A mesenchymal stem cell microsphere, its preparation, and application in the preparation of a drug for neurogenic erectile dysfunction

Abstract

The present invention relates to the field of cell culture technology, and in particular to a mesenchymal stem cell microsphere, its preparation and application in the preparation of a neurogenic erectile dysfunction drug. The present invention utilizes a multi-compartment synchronous rotating bioreactor to culture mesenchymal stem cell microspheres in a three-dimensional scale. The method includes culturing cells, preparing three-dimensional cell culture conditions and culturing three-dimensional cells. The method is simple, convenient, economical, and has a high degree of scale. It can also significantly improve work efficiency, reduce the time for exploring optimal conditions during culture, and the method can significantly improve the paracrine function of MSC. Microspheres obtained by the method and their application in the preparation of a neurogenic erectile dysfunction drug are also proposed.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and particularly relates to a mesenchymal stem cell microsphere, its preparation method, and its application in the preparation of drugs for neurogenic erectile dysfunction. Background Art

[0002] Existing studies have shown that culturing MSCs (mesenchymal stem cells) into three-dimensional stem cell microspheres can significantly activate their paracrine function, which is of great significance for improving the therapeutic potential of MSCs. Currently, the classic method for culturing scaffold-free three-dimensional stem cell microspheres is the hanging drop culture method. By dropping the cell suspension onto the bottom of the culture medium, about 30 uL - 50 uL per drop, and inverting it so that the droplet hangs down, the cells form microspheres at the top of the droplet under the action of gravity (see Figure 1 ). This method is extremely cumbersome and time-consuming. Only one microsphere can be formed in each droplet. Generally, only about 20 to 30 droplets can be cultured in a 10 cm diameter cell culture dish, that is, 20 to 30 microspheres are formed. And the preparation time of these droplets takes several minutes, and it takes about several hours to prepare 1000 droplets, with very low efficiency. Moreover, the subsequent recovery of stem cell microspheres is also troublesome. In addition, as a three-dimensional culture conditioned medium rich in cytokines, it has important application value, but the recovery rate is very low. To improve the large-scale culture of stem cell microspheres, some researchers have developed a 3D hanging drop culture plate, which is based on the principle of a conventional 96-well plate (see Figure 2 ). 96 channels for preparing hanging drops are constructed on a substrate similar to a 96-well plate, and batch operations can be carried out with a multi-channel pipette, which improves the number of hanging drop cultures to a certain extent and reduces the cumbersome operation, but there are still obvious problems of cumbersome operation and low efficiency. In the latest research, by changing the design of the hanging drop culture plate and designing a large number of microarray pits on the bottom of the culture plate, the cells settle in the pits to form microspheres. The yield of stem cell microsphere culture is ten times higher than that of the above-mentioned commercial hanging drop culture plate, and the operation is relatively simplified. However, the large-scale level of this culture method is still limited, and both this culture method and the previous methods have the problem that the culture plate cannot be easily moved after cell inoculation, and the culture medium cannot be replaced or recovered in the middle, so as to avoid the aggregation of the formed stem cell microspheres. The corresponding methods can only recover the culture medium and stem cell microspheres once at the end of the culture, and the available cell conditioned medium is very limited. Therefore, developing a method for culturing mesenchymal stem cell microspheres with a higher large-scale culture level, simpler operation, and capable of continuously obtaining a large amount of conditioned medium has become a technical problem that urgently needs to be solved at present. Summary of the Invention

[0003] In view of the deficiencies of the prior art, the present invention provides a mesenchymal stem cell microsphere, its preparation method, and its application in the preparation of drugs for neurogenic erectile dysfunction. The technical solution of the present invention mainly includes: using a multi-compartment synchronous rotation bioreactor for three-dimensional large-scale culture of mesenchymal stem cell microspheres. This method includes culturing cells, preparing three-dimensional cell culture conditions, and culturing three-dimensional cells. It is simple, convenient, economical, and highly scalable. It can also significantly improve work efficiency, reduce the time for exploring the optimal conditions during culture, and can significantly improve the paracrine function of MSCs. Specifically, it includes:

[0004] One of the purposes of the present invention is to provide a method for preparing a mesenchymal stem cell microsphere, including:

