Method for promoting differentiation of primary neural stem cells based on ultrasonic patterning

Abstract

The invention discloses a method for promoting differentiation of primary neural stem cells based on ultrasonic patterning. Putting the cell slide into a cell culture dish, adding a poly-D-lysine solution to immerse the cell slide, preserving in a dark place at room temperature, completely absorbing the solution, cleaning the slide, and airing for later use; placing the cell culture dish in a sound field generator in an ultrasonic device, and adding sterile water into a gap area; emitting ultrasonic waves by using an ultrasonic device, and adding the cell suspension containing the primary neural stem cells into a cell culture dish; and stopping emitting the ultrasonic waves, transferring the cell culture dish into an incubator for culturing, and then replacing the culture medium for culturing to obtain mature nerve cells. According to the method, the one-dimensional ultrasonic standing wave field is applied in the early stage of the cell culture process to remarkably promote the differentiation rate of in-vitro culture of the neural stem cells, the spatial arrangement of the primary neural stem cells is accurately controlled by adopting a non-contact method, ultrasonic field patterning is realized, damage to the cells is reduced, and operation safety and cell activity are ensured.

Description

Technical Field

[0001] The present invention belongs to the field of nerve cell culture, and particularly relates to a method for promoting the differentiation of primary neural stem cells based on ultrasonic patterning. Background Art

[0002] Neural stem cells (NSCs) are the sources of neurons, oligodendrocytes, and astrocytes in the nervous system and have extremely high differentiation potential. Most nerve cells are formed by the differentiation of neural stem cells during development and are responsible for sensing stimuli and conducting nerve impulses, playing an important role in the development and repair of the nervous system.

[0003] Currently, in the field of nerve cell research, the differentiation process of in vitro neural stem cells usually relies on methods such as biologic agent stimulation and the addition of pro-differentiation factors. However, this process often takes weeks or even months, and due to delays and complexities, it greatly limits the yield of neuron populations available for treatment, thus restricting the treatment effect. For example, Chinese patent document CN116536261A discloses a nerve cell growth and differentiation promoter, which contains cistanche peptide, xylooligosaccharide, and Jerusalem artichoke extract, and can effectively improve nerve cell viability and promote the differentiation of axons similar to neurons at specific concentrations. However, this method is only applicable to human neuroblastoma SH-SY5Y cells and lacks universality. Another patent CN118389434A discloses a method for promoting nerve cell growth based on magnetic field synergistic cell protectant, in which the NR2B9c protectant can not only protect nerve cells but also promote their growth under a 50Hz magnetic field. However, this method requires precise control of the dose and action time and is only applicable to the magnetic field environment, with certain application limitations.

[0004] Cell patterning technology, as a rapidly developing field, has achieved precise cell alignment on different substrates through techniques such as lithography and 3D bioprinting, and has been widely applied in fields such as stem cell therapy and tissue engineering. However, this technology still faces challenges. For example, the multi-scale patterning method based on lithography technology disclosed in Chinese patent document CN119064264A can quickly and accurately measure the single-cell adhesion force between cells and the substrate. However, the hydrophobic modification and antibody modification in the pretreatment steps may cause damage to cells and affect the natural behavior of cells. In addition, the culture chip based on the open-chamber patterned array microfluidics proposed in Chinese patent document CN118440822A promotes the uniform distribution of cells and the formation of consistent spheres. However, the patterning type is single, mainly limited to spherical structures, and its applicability in tissue engineering is low, making it difficult to achieve fine positioning or directional alignment of cells. Therefore, although cell patterning technology provides a powerful tool for cell behavior research and tissue construction, it still needs to be further optimized to reduce potential damage to cells and expand the diversity and precision of patterning to meet the more complex needs of tissue engineering. Summary of the Invention

[0005] In view of the deficiencies and drawbacks in the above background art, the present invention provides a method for promoting the differentiation of primary neural stem cells based on ultrasonic patterning.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A method for promoting the differentiation of primary neural stem cells based on ultrasonic patterning includes the following steps:

[0008] Step S1: In a laminar flow hood, place a cell slide into a cell culture dish, add a poly-D-lysine solution to submerge the cell slide, store it in the dark at room temperature, then manually aspirate the poly-D-lysine solution and then wash the cell slide twice with sterile water, and dry the cell slide in the cell culture dish.

