatory microenvironment, prevent tissue fibrosis, and differentiate into blood vessels to provide nutritional support to improve tissue function repair. Therefore, stem cell transplantation provides a new direction for the clinical treatment of premature ovarian failure. However, due to the uneven composition of stem cell preparations and insufficient activity, the treatment effect still needs to be improved. Now there is an urgent need for a stem cell preparation for the treatment of premature ovarian failure and its preparation method to solve the above problems.

[0004] Human albumin: provides nutrition for cells, maintains cell activity, avoids cell aggregation, and prevents embolism caused by the reinfusion process; Compound amino acid injection (18AA): Compound amino acid 18aa contains a variety of essential amino acids for the human body. It meets the needs of the body by providing a complete amino acid spectrum, thereby playing its role in nutritional support, stimulating cell regeneration and tissue repair, and has the function of regulating the activity of the immune system, which can enhance the body's defense against pathogens; Ginkgo biloba extract injection: The main ingredients are ginkgo biloba extract and ginkgo flavonoids, which can help remove excessive free radicals in the body and inhibit the peroxidation reaction of cell membrane lipids, thereby protecting the cell membrane and preventing free radicals from causing a series of damages to the body. It can increase the supply of oxygen and glucose to ischemic tissues, increase the number of certain neurotransmitter receptors, improve blood circulation, etc. Reduced glutathione: It has reducing antioxidant, detoxification, protects cells from free radical damage, participates in a variety of biochemical reactions, plays an important role in protein synthesis, metabolic regulation, etc., and is of great significance for maintaining the normal physiological functions of the body; Coenzyme I for injection: NAD+, the full name of nicotinamide adenine dinucleotide, is a molecule that plays a key role in cells and is essential for the life activities of cells. It not only plays a role in energy metabolism, DNA repair and cell signal transduction, but also plays an important role in maintaining the normal physiological functions of the human body. Increasing the content of NAD+ can enhance mitochondrial function and improve energy metabolism.

[0005] Dextran 40 Glucose Injection: It can improve microcirculation, prevent or eliminate red blood cell aggregation and thrombosis in blood vessels, and also has the effect of expanding blood volume; Glucose is a monosaccharide and one of the most important energy sources in human cells. Glucose can provide cells with the required energy and nutritional support, helping to maintain cell survival and function. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a stem cell preparation for treating premature ovarian failure and a preparation method thereof, so as to solve the problems raised in the above-mentioned background technology. The present invention has clear ingredients, does not contain xenobiotic animal serum, and after being stored at 4-8°C for 96 hours, the cell morphology is uniform and the survival rate is still maintained at more than 90%, which has a good therapeutic effect on premature ovarian failure.

[0007] In order to achieve the above-mentioned object, the present invention is realized by the following technical scheme: a stem cell preparation for treating premature ovarian failure, comprising human amniotic membrane stem cells and a matrix solution, wherein the matrix solution comprises dextran 40 glucose injection, 1-3% human serum albumin (V / V), 1-10% compound amino acid injection (18AA) (V / V), 1-5% ginkgo leaf extract injection (V / V), 1-5uM reduced glutathione (600mg / vial) and 10-20ug / ml coenzyme I for injection (NAD+; 5mg / vial), human amniotic membrane stem cells are obtained by 3D gelatin microcarrier culture, the cell generation is controlled at P4-6, and the concentration of the matrix solution in the preparation is 1-4×10 6 / ml.

[0008] Furthermore, the dextran concentration in the dextran 40 glucose injection is 6%, and the glucose concentration is 5%.

[0009] Furthermore, the optimal concentration of the stem cell preparation is: 2% human albumin (V / V), 5% compound amino acid injection (18AA) (V / V), 3% Ginkgo biloba extract injection (V / V), 3uM reduced glutathione (600mg / vial), 15ug / ml coenzyme I for injection (NAD+; 5mg / vial), 2.5×10 6 / ml human amniotic stem cells.

