Method and device for inducing pluripotent stem cells

Abstract

The invention discloses a method and device for inducing pluripotent stem cells, and relates to the technical field of cell culture.The inducing method specifically comprises the following steps that S1, target cells are separated and treated and inoculated into a three-dimensional support containing a culture medium, and meanwhile a space gradient microenvironment is established; s2, performing periodic physical stimulation on each target body cell by adopting low-intensity pulse ultrasound, and activating a signal channel corresponding to mechanical transduction in cooperation with a small molecule inducer; s3, adding a photosensitive metabolism intermediate precursor or a photoresponsive regulating agent into the three-dimensional bracket, and performing time-limited irradiation; by simulating a signal environment of embryonic development, directional migration and gradual reprogramming of each target body cell are effectively guided, accurate intervention on different target body cells is realized, meanwhile, a reprogramming effect is verified from multiple angles, and the quality and stability of a pluripotent stem cell product are guaranteed.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and in particular to a method and apparatus for inducing pluripotent stem cells. Background Technology

[0002] Induced pluripotent stem cells (iPSCs) have become an important technology in regenerative medicine, tissue engineering, and disease model research in recent years. Traditional iPSC induction methods mainly rely on virus-mediated transgene expression. Although this method is relatively efficient, it has safety risks such as viral integration leading to gene mutations, potential tumorigenicity, and uncontrolled transcription factor expression, which seriously limits its clinical application. With a deeper understanding of the reprogramming mechanism, researchers have gradually developed virus-free induction strategies. Although these methods can avoid the risk of genome insertion, their induction efficiency is generally low, the reaction time is long, and the reprogramming process is easily affected by external microenvironmental factors. Therefore, improving the efficiency and controllability of non-viral methods has become a key challenge.

[0003] Existing methods and devices for inducing pluripotent stem cells lack effective guidance for the directed migration and gradual reprogramming of target somatic cells, resulting in insufficient precision in intervention. At the same time, the verification dimensions for reprogramming effects are relatively singular, making it difficult to comprehensively guarantee the stability and quality consistency of the finally obtained pluripotent stem cells. To address this, we propose a method and device for inducing pluripotent stem cells. Summary of the Invention

[0004] The purpose of this invention is to address the deficiencies in the existing technology by proposing a method and apparatus for inducing pluripotent stem cells.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for inducing pluripotent stem cells, the specific steps of which are as follows:

[0007] Step S1: Isolate and process each target somatic cell, and seed each target somatic cell into a three-dimensional scaffold containing culture medium, while establishing a spatial gradient microenvironment;

[0008] Step S2: Use low-intensity pulsed ultrasound to periodically physically stimulate each target somatic cell, and in conjunction with a small molecule inducer, activate the signaling pathway corresponding to mechanotransduction;

[0009] Step S3: Add a photosensitive metabolic intermediate precursor or a photoresponsive modulator to the three-dimensional scaffold and irradiate for a specified time.

[0010] Step S4: During the simultaneous or alternating administration of sound wave and light interventions, add epigenetic regulatory small molecules;

[0011] Step S5: Monitor the expression of pluripotency markers of each target somatic cell in real time during the reprogramming cycle, and purify them by magnetic bead enrichment to establish a stable iPSC line.

[0012] As a further aspect of the present invention, the specific steps for establishing the spatial gradient microenvironment in step S1 are as follows:

[0013] Based on the isolated target somatic cell types, a three-dimensional biological scaffold and microfluidic chip for constructing a gradient environment are selected, and the type and parameters of the gradient to be established are set.

[0014] By controlling the convergence and diffusion of liquids of different concentrations in the chip channel through a microfluidic chip, different concentrations of factor solutions are dripped or injected layer by layer from the bottom layer of the three-dimensional biological scaffold. Each layer of gel is cross-linked before the next layer is added to establish a continuous or segmented chemical gradient.