[0009] Before step S1, the cell slide and the cell culture dish need to be sterilized. The sterilization steps are as follows: Place the clean cell slide and cell culture dish in an autoclave for high-temperature and high-pressure sterilization, dry them in an oven at 60 °C, and then take them into the laminar flow hood for ultraviolet irradiation.

[0010] Step S2: In a laminar flow hood, place the cell culture dish containing the cell slide in the main chamber of an ultrasonic device, and then add sterile water to the gap area between the side wall of the main chamber of the ultrasonic device and the cell culture dish.

[0011] Before step S2, the sound field generator in the ultrasonic device needs to be disinfected and sterilized. The disinfection and sterilization steps are as follows: Sprinkle alcohol on the surface of the sound field generator and then take it into the laminar flow hood for ultraviolet irradiation.

[0012] Step S3: Use an ultrasonic device to emit ultrasonic waves, and make the emitted ultrasonic waves form a standing wave field in the cell culture dish for patterning and promoting the differentiation of primary neural stem cells.

[0013] Ultrasonic standing wave field patterning is a non-contact cell manipulation technology based on the principle of acoustofluidics. It refers to a technology that forms a stable standing wave field in the culture medium by emitting ultrasonic waves of a specific frequency, and uses the acoustic radiation force to drive cells to migrate directionally to the nodes or antinodes of the standing wave, thus forming a periodic one-dimensional linear arrangement.

[0014] Among them, the standing wave field is formed by the superposition of two ultrasonic waves with the same frequency but opposite directions, and has the characteristics of periodic changes in spatial amplitude, fixed positions of nodes (minimum amplitude) and antinodes (maximum amplitude). Since the cell density is slightly greater than that of the liquid culture medium, when the cells are in the standing wave field, the acoustic radiation force generated by the ultrasonic waves will push the cells to gather at the nodes of the standing wave, thereby making the originally disordered cells form a periodic one-dimensional linear cell array, that is, patterning.

[0015] Step S4: Suspend the isolated primary neural stem cells obtained by extraction and treatment from rat embryos in a culture medium to obtain a cell suspension, and then add the cell suspension to the cell culture dish.

[0016] The primary neural stem cells can usually be obtained by extraction and treatment from rat embryos.

[0017] Step S5: The ultrasonic device stops emitting ultrasonic waves, and the primary neural stem cells arranged in a one-dimensional linear array are obtained and the cell culture dish is transferred to a carbon dioxide cell incubator at 37 °C and a CO2 concentration of 5% for static culture. Subsequently, the culture medium is replaced with a neural cell differentiation medium, and mature neural cells are cultured, that is, promoting the differentiation of primary neural stem cells into mature neural cells.

[0018] The sterile water is obtained by sterilizing ultrapure water in an autoclave.

[0019] In step S1, the concentration of the poly-D-lysine solution is 80 - 120 ug / mL.

[0020] In step S1, the light-shielded storage time is at least 2 hours or overnight.

[0021] The ultrasonic device in step S2 mainly consists of an acoustic field generator, a piezoelectric ceramic, four L-shaped limiting brackets, a signal generator, and a power amplifier.

[0022] In the middle of the upper surface of the sound field generator, there is a chamber vertically penetrating up and down. The chamber is mainly composed of a main chamber, a central chamber, and a glass cover slip mounting groove opened in sequence from top to bottom. The main chamber, the central chamber, and the glass cover slip mounting groove are interconnected. A glass cover slip is installed in the glass cover slip mounting groove. Both the main chamber and the central chamber are square cavities. Four L-shaped limiting brackets are integrally formed at the four corners of the main chamber. The outer corner parts of the four L-shaped limiting brackets are respectively attached to the four corners of the main chamber for horizontal positioning of the cell culture dish. The cell culture dish is placed in the main chamber, and the outer side wall of the cell culture dish is in contact connection with all four L-shaped limiting brackets. The lower surface of the cell culture dish is in contact connection with the step surface between the main chamber and the central chamber. The sound field generator is placed on the microscope stage to observe the cell arrangement state in the cell culture dish and prevent the sterile water added to the gap between the cavity and the cell culture dish from leaking.