[0010] Furthermore, a method for preparing a stem cell preparation for treating premature ovarian failure comprises the following steps:

[0011] Step 1: Prepare 100 ml of matrix solution, add 1-3 ml of human albumin, 1-10 ml of compound amino acid injection (18AA) (v / v), 1-5 ml of ginkgo leaf extract injection, 0.1-0.5 ml of reduced glutathione, 0.5-1 ml of coenzyme I for injection, add dextran 40 glucose injection to 100 ml, put it in a micro oscillator (VORTEX GENIE) and shake it thoroughly at 150-180 rpm for 30 seconds to 1 minute to mix, and store it at 4°C;

[0012] Step 2: Grow P4-P6 cells to a sufficient number, remove about 80% of the supernatant from the culture system, add 5× lysis buffer to make the lysis enzyme concentration close to 1 mg / ml; lyse at 37°C for 30-40 min, collect the cell suspension after the microcarrier is fully lysed; centrifuge at 1500 rpm for 5 min, discard the supernatant, wash with physiological saline, resuspend by centrifugation 2-3 times, count, pass through a 100-mesh sieve, and add human amniotic stem cells to the prepared matrix solution to make the stem cell concentration in the preparation 1-4×10 6 / ml.

[0013] Compared with the prior art, the present invention has the following beneficial effects: the stem cell preparation provided by the present invention can well maintain the activity, dryness and uniformity of stem cells, and the present invention has clear ingredients and does not contain xenobiotic animal serum. After being stored at 4-8°C for 96 hours, the cell morphology is uniform and the survival rate remains above 90%, which has a good therapeutic effect on premature ovarian failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0015] Figure 1 This is a morphological diagram of cells under 3D culture conditions in a stem cell preparation for treating premature ovarian failure and a preparation method thereof of the present invention;

[0016] Figure 2 This is a morphological diagram of cells under 2D culture conditions in a stem cell preparation for treating premature ovarian failure and a preparation method thereof of the present invention. DETAILED DESCRIPTION

[0017] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0018] Example 1: Amniotic membrane stem cell culture: The P3 cells frozen in the working bank were thawed in a 37-degree water bath, and then added to a 50-ml centrifuge tube containing 10 ml of normal saline. The cells were centrifuged at 1400 rpm for 5-8 min, and the cells at the bottom were collected. Serum-free medium (DMEM / F12+10% serum replacement+20 ng / ml bFGF+10 ng / ml EGF+1×NEAA+5-10 umolRA) was added, and the cells were resuspended and counted. Serum-free medium was added to the culture flask, and then gelatin microcarriers were added at a ratio of 2 mg / ml to fully swell the microcarriers. The cells were cultured at a rate of 0.3-0.5×10 5 / ml density was inoculated in a bioreactor equipped with fully swollen microcarriers, and the bioreactor process control parameters were set, including temperature of 37°C, pH 7.2-7.4, dissolved oxygen concentration of 20-25%, intermittent stirring mode: stirring at a speed of 25-35 rpm for 10-15 minutes, stopping for 30-40 minutes, stirring cycle times of 1 to 5 times, and amplification culture.

[0019] Cell enzymatic hydrolysis and harvesting: When the cells grow to a sufficient number, remove about 80% of the supernatant from the culture system, add 5× lysis buffer at a mass ratio of 1:5 between the lysis enzyme and the microcarrier, and add physiological saline to make the volume about 40% of the original culture system, so that the concentration of the lysis enzyme is close to 1 mg / ml; lyse at 37°C for 30-40 min, collect the cell suspension after the microcarrier is fully lysed; centrifuge at 1500 rpm for 5 min, remove the supernatant, wash with physiological saline, resuspend by centrifugation 2-3 times, and count; perform continuous cell passage or cell cryopreservation, and sample cells of different generations for cell morphology observation, quantity determination, and cell phenotype detection.

[0020] Flow cytometry phenotyping: Wash the cell pellet twice with DPBS and take 0.8-1×10 6 / 100ul cell suspension was placed in an EP tube, 15ul of primary antibody was added, and no primary antibody was added to the blank tube. The tube was incubated at 4℃ in the dark for 30min, 400ul of DPBS was added, and the tube was centrifuged at 1500rpm for 6min. The supernatant was discarded, 10ul of secondary antibody was added, and the tube was incubated at 4℃ in the dark for 30min. 400ul of DPBS was added, and the tube was centrifuged at 1500rpm for 6min. The supernatant was discarded, 500ul of DPBS was added, mixed, and transferred to the tube on the flow cytometer for detection.

