C2BBe1 intestinal cell reprogramming method and system

Abstract

The invention provides a C2BBe1 intestinal cell reprogramming method and system, and aims to solve the technical problems of low reprogramming efficiency, unstable reprogramming result, difficulty in truly simulating in-vivo real conditions and difficulty in driving intestinal epithelial cell physiological morphology and differentiation maintenance in current intestinal tissue modeling. An in-vivo intestinal canal model and an in-vitro gas-liquid exchange interface model are constructed, real in-vivo conditions can be simulated, a crypt-villus topological structure can be reconstructed spontaneously, and spatial heterogeneity and cell lineage distribution of intestinal epithelium can be simulated truly. After specific mechanical stimulation characteristic verification of the intestinal canal at different development stages is carried out, a verification result is obtained, and actual in-vitro inoculation is carried out on the basis of the verification result. And finally undifferentiated cells can be directly reprogrammed, so that the dependence on pluripotent stem cells and a complex induction process in a traditional method is avoided, and the reprogramming efficiency and stability are remarkably improved.

Description

TECHNICAL FIELD

[0001] The application belongs to a cell reprogramming method, and particularly relates to a C2BBe1 intestinal cell reprogramming method and system. BACKGROUND

[0002] The intestine has significant regenerative capacity, which depends on the dynamic interaction between stem cells and their microenvironment to maintain the continuous update and homeostasis of the tissue. With the rapid development of regenerative medicine and organoid technology, establishing an epithelial cell model with stemness characteristics in vitro has become an important means for studying intestinal development, disease mechanisms and drug screening. However, most current in vitro models have many limitations in structural complexity, repeatability and clinical translation: (1) The existing reconstruction method for inducing part of the epithelial cells to reverse to the stemness state generally has the technical bottlenecks of low reprogramming efficiency, unstable cell state or dependence on complex growth factors; (2) Due to the inversion of the crypt and villus topological relationship, the crypt faces the external stimulus and cannot truly simulate the spatial drug response and pathogen interaction in vivo; (3) The existing system generally lacks simulation of complex in vivo mechanics and metabolic microenvironment, making it difficult to drive the physiological morphogenesis and differentiation maintenance of intestinal epithelial cells. SUMMARY

[0003] The application provides a C2BBe1 intestinal cell reprogramming method and system to solve the technical problems of low reprogramming efficiency and unstable reprogramming results, difficulty in truly simulating the in vivo actual situation, and difficulty in driving the physiological morphology and differentiation maintenance of intestinal epithelial cells in the current intestinal tissue modeling.

[0004] To achieve the above-mentioned purpose, the application adopts the following technical solutions: In a first aspect, the application provides a C2BBe1 intestinal cell reprogramming method, comprising: The mechanical stimulation characteristics specific to different development stages of the intestinal tube are verified in combination with an in vivo intestinal tube model and an in vitro gas-liquid exchange interface model to obtain verification results; The in vivo intestinal tube model includes an early endoderm intestinal tube model corresponding to the early development stage of the intestinal tube, and a late endoderm intestinal tube model corresponding to the late development stage of the intestinal tube; In the in vitro gas-liquid exchange interface model, the cells at the early development stage of the intestinal tube form a rectangular structure on the gas-liquid exchange interface, and the cells at the late development stage of the intestinal tube construct a three-dimensional crypt-villus structure model on the gas-liquid exchange interface; According to the verification results, C2BBe1 intestinal cells are inoculated in a double-layer culture chamber until the C2BBe1 intestinal cells are reprogrammed to a stemness precursor state.

[0005] Further, the construction of the in-vivo intestinal canal model is implemented in a multi-physics simulation software; When the in-vivo intestinal canal model is constructed, the curve expression input by the multi-physics simulation software is:

[0006]

[0007] wherein, is the horizontal coordinate, representing the length of the cross section of the in-vivo intestinal canal, is the vertical coordinate, representing the width of the cross section of the in-vivo intestinal canal, is a parameter of curve progression, used to generate the index or progress variable of the curve point, is the central circle radius, is the wrinkle amplitude, is the wrinkle wave number, is a constant.

[0008] Further, the construction of the in-vitro gas-liquid exchange interface model is implemented in a multi-physics simulation software; When the in-vitro gas-liquid exchange interface model is constructed, the curve expression input by the multi-physics simulation software is:

[0009]

[0010] wherein, is the horizontal coordinate of the cross section of the three-dimensional structure of the intestinal canal formed in the gas-liquid exchange interface model, representing the length of the cross section of the three-dimensional structure, is the vertical coordinate of the cross section of the three-dimensional structure of the intestinal canal formed in the gas-liquid exchange interface model, representing the height of the three-dimensional structure, is a parameter of curve progression, used to generate the index or progress variable of the curve point, is the wrinkle wave number, , is the total length of the cross section of the three-dimensional structure of the intestinal canal formed in the gas-liquid exchange interface model.

[0011] Further, the in-vivo intestinal canal model and the in-vitro gas-liquid exchange interface model are visualized: In the multi-physics simulation software, the physical field is configured according to the following formula:

[0012]

[0013] wherein, is the horizontal coordinate of the configured physical field, To configure the ordinate of the physical field, This is the distance from the point to the origin.

[0014] Furthermore, the method for seeding C2BBe1 intestinal cells in a double-layer culture chamber includes: Both the upper and lower chambers of the double-layer culture chamber were filled with Durbeco modified Eagle complete medium, so that C2BBe1 intestinal cells could be cultured at the liquid-liquid exchange interface to form an intestinal epithelial monolayer. The upper and lower chambers of the double-layer culture chamber were then filled with a mixed medium consisting of Durbeco modified Eagle complete medium, fetal bovine serum complete medium, and penicillin-streptomycin mixture complete medium, so that the C2BBe1 intestinal cells were cultured at the gas-liquid exchange interface until the C2BBe1 intestinal cells were reprogrammed into a stem precursor state.