[0015] By adjusting the material thickness, ventilation channels, or local sealing, oxygen diffusion in the three-dimensional biological scaffold is restricted, creating a difference in oxygen concentration between the inner and outer layers to establish a physical gradient. This forms a continuous or segmented microenvironmental region containing both chemical and physical gradients, allowing target somatic cells to be seeded onto the induction culture medium in each microenvironmental region.

[0016] As a further aspect of the present invention, the gradient types include chemical gradients and physical gradients, and the gradient parameters include initial concentration, gradient range, gradient direction, and gradient duration.

[0017] As a further aspect of the present invention, the specific steps for activating the signal pathway corresponding to mechanical transduction in step S2 are as follows:

[0018] Add specific small molecule inducers to the induction culture medium inoculated with each target somatic cell, mix gently, select a low-intensity pulse ultrasound device, and set the parameters of the low-intensity pulse ultrasound device according to experimental requirements.

[0019] The target somatic cells were stimulated with low-intensity pulsed ultrasound for a set time period. Simultaneously, the flattening, contraction and skeletal reconstruction of the target somatic cells were observed under a microscope. The results were evaluated to determine whether the sound waves induced the rearrangement or enhanced the migration of the target somatic cells and activated the signaling pathways corresponding to mechanotransduction.

[0020] Sound wave stimulation was applied according to a pre-set cycle plan. The formulation and concentration of small molecule inducers and the induction culture medium were changed according to different stages of the pre-set cycle plan. After the pre-set induction cycle was reached, the synergistic effect of sound wave combined with small molecule stimulation on the reprogramming of each target somatic cell was evaluated.

[0021] As a further aspect of the present invention, the specific small molecule inducer is specifically an HDAC inhibitor, a GSK3β inhibitor, or a TGF-β pathway inhibitor.

[0022] As a further aspect of the present invention, the specific steps for performing time-limited irradiation in step S3 are as follows:

[0023] Based on the metabolic requirements during the induced pluripotent stem cell experiment, the types of targeted metabolic pathways that need to be regulated are selected, and the metabolic enzymes, metabolic intermediates, and regulatory factors of each targeted metabolic pathway are taken as node molecules.

[0024] Photosensitive metabolic intermediate precursors or photosensitive enzyme inhibitors were selected as photosensitive regulatory molecules. The photosensitive regulatory molecules were dissolved in DMSO solvent, and then the diluted photosensitive regulatory molecules were added to the induction culture medium inoculated with each target somatic cell. The mixture was gently mixed, and then an tunable wavelength LED light source was selected, and the light intensity, irradiation time and irradiation cycle were set.

[0025] The LED light source is moved above the induction culture medium for illumination, and the photosensitizing regulatory molecules in each target somatic cell absorb specific wavelengths of light energy, activating the molecular structure of the photosensitizing regulatory molecules. The activated molecular structure then binds to the target enzyme in the target metabolic pathway, so as to instantaneously turn the target metabolic pathway of each target somatic cell on or off.

[0026] As a further aspect of the present invention, the targeted metabolic pathways include glycolysis, oxidative phosphorylation, acetyl-CoA, and one-carbon metabolism / NAD+ cycle.

[0027] As a further aspect of the present invention, the specific steps for adding the epigenetic regulatory small molecule in step S4 are as follows:

[0028] DNA methyltransferase inhibitors or DNA methyltransferase inhibitors were selected as small molecule regulators and diluted with sterile water. Then, the parameters of the low-intensity pulsed ultrasound instrument and the illumination parameters of the LED light source were adjusted to set two intervention schemes: simultaneous sound stimulation and light stimulation, and alternating sound stimulation and light stimulation.

[0029] The diluted small molecule regulator was added to the induction medium inoculated with each target somatic cell, and then acoustic stimulation and light stimulation were performed. The concentration of the small molecule regulator was maintained while acoustic stimulation and light stimulation were performed simultaneously or alternately. At the same time, the cell morphology, proliferation rate and survival rate of each target somatic cell in the induction medium were monitored in real time. The degree of response of each target somatic cell to the small molecule regulator was determined by molecular biological detection.