[0021] Reagent configuration:

[0022] Lysis enzyme preparation (5×): Take 0.5 g of lysase, add 100 ml of normal saline to prepare 5 mg / mL 5× lysis solution, filter with 0.22 μm filter, and store in aliquots at -20°C.

[0023] Preparation of reduced glutathione (1000uM): take 1 vial of 600mg reduced glutathione freeze-dried powder, add 19.52ml sterile water to dissolve, shake and mix thoroughly, prepare a 100000uM stock solution, dispense 1ml / vial, take 1ml of the stock solution and add 100ml of normal saline to dilute it to 1000uM, and store it at -20 degrees;

[0024] Coenzyme I for injection: Take a 5mg / ml Coenzyme I for injection and add 2.5ml normal saline to fully dissolve, divide into packages, and store at 4 degrees away from light for later use;

[0025] Comparison of cell morphology under 2D and 3D culture conditions: Figure 1 and Figure 2 (Morphology of cells after digestion). From the figure, we can see that compared with 2D, the cell morphology of 3D culture is more uniform. The average cell diameter is measured to be 15.84um, while the average cell diameter under 2D culture conditions is 16.53um.

[0026] 3D culture cell phenotype detection

[0027] FLK1 CD73 CD90 CD105 CD34 HLA-DR P3 99.87％ 97.64％ 98.42％ 99.18％ 0.21％ 0.16％ P4 98.74％ 98.27％ 96.26％ 98.61％ 0.34％ 0.13％ P5 97.68％ 97.54％ 97.14％ 99.07％ 0.18％ 0.25％

[0028] The results showed that the stem cells expressed FLK1, CD73, CD90, and CD105, but did not express CD34 and HLA-DR.

[0029] Preparation of cell suspension: centrifuge the digested cells at 1200-1500 rpm for 6-8 min, resuspend with physiological saline, wash 2-3 times, pass the cells through a 100-mesh sieve, centrifuge, resuspend the cells at the bottom with matrix solution and set aside.

[0030] Example 2: Preparation of amniotic stem cell preparation, preparation of 100 ml matrix solution: add 1 ml human albumin, 1 ml compound amino acid injection (18AA), 1 ml ginkgo leaf extract injection, 0.1 ml reduced glutathione, and 0.5 ml coenzyme I for injection to 90 ml dextran 40 glucose injection, add dextran 40 glucose injection to 100 ml, put it in a micro oscillator (VORTEX GENIE) and shake it thoroughly at 180 rpm for 30 seconds to mix, put it at 4°C for use, and add 1×10 6 / ml and resuspend in the matrix solution.

[0031] Example 3: Preparation of amniotic stem cell preparation, preparation of 100 ml matrix solution: add 2 ml human albumin, 5 ml compound amino acid injection (18AA), 3 ml ginkgo leaf extract injection, 0.3 ml reduced glutathione, 0.75 ml coenzyme I for injection to 80 ml dextran 40 glucose injection, add dextran 40 glucose injection to 100 ml, put it in a micro oscillator (VORTEX GENIE) and shake it thoroughly at 180 rpm for 30 s-1 min to mix, and mix the amniotic stem cells prepared in Example 1 at 2.5×10 6 / ml and resuspend in the matrix solution.

[0032] Example 4: Preparation of amniotic stem cell preparation, preparation of 100 ml matrix solution: add 3 ml human albumin, 10 ml compound amino acid injection (18AA), 5 ml ginkgo leaf extract injection, 0.5 ml reduced glutathione, 1 ml coenzyme I for injection into 70 ml dextran 40 glucose injection, add dextran 40 glucose injection to 100 ml, put it in a micro oscillator (VORTEX GENIE) and shake it thoroughly at 180 rpm for 30 s-1 min to mix, and mix the amniotic stem cells prepared in Example 1 at 4×10 6 / ml and resuspend in the matrix solution.