[0005] S1. Culturing cells: putting mesenchymal stem cells into a culture dish containing a culture medium, and placing the culture dish in an incubator at 37°C and 5% CO 2 . When the cell density in the culture dish reaches 80%-90%, passage is carried out. After digestion, centrifugation, and resuspension, a cell suspension for three-dimensional cell culture is obtained;

[0006] S2. Preparing three-dimensional cell culture conditions: counting the cell suspension, and adjusting the cell concentration to 0.2×10 5 / ml - 1.2×10 5 / ml;

[0007] S3. Culturing three-dimensional cells: transferring the cell suspension to a low-adhesion cell culture dish, placing the culture dish on a variable-speed three-dimensional rotating shaker with an inclination angle, adjusting the shaker speed to 7 rpm - 30 rpm, and placing it in an incubator for culture. After culture, three-dimensional stem cell microspheres are obtained.

[0008] Further, the preparation process is completed using a multi-compartment synchronous rotation bioreactor. The lower part of the multi-compartment synchronous rotation bioreactor is a fixed-inclination-angle mechanical three-dimensional rotating shaker, and the upper part is a parallel synchronous compartment capable of carrying culture dishes. A protective cover is provided on the outside of the shaker.

[0009] Further, the culture medium is DMEM culture medium containing 5%-15% fetal bovine serum by volume percentage.

[0010] Further, in S2, the cell concentration is preferably 0.6×10 5 / ml - 1.0×10 5 / ml.

[0011] Further, mesenchymal stem cell microspheres of different specifications can be prepared according to the cell concentration.

[0012] Furthermore, the preferred shaker speed is 8 rpm - 20 rpm; the culture time is 24 h - 72 h.

[0013] Furthermore, the culture dish is a cell culture dish with a diameter of 35 mm - 150 mm.

[0014] Furthermore, the preparation method is a scaffold-free three-dimensional large-scale culture method for mesenchymal stem cell microspheres.

[0015] The second object of the present invention is to provide a mesenchymal stem cell microsphere, comprising:

[0016] The mesenchymal stem cell microsphere is prepared according to the preparation method of the mesenchymal stem cell microsphere.

[0017] The third object of the present invention is to provide an application of a mesenchymal stem cell microsphere in the preparation of a drug for neurogenic erectile dysfunction, comprising:

[0018] The microsphere is prepared according to the preparation method of the mesenchymal stem cell microsphere, or is the microsphere.

[0019] Compared with the prior art, the present invention provides a mesenchymal stem cell microsphere and its preparation and application in the preparation of a drug for neurogenic erectile dysfunction, having the following beneficial effects:

[0020] 1. The present invention proposes a method for scaffold-free three-dimensional large-scale culture of mesenchymal stem cell microspheres, which can simply and large-scale perform three-dimensional culture of mesenchymal stem cells; at the same time, the culture obtained by this method has high viability.

[0021] 2. The present invention provides a model for research in the field of scaffold-free three-dimensional cell culture. The model in the present invention is not only simple, convenient, economical and highly scalable, but also can significantly improve work efficiency and reduce the time for exploring the optimal conditions during culture; and this method can significantly improve the paracrine function of MSCs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shows a schematic diagram of the hanging drop culture method in the prior art, wherein, Figure 1 A is a side schematic diagram of the hanging drop culture method; Figure 1 B is a top view and enlarged view of the hanging drop culture method (scale = 500 μm);

[0023] Figure 2 Shows a schematic diagram of the 3D suspension culture plate method in the prior art;

[0024] Figure 3 Shows a schematic diagram of a multi-compartment synchronous rotating bioreactor according to an embodiment of the present invention;

[0025] Figure 4 The top view and side view of a low - adhesion cell culture dish for dynamically culturing cell microspheres according to an embodiment of the present invention are shown;

[0026] Figure 5 A diagram showing the three - dimensional shaker culture of adipose - derived MSCs according to an embodiment of the present invention is shown; wherein, Figure 5 Condition A is 8 rpm, 1×10 5 / ml, 48 h; Figure 5 Condition B is 12 rpm, 1×10 5 / ml, 48 h; Figure 5 Condition C is 16 rpm, 1×10 5 / ml, 48 h; Figure 5 Condition D is 20 rpm, 1×10 5 / ml, 48 h;

