A flow shear force loading system and a method for promoting neural differentiation of stem cells by shear force

Abstract

The present invention belongs to the field of regenerative medicine and cell therapy, and specifically relates to a flow shear force loading system and a method for promoting neural differentiation of stem cells by shear force. The flow shear force loading system includes a liquid storage device, a peristaltic device, a flow stabilization device and a parallel plate flow chamber device which are connected in a circular manner. The system and method of the present invention can accurately control the magnitude of the flow shear force by adjusting the flow rate of the fluid, and its advantages include low cost, no need for chemical additives and repeatability of operation. The use of flow shear force can significantly increase the expression of neural differentiation gene markers (such as Nestin and NeuroD1) of stem cells in a short time (such as 4 hours), thereby improving the efficiency of neural differentiation. Compared with the traditional chemical induction method that takes up to 5 to 7 days, this method is not only more efficient, but also safer and more reliable. This provides a new and safer cell culture strategy for the clinical application of human dental pulp stem cells.

Description

Technical Field

[0001] The invention belongs to the field of regenerative medicine and cell therapy, and specifically relates to a flow shear force loading system and a method for promoting neural differentiation of stem cells by shear force. Background Art

[0002] Stem cell technology is the core of modern regenerative medicine. Human dental pulp stem cells are derived from the dental pulp tissue of the neural crest. Due to this unique origin, these stem cells have significant natural advantages in neural cell differentiation. Therefore, human dental pulp stem cells are highly valued in the field of neural regeneration and show significant application potential in the development of new strategies for the treatment of neurodegenerative diseases and nerve damage.

[0003] Although human dental pulp stem cells have natural advantages in neural cell differentiation, current differentiation techniques mainly rely on chemical inducers, such as growth factors and cytokines. Although these methods are effective, they face limitations such as high cost, operational complexity, and potential side effects. In addition, the use of these chemical inducers may also cause uncontrollable cell responses and biosafety issues.

[0004] Current technical solutions mainly involve the use of biochemical substances such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) to promote the neural differentiation of stem cells. These substances activate signal transduction pathways by binding to cell surface receptors, thereby promoting the expression of neural cell characteristics. However, these methods are not only costly, but also require precise control of dosage and duration of action to avoid non-specific effects. Long-term use may also affect the genetic stability of cells.

[0005] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a flow shear force loading system and a method for promoting the neural differentiation of stem cells by shear force. The purpose is to quickly and effectively promote the neural differentiation of human dental pulp stem cells through precisely controlled flow shear force. Compared with the traditional chemical induction method, this technology stimulates and accelerates the expression of neural markers through physical means to improve differentiation efficiency and cell safety. More specifically:

[0007] The present invention provides a flow shear force loading system, which comprises a liquid storage device, a peristaltic device, a flow stabilization device and a parallel plate flow chamber device which are cyclically connected in sequence; wherein:

[0008] The liquid storage device can be regarded as the starting point of the system, which is used to store the culture medium. The culture medium is stored in the liquid storage device and circulates through the system;

[0009] The peristaltic device is used to promote the circulation of the culture medium in the whole system, which can be regarded as the power source of the system. The peristaltic device can ensure the continuous and uniform flow of the fluid (culture medium), which is essential for maintaining the required shear force;

[0010] The flow stabilizing device is used to balance and stabilize the flow rate of the culture medium to ensure that there will be no fluctuations during the flow process, thereby providing a stable shear environment for the cells;

[0011] The parallel plate flow chamber device is used for cell culture, and the cells are directly exposed to the flow shear force passing through the parallel plate flow chamber device. The cells are planted on a glass slide and placed in the parallel plate flow chamber device.

[0012] To facilitate understanding of the system of the present invention, the following description is given:

[0013] The flow shear force loading system of the present invention works in coordination through its carefully designed components to promote the neural differentiation of human dental pulp stem cells. The core of the system is to achieve precise shear force control through the organic combination of a liquid storage device, a peristaltic device, a flow stabilization device and a parallel plate flow chamber device. First, the peristaltic device drives the culture medium in the liquid storage device to circulate in the system, ensuring the continuous flow of the fluid. This continuous medium flow is the basis for achieving flow shear force and is crucial to the mechanical stimulation of cells. Then, the flow stabilization device plays its role to ensure the consistency and stability of the flow rate and avoid any fluctuations during the flow process, which is extremely important for maintaining the uniformity of the shear force. Finally, the culture medium flows through the parallel plate flow chamber device, and the cells in the device are planted on a glass slide and directly exposed to the flow shear force. Through this setting, the cells not only receive continuous and uniform mechanical stimulation, but also this mechanical stimulation is safer and more effective than the traditional chemical induction method, which can stimulate and accelerate the expression of neural markers and improve the differentiation efficiency.