[0015] Furthermore, the addition ratios of the Durbeco modified Eagle complete medium, fetal bovine serum complete medium, and penicillin-streptomycin mixed complete medium are 90%, 10%, and 1%, respectively.

[0016] Furthermore, the step of seeding C2BBe1 intestinal cells in a double-layered culture chamber further includes: Oxygen is introduced at the gas-liquid exchange interface, so that the C2BBe1 intestinal cells are in an oxygen-infused state.

[0017] Furthermore, the expression for the concentration distribution of oxygen includes:

[0018] in, This represents the number of moles of oxygen consumed per unit time. This refers to the oxygen concentration in a CO2 incubator at 37 ℃. It is a hyperbolic cosine function. The first-order rate constant for the oxygen decomposition reaction is... L It is a constant value. z The height of the three-dimensional intestinal structure formed in the gas-liquid exchange interface model.

[0019] Furthermore, the method for solving the oxygen concentration distribution expression includes: Solve the following equations in conjunction with the boundary conditions:

[0020] in, denoted as the diffusion coefficient of oxygen in C2BBe1 intestinal cells.

[0021] Secondly, this application proposes a C2BBe1 intestinal cell reprogramming system, comprising: The verification module is used to verify the specific mechanical stimulation characteristics of the intestine at different developmental stages by combining in vivo intestinal models and in vitro gas-liquid exchange interface models, and obtain the verification results. The in vivo intestinal model includes an early endoderm intestinal model corresponding to the early development stage of the intestinal tract, and a late endoderm intestinal model corresponding to the late development stage of the intestinal tract. In the in vitro gas-liquid exchange interface model, cells in the early development stage of the intestinal tract form a rectangular structure on the gas-liquid exchange interface, while cells in the late development stage of the intestinal tract construct a three-dimensional crypt-village structure model on the gas-liquid exchange interface. The inoculation module is used to inoculate C2BBe1 intestinal cells in a double-layer culture chamber according to the verification results, until the C2BBe1 intestinal cells are reprogrammed into a stem precursor state.

[0022] Compared with the prior art, this application has the following beneficial effects: This application proposes a C2BBe1 intestinal cell reprogramming method, which constructs an in vivo intestinal tract model and an in vitro gas-liquid exchange interface model, enabling simulation of real-world in vivo conditions. It can spontaneously reconstruct the crypt-village topology, realistically simulating the spatial heterogeneity and cell lineage distribution of the intestinal epithelium. After verifying the specific mechanical stimulation characteristics of different developmental stages of the intestinal tract, validation results are obtained, and actual in vitro seeding is performed based on these results. This method allows direct reprogramming of terminally differentiated cells, avoiding the dependence on pluripotent stem cells and complex induction procedures in traditional methods. It significantly improves reprogramming efficiency and stability, filling the gap in reprogramming techniques specifically for terminally differentiated intestinal cells, which lacks effective strategies for stable and reproducible induction of stemness recovery.

[0023] This application also proposes a C2BBe1 intestinal cell reprogramming system, which possesses all the advantages of the aforementioned C2BBe1 intestinal cell reprogramming methods. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a flowchart illustrating one aspect of the C2BBe1 intestinal cell reprogramming method of this application; Figure 2 This is a schematic diagram illustrating the stage-specific mechanical stimulation distribution analysis during the culture process of intestinal morphogenesis and gas-liquid exchange interface based on modeling, as described in this application embodiment. Figure 3 This is a schematic diagram illustrating the results of the pseudo-temporal expression dynamic analysis of PIEZO1 and CAMK2G in intestinal stem cells using single-cell RNA sequencing in an embodiment of this application. Figure 4 This is a schematic diagram showing the expression changes at the gas-liquid exchange interface from day 0 to day 4 as displayed by immunoblotting analysis in an embodiment of this application. Figure 5 As an embodiment of this application, for Figure 4 A schematic diagram showing the results of quantitative analysis of Western blot results; Figure 6 This is a schematic diagram illustrating the changes in transcriptional levels of CTGF and CAMK2G at the gas-liquid exchange interface from day 0 to day 4 during culture, as described in this embodiment of the application. Figure 7 This is a schematic diagram of immunoblotting analysis of YAP1 / TAZ and its phosphorylated form at the gas-liquid exchange interface from day 0 to day 4 of culture in this application embodiment. Figure 8 This is a schematic diagram of the immunoblotting quantitative results of YAP1 / TAZ and its phosphorylated form at the gas-liquid exchange interface from day 0 to day 4 of culture in this application embodiment. Figure 9 This is a schematic diagram of immunofluorescence staining of YAP1 protein expression at the gas-liquid exchange interface from day 0 to day 4, as described in this application embodiment. Figure 10 This is a schematic diagram illustrating the quantitative analysis of the fluorescence intensity ratio of YAP1 protein between the cell nucleus and cytoplasm on days 0 to 4 at the gas-liquid exchange interface, as described in this application embodiment. Figure 11 This is a schematic diagram of the immunofluorescence staining results of YAP1 protein in a frozen section of tissue at the gas-liquid exchange interface on day 4, as described in this embodiment of the application. Figure 12 This is a schematic diagram illustrating the expression changes of typical YAP1 target genes detected by qPCR at the gas-liquid exchange interface from day 0 to day 10 of culture, as described in this embodiment of the application. Figure 13 This is a schematic diagram of the reprogrammed intestinal stemness markers in an embodiment of this application; Figure 14 This is a schematic diagram of the intestinal adequacy potential markers after reprogramming, as shown in the embodiments of this application. Figure 15 This is a schematic diagram illustrating the induction of YAP1 activation and transcriptional reprogramming through gas-liquid exchange interface culture in an embodiment of this application. Figure 16 This is a schematic diagram of the oxygen distribution model in an embodiment of this application; Figure 17This is a schematic diagram illustrating the dynamic gene expression and metabolic transformation during the early reprogramming process of the gas-liquid exchange interface as revealed by transcriptome analysis in this application embodiment. Figure 18 This is a heatmap of bulk RNA sequencing from day 0 to day 4 of culture at the gas-liquid exchange interface, as shown in this embodiment of the application. Figure 19 This is a schematic diagram of the molecular network that, in the embodiments of this application, jointly promotes proliferation and differentiation, skeletal rearrangement, and anti-apoptosis through oxygen and mechanical stimulation. Figure 20 This embodiment of the application provides a schematic diagram of the cell composition and differentiation trajectory between an in vitro gas-liquid exchange interface model and in vivo parenteral tissue revealed by single-cell RNA sequencing. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0027] Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of the application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0028] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0029] In the description of the embodiments of this application, it should be noted that if terms such as "upper," "lower," "horizontal," or "inner" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the product of the invention is in use, they are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on this application. In addition, terms such as "first" and "second" are only used to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0030] Furthermore, the use of the term "horizontal" does not imply that the component must be absolutely horizontal, but rather that it can be slightly tilted. For example, "horizontal" simply means that its direction is more horizontal than "vertical," and does not mean that the structure must be completely horizontal, but can be slightly tilted.