[0027] Figure 6 A diagram showing the comparison of cytokine gene expression between three - dimensional shaker - cultured adipose MSC microspheres and 2D - cultured MSCs according to an embodiment of the present invention is shown; wherein, Figure 6 A is a comparison experimental diagram of cell seeding concentration and culture time during the culture process; Figure 6 B is a comparison experimental diagram of shaker rotation speed during the culture process;

[0028] Figure 7 A diagram showing the use of conditioned medium for scratch experiments on HUVECs according to an embodiment of the present invention is shown; wherein, Figure 7 A is HUVEC medium; Figure 7 B is 50% HUVEC medium + 50% conditioned medium of ADSC 2D - cultured for 48 h; Figure 7 C is 50% HUVEC medium + 50% conditioned medium of ADSC shaker - cultured at 1×10 5 / ml for 48 h; Figure 7 D is a comparison diagram of conditions A, B, and C (**, compared with group A, P < 0.01; &: compared with group B, P < 0.05);

[0029] Figure 8 A diagram showing the determination of the use of the microspheres prepared in the present invention for the treatment of neurogenic erectile dysfunction (ED) in SD rats is shown; wherein, the ICPmax / MAP ratio of the rat penis under 5V electrical stimulation (n = 6); Figure 8 A is a representative original record of ICP, MAP, and ICP / MAP; Figure 8 B is a statistical diagram of the changes in ICPmax / MAP of the sham - operation group (Sham), nerve - injury group (BCNI), adipose - stem - cell treatment group (T1), and adipose - stem - cell microsphere treatment group (T2) after 5V electrical stimulation. Each bar represents (Compared with the nerve injury group, **: P < 0.01; compared with the adipose stem cell group, #: P < 0.01);

[0030] Figure 9 The pathological detection diagrams of rats in each group after microsphere treatment according to an embodiment of the present invention are shown; among them, Figure 9 A shows the results of the smooth muscle / collagen ratio of the corpus cavernosum in the sham operation group (Sham), nerve injury group (BCNI), adipose stem cell group (T1), and adipose stem cell microsphere group (T2) in Masson trichrome staining, scale bar = 500 μm; Figure 9 B shows the statistical data analysis of the smooth muscle / collagen tissue ratio in different groups, and each bar represents Figure 9 C shows the staining results of smooth muscle / corpus cavernosum tissue in rats in the sham operation group (Sham), nerve injury group (BCNI), adipose stem cell group (T1), and adipose stem cell microsphere group (T2) in immunofluorescence staining. The upper part is smooth muscle cells, the middle part is the nuclear staining of the same part, and the lower part is the corresponding composite picture, scale bar = 100 μm; Figure 9 D shows the statistical situation of the staining of smooth muscle / corpus cavernosum tissue in rats in different groups (n = 6), and each bar represents Figure 9 E shows the staining results of smooth muscle / corpus cavernosum tissue in rats in the sham operation group (Sham), nerve injury group (BCNI), adipose stem cell group (T1), and adipose stem cell microsphere group (T2) in immunohistochemical staining, scale bar = 100 μm; Figure 9 F shows the statistical data analysis diagram of the smooth muscle / corpus cavernosum ratio in different groups, and each bar represents (Compared with the nerve injury group, **: P < 0.01, *: P < 0.05; compared with the adipose stem cell group, #: P < 0.01, &: P < 0.05). Detailed implementation manners

[0031] To better understand the present invention, specific embodiments will be given to further illustrate the present invention. However, it should be understood that the described embodiments are exemplary embodiments, and the present invention can be implemented in various forms and should not be limited by the embodiments described here. On the contrary, these embodiments are provided to more thoroughly understand the present invention and to be able to fully convey the scope of the present invention to those skilled in the art.

[0032] Note that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular forms also include the plural forms. In addition, it should also be understood that when the terms "comprise" and / or "include" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0033] In the embodiments of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art, and the reagents and materials in the present invention are obtained from the market or other public channels.