[0014] It should be emphasized that the calculation and adjustment strategy of flow shear force is as follows:

[0015] The magnitude of the flow shear force is determined by the flow rate. The required flow rate Q is calculated to achieve the predetermined shear force τ using the formula: τ = 6μQ / H 2 W, where μ is the viscosity of the culture medium (cp), H is the chamber height of the parallel plate flow chamber device (mm), and W is the chamber width of the parallel plate flow chamber device (mm). In the flow shear force loading system, μ, H, and W are fixed values, so the flow rate (Q) is proportional to the shear force magnitude τ. That is, the shear force can be accurately adjusted by adjusting the flow rate of the culture medium, and the magnitude of the flow rate can be controlled by adjusting the peristaltic device.

[0016] Preferably, the liquid storage device is a liquid storage bottle;

[0017] And / or, the peristaltic device is a peristaltic pump;

[0018] And / or, the flow stabilizing device is a flow stabilizer;

[0019] And / or, the parallel plate flow chamber device is a parallel plate flow chamber.

[0020] Based on the same technical concept, another solution of the present invention is to provide a method for promoting neural differentiation of stem cells by shear force, wherein the method adopts the flow shear force loading system and comprises the following steps:

[0021] (1) Seeding stem cells onto a glass slide;

[0022] (2) placing the stem cells and the glass slide in the parallel plate flow chamber device;

[0023] (3) starting the peristaltic device and adjusting the flow stabilizing device to allow the culture medium in the liquid storage device to circulate and generate shear force;

[0024] (4) The generated shear force continuously acts on the stem cells in the parallel plate flow chamber device, causing them to differentiate and allowing the cells to grow to a density of 80-90% under standard culture conditions.

[0025] Preferably, in step (1), the stem cells include but are not limited to human dental pulp stem cells, embryonic stem cells, induced pluripotent stem cells, neural stem cells, and mesenchymal stem cells.

[0026] Preferably, in step (3), the culture medium is α-DMEM medium containing 10% serum.

[0027] Preferably, in step (4), the shear force generated is 3 dyn / cm 2 The duration of action (cell culture time) is 1~8h.

[0028] Preferably, in step (4), the shear force generated is 6 dyn / cm 2 The duration of action (cell culture time) is 1~8h.

[0029] Preferably, in step (4), the shear force generated is 9 dyn / cm 2 The duration of action (cell culture time) is 1~8h.

[0030] The beneficial effects of the present invention are:

[0031] The system and method of the present invention can accurately control the magnitude of the flow shear force by adjusting the flow rate of the fluid, and its advantages include low cost, no need for chemical additives and repeatability of operation. The use of flow shear force can significantly improve the efficiency of neural differentiation in a short time (e.g., 4 hours). Compared with the traditional chemical induction method that takes up to 5 to 7 days, it is not only efficient but also safer and more reliable. This provides a new and safer cell culture strategy for the clinical application of human dental pulp stem cells, especially for achieving effective neural differentiation without the need for chemical factors. More specifically:

[0032] 1. Rapid efficiency improvement: Traditional methods for neural cell differentiation usually rely on chemical inducers, which often take several days or even a week to observe a significant increase in the expression of neural markers. In contrast, the present invention uses flow shear force as a physical stimulation method, which is 3-9 dyn / cm 2 In the range of 6dyn / cm 2 After 4 hours of shear force, the expression of neural markers of human dental pulp stem cells increased significantly. This rapid differentiation efficiency significantly shortens the experimental cycle and greatly improves the efficiency of research and clinical application.

[0033] 2. Safety and biocompatibility: The physical stimulation method of the present invention avoids the use of exogenous chemicals, thereby reducing the risk of cytotoxicity and potential side effects. The flow shear force used only exerts its influence by adjusting the fluid dynamic conditions in the cell culture environment, without adding any chemical inducers, ensuring higher cell biosafety. This method is particularly suitable for clinical applications with extremely high safety requirements.

[0034] 3. Precise control and repeatability: The flow shear force loading system of the present invention is equipped with advanced control equipment, which can accurately adjust the flow rate and shear force of the fluid, thereby ensuring the consistency and repeatability of experimental conditions. This precise control helps to obtain reliable and consistent results in scientific research and provides a solid technical foundation for future clinical applications. The introduction of the automated control system has also greatly improved the convenience and standardization of operations.