[0031] In the description of the embodiments of this application, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0032] The human gut possesses remarkable regenerative capacity, with stem cells dynamically interacting with their microenvironment to maintain continuous tissue renewal and homeostasis. Intestinal tissue is spatially ordered along the crypt-villi axis, where proliferating progenitor cells reside in the crypts and differentiate as they migrate towards the villi apex. With the rapid development of regenerative medicine and organoid technology, establishing in vitro epithelial cell models with stem cell characteristics has become an important tool for studying intestinal development, disease mechanisms, and drug screening. However, most current in vitro models, including organoids derived from adult stem cells or pluripotent stem cells, have limitations in terms of structural complexity, reproducibility, and clinical translation. Specifically: (1) The stem cell dependency paradox: In recent years, studies have attempted to induce partial reversal of epithelial cells to a stem state through methods such as the introduction of exogenous transcription factors, activation of the Wnt (Wingless-Type MMTV Integration Site Family) pathway, and reconstruction of three-dimensional culture systems. However, these methods generally suffer from technical bottlenecks such as low reprogramming efficiency, unstable cell state, or dependence on complex growth factors. Currently, more than 80% of systems rely on the intermediate state of pluripotent stem cells and a multi-step induction differentiation process. The entire process usually takes 2 to 8 weeks, is cumbersome, has large batch variations, and involves a large number of uncertainties, which limits its application in regenerative medicine and high-throughput drug screening.

[0033] (2) Insufficient structural fidelity: Traditional organoids usually exhibit closed cavity structures with inverted topological relationships between crypts and villi, causing the crypts to face external stimuli, thus failing to realistically simulate spatial drug responses and pathogen interactions in vivo.

[0034] (3) Lack of mechanical-metabolic microenvironment: Existing systems generally lack the simulation of complex in vivo mechanical and metabolic microenvironments (such as oxygen gradient and mechanical signals), making it difficult to drive the physiological morphological formation and differentiation maintenance of intestinal epithelial cells.

[0035] Based on the above, this application proposes a C2BBe1 intestinal cell reprogramming method and system, which will be described in detail below with reference to embodiments and accompanying drawings.

[0036] like Figure 1 The diagram shown is a flowchart of one method for reprogramming C2BBe1 intestinal cells according to this application, which may include: S101, combining in vivo intestinal models and in vitro gas-liquid exchange interface models, was used to verify the specific mechanical stimulation characteristics of different developmental stages of the intestine, and the verification results were obtained.

[0037] By constructing in vivo intestinal models and in vitro gas-liquid exchange interface models, we can verify whether the in vitro gas-liquid exchange interface model can accurately reproduce the specific mechanical stimulation characteristics of different developmental stages of the intestine, providing a mechanical microenvironment basis consistent with in vivo physiological state for subsequent cell reprogramming.

[0038] The in vivo intestinal models include an early endoderm intestinal model corresponding to the early developmental stage of the intestinal tract, and a late endoderm intestinal model corresponding to the late developmental stage of the intestinal tract. In the in vitro gas-fluid exchange interface model, cells in the early developmental stage of the intestinal tract form rectangular structures at the gas-fluid exchange interface, while cells in the late developmental stage of the intestinal tract construct a three-dimensional crypt-village structure model at the gas-fluid exchange interface.

[0039] It is important to note that there are significant mechanical differences between the early and late stages of intestinal development. For example, mechanical forces are uniform in the early stages, while local stress increases in the later stages. These differences are key signals regulating cell differentiation. If in vitro models cannot simulate these characteristics, the microenvironment for subsequent cell culture will not match that in vivo, and reprogramming efficiency or structural development will deviate from the physiological state.

[0040] This application constructs corresponding models for the early and late stages of intestinal development. The early endoderm intestinal model simulates the early stages of intestinal development, typically presenting as a simple tubular structure. In practical applications, it can be constructed using parametric curves in COMSOL software, such as circular equations incorporating the basic radius and initial folds, to match the mechanical properties of in vivo intestinal tissue. The late endoderm intestinal model simulates the structural features of late-stage intestinal development, focusing on the complex morphology after crypt-villi formation. It is constructed using more complex parametric curves and adjusted material properties to reflect the mechanical changes following muscle layer development.

[0041] Furthermore, the in vitro gas-fluid exchange interface model is constructed based on a bilayer culture chamber, corresponding one-to-one with the stages of the in vivo model. In the early stage, cells form rectangular structures on the gas-fluid exchange interface, simulating the simple epithelial morphology of the early intestinal tract before the formation of crypts and villi, and uniform mechanical stimulation is generated by controlling the hydraulic pressure difference between the upper and lower chambers. In the late stage, cells spontaneously construct a three-dimensional crypt-villi structure model on the gas-fluid exchange interface, and the spatial heterogeneity of the late intestinal tract is simulated through parametric curves. At this stage, the mechanical stimulation shows a local increase due to the structural folds.