[0030] Based on the monitoring and judgment results, the concentration of small molecule regulators was adjusted, and the frequency of small molecule regulator addition was adjusted according to the reprogramming process of each target somatic cell and the preset physical stimulation cycle. The dosage, time and cell response of each adjustment were recorded in detail, and a dynamic regulation database was established. The intervention strategy was gradually adjusted based on the data feedback.

[0031] As a further aspect of the present invention, a predetermined number of target somatic cells are extracted from the induction culture medium, the total RNA of each target somatic cell is obtained, and reverse transcribed into cDNA. Primers are then set for the pluripotency marker gene, and qPCR is performed to detect changes in expression levels. Immunofluorescence staining is used to detect the expression and localization of the protein encoded by the corresponding pluripotency gene. RNA-seq analysis is then performed to observe changes in gene expression in each target somatic cell.

[0032] By using transposases to cut open chromatin regions, the accessibility of chromatin in the genomes of various target somatic cells was measured, the sensitivity of DNA to enzyme digestion was assessed, the changing trends of pluripotent gene expression and chromatin accessibility were compared at different time points and under different treatment conditions, and the synergistic effect of small molecule regulators and physical stimulation was evaluated to significantly improve gene region accessibility and expression levels.

[0033] A device for inducing pluripotent stem cells includes a piezoelectric transducer, a radio frequency signal generator, an LED array light source, a light source driver controller, a microfluidic pump, and a temperature-controlled heater;

[0034] The piezoelectric transducer is used to generate low-intensity pulsed ultrasound and convert it into mechanical vibrations to periodically stimulate cells;

[0035] The radio frequency signal generator is used to control the frequency, pulse interval, and duration of the sound wave;

[0036] The LED array light source is used to activate photoresponsive small molecules or photocontrolled metabolic enzymes to control the opening or closing of cellular metabolic pathways.

[0037] The light source driver controller is used to adjust the current, voltage, and on / off time of the LED array light source;

[0038] The microfluidic pump is used to drive fluid transport within the microfluidic chip and control the flow of nutrient solution.

[0039] The temperature-controlled heater is used to maintain a constant temperature inside the induction culture medium chamber.

[0040] As a further embodiment of the present invention, it also includes a microcontroller, an image sensor, a power supply module, and a data recording and communication module;

[0041] The microcontroller is used to coordinate the signal inputs of all sub-modules and execute preset programs;

[0042] The image sensor is used to monitor the morphology, adhesion status, and fluorescent labeling signal of each target somatic cell in real time.

[0043] The power module is used to provide a stable power supply;

[0044] The data recording and communication module is used to store and export experimental parameters and process data.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] This invention establishes a microenvironment with chemical and physical gradients using microfluidic chips and a three-dimensional scaffold, guiding the migration and state transition of target somatic cells. Simultaneously, small molecule inducers are added and acoustic stimulation is applied to promote skeletal reconstruction and signaling pathway activation in target somatic cells. Then, by adding photosensitive metabolic regulators and combining them with tunable wavelength LED light sources, the instantaneous activation or deactivation of targeted metabolic pathways is achieved. Epigenetic regulators are added during this process to enhance the accessibility of pluripotent gene regions under acoustic and photostimulation. The entire process involves real-time monitoring of the responses of each target somatic cell and adjustment of small molecule dosages and intervention protocols. Finally, qPCR, immunostaining, and chromatin accessibility analysis are used to verify the reprogramming effect and the enhancement of gene expression levels. This induces somatic cell reprogramming into pluripotent stem cells, mimicking the signaling environment of embryonic development, effectively guiding the directional migration and gradual reprogramming of target somatic cells, achieving precise intervention for different target somatic cells. Simultaneously, the reprogramming effect is verified from multiple angles, ensuring the quality and stability of pluripotent stem cell products. Attached Figure Description