[0023] On the upper surface of the sound field generator, there are four rectangular piezoelectric ceramic grooves. The piezoelectric ceramic grooves are symmetrically distributed around the main chamber. A piezoelectric ceramic is attached to the side wall of each piezoelectric ceramic groove close to the chamber. Both poles of each piezoelectric ceramic are connected to a signal generator and a power amplifier.

[0024] The signal generator is used to generate a sine wave signal, and the power amplifier is used to amplify the sine wave signal of the signal generator and act on the piezoelectric ceramic, so that two non-adjacent piezoelectric ceramics generate ultrasonic waves.

[0025] The specific content of step S2 is: In the ultra-clean bench, place the cell culture dish with a cell slide in the main chamber of the sound field generator, and then add sterile water to the gap area between the side wall of the main chamber of the sound field generator and the cell culture dish.

[0026] The gap area between the side wall of the main chamber of the ultrasonic device and the cell culture dish is specifically the area surrounded by two adjacent L-shaped limiting brackets, the side wall of the main chamber, and the side wall of the cell culture dish.

[0027] The specific operation of generating ultrasonic waves by the ultrasonic device in step S3 is: The two relatively arranged piezoelectric ceramics form a group of piezoelectric ceramic groups. Therefore, the ultrasonic device has a total of two groups of piezoelectric ceramic groups. The two piezoelectric ceramics in the same group have the same working frequency, and the piezoelectric ceramics in different groups have different working frequencies.

[0028] The signal generator generates two groups of sine wave signals respectively with the same working frequencies as the piezoelectric ceramics in the two groups of piezoelectric ceramic groups, and then after being amplified by the power amplifier, they are input to the piezoelectric ceramic group with the same working frequency as itself. The two groups of piezoelectric ceramic groups, that is, the piezoelectric ceramics are paired and configured to generate ultrasonic waves through the drive of the sine wave signal and act on the cell culture dish. Ultrasonic waves form a standing wave field in the cell culture dish for patterning and promoting the differentiation of primary neural stem cells.

[0029] The working frequency of the sine wave signal generated by the signal generator is 3.0 - 3.5 MHz, the peak-to-peak voltage is 10 - 12 Vpp, and the gain of the power amplifier is 10 - 12 dB.

[0030] In step S4, the ultrasonic device stops emitting ultrasonic waves specifically as follows: Starting from the moment when the cell suspension is added to the cell culture dish, the power amplifier and the signal generator in the ultrasonic device act together for 12 - 15 minutes, and then only the signal generator acts for 55 - 65 minutes before the ultrasonic device stops emitting ultrasonic waves.

[0031] The culture medium in step S3 is mainly obtained by adding fetal bovine serum (FBS), horse serum (HS), and glutamine (Glutamine) to DMEM medium. The volume fraction of the fetal bovine serum is 10%, the volume fraction of the horse serum is 5%, and the concentration of glutamine is 2 mM;

[0032] The neural cell differentiation medium in step S4 is mainly obtained by adding B-27 additive, N-2 additive, and glutamine to Neurobasal medium. The volume fraction of the B-27 additive is 2%, the volume fraction of the N-2 additive is 1%, and the concentration of glutamine is 2 mM.

[0033] In step S3, the cell density in the cell suspension is 80 - 120 w / mL, and the volume of the cell suspension is 250 - 300 μL.

[0034] In step S4, after static culture in a carbon dioxide cell incubator for 24 h, the culture medium is replaced with a neural cell differentiation medium, and mature neural cells are obtained after culturing for 10 - 14 days.

[0035] Neural stem cells are different from ordinary cells and are not easy to adhere and grow. The substrate used needs to be modified in advance. The poly-D-lysine used is a positively charged natural polymer, which can form a polyelectrolyte in water. By coating the cell slide with a poly-D-lysine solution (PDL solution), positive charges are introduced onto the surface of the slide. During the process of the neural stem cells adhering to the cell slide, the positive charges interact with the negative charges on the cell surface to form an electrostatic adsorption force. This electrostatic adsorption force can cause an adhesion effect between the neural stem cells and the slide, significantly improving the adhesion effect of the neural stem cells.