[0035] 4. Although the present invention takes the neural differentiation of human dental pulp stem cells as the main application example, its application potential is not limited to this. As a widely adjustable physical factor, flow shear force is theoretically applicable to the culture and differentiation research of various types of stem cells and tissues. Therefore, this technical solution can be widely used in multiple fields such as regenerative medicine, drug testing, disease model construction, etc., and has broad market and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 It is a structural schematic diagram of the flow shear force loading system of the present invention.

[0038] Figure 2 is a top view of the parallel plate flow chamber device.

[0039] Figure 3 It is the shear force distribution diagram in the parallel plate flow chamber.

[0040] Figure 4 Schematic diagram of human dental pulp stem cells cultured under exposure to flow shear stress.

[0041] Figure 5 When the shear force is 3dyn / cm 2 The expression level data of Nestin and NeuroD1 genes when .

[0042] Figure 6 When the shear force is 6dyn / cm 2 The expression level data of Nestin and NeuroD1 genes when .

[0043] Figure 7 When the shear force is 9dyn / cm 2 The expression level data of Nestin and NeuroD1 genes when .

[0044] Figure 8 is a diagram of the experimental layout and cell culture timeline.

[0045] Fig. 9 This is a graph showing the relative mRNA expression of Nestin at different time points.

[0046] Fig.10 This is a graph showing the relative mRNA expression of NeuroD1 at different time points.

[0047] The accompanying drawings in the figure are marked as follows:

[0048] 1-liquid storage device; 2-peristaltic device; 3-flow stabilization device; 4-parallel plate flow chamber device. DETAILED DESCRIPTION

[0049] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0050] Example 1

[0051] like Figure 1 , Figure 2 As shown, this embodiment provides a flow shear force loading system, which includes a liquid storage device 1, a peristaltic device 2, a flow stabilization device 3 and a parallel plate flow chamber device 4 which are cyclically connected in sequence; wherein:

[0052] The liquid storage device 1 is used to store a culture medium (fluid), and the culture medium is stored in the liquid storage device 1 and circulates through the system;

[0053] The peristaltic device 2 is used to promote the circulation of the culture medium in the whole system;

[0054] The flow stabilizing device 3 is used to balance and stabilize the flow rate of the culture medium;

[0055] The parallel plate flow chamber device 4 is used for cell culture, and the cells are directly exposed to the flow shear force passing through the parallel plate flow chamber device 4 .

[0056] As an optional embodiment, the liquid storage device 1 is a liquid storage bottle;

[0057] And / or, the peristaltic device 2 is a peristaltic pump;

[0058] And / or, the flow stabilizing device 3 is a flow stabilizer;

[0059] And / or, the parallel plate flow chamber device 4 is a parallel plate flow chamber.

[0060] Example 2

[0061] This embodiment provides a method for promoting neural differentiation of stem cells by shear force, the method adopts the flow shear force loading system described above, and the method comprises the following steps:

[0062] (1) Inoculate human dental pulp stem cells onto a glass slide;

[0063] (2) placing human dental pulp stem cells together with a glass slide in the parallel plate flow chamber;

[0064] (3) starting the peristaltic pump and adjusting the flow rate so that the culture medium in the liquid storage bottle circulates and generates a certain amount of shear force;

[0065] (4) Control the shear force to 3 dyn / cm 2 , continuously acting on the human dental pulp stem cells in the parallel plate flow chamber device for 1 hour to differentiate them.

[0066] Embodiment 3 to Embodiment 13

[0067] Embodiments 3 to 13 differ from Embodiment 2 only in shear force and / or duration of action, and the rest of the operations are the same, as shown in Table 1.

[0068] Table 1

[0069]

[0070] Effect verification example (one)

[0072] Figure 3 The three-dimensional computational simulation using FLUENT 5.5 (Fluent, USA) software was presented to analyze the shear force distribution in the parallel plate flow chamber. The quantitative analysis results showed that the shear force distribution in the cell culture area (parallel plate flow chamber) was very uniform, which is of great significance for optimizing cell culture conditions and improving cell differentiation efficiency. (two)

[0074] Figure 4~Figure 7 Demonstrated neural differentiation of human dental pulp stem cells under different flow shear stress conditions. More specifically:

[0075] Figure 4 The basic setup of the experiment is shown in the schematic diagram:

[0076] Flow shear stress: Human dental pulp stem cells were exposed to 3, 6, or 9 dyn / cm 2 flow shear force.