[0047] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0048] Figure 1 This is a flowchart of a method for inducing pluripotent stem cells proposed in this invention;

[0049] Figure 2 This is a system block diagram of an induced pluripotent stem cell device proposed in this invention. Detailed Implementation

[0050] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0051] Example 1

[0052] Reference Figure 1 This embodiment discloses a method for inducing pluripotent stem cells, the specific steps of which are as follows:

[0053] Each target somatic cell was isolated and processed, and then seeded into a three-dimensional scaffold containing culture medium, while simultaneously establishing a spatial gradient microenvironment.

[0054] Specifically, based on the isolated target somatic cell types, a three-dimensional biological scaffold and microfluidic chip for constructing a gradient environment are selected, and the types and parameters of the gradients to be established are set. The microfluidic chip controls the convergence and diffusion of liquids of different concentrations in the chip channels. At the same time, different concentrations of factor solutions are dropped or injected layer by layer from the bottom layer of the three-dimensional biological scaffold. Each layer of gel is cross-linked before the next layer is added to establish a continuous or segmented chemical gradient. By adjusting the material thickness, ventilation channels, or local sealing, oxygen diffusion in the three-dimensional biological scaffold is restricted to form a difference in oxygen concentration between the inner and outer layers, thereby establishing a physical gradient. This forms a continuous or segmented microenvironmental region containing both chemical and physical gradients. The target somatic cells are then seeded on the induction culture medium of each microenvironmental region.

[0055] It should be further explained that gradient types include chemical gradients and physical gradients, and gradient parameters include initial concentration, gradient range, gradient direction, and gradient duration.

[0056] Low-intensity pulsed ultrasound was used to periodically physically stimulate the target somatic cells, and small molecule inducers were used in conjunction to activate the signaling pathways corresponding to mechanotransduction.

[0057] Specifically, a specific small molecule inducer was added to the induction culture medium inoculated with each target somatic cell, and gently mixed. A low-intensity pulsed ultrasound device was selected, and the parameters of the low-intensity pulsed ultrasound device were set according to experimental requirements. The target somatic cells were stimulated with sound waves using the low-intensity pulsed ultrasound device within a set time. At the same time, the flattening, contraction, and skeletal remodeling signs of each target somatic cell were observed under a microscope. The effects of sound waves on the rearrangement or enhanced migration of each target somatic cell were evaluated, as well as the activation of signaling pathways corresponding to mechanotransduction. Sound wave stimulation was applied according to a preset cycle plan. The formulation and concentration of the small molecule inducer and the induction culture medium were changed according to different stages of the preset cycle plan. After the preset induction cycle was reached, the synergistic effect of sound waves combined with small molecule stimulation on the reprogramming of each target somatic cell was evaluated.

[0058] It should be further noted that the specific small molecule inducers are HDAC inhibitors, GSK3β inhibitors, or TGF-β pathway inhibitors.

[0059] Add photosensitive metabolic intermediate precursors or photoresponsive modulators to a three-dimensional scaffold and subject to time-limited irradiation.

[0060] Specifically, based on the metabolic needs during the induced pluripotent stem cell experiment, the types of targeted metabolic pathways to be regulated are selected. The metabolic enzymes, metabolic intermediates, and regulatory factors of each targeted metabolic pathway are used as node molecules. Photosensitive metabolic intermediate precursors or photosensitive enzyme inhibitors are selected as photosensitive regulatory molecules. The photosensitive regulatory molecules are dissolved in DMSO solvent, and then the diluted photosensitive regulatory molecules are added to the induction culture medium seeded with each target somatic cell. After gentle mixing, an tunable wavelength LED light source is selected, and the light intensity, irradiation duration, and irradiation cycle are set. The LED light source is moved above the induction culture medium for illumination, inducing the photosensitive regulatory molecules in each target somatic cell to absorb specific wavelength light energy, activating the molecular structure of the photosensitive regulatory molecules, and then binding the activated molecular structure to the target enzyme in the targeted metabolic pathway to instantaneously turn the targeted metabolic pathway of each target somatic cell on or off.