[0036] After the cell culture dish is placed in the acoustic field generator chamber, the four walls of the culture dish are parallel to the four walls of the chamber and there are gaps. The gaps between the inner wall of the chamber and the outer wall of the culture dish are filled with sterile water to ensure normal ultrasonic wave conduction.

[0037] In the present invention, four piezoelectric ceramics are provided. Two opposite piezoelectric ceramics receive signals emitted by a signal generator (a power amplifier can be connected as required) to form a standing wave field in a cell culture dish, realizing a one-dimensional ultrasonic sound field. When the signal amplitude is maximum, neural stem cells form a one-dimensional linear array parallel to the x-axis or y-axis in the xoy plane of the culture dish, triggering the cells to form different contact orientations, and the cells on the array show a morphology arranged along the array direction. In addition, the array realizes focused adhesion among neural stem cells in a local area, and this phenomenon significantly promotes the differentiation of neural stem cells. The xoy plane of the culture dish is parallel to the cross-section of the ultrasonic device, where the x-axis is parallel to the long side of the ultrasonic device and the y-axis is parallel to the short side of the ultrasonic device.

[0038] Aiming at the deficiencies of existing traditional methods for promoting the differentiation of neural stem cells, the present invention provides a culture method for promoting the differentiation of primary neural stem cells based on ultrasonic patterning. This method precisely manipulates the spatial arrangement of cells through non-invasive operations, realizes the patterned arrangement of cells in an ultrasonic standing wave field, and at the same time triggers the neural stem cell signaling pathway to participate in regulating the differentiation of neural stem cells by changing the activity of growth factors and their protein kinases in cells, promoting the differentiation of neural stem cells and significantly shortening the time required for the differentiation and maturation of neural stem cells.

[0039] The beneficial effects of the present invention are as follows:

[0040] The present invention provides a culture method for promoting the differentiation of primary neural stem cells based on ultrasonic patterning. By applying a one-dimensional ultrasonic standing wave field in the early stage of neural stem cell culture, the differentiation and maturation time of neural stem cells is shortened from 14 days in the traditional method to 7 - 10 days, and the differentiation efficiency is increased by 60%, significantly promoting the differentiation rate of neural stem cells in vitro culture.

[0041] The present invention uses a non-contact method to precisely manipulate the spatial arrangement of primary neural stem cells, realizes cell patterning through ultrasonic acoustic radiation force, and causes less damage to neural stem cells. After 24 hours of culture, the survival rate of neural stem cells reaches over 80% under both the culture method with an ultrasonic sound field and the culture method without an ultrasonic sound field.

[0042] The present invention can achieve high-throughput in vitro promotion of the differentiation and maturation of primary neural stem cells, and has broad application prospects in medical fields such as stem cell therapy and spinal cord injury repair. Description of the Drawings

[0043] Figure 1 Time-series optical microscope photographs of the patterning of primary neural stem cells cultured by ultrasound for 15 minutes;

[0044] Figure 2Microscopic photographs after 24 hours of primary neural stem cell culture, where (a) is the microscopic photograph of the ultrasound group and (b) is the microscopic photograph of the control group;

[0045] Figure 3 Microscopic photographs after 3 days of primary neural stem cell culture, where (a) is the microscopic photograph of the ultrasound group and (b) is the microscopic photograph of the control group;

[0046] Figure 4 Scatter plot of KEGG enrichment pathway analysis of up-regulated genes in the RNA-seq of the neural cell ultrasound group and the control group, where (a) is the result after 24 h of culture, (b) is the result after 10 days of culture, and (c) is the result after 13 days of culture;

[0047] Figure 5 Schematic diagram of the device, where (a) is the schematic diagram of the ultrasonic device and (b) is the three-dimensional split diagram of each component of the ultrasonic device;

[0048] Reference numerals: cell culture dish 1, L-shaped limit bracket 2, piezoelectric ceramic 3, sound field generator 4, piezoelectric ceramic groove 5, glass cover slip 6. Detailed implementation manners

[0049] The content of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but it should not be construed as a limitation to the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well-known to those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0050] In the embodiments of the present invention, the steps for extracting and culturing primary neural stem cells are as follows:

[0051] S1: Take the spinal cord tissue of a precisely pregnant 16-day-old SD rat embryo. Anesthetize and sacrifice the pregnant E16 rat with carbon dioxide, fix the four limbs of the rat, disinfect the abdominal fur of the rat with 75% alcohol and then remove the abdominal hair. Open the abdomen, take out the embryo string and place it in pre-cooled PBS solution. After stripping and taking out the formed embryo, place it in pre-cooled Leibovitz's L-15 medium. In the ultra-clean bench, divide the embryo head and body into two, open the vertebral lamina, separate the spinal cord tissue with a length of 5-10 mm, remove the surface spinal meninges, and then store the clean spinal cord tissue in Hibernate E medium (containing 2% B-27, 50 μg / ml Gentamicin, 250 ng / ml Fungizone), and perform digestion within one week.

[0052] S2: Digest the spinal cord tissue to obtain primary neural stem cells. In the laminar flow hood, transfer the stored spinal cord tissue into a small culture dish and cut it into small pieces with scissors. Add pre-warmed 0.05% Trypsin-EDTA into a 15 mL centrifuge tube, evenly divide the small spinal cord pieces into the tube, and then place it in a CO2 incubator for 15 min, shaking it every 3 min to break up the clumps. After that, add 3 mL of DMEM medium (containing 10% FBS, 5% HS, 2 mM Glutamine) to terminate the digestion, and centrifuge at a speed of 1200 r / min for 5 min. Use a Pasteur pipette to directly pick up the cell pellet at the bottom and transfer it to a new centrifuge tube (containing 3 ml of DMEM culture medium). After the remaining cell suspension is centrifuged at a speed of 1200 r / min for 5 min for the second time, use a Pasteur pipette to pick up the cells at the bottom and place them in the centrifuge tube, and pipette to disperse the cells evenly. Centrifuge twice again, transfer the obtained cell pellet into a 50 mL centrifuge tube containing an appropriate amount of DMEM medium and pipette to mix evenly. After mixing, filter it through a 140 uM nylon membrane into a new 50 mL centrifuge tube. Pour the cell suspension evenly into the culture dish and let it stand in the incubator. After 30 min, collect the supernatant and use a cell counter to count the cells.

[0053] S3: Culture neural stem cells. Coat the well plate with PDL in advance, adjust the appropriate cell density, inoculate the cells in the well plate, shake it evenly and then place it in the incubator. After culturing for 24 h, carefully aspirate the supernatant, add an appropriate amount of pre-warmed Neurobasal medium, and then continue to culture in the incubator. Subsequently, change the Neurobasal medium every 3 - 4 days until the nerve cells mature.

[0054] Example 1: A culture method for promoting the differentiation of primary neural stem cells based on ultrasonic patterning:

[0055] S1: Sterilize the cell slide, cell culture dish 1, and ultrasonic device: Put the clean cell slide, cell culture dish 1, and ultrasonic device into the autoclave for high-temperature and high-pressure sterilization, dry them in an oven at 60 °C, and then take them into the laminar flow hood and irradiate with ultraviolet light.

[0056] S2: Place the cell slide into cell culture dish 1 in the laminar flow hood, add 100 ug / mL poly-D-lysine (PDL) solution to immerse the cell slide, store it in the dark at room temperature overnight, then aspirate the PDL solution, and then wash the cell slide twice with sterile water and let it dry for later use;

[0057] S3: In the laminar flow hood, place cell culture dish 1 with the dried cell slide in the cavity of the sound field generator 4 of the ultrasonic device, and add sterile water to the gap area between the cavity of the sound field generator 4 and cell culture dish 1.

[0058] S4: Inside the laminar flow hood, connect the piezoelectric ceramic 3 used by the sound field generator 4 of the ultrasonic device to the power amplifier, and connect the power amplifier to the signal generator. Start the signal generator and the power amplifier, set the power amplifier parameter to 11 dB, and the signal generator parameters to 3.0 MHz and 10 Vpp. The signal generator generates a sine wave signal and acts on a pair of piezoelectric ceramics 3, causing the pair of piezoelectric ceramics 3 to generate ultrasonic waves in the cavity of the sound field generator 4 and act on the cell culture dish 1. Ultrasonic waves form a standing wave field in the cell culture dish 1 for patterning and promoting the differentiation of primary neural stem cells.