[0042] Meanwhile, COMSOL software can be used to simulate and calculate the mechanical stimulation characteristics of in vivo and in vitro models, such as stress range verification, spatial distribution verification, and reliability verification.

[0043] Validation shows that the external gas-liquid exchange interface model can effectively simulate the mechanical stimulation characteristics of different developmental stages of the intestinal tract in vivo, providing physiologically validated mechanical parameters for subsequent cell culture and avoiding reprogramming failures due to incompatibility between the in vitro and in vivo environments. Furthermore, a correlation model between structural morphology, mechanical stimulation, and developmental stage is established, providing a mechanical environmental basis for explaining the molecular mechanisms of subsequent cell reprogramming.

[0044] S102, based on the validation results, C2BBe1 intestinal cells were seeded in a double-layer culture chamber until the C2BBe1 intestinal cells were reprogrammed into a stem precursor state.

[0045] C2BBe1 cells are a monoclonal cell subline isolated from the human colon cancer cell line Caco-2. They possess more stable epithelial polarity and intestinal differentiation characteristics, but as highly differentiated epithelial cells, they naturally lack the proliferative potential and multi-lineage differentiation ability of intestinal stem cells, limiting their application in stem cell and regeneration research. C2BBe1 cells are terminally differentiated intestinal epithelial cells, lacking the proliferative and multi-lineage differentiation capabilities of stem cells. By regulating the in vitro mechanical environment, i.e., the verification results obtained in step S101, intracellular stemness-related signaling pathways are activated, achieving stemness restoration without the intervention of exogenous stem cells.

[0046] In practical applications, appropriate inoculation conditions and parameters can be selected according to the actual situation, such as the culture medium formula, gas-liquid exchange interface conditions, and inoculation parameters.

[0047] By inoculating cells based on validation results, this application demonstrates the ability to reprogram terminally differentiated C2BBe1 cells into a stem-like precursor state, restoring their self-renewal capacity and multi-lineage differentiation potential. This reprogramming can be achieved solely through biomechanical microenvironment regulation without the need for exogenous stem cells or gene editing, reducing ethical risks and operational complexity, and possessing greater clinical translational potential. Furthermore, because the reprogramming process aligns with the biomechanical regulation mechanisms of in vivo intestinal development, the physiological relevance and functional reliability of the precursor cells can be ensured.

[0048] The following specific example will further illustrate this application in detail: (1) Cell reprogramming is initiated.

[0049] like Figure 2 The diagram shows a schematic analysis of stage-specific mechanical stimulation distribution during intestinal morphogenesis and gas-liquid exchange interface culture based on modeling. In row a, the left image shows a schematic diagram of the cavity structure in the early stage of intestinal development, and the right image shows a schematic diagram of the corresponding mechanical stress distribution simulated using COMSOL Multiphysics software. In row b, the left image shows a schematic diagram of the structure in the late stage of intestinal development, the middle image shows the corresponding mechanical stress simulation results, and the right image shows a magnified detail of the mechanical stress in the magnified area. In row c, the left image shows a schematic diagram of the early intestinal epithelial cell arrangement in the in vitro gas-liquid exchange interface model, the middle image shows a schematic diagram of the mechanical stress distribution simulation results within the Transwell system of the gas-liquid exchange interface, and the right image shows a locally magnified stress distribution based on the geometry of the gas-liquid exchange interface. In row d, the left image shows a schematic diagram of the late-stage crypt-villi three-dimensional structure in the in vitro gas-liquid exchange interface model, the middle image shows a schematic diagram of the corresponding spatial heterogeneity mechanical stress simulation results, and the right image shows a locally (middle image) magnified stress distribution. Based on the mechanical stimulation methods and ranges experienced at different stages of real intestinal development, this application utilizes the gas-liquid exchange interface to simulate in vivo mechanical stimulation and intestinal development. In the Transwell bilayer culture chamber system, 500 μL of complete culture medium is added to the lower chamber, while no liquid culture medium is added to the upper chamber. As an example, the complete culture medium formulation used is 90% DMEM, 10% FBS, and 1% PS. This method automatically converts the hydraulic pressure difference between the upper and lower chambers into mechanical stimulation. It should be noted that this culture medium can be used in both the early and late stages of the culture process.

[0050] Specifically: As an example, in this embodiment, the parameters used in the in vivo intestinal model at the early and late stages can be set based on existing research, and the relevant parameter settings can be directly implemented in COMSOL Multiphysics software. For example, in the early stage (day 7), the outer diameter of the in vivo intestinal model is 500 μm, the inner diameter is 460 μm, and the wall thickness is 20 μm. In the late stage (day 12), the outer perimeter of the in vivo intestinal model is 1 mm. The three-dimensional structure is constructed in COMSOL Multiphysics software using the "parametric curve" function in the "Geometry" module. The parametric curve expression input into COMSOL Multiphysics software is:

[0051]

[0052] in, The x-axis represents the length of the cross-section of the intestine within the body. The vertical axis represents the width of the cross-section of the intestine within the body. These are parameters for curve advancement, used to generate "index" or "progress" variables for curve points. The radius of the central circle, For the amplitude of the fold, For the fold wave number, It is a constant.

[0053] In this embodiment, the specific parameter settings can be as follows: The range is from 0 to 2π. =60 μm, =60μm, =18, n=1.5. Meanwhile, the material properties are set as follows: density 1.05 g / cm³, Young's modulus 980 Pa, Poisson's ratio 0.3.

[0054] The parameters of the in vitro gas-liquid exchange interface model can be determined based on experimental measurements and data provided by the manufacturer. As an example, in the early stages of intestinal development, C2BBe1 intestinal cells form a rectangular structure at the gas-liquid interface of the Transwell membrane, with a width of 6.5 mm and a height of 10 μm. In the later stages of intestinal development, a model with a specific three-dimensional crypt-village structure can be created using the "parameter curve" function. The parameter curve expression input into the COMSOL Multiphysics software is as follows:

[0055]

[0056] in, The x-coordinate represents the cross-sectional length of the three-dimensional intestinal structure formed in the fluid exchange interface model, indicating the length of the three-dimensional structure profile. The vertical coordinate of the cross-section of the three-dimensional intestinal structure formed in the gas-liquid exchange interface model represents the height of the three-dimensional structure. These are parameters for curve advancement, used to generate "index" or "progress" variables for curve points. The fold wave number.