[0034] Reagents: DMEM-basic basal medium (Wuhan Punosai Co., Ltd.); fetal bovine serum, 1% penicillin-streptomycin double antibody, 0.25% trypsin digestion solution (Biological Industries, Israel); phosphate buffer solution (PBS, Gibco, USA); Masson trichrome staining solution (Solarbio, Beijing); α-smooth muscle actin (α-SMA, rabbit polyclonal IgG antibody), Alexa Fluor-594 goat anti-rabbit fluorescent secondary antibody (Abcam, USA); Hoechst 33342 (Solarbio, Beijing); anti-fluorescence quenching mounting medium (Beyotime, Beijing); SP immunohistochemistry kit, concentrated DAB kit (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing); 4% paraformaldehyde fixative (Wuhan Sevier Biotechnology Co., Ltd.).

[0035] Instruments: MP150 multi-channel physiological instrument (Biopac, USA); biological safety cabinet, cell culture incubator (Thermo, USA); laser confocal microscope (Nikon, Japan); ordinary PCR instrument (Bio-Rad, USA); fluorescence quantitative PCR instrument (Applied Biosystem, USA).

[0036] Organisms: Adipose-derived stem cells (rADSCs) were isolated from the periepididymal adipose tissue of rats according to the method in the reference. rADSCs were cultured in DMEM-basic basal medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibody (Pen-Strep Solution). Human umbilical vein endothelial cells (HUVEC) were cultured in DMEM-high glucose medium containing 10% FBS and 1% double antibody at 37°C and 5% CO2 Cultured in an incubator.

[0037] The present invention provides a three-dimensional large-scale culture method for mesenchymal stem cell microspheres without a scaffold, including:

[0038] S1. Cell culture: Put mesenchymal stem cells into a culture dish containing a culture medium, and place the culture dish in an incubator at 37°C and 5% CO 2 . When the cell density in the culture dish reaches 80 - 90%, passage is carried out. After digestion, centrifugation, and resuspension, a cell suspension for three-dimensional cell culture is obtained;

[0039] S2. Preparation for three-dimensional cell culture: Count the cell suspension obtained in S1, and adjust the final cell concentration to 2.0×10 4 -1.2×10 5 / ml;

[0040] S3. Three-dimensional cell culture: Transfer the cell suspension to a low-adhesion cell culture dish, place it on a variable-speed three-dimensional rotating shaker with an inclination angle, adjust the shaker speed to 8 rpm - 16 rpm, and place it in an incubator for culture. After culture, three-dimensional stem cell microspheres are obtained.

[0041] Example 1

[0042] The present invention proposes a multi-compartment synchronous rotating bioreactor.

[0043] See Figure 3 , the present invention prepares a multi-compartment synchronous rotating bioreactor. The lower part of the reactor is a fixed-inclination-angle mechanical three-dimensional rotating shaker with an inclination angle of 7°. The upper part is a parallel synchronous compartment capable of carrying culture dishes. As Figure 3 shows four different frontal views when the shaker rotates. A protective cover is provided on the outside of the shaker; see Figure 4 , the culture dish in the present invention is a low-adhesion cell culture dish for dynamically culturing cell microspheres, and can be a commercial low-adhesion culture dish, a glass culture dish, or a conventional culture dish coated with agarose on the surface. The culture dish is a different-type cell culture dish with a diameter of 35 mm - 150 mm, and the characteristic of this dish is low adhesion; the rotating shaker can be adjusted in speed from 7.0 rpm to 30.0 rpm. By adjusting the cell suspension density and speed, stem cell microspheres with relatively uniform sizes can be formed.

[0044] Results: The design of multiple compartments not only has a high degree of scalability, greatly improving work efficiency and reducing the time for exploring optimal conditions during cultivation, but also significantly enhancing the paracrine function of MSCs. The design of the protective cover reduces the interference of the external environment on microsphere formation. The low-adhesion cell culture dish used for dynamically culturing cell microspheres ensures that MSCs do not adhere during dynamic cultivation on a shaker, and the low-adhesion cell culture dish used for dynamically culturing cell microspheres is a common bacterial culture dish, which is easily obtained. Below the reactor is a fixed-inclination-angle mechanical three-dimensional rotating shaker. Due to the fixed inclination angle, the three-dimensional rotating shaker can reduce the adhesion phenomenon of adipose stem cells due to their own gravity, keep the cells and the cell microspheres formed over time in a continuous passive motion state, and form microspheres relying on the centrifugal force given by the shaker and the cell-cell adhesion force. Further, comparing from no inclination angle to a 15° inclination angle, a 7° - 15° inclination angle is a more suitable inclination angle condition for culturing adipose stem cells. When the angle is large, the culture medium is unstable and easy to shake; when the angle is small, the stirring effect is poor and the cells cannot be fully contacted with the culture medium. Setting parallel synchronous compartments that can carry culture dishes can achieve simple, convenient, economical, and large-scale culturing of mesenchymal stem cells, and further improve work efficiency. This reactor can batch-produce conditioned medium for the enrichment of related factors and the preparation of various other biological agents.