[0059] S5: Take 250 uL of the primary neural stem cell suspension with a cell density of 100 w / mL and inoculate it into the cell culture dish 1. Incubate it statically for 13 min, then turn off the power amplifier, directly connect the signal generator instead, and place it in a carbon dioxide incubator at 37 °C with a CO2 concentration of 5% and incubate it statically for 1 hour. Then turn off the signal generator. After transferring the cell culture dish 1 into the well plate, continue to incubate it statically in the carbon dioxide incubator.

[0060] S6: After culturing for 24 hours, carefully aspirate the supernatant, add 400 uL of Neurobasal medium (containing 2% B-27, 1% N-2, 2 mM Glutamine), and then place it back in the incubator for continuous culture. Replace the Neurobasal medium every 3 - 4 days hereafter. After 10 - 14 days, mature differentiated nerve cells are obtained through culture;

[0061] S7: Extract cell RNA. Respectively select the nerve cells cultured on the 1st, 10th, and 13th days. Carefully aspirate the supernatant in the cell culture dish 1, and use phosphate buffered saline (PBS) to thoroughly wash the cells twice. Subsequently, add an appropriate amount of Trizol extraction solution to each cell culture dish 1. Gently and repeatedly pipette the cell suspension with a pipette until it is observed that the viscosity of the extraction solution is significantly reduced. Transfer the suspension to a centrifuge tube, seal it, and store it in a -80 °C refrigerator for freezing preservation, which is convenient for sending samples for RNA-seq detection.

[0062] In Example 1, under the action of the sound field generated by the ultrasonic device for 15 min, the primary neural stem cells gradually formed a clear and orderly patterned linear array on the glass slide, and this process was visually demonstrated in the Figure 1 time-series optical microscope images. Figure 2 (a) and Figure 3 (a) respectively show the microscope images of the cell arrays of the primary neural stem cells in the ultrasonic group after culturing for 24 hours and three days. The results show that under the influence of ultrasonic patterning, the focal adhesion effect among the primary neural stem cells is enhanced, a clear patterned array is formed, and the array still remains unchanged after long-term culture.

[0063] Comparative Example 1: Method for differentiating primary neural stem cells without one-dimensional ultrasonic sound field:

[0064] S1: Place the cell slide in cell culture dish 1, coat the slide with PDL, aspirate the solution after overnight incubation in the dark, wash twice with sterile water, and air dry for later use.

[0065] S2: Place cell culture dish 1 in a well plate, take 250 μL of primary neural stem cell suspension with a cell density of 1×10⁶ / mL and inoculate it into cell culture dish 1, then place it in a carbon dioxide incubator at 37°C with a CO₂ concentration of 5% for static culture.

[0066] S3: After culturing for 24 hours, carefully aspirate the supernatant, add an appropriate amount of pre-warmed Neurobasal medium, and then place it back in the incubator for continuous culture. Replace the Neurobasal medium every 3 - 4 days subsequently to obtain maturely differentiated nerve cells.

[0067] S4: Extract cell RNA. Perform RNA extraction and preservation according to the method of S7 in Example 1.

[0068] In this comparative example, without the action of the ultrasonic device Figure 2 (b) and Figure 3 (b) respectively show the microscopic images of primary neural stem cells in the control group cultured for 24 hours and 3 days. The results show that the layout of primary neural stem cells on the slide without ultrasonic sound field patterning is disordered and lacks obvious directional arrangement characteristics.

[0069] As Figure 2 shown, after culturing for 24 hours, the primary neural stem cells in both the ultrasonic group and the control group have adhered to the wall, and most cells are spherical, without showing synaptic differentiation. A few neural stem cells begin to extend synapses, and the differentiation status of the two groups is similar. However, after culturing for 3 days, as Figure 3 shown, in the ultrasonic treatment group ( Figure 3 (a)), the number of neural stem cells with long protrusions observed is significantly more than that in the control group ( Figure 3 (b)). This result indicates that the ultrasonic sound field has a significant promoting effect on the differentiation of neural stem cells.