[0057] In this embodiment, the specific parameter settings can be as follows: The range is from 0 to 1.15. , , L represents the total length of the cross-section of the three-dimensional intestinal structure formed in the gas-liquid exchange interface model. The bottom boundary of this parameter curve connects to the left and right vertices. The material properties are: density 1.0 g / cm³, Young's modulus 1000 Pa, and Poisson's ratio 0.4.

[0058] In practical applications, during the geometric modeling process of the two models mentioned above, the visualization output can be selected using either the "2D" or "2D axisymmetric" component based on the model parameters. Under the "Structural Mechanics" module, the "Solid Mechanics" function can be enabled. The physical fields used to characterize the forces acting on the model can be configured according to the following formula to ensure full coverage of the model range:

[0059]

[0060] in, To configure the x-coordinate of the physical field, To configure the ordinate of the physical field, This is the distance from the point to the origin (i.e., the magnitude of the vector). It is a two-parameter arctangent function.

[0061] In addition, the final modeling and simulation results can be visualized using COMSOL Multiphysics software.

[0062] Based on the obtained model, this embodiment verifies the specific mechanical stimulation characteristics of different developmental stages of the intestine. Simulation results reveal the specific mechanical stimulation characteristics of different developmental stages of the intestine: in the early stages of intestinal development, the intestinal epithelial cells are subjected to a uniform unidirectional force, with a stress range of 2.5 to 4.1 × 10⁻⁶. -4 N / m². In the later stages of intestinal development, with the formation of muscle layer constraints and three-dimensional structure, significant spatial heterogeneity emerges, with stress ranging from 0 to 15 × 10⁻⁶ N / m². - ³ N / m², this trend is consistent with the stress distribution characteristics of stem cell sites in vivo.

[0063] The model established in this application successfully reproduced the aforementioned mechanical characteristics: in the early stages of intestinal development, before the three-dimensional crypt-villi structure is formed, the mechanical stimulation received by C2BBe1 intestinal cells is relatively uniform, with the simulated stress range from 5 to 25 × 10⁻⁶. -4 N / m². During the late stages of intestinal development, the simulated stress ranged from 2 to 12 × 10⁻⁶ N / m². - The stress value of ³ N / m² is consistent with in vivo observations. These results also indicate that the model constructed in this application can effectively reproduce the distribution characteristics of mechanical stimulation at different stages of intestinal development.

[0064] Further enhancing the reliability of this model is its ability to simulate the increased local mechanical stress at the base of the crypts during the late stages of in situ intestinal development (see details). Figure 2 (See the right-hand figures in rows b and d) to verify the reliability of the in vitro gas-liquid exchange interface model in this application in reconstructing physiologically relevant mechanical microenvironments.

[0065] After the above verification, C2BBe1 cells were seeded into the Transwell bilayer culture chamber system. The seeding protocol can specifically follow the scheme described in Chinese invention patent application publication number CN118703421A. The specific procedure is as follows: the seeding density of C2BBe1 cells is 10-1. 5 Cells were cultured in complete DMEM medium in wells, with each well containing one cell. Both the upper and lower chambers were filled to establish a liquid-liquid exchange interface. After overnight equilibration in the liquid-liquid exchange interface, the intestinal epithelial monolayer rapidly polarized. A TEER (Trans-Epithelial Electrical Resistance) reading above 500 Ω was considered indicative of intestinal epithelial monolayer formation. C2BBe1 cells were then cultured for 4 days in complete medium containing 90% DMEM (Dulbecco's Modified Eagle Medium), 9% FBS (Fetal Bovine Serum), and 1% PS (Penicillin-Streptomycin) with an air-liquid exchange interface. The expression of reprogramming markers and intestinal stem markers was monitored daily using qPCR (Quantitative Polymerase Chain Reaction), immune imprinting, and immunofluorescence.

[0066] like Figure 3The image shows a schematic diagram illustrating the results of the pseudo-temporal expression dynamic analysis of PIEZO1 and CAMK2G in intestinal stem cells (intStemL) using single-cell RNA sequencing (scRNA-seq). Figure 3 In the diagram, the solid red line represents PIEZO1, the solid blue line represents the smooth expression curve of CAMK2G, and the dashed lines represent the peak expression time points of each gene. For example... Figure 4 The image shows a schematic diagram of expression changes at the gas-liquid exchange interface from day 0 to day 4, as displayed by Western blot analysis. CAMK2G represents calcium / calmodulin-dependent protein kinase IIγ, and phos indicates phosphorylation. Figure 5 As shown, this is for Figure 4 A schematic diagram illustrating the results of quantitative analysis of Western blot results. (Example: ...) Figure 6 The diagram shows the changes in transcriptional levels of CTGF and CAMK2G at the gas-liquid exchange interface from day 0 to day 4, analyzed using real-time quantitative PCR (qPCR). Figure 7 The image shows a schematic diagram of the immunoblotting analysis of YAP1 / TAZ and its phosphorylated form (phos-YAP1 / TAZ) at the gas-liquid exchange interface from day 0 to day 4. Figure 8 The image shows a schematic diagram of the immunoblotting quantitative results of YAP1 / TAZ and its phosphorylated form (phos-YAP1 / TAZ) at the gas-liquid exchange interface from day 0 to day 4. Figure 9 The image shows a schematic diagram of immunofluorescence staining for the expression of YAP1 protein at the gas-liquid exchange interface from day 0 to day 4. Figure 9 In the image, the top row shows the original 60× field-of-view image, and the bottom row shows the 120× magnified image. (Example) Figure 10 The diagram illustrates the quantitative analysis of the fluorescence intensity ratio of YAP1 protein between the cell nucleus and cytoplasm at the gas-liquid exchange interface from day 0 to day 4. 100 cells were measured at each time point, and the experiment was repeated three times. Figure 11 The image shows a schematic diagram of the immunofluorescence staining results of YAP1 protein in a frozen section of tissue from the gas-liquid exchange interface on day 4. Figure 11 In the image, white arrows indicate regions of enhanced YAP1 expression. (For example...) Figure 12 The diagram shows the expression changes of typical YAP1 target genes detected by qPCR at the gas-liquid exchange interface from day 0 to day 10. Figure 12In this study, CDK4, SOX2, AREG, MYC, and AFP represent cyclin-dependent kinase 4, SRY-box transcription factor 2, amphiregulin, MYC proto-oncogene, and alpha-fetoprotein, respectively, all downstream effector molecules of YAP1. Data are expressed as mean ± standard error (SEM). Statistical significance was tested using one-way ANOVA and Dunnett's multiple comparison test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (applicable to...). Figure 5 , Figure 6 , Figure 8 , Figure 10 and Figure 12 In the immunofluorescence image, the corresponding colors and scales are: cell nucleus (blue), YAP1 (green), F-actin (red), and the scale bar is 20 μm.