[0061] It should be further noted that the types of targeted metabolic pathways include glycolysis, oxidative phosphorylation, acetyl-CoA, and one-carbon metabolism / NAD+ cycle.

[0062] Epigenetic regulatory molecules are added during the simultaneous or alternating use of sound waves and light.

[0063] Specifically, DNA methyltransferase inhibitors were selected as small molecule regulators and diluted with sterile water. The parameters of the low-intensity pulsed ultrasound device and the LED light source were then adjusted. Two intervention schemes were set: simultaneous acoustic and light stimulation, and alternating acoustic and light stimulation. The diluted small molecule regulator was added to the induction culture medium inoculated with the target somatic cells, followed by acoustic and light stimulation. The concentration of the small molecule regulator was maintained during the simultaneous or alternating acoustic and light stimulation. Simultaneously, the cell morphology, proliferation rate, and survival rate of each target somatic cell in the induction culture medium were monitored in real time. Molecular biological assays were then used to determine the response of each target somatic cell to the small molecule regulator. Based on the monitoring and assessment results, the concentration of the small molecule regulator was adjusted, and the reprogramming process of each target somatic cell and the preset physical stimulation cycle were followed. The frequency of small molecule regulator addition was adjusted, and the dosage, time, and cellular response of each adjustment were recorded in detail. A dynamic regulation database was established. Based on the data feedback, the intervention strategy was gradually adjusted. A predetermined number of target somatic cells were extracted from the induction culture medium, and total RNA from each target somatic cell was obtained and reverse transcribed into cDNA. Primers were then set for pluripotency marker genes, and qPCR was used to detect changes in expression levels. Immunofluorescence staining was used to detect the expression and localization of proteins encoded by corresponding pluripotent genes. RNA-seq analysis was performed to observe changes in gene expression in each target somatic cell. Transposases were used to cut open chromatin regions to determine the accessibility of chromatin in the genome of each target somatic cell and to assess the sensitivity of DNA to enzyme digestion. The trends of pluripotent gene expression and chromatin accessibility were compared at different time points and under different treatment conditions to evaluate whether the synergistic effect of small molecule regulators and physical stimulation significantly improved gene region accessibility and expression levels.

[0064] The expression of pluripotency markers of each target somatic cell was monitored in real time during the reprogramming cycle, and purified by magnetic bead enrichment to establish a stable iPSC line.

[0065] Example 2

[0066] Reference Figure 2 This embodiment discloses a device for inducing pluripotent stem cells, including a piezoelectric transducer, a radio frequency signal generator, an LED array light source, a light source driver controller, a microfluidic pump, a temperature-controlled heater, a microcontroller, an image sensor, a power supply module, and a data recording and communication module;

[0067] The piezoelectric transducer is used to generate low-intensity pulsed ultrasound and convert it into mechanical vibrations to periodically stimulate cells;

[0068] The radio frequency signal generator is used to control the frequency, pulse interval, and duration of the sound wave;

[0069] The LED array light source is used to activate photoresponsive small molecules or photocontrolled metabolic enzymes to control the opening or closing of cellular metabolic pathways.

[0070] The light source driver controller is used to adjust the current, voltage, and on / off time of the LED array light source;

[0071] The microfluidic pump is used to drive fluid transport within the microfluidic chip and control the flow of nutrient solution.

[0072] The temperature-controlled heater is used to maintain a constant temperature inside the induction culture medium chamber;

[0073] The microcontroller is used to coordinate the signal inputs of all sub-modules and execute preset programs;

[0074] The image sensor is used to monitor the morphology, adhesion status, and fluorescent labeling signal of each target somatic cell in real time.

[0075] The power module is used to provide a stable power supply;

[0076] The data recording and communication module is used to store and export experimental parameters and process data.