[0070] For the RNA-seq results of the ultrasonic group and the control group on the 1st, 10th, and 13th days, the present invention performed KEGG pathway enrichment analysis and selected 20 KEGG pathways to draw a scatter plot. The size of the points in the scatter plot represents the number of differential genes enriched in the pathway. The larger the size of the point, the more enriched gene numbers; the color from red to purple represents the enrichment significance degree. The more purple the color, the higher the significance of the functional pathway.

[0071] Figure 4(a), (b), and (c) respectively represent the scatter plots of KEGG pathway enrichment analysis on the 1st, 10th, and 13th days. For the neural stem cells treated with ultrasound, the number of differential genes increased significantly on the 10th day compared to the 1st and 13th days, especially showing significant differences in the neural cell differentiation function pathways related to excitatory synapses, axon guidance, calcium signaling, etc. This indicates that on the 10th day, the differentiation degree of neural stem cells in the ultrasound treatment group was significantly higher than that in the control group. Among them, the number of genes enriched in the axon guidance pathway in the ultrasound group reached 32, and the significance level was p = 2.0×10- 7 , and the differential genes shown in the original data included Slit2, Robo1, EphB2, etc. The proteins encoded by the above genes directly participate in the directional growth of nerve axons by regulating cell polarity and the direction of synaptic extension. The one-dimensional linear array induced by the ultrasonic standing wave field ( Figure 1 , Figure 3 a) enhances the cell contact guidance effect through mechanical microenvironment regulation, promoting the extension of synapses of neural stem cells along the array direction. This spatial arrangement feature is highly consistent with the activation of the Slit2 / Robo1 signaling pathway, indicating that patterning significantly accelerates the axon guidance process through a mechanical-biochemical coupling mechanism. In the KEGG pathway enrichment analysis on the 1st and 13th days, no significant differences were found in the pathways related to neural cell differentiation. This may mean that on the 1st day, neither the neural stem cells in the ultrasound treatment group nor those in the control group had started to differentiate, and their differentiation degrees were similar; while on the 13th day, the neural stem cells in both groups had reached a differentiated and mature state, and the cells tended to be stable. Therefore, the significant differences in the functional pathways related to neural cell differentiation and patterning decreased. In summary, the promotion effect of ultrasonic standing wave field patterning on the differentiation of neural stem cells is significant.

[0072] The above-described embodiments have detailed the technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, supplements, equivalent replacements, etc. made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for promoting the differentiation of primary neural stem cells based on ultrasonic patterning, characterized in that, The method comprises the following steps: Step S1: In a laminar flow hood, place the cell slide into the cell culture dish (1), add poly-D-lysine solution to submerge the cell slide, store it in the dark at room temperature, then aspirate the poly-D-lysine solution and further wash the cell slide with sterile water, and air-dry the cell slide in the cell culture dish (1); Step S2: In a laminar flow hood, place the cell culture dish (1) containing the cell slide into the main chamber of the ultrasonic device, and then add sterile water into the gap area between the side wall of the main chamber of the ultrasonic device and the cell culture dish (1); Step S3: Use the ultrasonic device to emit ultrasonic waves, and make the emitted ultrasonic waves form a standing wave field in the cell culture dish (1) for promoting the differentiation of primary neural stem cells; Step S4: Suspend the primary neural stem cells in the culture medium to obtain a cell suspension, and then add the cell suspension into the cell culture dish (1); Step S5: The ultrasonic device stops emitting ultrasonic waves, and transfer the cell culture dish (1) to a carbon dioxide cell incubator for static culture, and then replace the culture medium with a neural cell differentiation medium to culture mature neural cells.

2. The method for promoting the differentiation of primary neural stem cells based on ultrasonic patterning according to claim 1, wherein: In the step S1, the concentration of the poly-D-lysine solution is 80 - 120 μg / mL.

3. The method for promoting the differentiation of primary neural stem cells based on ultrasonic patterning according to claim 1, wherein: In the step S1, the storage time in the dark is at least 2 hours.