[0033] Comparative Example 5: Add human albumin to the dextran 40 glucose injection at a volume ratio of 1-3%, and mix thoroughly to prepare a matrix solution. Place the prepared matrix solution in a 100 ml sterile culture medium bottle and store at 4°C. 6 / ml and resuspend in the matrix solution.

[0034] Comparative Example 6: Matrix solution without stem cells

[0035] The quality evaluation of cells and preparations is shown in the following table:

[0036]

[0037] Cell morphology: A+: round or oval, evenly distributed, with a diameter between 14-20um and good refractive index; there is slight aggregation after standing, and it disperses after shaking. A: round or oval, evenly distributed, with good refractive index; it aggregates when standing, and there are still small clusters after shaking; B: individual cells become larger, the refractive index decreases, the cells become darker under the microscope, and a small number of dead clusters appear after standing for a long time. -: indicates that no cells were detected. After 96 hours of preparation, the cells in the preparations prepared in Examples 3-5 had uniform morphology, strong refractive index, and a cell survival rate higher than 90%, which was significantly higher than that of the control group. The bacteria and fungi tests were negative, and the endotoxin test was less than 0.1EU / ml.

[0038] After 96 hours, the cell flow cytometry phenotype was determined. The cells in Examples 2-4 and Comparative Example 5 were divided into 2.5×10 6 The cell suspension was prepared at a density of 100 cells / ml. After being stored for 96 hours, 1.5 ml of each sample was taken and labeled for flow cytometry phenotyping. The results in the table show that the mesenchymal stem cell-specific surface markers FLK1, CD73, CD90, and CD105 were all higher than 95%, while the negative indicators CD34 and HLA-DR were lower than 2%, indicating that the cells still had good stemness after being stored in the protective agent for 96 hours and had not differentiated. The positive rates of FLK1, CD73, CD90, and CD105 in the control group were lower than 90%, while the negative indicators CD34 and HLA-DR were 5-10%.

[0039] Example FLK1 CD73 CD90 CD105 CD34 HLA-DR 2 95.62％ 95.07％ 95.81％ 95.79％ 1.92％ 0.36％ 3 98.55％ 97.39％ 96.08％ 96.38％ 1.34％ 1.59％ 4 96.83％ 96.28％ 95.64％ 95.01％ 1.89％ 1.73％ 5 85.61％ 88.24％ 81.37％ 78.68％ 6.42％ 8.65％

[0040] Cell adhesion rate after 96h

[0041] Example Paste ratio 2 85.17％ 3 88.69％ 4 84.52％

[0042] Experiment 1: Treatment of premature ovarian failure. 60 mouse models of premature ovarian failure were purchased from the animal center and divided into 6 groups, 10 mice in each group, and the following reagents were administered respectively:

[0043] 1. Inject the stem cell preparation in Example 2;

[0044] 2. Injecting the stem cell preparation in Example 3;

[0045] 3. Injecting the stem cell preparation in Example 4;

[0046] 4. No treatment, normal diet (blank group);

[0047] 5. Injecting the stem cell preparation in Comparative Example 5;

[0048] 6. Inject the matrix solution in Comparative Example 6;

[0049] The injection method uses tail vein injection. During the injection, the mouse is fixed, the tail is pulled taut and straight, and the tail is wiped with an alcohol cotton ball to dilate the tail blood vessels. 0.05-0.1ml of cell suspension is injected with a 1ml syringe at a distance of one-third to one-half of the distance from the tail tip. The injection is repeated the next day, for a total of 2 injections. After the injection is completed, wait for the mouse to recover for 3-5 days before counting the number of follicles and ovulation.

[0050] 1. Follicle counting method: The follicle is the most basic functional unit of the ovary, which is composed of the oocyte and the surrounding granulosa cells and theca cells. In a normally growing follicle, the growth, maturation of the oocyte, the proliferation and development of the granulosa cells and theca cells are harmonious, forming their own follicle units. Follicles are divided into primordial follicles, primary follicles, secondary follicles, antral follicles and mature follicles.