[0067] In summary Figures 3 to 12 It can also be seen that this application activates the mechanosensitive ion channel PIEZO, causing an influx of calcium ions. Figures 3 to 6 ), inhibiting the Hippo signaling pathway ( Figures 3 to 6 ,as well as Figure 12 ), induces nuclear translocation of Yes-related protein 1 (YAP1) ( Figures 9 to 11 This process reprograms terminally differentiated C2BBe1 intestinal cells into a precursor state with stemness (marked by LGR5). Figure 13 The image shown is a schematic diagram of intestinal dryness markers after reprogramming. Figure 13 In the middle, the first row is ALI 44 A schematic diagram of LGR5 Z-stack immunofluorescence staining of the 3D structure of a bud (obtained from the crypt to the villi direction), with the lower rows showing the corresponding lateral projections. White arrows indicate LGR5 enrichment in the crypts. Color differentiation is blue for the nucleus, green for LGR5, and red for F-actin. Scale bar is 20 micrometers. Figure 14 The image shown is a schematic diagram of the potential markers for gut adequacy after reprogramming. Figure 14In the qPCR analysis of Ki67, LYZ, CDX2, and MUC2 expression after gas-liquid interface treatment, data are expressed as mean ± standard error; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, and Dunnett's test (b, c, h) were performed after one-way ANOVA. The ability to differentiate into other intestinal cell lineages was confirmed, with Ki67 (intestinal proliferation), MUC2 (mucosal layer), CDX2 (intestinal epithelium), and LYZ (endocrine function) as marker genes.

[0068] like Figure 15 The diagram illustrates how gas-liquid exchange interface culture induces YAP1 activation and transcriptional reprogramming. In conventional culture, the PIEZO channels in C2BBe1 cells are closed, preventing calcium ion influx and resulting in phosphorylated YAP1 that cannot enter the nucleus. However, the mechanical stimulation from ALI promotes the opening of the PIEZO pathway in C2BBe1 cells, leading to calcium ion influx and inducing YAP1 dephosphorylation, high expression, and nuclear translocation. This reprograms the cells to produce intestinal stem cell-related characteristics.

[0069] (2) Activation of the proliferation axis.

[0070] In this embodiment, the oxygen microenvironment in vivo was further simulated in the gas-liquid exchange interface culture method. For example... Figure 16 The figure shown is a schematic diagram of the oxygen distribution model. Figure 16 In the figures, (a) is a schematic diagram of oxygen distribution within the intestinal lumen during the early developmental stages of the intestinal tract (before crypt-villi structure formation) and the late developmental stages of the intestinal tract (after crypt-villi structure formation). (b) is a theoretical diagram of simulated oxygen distribution and diffusion in the in vitro gas-liquid exchange interface model during the early and late developmental stages of the intestinal tract. (c) is a graph showing the simulated and fitted oxygen diffusion results in the in vitro gas-liquid exchange interface model during the early (left) and late (right) developmental stages of the intestinal tract. The results indicate that in the model of this application, cells are rich in oxygen (oxygen concentration higher than 7.0 mmol / L) during both the early and late developmental stages, suggesting that its oxygen environment is similar to the eutrophic environment observed in vivo.

[0071] The specific calculation process is as follows: This application introduces oxygen at the gas-liquid exchange interface, placing the cultured tissue in an oxygen-immersed state. Modeling is performed by combining oxygen diffusion in the liquid and cells with aerobic metabolic reactions. In the figure, A represents oxygen, and B represents the cell. The cell is approximated as a liquid; therefore, it can be considered a binary solution composed of A and B. For a liquid phase thickness of... Mass conservation analysis of component A within the specified range yields the following results:

[0072] in, and These represent the positions of component A. and The quality of the work. Let A be the first-order rate constant for the decomposition reaction of A. This represents the cross-sectional area of ​​the liquid phase. Experimental data show that, in the in vitro gas-liquid exchange interface model of this application, oxygen is transferred from the cells at a constant rate. break down. This represents the number of moles of A consumed per unit time. When taking the limit... Then, the above equation can be transformed into:

[0073] in, Let be the molar flux of component A in the z-direction.

[0074] Assuming the concentration of component A is low, the equation can be simplified to an approximation:

[0075] in, Let represent the diffusion coefficient of A in B. Since the total molar concentration in the liquid phase remains essentially uniform, substituting the aforementioned expression into the mass conservation equation yields:

[0076] The equation can be solved by considering the following two boundary conditions: BC1: at ,

[0077] BC2: at ,

[0078] The value of L was determined experimentally, with 20 μm for the early stage of intestinal development and 150 μm for the late stage of intestinal development. The oxygen concentration in the 37 ℃ CO2 incubator was approximately 7.6 mmol / L.