4. A method for promoting the differentiation of primary neural stem cells based on ultrasonic patterning according to claim 1, characterized in that: The ultrasonic device in the step S2 mainly consists of a sound field generator (4), a piezoelectric ceramic (3), four L-shaped limiting brackets (2), a signal generator and a power amplifier. The middle of the sound field generator (4) is provided with a vertically penetrating chamber, which is mainly composed of a main chamber, a central chamber and a glass cover slip mounting groove opened from top to bottom in sequence. The main chamber, the central chamber and the glass cover slip mounting groove are interconnected. A glass cover slip (6) is installed in the glass cover slip mounting groove. The main chamber is a square cavity. Four L-shaped limiting brackets (2) are respectively integrally formed at the four corners of the main chamber. The cell culture dish (1) is placed in the main chamber, and the outer side wall of the cell culture dish (1) is in contact connection with all four L-shaped limiting brackets (2), and the lower surface of the cell culture dish (1) is in contact connection with the step surface between the main chamber and the central chamber; Four piezoelectric ceramic grooves (5) are opened on the upper surface of the sound field generator (4). The piezoelectric ceramic grooves (5) are symmetrically distributed around the main chamber. A piezoelectric ceramic (3) is attached to the side wall of each piezoelectric ceramic groove (5) close to the chamber. Each piezoelectric ceramic (3) is connected to the signal generator and the power amplifier; The step S2 is specifically: In a laminar flow hood, place the cell culture dish (1) containing the cell slide into the main chamber of the sound field generator (4), and then add sterile water into the gap area between the side wall of the main chamber of the sound field generator (4) and the cell culture dish (1).

5. A method for promoting the differentiation of primary neural stem cells based on ultrasonic patterning according to claim 4, characterized in that: The specific operation of using the ultrasonic device to emit ultrasonic waves in the step S3 is: The two oppositely arranged piezoelectric ceramics (3) form a group of piezoelectric ceramic groups. The working frequencies of the two piezoelectric ceramics (3) in the same group are the same, and the working frequencies of different groups of piezoelectric ceramics (3) are different. The signal generator generates two sets of sine wave signals with the same working frequencies as those of the piezoelectric ceramics (3) in the two sets of piezoelectric ceramic groups respectively, and then amplifies them through a power amplifier and inputs them to the piezoelectric ceramic group with the same working frequency as itself. The two sets of piezoelectric ceramic groups are driven by the sine wave signals to generate ultrasonic waves and act on the cell culture dish (1). Standing wave fields that promote the differentiation of primary neural stem cells are formed in the cell culture dish (1).

6. A method for promoting the differentiation of primary neural stem cells based on ultrasonic patterning according to claim 5, characterized in that: The working frequency of the sine wave signals generated by the signal generator is 3.0 - 3.5 MHz, the peak-to-peak voltage is 10 - 12 Vpp, and the gain of the power amplifier is 10 - 12 dB.

7. A method for promoting the differentiation of primary neural stem cells based on ultrasonic patterning according to claim 5, characterized in that: In step S4, the ultrasonic device stops emitting ultrasonic waves specifically as follows: The power amplifier and the signal generator in the ultrasonic device act together for 12 - 15 minutes, and then only the signal generator acts for 55 - 65 minutes before the ultrasonic device stops emitting ultrasonic waves.

8. A method for promoting the differentiation of primary neural stem cells based on ultrasonic patterning according to claim 1, characterized in that: The culture medium in step S3 is mainly obtained by adding fetal bovine serum, horse serum and glutamine to DMEM medium. The volume fraction of the fetal bovine serum is 10%, the volume fraction of the horse serum is 5%, and the concentration of glutamine is 2 mM. The neural cell differentiation medium in step S4 is mainly obtained by adding B-27 additive, N-2 additive and glutamine to Neurobasal medium. The volume fraction of the B-27 additive is 2%, the volume fraction of the N-2 additive is 1%, and the concentration of glutamine is 2 mM.

9. A method for promoting the differentiation of primary neural stem cells based on ultrasonic patterning according to claim 1, characterized in that: In step S3, the cell density in the cell suspension is 80 - 120 w / mL, and the volume of the cell suspension is 250 - 300 μL.

10. A method for promoting the differentiation of primary neural stem cells based on ultrasonic patterning according to claim 1, characterized in that: In step S4, after static culture in a carbon dioxide cell incubator for 24 h, the culture medium is replaced with the neural cell differentiation medium, and mature neural cells are obtained after culturing for 10 - 14 days.

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