[0051] Primordial follicle: The oocyte is surrounded by only a layer of flat granulosa cells;

[0052] Primary follicle: The oocyte is surrounded by only one layer of cuboidal granulosa cells;

[0053] Secondary follicle: There are 2-3 layers of cubic granulosa cells around the oocyte and no antrum;

[0054] Early antral follicle: Antral cavity appears;

[0055] Preovulatory follicle: It is the largest follicle and has a clearly packed granulosa cell layer.

[0056] Changes in ovulation: One month after the injection, mice in each group were superovulated and their eggs were collected. PMSG was injected intraperitoneally before egg collection, HCG was injected 48 hours later, and mice were killed and their eggs were collected 12-16 hours later. The results of follicle and ovulation counts are shown in Table 1.

[0057]

[0058] Table 1 (average value)

[0059] As can be seen from Table 1, one week after the injection of the preparations of Example 3-5 and Comparative Example 5-6, the number of follicles at all levels increased, but the number of follicles in the group injected with Example 3-5 increased significantly compared with the group injected with Comparative Example 5-6, indicating that the stem cell preparation of Example 3-5 has a better therapeutic effect on premature ovarian failure.

[0060] The number of ovulations was calculated, and it was found that one month after the injection of the preparations of Examples 2-4 and Comparative Examples 5-6, the number of ovulations in the blank group and Comparative Example 6 group was still at a low level, and some eggs showed irregular morphology, with fragments or apoptosis in the cytoplasm; compared with the blank group and Comparative Example 6 group, the number of ovulations in Comparative Example 5 group and Example 2-4 group increased significantly one month after transplantation, but the number of ovulations in Example 2-4 group was more, which was significantly different from that in Comparative Example 5 group, indicating that the stem cell preparation of Example 2-4 has a better therapeutic effect on premature ovarian failure.

[0061] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.

[0062] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A stem cell preparation for treating premature ovarian failure, comprising human amniotic membrane stem cells and a matrix solution, wherein the matrix solution comprises dextran 40 glucose injection, 1-3% human serum albumin (V / V), 1-10% compound amino acid injection (18AA) (V / V), 1-5% ginkgo leaf extract injection (V / V), 1-5uM reduced glutathione (600mg / vial) and 10-20ug / ml coenzyme I for injection (NAD+; 5mg / vial), human amniotic membrane stem cells are cultured on 3D gelatin microcarriers, the cell generation is controlled at P4-6, and the concentration of the matrix solution in the preparation is 1-4×10 6 / ml.

2. A stem cell preparation for treating premature ovarian failure according to claim 1, characterized in that: The dextran 40 glucose injection has a dextran concentration of 6% and a glucose concentration of 5%.

3. The stem cell preparation for treating premature ovarian failure according to claim 1, characterized in that: The optimal concentration of the stem cell preparation is: 2% human albumin (V / V), 5% compound amino acid injection (18AA) (V / V), 3% ginkgo leaf extract injection (V / V), 3uM reduced glutathione, 15ug / ml coenzyme I for injection (NAD+; 5mg / vial), 2.5×10 6 / ml human amniotic stem cells.

4. A method for preparing a stem cell preparation for treating premature ovarian failure, characterized in that: The following steps are involved: Step 1: Prepare 100 ml of matrix solution, add 1-3 ml of human albumin, 1-10 ml of compound amino acid injection (18AA) (v / v), 1-5 ml of ginkgo leaf extract injection, 0.1-0.5 ml of reduced glutathione, 0.5-1 ml of coenzyme I for injection, add dextran 40 glucose injection to 100 ml, put it in a micro-oscillator (VORTEX GENIE) and shake it thoroughly at 180 rpm for 30 seconds to 1 minute to mix, and store it at 4°C; Step 2: Grow P4-P6 cells to a sufficient number, remove about 80% of the supernatant from the culture system, add 5× lysis buffer to make the lysis enzyme concentration close to 1 mg / ml; lyse at 37°C for 30-40 min, collect the cell suspension after the microcarrier is fully lysed; centrifuge at 1500 rpm for 5 min, discard the supernatant, wash with physiological saline, resuspend by centrifugation 2-3 times, count, pass through a 100-mesh sieve, and add human amniotic stem cells to the prepared matrix solution to make the stem cell concentration in the preparation 1-4×10 6 / ml.

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