[0045] Example 2

[0046] The present invention proposes an experimental comparison of data on different cell spheroid formation methods.

[0047] By using the multi-compartment synchronous rotation bioreactor of the present invention, the differences and effects of different cell spheroid formation methods in terms of sample loading time, MSC microsphere yield, culture medium acquisition, and cell paracrine are compared.

[0048] Results: As shown in Table 1, the sample loading time of the present invention is shorter, the MSC microsphere yield is significantly improved compared with previous methods, the culture medium acquisition amount has also increased exponentially, and at the same time, while significantly enhancing cell paracrine, the present invention can obtain more cell microspheres and conditioned medium; the predicted results using the prediction model in the present invention have little difference from the measured values, can better predict the activity value, and using this prediction model can reduce the determination experiments of parameters in the same experiment, reduce repetitive labor, and improve the culture efficiency.

[0049] Table 1. List of data comparison of different cell spheroid formation methods

[0050] Different methods Loading time MSC microsphere yield Culture medium acquisition Cell paracrine Hanging drop 2 - 3 min 20-40 ~1 mL Significantly up - regulated Hanging drop plate 1 - 2 min 96 ~3 - 4 mL Significantly up - regulated Microarray plate 0.5 - 1 min 960 40 - 50 mL Significantly up - regulated The present invention 0.5 - 1 min 2000～3000 10 mL×n Significantly up - regulated

[0051] Example 3

[0052] The present invention proposes an experimental study on the spheroid formation of adipose stem cells at different rotation speeds on a three-dimensional rotating shaker.

[0053] Using the three-dimensional rotary shaker of the present invention, adipose-derived mesenchymal stem cells are cultured in the three-dimensional shaker (the culture medium is DMEM medium containing 5%-15% fetal bovine serum, and the optimal concentration in the experiment is the medium containing 10% fetal bovine serum, which is a commonly used concentration for cell culture). The selectable rotation speed range of the shaker is 7 rpm - 30 rpm. In the experiment, rotation speeds of 8 rpm, 12 rpm, 16 rpm, and 20 rpm are respectively selected. The results show that too low or too high speeds are not conducive to microsphere formation. Therefore, the optimal speed in the experiment is 12 rpm. For the cell concentration experiment, concentrations of 0.2×10 5 / mL - 1.2×10 5 / mL can be used. If the concentration is too low, fewer spheres are formed. If the concentration is too high, the cell microspheres will also aggregate with each other to form super microspheres, which is not conducive to subsequent utilization. The optimal concentration in this experiment is 1×10 5 / mL, with more cell microspheres obtained and significant paracrine. The culture time is 48 h, and the spheroid formation of the cells after culture is observed; different-sized microspheres are prepared according to different densities, and conditioned medium is obtained in batches using a multi-compartment synchronous rotation bioreactor at the same time.

[0054] Results: See Figure 5 A - Figure 5 D. Adipose stem cells can form spheres well at different rotation speeds of the three-dimensional rotary shaker, and the spheroid formation is good at 48 h; see Figure 1 the hanging drop mode diagram of A and Figure 1 the actual hanging drop diagram of B. The operation time of the ordinary hanging drop is long, the technical requirements for inverting the culture dish lid are high, and there is more loss of microspheres obtained. Compared with the ordinary hanging drop, the operation of the present invention has a short time, only requires cell culture technology, and has less loss of microspheres obtained. See Figure 2 , this method for culturing cell microspheres consumes a large amount of consumables, has a high cost, and has a small number of spheres formed. The present invention is faster / more convenient. Figure 5 Photographed at 50×.

[0055] Example 4

[0056] The present invention proposes an experimental comparison of the cytokine gene expression between adipose MSC microspheres cultured by a three-dimensional shaker and MSC cultured in 2D.