[0077] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

[0078] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for inducing pluripotent stem cells, characterized in that, The specific steps of this induction method are as follows: Step S1: Isolate and process each target somatic cell, and seed each target somatic cell into a three-dimensional scaffold containing culture medium, while establishing a spatial gradient microenvironment; Step S2: Use low-intensity pulsed ultrasound to periodically physically stimulate each target somatic cell, and in conjunction with a small molecule inducer, activate the signaling pathway corresponding to mechanotransduction; Step S3: Add a photosensitive metabolic intermediate precursor or a photoresponsive modulator to the three-dimensional scaffold and irradiate for a specified time. Step S4: During the simultaneous or alternating administration of sound wave and light interventions, add epigenetic regulatory small molecules; Step S5: Monitor the expression of pluripotency markers of each target somatic cell in real time during the reprogramming cycle, and purify them by magnetic bead enrichment to establish a stable iPSC line.

2. The method for inducing pluripotent stem cells according to claim 1, characterized in that, The specific steps for establishing the spatial gradient microenvironment described in step S1 are as follows: Based on the isolated target somatic cell types, a three-dimensional biological scaffold and microfluidic chip for constructing a gradient environment are selected, and the type and parameters of the gradient to be established are set. By controlling the convergence and diffusion of liquids of different concentrations in the chip channel through a microfluidic chip, different concentrations of factor solutions are dripped or injected layer by layer from the bottom layer of the three-dimensional biological scaffold. Each layer of gel is cross-linked before the next layer is added to establish a continuous or segmented chemical gradient. By adjusting the material thickness, ventilation channels, or local sealing, oxygen diffusion in the three-dimensional biological scaffold is restricted, creating a difference in oxygen concentration between the inner and outer layers to establish a physical gradient. This forms a continuous or segmented microenvironmental region containing both chemical and physical gradients, allowing target somatic cells to be seeded onto the induction culture medium in each microenvironmental region.

3. The method for inducing pluripotent stem cells according to claim 1, characterized in that, The specific steps for activating the signal pathway corresponding to mechanotransduction in step S2 are as follows: Add specific small molecule inducers to the induction culture medium inoculated with each target somatic cell, mix gently, select a low-intensity pulse ultrasound device, and set the parameters of the low-intensity pulse ultrasound device according to experimental requirements. The target somatic cells were stimulated with low-intensity pulsed ultrasound for a set time period. Simultaneously, the flattening, contraction and skeletal reconstruction of the target somatic cells were observed under a microscope. The results were evaluated to determine whether the sound waves induced the rearrangement or enhanced the migration of the target somatic cells and activated the signaling pathways corresponding to mechanotransduction. Sound wave stimulation was applied according to a pre-set cycle plan. The formulation and concentration of small molecule inducers and the induction culture medium were changed according to different stages of the pre-set cycle plan. After the pre-set induction cycle was reached, the synergistic effect of sound wave combined with small molecule stimulation on the reprogramming of each target somatic cell was evaluated.

4. The method for inducing pluripotent stem cells according to claim 1, characterized in that, The specific steps for performing time-limited irradiation as described in step S3 are as follows: Based on the metabolic requirements during the induced pluripotent stem cell experiment, the types of targeted metabolic pathways that need to be regulated are selected, and the metabolic enzymes, metabolic intermediates, and regulatory factors of each targeted metabolic pathway are taken as node molecules. Photosensitive metabolic intermediate precursors or photosensitive enzyme inhibitors were selected as photosensitive regulatory molecules. The photosensitive regulatory molecules were dissolved in DMSO solvent, and then the diluted photosensitive regulatory molecules were added to the induction culture medium inoculated with each target somatic cell. The mixture was gently mixed, and then an tunable wavelength LED light source was selected, and the light intensity, irradiation time and irradiation cycle were set. The LED light source is moved above the induction culture medium for illumination, and the photosensitizing regulatory molecules in each target somatic cell absorb specific wavelengths of light energy, activating the molecular structure of the photosensitizing regulatory molecules. The activated molecular structure then binds to the target enzyme in the target metabolic pathway, so as to instantaneously turn the target metabolic pathway of each target somatic cell on or off.