[0079] Under the above conditions, the oxygen concentration can be calculated. The distribution expression is:

[0080] Next, the conditions for oxygen concentration during the early (day 0) and late (day 10) developmental stages will be analyzed. The distribution expression was plotted. The results showed that the cells in the model were in an oxygen-rich state in both stages, indicating that its oxygen metabolism characteristics were similar to those in vivo (see [reference]). Figure 16 (line c).

[0081] Cellular oxygen metabolism patterns are crucial for reprogrammed cell self-organization and differentiation. RNA sequencing (RNA-seq) results showed that in an oxygen-rich environment, genes related to the cAMP signaling pathway were significantly upregulated, SOX9 expression levels were significantly increased, and proliferation-related markers were also significantly enhanced. Figure 17 The diagram shown illustrates the dynamic gene expression and metabolic transformation during the early reprogramming process at the gas-liquid exchange interface, as revealed by transcriptome analysis. Figure 17 Figure a shows a heatmap of bulk RNA sequencing from day 0 to day 4 of culture at the gas-liquid exchange interface, displaying differentially expressed genes (DEGs) related to oxygen-related cAMP signaling pathways and proliferation-related features. Figure 17 Figure b shows the FPKM expression value of the GAPDH gene under different culture conditions, demonstrating that the oxygen metabolism pattern changed. The experiment found that the gas-liquid exchange interface culture from day 0 to day 4 (corresponding to...) Figure 17 The genes shown in the diagram are: ALI0, ALI1, ALI2, ALI3, and ALI4. Gene expression in the cAMP signaling pathway, which is highly associated with oxygen metabolism, gradually increases. Figure 17 Figure a) suggests that the cell has switched from anaerobic metabolism to aerobic metabolism, which produces more energy, and the expression of GAPDH corroborates this. Figure 17 Figure b). Genes related to proliferation showed significantly high expression on the first day of culture at the gas-liquid exchange interface. Figure 17 Figure a shows that the cells have begun to proliferate.

[0082] like Figure 18 The image shows a heatmap of bulk RNA sequencing from day 0 to day 4 of culture at the gas-liquid exchange interface. Row a is a schematic diagram of downstream marker genes of PIEZO and YAP1, and row b is a schematic diagram of the NOTCH, Ras, and PI3K / AKT signaling pathways, as well as differentially expressed genes (DEGs) related to skeletal rearrangement and apoptosis. In this application, continuous mechanical stimulation not only activated downstream markers of PIEZO and YAP1 (… Figure 18 (a) also inhibited gene expression in the NOTCH signaling pathway ( Figure 18 (b) The NOTCH signaling pathway is inhibited, and the mechanically regulated PIEZO ion channel can maintain cell self-renewal and proliferation by regulating the balance of the NOTCH signaling pathway. Activation of PIEZO can induce inactivation of the Hippo pathway (leading to increased expression of YAP1 target genes) Figure 18(a line), and further activated the Ras-related signal cascade reaction ( Figure 18 (b line) The expression of these transcription factors is closely related to enhanced cell survival and anti-apoptotic mechanisms.

[0083] like Figure 19 The diagram illustrates the molecular network that promotes proliferation and differentiation, cytoskeleton rearrangement, and anti-apoptosis through the combined effects of oxygen and mechanical stimulation. This confirms that the model presented in this application achieves a balance between cell proliferation and differentiation by inhibiting the NOTCH signaling pathway. In summary, under oxygen tension regulation and continuous mechanical stimulation, the SOX9-mediated cell proliferation program is synergistically activated by cAMP and the YAP1–SOX9 signaling axis, while activation of the PIEZO pathway also inhibits the NOTCH signaling pathway, thus achieving a dynamic balance between proliferation and redifferentiation. Figure 19 The diagram shows a molecular network that combines oxygen and mechanical stimulation to promote proliferation and differentiation, skeletal rearrangement, and anti-apoptosis.

[0084] (3) Structure and survival support.

[0085] This application enables cells to spontaneously form a three-dimensional intestinal-like tissue with a crypt-village axial structure through signal integration without the need for exogenous scaffolds or morphological factor gradients. The Ras signaling pathway regulates the cytoskeleton's tissue structure via the RAC1–RHOA–ROCK1 axis and upregulates the anti-apoptotic gene BIRC5 through the PI3K–AKT pathway, while simultaneously inhibiting the expression of pro-apoptotic factors (such as BCL2L11, FOXO1, BBC3, and FOXO4). Figure 18 (b line).

[0086] Therefore, this application establishes an "oxygen-mechanical force" synergistic regulatory network to drive the formation of the three-dimensional structure of intestinal tissue. Figure 19 In terms of mechanostimulation, PIEZO-Ca² + The pathway promotes cell proliferation and anti-apoptotic function by inhibiting NOTCH and activating the YAP1–SOX9 axis and the Ras–PI3K / AKT pathway cross-signaling. In terms of energy sensing, the oxygen-dependent ATP–cAMP axis maintains SOX9-mediated sustained cell proliferation. At the matrix feedback level, Ras–RHOA–ROCK signaling synergistically regulates cytoskeleton remodeling. The SOX9 signaling node acts as a key regulatory hub in this process, integrating mechanical and metabolic signals and dominating structural reconstruction and functional remodeling during intestinal development.

[0087] like Figure 20As shown, this diagram illustrates the cell composition and differentiation trajectory between the in vitro air-fluid exchange interface model and the in vivo parenteral tissue, revealed by single-cell RNA sequencing. Row a shows the UMAP visualization results based on single-cell RNA sequencing (scRNA-seq), displaying the annotated cell types in human colon tissue (hColon) and intestinal organoids on day 10 at the air-fluid exchange interface. Cell types with the "like" suffix in the annotations represent the populations cultured in the in vitro model. Row B is a schematic diagram of cell differentiation trajectories derived from single-cell pseudo-time analysis. Therefore, this application also verifies that C2BBe1 cells regain stemness and can differentiate into a multifunctional intestinal lineage (…). Figure 20 (Line a). Intestinal cell lineages were detected, twice that of traditional organoids, demonstrating the high degree of realism of this application. Furthermore, the developmental trajectory of cells redifferentiated into other intestinal cell lineages after reprogramming is similar to the in vivo developmental trajectory. Figure 20 (b line).