[0077] Time points: cells were incubated under shear stress for 1 hour, 2 hours, 4 hours, and 8 hours.

[0078] Outcome indicators: The expression of neural differentiation genes - Nestin and NeuroD1.

[0079] Figure 5~Figure 7 The results show that the Nestin and NeuroD1 genes are expressed at different shear stresses (3, 6, 9 dyn / cm 2The control group was not treated with shear stress and used as a baseline comparison. The time series data included gene expression after 1 hour, 2 hours, 4 hours and 8 hours of shear stress. The results showed that at 3, 6, and 9 dyn / cm 2 Under the shear stress of 6 dyn / cm, 4 hours of loading time is sufficient to promote the neural differentiation of human dental pulp stem cells. 2 The shear stress had the most significant differentiation effect at 4 hours of loading. These findings confirmed that appropriate flow shear stress significantly promoted the expression of neural markers in human dental pulp stem cells, which provides a new approach and strategy for guiding stem cell differentiation using physical and mechanical stimulation. Compared with traditional chemical induction methods, this physical stimulation method provides an effective alternative strategy for achieving efficient and safe cell differentiation. (three)

[0081] Figure 8 The experimental layout and cell culture timeline are shown.

[0082] Cell type: Human dental pulp stem cells are used.

[0083] Culture medium:

[0084] Basal culture medium: α-DMEM medium containing 10% serum, indicated by red bars.

[0085] Neural differentiation induction medium: α-DMEM medium containing 10% serum, 20ng / mL EGF, 20ng / mL FGFb, and 1×B27, represented by pink bars.

[0086] Culture time: extended from 0 hours (control) to 7 days, and specific time points included 4 hours, 8 hours, 12 hours, 1 day, 3 days, 5 days and 7 days.

[0087] Figure 9~Figure 10 The relative mRNA expression levels of two key neural differentiation genes, Nestin and NeuroD1, at different time points are shown. Control group: represents cell samples without any treatment.

[0088] Data represent: Comparison of the expression levels of Nestin and NeuroD1 genes in basal culture medium (red bars) and neural differentiation induction medium (pink bars), respectively.

[0089] Statistical significance: The p value indicates the significant difference in gene expression levels at different time points compared with the control group, and P < 0.05 indicates that the difference is statistically significant.

[0090] Analysis: In the traditional neural differentiation induction culture medium, the neural differentiation gene Nestin of human dental pulp stem cells was highly expressed on the 5th day, and the neural differentiation gene NeuroD1 of human dental pulp stem cells was highly expressed on the 7th day, showing signs of neural differentiation. This further confirms that flow shear force, as a physical stimulation method, can provide an effective alternative strategy for efficient and safe cell differentiation compared to chemical induction.

[0091] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for promoting neural differentiation of stem cells by shear force, characterized in that: The method adopts a flow shear force loading system, which includes a liquid storage device, a peristaltic device, a flow stabilization device and a parallel plate flow chamber device that are cyclically connected in sequence; wherein: The liquid storage device is used to store culture medium, and the culture medium is stored in the liquid storage device and circulates through the system; The peristaltic device is used to promote the circulation of the culture medium in the whole system; The flow stabilizing device is used to balance and stabilize the flow rate of the culture medium; The parallel plate flow chamber device is used for cell culture, and the cells are directly exposed to the shear force of the flow through the parallel plate flow chamber device; The method comprises the following steps: (1) Seeding stem cells onto a glass slide; (2) placing the stem cells and the glass slide in the parallel plate flow chamber device; (3) starting the peristaltic device and adjusting the flow stabilizing device to allow the culture medium in the liquid storage device to circulate and generate shear force; (4) The generated shear force continuously acts on the stem cells in the parallel plate flow chamber device and causes them to differentiate; in: In step (1), the stem cells are human dental pulp stem cells; In step (4), the shear force generated is 6 dyn / cm 2 The duration of action is 4 hours.

2. The method for promoting neural differentiation of stem cells by shear force according to claim 1, characterized in that: The liquid storage device is a liquid storage bottle; And / or, the peristaltic device is a peristaltic pump; And / or, the flow stabilizing device is a flow stabilizer; And / or, the parallel plate flow chamber device is a parallel plate flow chamber.

3. The method for promoting neural differentiation of stem cells by shear force according to claim 1, characterized in that: In step (3), the culture medium is α-DMEM culture medium containing 10% serum.

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