[0057] After obtaining the cell microspheres, TRIzol method was used to extract cell RNA and measure the RNA concentration. The total RNA was reverse transcribed using a reverse transcription kit (TransGen Biotech) and reverse transcribed by a PCR instrument (55 °C for 5 min, 85 °C for 5 s, stored in a -20 °C refrigerator); SYBR Green fluorescent dye method (GenStar) was used to detect vascular endothelial growth factor (VEGF), placenta growth factor (PLGF), hepatocyte growth factor (HGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and nerve growth factor. There were 3 replicates for each sample, and a melting curve was obtained using a q-PCR instrument. With β-actin as the internal reference, the relative mRNA content was calculated. The nucleotide primer sequences of different genes are shown in Table 2, and the nucleotide sequence numbers are SEQ ID NO:1 to SEQ ID NO:14.

[0058] Table 2. Nucleotide primer sequences of different genes

[0059]

[0060]

[0061] Results: From Figure 5 、 Figure 6 A and Figure 6 B, it can be seen that the optimal conditions for cell seeding concentration, culture time, and rotation speed during the culture process are 1.0×10 5 / ml, 48 h, and 12 rpm respectively.

[0062] Example 5

[0063] The present invention proposes a conditioned medium for use in a scratch assay for HUVEC.

[0064] For the scratch assay of HUVEC using the conditioned medium, HUVEC medium, 50% HUVEC medium + 50% ADSC 2D culture for 48 h conditioned medium, and 50% HUVEC medium + 50% ADSC 1×10 5 / ml shaker culture for 48 h conditioned medium were respectively selected to analyze and compare the differences between different media.

[0065] Results: Refer to Figure 7 A to Figure 7 D, the best effect was obtained by culturing in a shaker for 48 h in 50% HUVEC medium + 50% ADSC 1×10 5 / ml conditioned medium; three-dimensional cell culture by this method can significantly reduce the operation time of three-dimensional culture, reduce the time cost, and without special culture dishes, reduce the production cost, and can scale up three-dimensional cell culture and improve the yield.

[0066] Example 6

[0067] The present invention proposes a preparation of microspheres for the determination of neurogenic erectile dysfunction (ED) in SD rats.

[0068] The microspheres with scaled-up three-dimensional culture were used to verify in rats with neurogenic erectile dysfunction. After 28 days of treatment, the erectile function of each group of rats was detected. The rats were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (30 mg / kg). Prepare 2 24G puncture needles, filled with 250 IU / ml heparin, and one end is connected to a multi-channel electrophysiological instrument (MP150) through a PE-50 tube; the right common carotid artery was dissected and a puncture needle was inserted to monitor the mean arterial pressure (MAP) of the rats; the skin at the penis was incised to fully expose the corpus cavernosum of the rats, and another puncture needle was inserted into the sinus of the corpus cavernosum; laparotomy was performed and the cavernous nerve located posterior and lateral to the prostate was found, and penile erection was induced by electrical stimulation of the cavernous nerve. The stimulation parameters were: 5 V, 1.5 mA, 20 Hz, pulse width 1 ms, duration 60 s, and the maximum value of the intracavernous pressure (ICP) and MAP were recorded. The penile erectile function of the rats was evaluated by comparing the ICPmax / MAP of each group.

[0069] Results: Refer to Figure 8 A and Figure 8 B. Compared with the simple adipose stem cell treatment group (T1), the adipose stem cell microsphere treatment group (T2) had better treatment effects.

[0070] Example 7

[0071] The present invention proposes a pathological detection experiment of each group of rats after treatment with mesenchymal stem cell microspheres.