5. The method for inducing pluripotent stem cells according to claim 1, characterized in that, The specific steps for adding epigenetic regulatory small molecules in step S4 are as follows: DNA methyltransferase inhibitors or DNA methyltransferase inhibitors were selected as small molecule regulators and diluted with sterile water. Then, the parameters of the low-intensity pulsed ultrasound instrument and the illumination parameters of the LED light source were adjusted to set two intervention schemes: simultaneous sound stimulation and light stimulation, and alternating sound stimulation and light stimulation. The diluted small molecule regulator was added to the induction medium inoculated with each target somatic cell, and then acoustic stimulation and light stimulation were performed. The concentration of the small molecule regulator was maintained while acoustic stimulation and light stimulation were performed simultaneously or alternately. At the same time, the cell morphology, proliferation rate and survival rate of each target somatic cell in the induction medium were monitored in real time. The degree of response of each target somatic cell to the small molecule regulator was determined by molecular biological detection. Based on the monitoring and judgment results, the concentration of small molecule regulators was adjusted, and the frequency of small molecule regulator addition was adjusted according to the reprogramming process of each target somatic cell and the preset physical stimulation cycle. The dosage, time and cell response of each adjustment were recorded in detail, and a dynamic regulation database was established. The intervention strategy was gradually adjusted based on the data feedback.

6. The method for inducing pluripotent stem cells according to claim 5, characterized in that, A predetermined number of target somatic cells were extracted from the induction culture medium, and the total RNA of each target somatic cell was obtained and reverse transcribed into cDNA. Primers were then set for the pluripotency marker gene, and qPCR was used to detect changes in expression levels. Immunofluorescence staining was used to detect the expression and localization of the corresponding pluripotency gene-encoded protein. RNA-seq analysis was then performed to observe changes in gene expression in each target somatic cell. By using transposases to cut open chromatin regions, the accessibility of chromatin in the genomes of various target somatic cells was measured, the sensitivity of DNA to enzyme digestion was assessed, the changing trends of pluripotent gene expression and chromatin accessibility were compared at different time points and under different treatment conditions, and the synergistic effect of small molecule regulators and physical stimulation was evaluated to significantly improve gene region accessibility and expression levels.

7. An apparatus for inducing pluripotent stem cells, used to implement the method for inducing pluripotent stem cells according to any one of claims 1-6, characterized in that, The device includes a piezoelectric transducer, a radio frequency signal generator, an LED array light source, a light source driver controller, a microfluidic pump, and a temperature-controlled heater; The piezoelectric transducer is used to generate low-intensity pulsed ultrasound and convert it into mechanical vibrations to periodically stimulate cells; The radio frequency signal generator is used to control the frequency, pulse interval, and duration of the sound wave; The LED array light source is used to activate photoresponsive small molecules or photocontrolled metabolic enzymes to control the opening or closing of cellular metabolic pathways. The light source driver controller is used to adjust the current, voltage, and on / off time of the LED array light source; The microfluidic pump is used to drive fluid transport within the microfluidic chip and control the flow of nutrient solution. The temperature-controlled heater is used to maintain a constant temperature inside the induction culture medium chamber.

8. The apparatus for inducing pluripotent stem cells according to claim 7, characterized in that, It also includes a microcontroller, an image sensor, a power supply module, and a data recording and communication module; The microcontroller is used to coordinate the signal inputs of all sub-modules and execute preset programs; The image sensor is used to monitor the morphology, adhesion status, and fluorescent labeling signal of each target somatic cell in real time. The power module is used to provide a stable power supply; The data recording and communication module is used to store and export experimental parameters and process data.

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