[0072] Pathological histological analysis was performed on each group of rats. For Masson staining, the middle segment tissue of the rat penis was taken, fixed with 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned at a thickness of 5 μm; staining was performed according to the instructions of the Masson trichrome staining kit, and sealed with neutral gum; images were observed and collected by an optical microscope, and analyzed and processed with Image J. For immunofluorescence staining, the rat penis tissue fixed with 4% paraformaldehyde was dehydrated with 10%, 20%, and 30% sucrose solutions for 12 h each; frozen sections (5 μm) were made after embedding in OCT; placed at room temperature for 30 min, rinsed 3 times with PBS, 5 min each time; perforated with 0.3% Triton for 15 min, rinsed 2 times with PBS, 5 min each time, and blocked with 5% BSA for 1 h; incubated with primary antibody α-SMA (1:300) overnight at 4 °C; rinsed 2 times with PBS, 5 min each time, incubated with fluorescently labeled secondary antibody (both 1:500) for 1 h in the dark, rinsed 2 times with PBS, 5 min each time; stained with Hoechst33342 (1:5000) for 10 min; rinsed 2 times with PBS, 5 min each time; sealed with an anti-fluorescence quenching mounting medium, and photographed and collected images under a laser confocal microscope after drying at room temperature. For immunohistochemical staining, paraffin sections of the middle segment tissue of the penis were baked at 65 °C for 60 min, and detected by immunohistochemical SP method according to the instructions after dewaxing. After the slides were air-dried, images were collected with an optical microscope, and the distribution of penile smooth muscle was evaluated using the smooth muscle / corpus cavernosum ratio.

[0073] Results: See Figure 9 A~ Figure 9 F. Compared with the simple adipose stem cell treatment group (T1), the adipose stem cell microsphere treatment group (T2) had a more significant expression level of the corpus cavernosum smooth muscle content in rats.

[0074] The above are only examples of the present invention and are not used to limit the present invention. For those skilled in the art, various changes and modifications can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the scope of the claims of this application.

Claims

1. A method for preparing mesenchymal stem cell microspheres, characterized in that, the preparation method includes: S1. Culture cells. Place mesenchymal stem cells in a culture dish containing a culture medium, and place the culture dish in an incubator at 37 °C and 5% CO 2 . Culture. When the cell density in the culture dish reaches 80%-90%, passage the cells. After digestion, centrifugation, and resuspension, obtain a cell suspension for three-dimensional cell culture; S2. Prepare three-dimensional cell culture conditions, count the cell suspension, and adjust the cell concentration to 2.0×10 4 / ml - 1.2×10 5 / ml; S3. Culturing three-dimensional cells, transferring the cell suspension to a low-adhesion cell culture dish, placing the culture dish on a variable-speed three-dimensional rotary shaker with an inclination angle, adjusting the shaker speed to 7 rpm - 30 rpm, and placing it in an incubator for culturing to obtain three-dimensional stem cell microspheres after culturing; the preparation method uses a multi-compartment synchronous rotary bioreactor, and the multi-compartment synchronous rotary bioreactor can batch obtain conditioned medium; a fixed-inclination-angle mechanical three-dimensional rotary shaker is provided below the multi-compartment synchronous rotary bioreactor, and the inclination angle is 7°; a parallel synchronous compartment capable of carrying a culture dish is provided above the multi-compartment synchronous rotary bioreactor; a protective cover is provided outside the fixed-inclination-angle mechanical three-dimensional rotary shaker; the culture dish is a low-adhesion cell culture dish for dynamically culturing cell microspheres; the time for transferring the cell suspension to the low-adhesion cell culture dish is the loading time; the loading time is 0.5 min to 1.0 min.

2. The method for preparing mesenchymal stem cell microspheres according to claim 1, characterized in that, the culture medium is DMEM culture medium with 5% - 15% fetal bovine serum by volume percentage.

3. The method for preparing mesenchymal stem cell microspheres according to claim 1, characterized in that, In S2, the cell concentration is 0.6×10 5 / ml - 1.0×10 5 / ml.

4. The method for preparing mesenchymal stem cell microspheres according to claim 1, characterized in that, mesenchymal stem cell microspheres of different specifications can be prepared according to the cell concentration.

5. The method for preparing mesenchymal stem cell microspheres according to claim 1, characterized in that, the shaker speed is 8 rpm - 20 rpm; the culture time is 24 h - 72 h.

6. The method for preparing mesenchymal stem cell microspheres according to claim 1, characterized in that, the diameter of the culture dish is a cell culture dish with a diameter of 35 mm - 150 mm.

7. The method for preparing mesenchymal stem cell microspheres according to any one of claims 1 to 6, characterized in that, the preparation method is a scaffold-free three-dimensional large-scale culture method for mesenchymal stem cell microspheres.

Images