[0088] This application directly reprograms terminally differentiated cells, avoiding the reliance on pluripotent stem cells and complex induction procedures in traditional methods. This significantly improves efficiency and stability, filling the gap in effective strategies for stable and reproducible induction of stemness recovery in terminally differentiated intestinal cells through specific reprogramming technology. Furthermore, the model proposed in this application can spontaneously reconstruct the crypt-village topology, realistically simulating the spatial heterogeneity and cell lineage distribution of the intestinal epithelium. Moreover, the oxygen-mechanical regulation network significantly enhances the proliferation capacity of C2BBe1 cells and the expression level of stem cell markers. The entire induction process is rapid, controllable, and xeno-free (e.g., viral vectors or complex protein factors), making it more suitable for clinical translation. Finally, by introducing oxygen at the air-liquid interface, the cultured tissue is kept in an oxygen-rich state, solving the problem that traditional culture methods cannot accurately simulate the oxygen metabolism of intestinal epithelial cells. It also integrates, for the first time, multiple pathway mechanisms such as PIEZO–YAP1–SOX9, reconstructing the regulation of intestinal epithelial homeostasis by the mechano-metabolic microenvironment, providing a more realistic platform for disease modeling and drug screening. This method possesses structural stability and long-term cell viability, making it suitable for the construction of chronic disease models and high-throughput pharmacodynamic evaluation.

[0089] Based on the above method, this application also proposes a C2BBe1 intestinal cell reprogramming system, comprising: The verification module is used to verify the specific mechanical stimulation characteristics of the intestine at different developmental stages by combining in vivo intestinal models and in vitro gas-liquid exchange interface models, and obtain the verification results. The in vivo intestinal model includes an early endoderm intestinal model corresponding to the early development stage of the intestinal tract, and a late endoderm intestinal model corresponding to the late development stage of the intestinal tract. In the in vitro gas-liquid exchange interface model, cells in the early development stage of the intestinal tract form a rectangular structure on the gas-liquid exchange interface, while cells in the late development stage of the intestinal tract construct a three-dimensional crypt-village structure model on the gas-liquid exchange interface. The inoculation module is used to inoculate C2BBe1 intestinal cells in a double-layer culture chamber according to the verification results, until the C2BBe1 intestinal cells are reprogrammed into a stem precursor state.

[0090] The above are merely preferred embodiments of this application and are not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method of reprogramming C2BBel intestinal cells, characterized in that, The method comprises the following steps: The verification result is obtained by combining the in-vivo intestinal tube model and the in-vitro gas-liquid exchange interface model to verify the mechanical stimulation characteristics specific to different development stages of the intestinal tube. The in-vivo intestinal tube model comprises an early endoderm intestinal tube model corresponding to an early development stage of the intestinal tube and a late endoderm intestinal tube model corresponding to a late development stage of the intestinal tube. In the in-vitro gas-liquid exchange interface model, cells in the early development stage of the intestinal tube form a rectangular structure on the gas-liquid exchange interface, and cells in the late development stage of the intestinal tube construct a three-dimensional crypt-villus structure model on the gas-liquid exchange interface. According to the verification result, C2BBe1 intestinal cells are inoculated in a double-layer culture chamber, and the C2BBe1 intestinal cells are reprogrammed to a precursor state with stemness.

2. The C2BBe1 intestinal cell reprogramming method of claim 1, wherein: The in-vivo intestinal tube model is constructed in a multi-physical field simulation software; When constructing the in-vivo intestinal tube model, the curve expression input by the multi-physical field simulation software is: wherein, is the abscissa representing the length of the cross section of the intestinal canal in the body, is the ordinate representing the width of the cross section of the intestinal canal in the body, is a parameter of the curve progression, used to generate an index or progress variable of the curve points, is the radius of the central circle, is the amplitude of the corrugation, is the wave number of the corrugation, is a constant.

3. The C2BBe1 intestinal cell reprogramming method according to claim 1, characterized in that: The in-vitro gas-liquid exchange interface model is constructed in a multi-physical field simulation software; When constructing the in-vitro gas-liquid exchange interface model, the curve expression input by the multi-physical field simulation software is: wherein, is the horizontal coordinate of the cross section of the three-dimensional structure of the intestine formed in the gas-liquid exchange interface model, representing the length of the cross section of the three-dimensional structure, is the vertical coordinate of the cross section of the three-dimensional structure of the intestine formed in the gas-liquid exchange interface model, representing the height of the three-dimensional structure, is the parameter of the curve progression, used to generate the index or progress variable of the curve points, is the wave number of the fold, , is the total length of the cross section of the three-dimensional structure of the intestine formed in the gas-liquid exchange interface model.

4. The method of claim 1, wherein the C2BBe1 intestinal cells are reprogrammed by, The in-vivo intestinal tube model and the in-vitro gas-liquid exchange interface model are visualized. The visualization is realized in the multi-physical field simulation software, and the physical field is configured according to the following formula: wherein, is the abscissa of the physical field to be configured, is the ordinate of the physical field to be configured, is the distance of the point from the origin.

5. The method of reprogramming C2BBe1 intestinal cells according to claim 1, wherein, The method of inoculating C2BBe1 intestinal cells in a double-layer culture chamber comprises the following steps: The upper chamber and the lower chamber of the double-layer culture chamber are filled with Dulbecco's modified Eagle's complete medium, so that the C2BBe1 intestinal cells are cultured under a liquid-liquid exchange interface to form an intestinal epithelial monolayer; The upper chamber and the lower chamber of the double-layer culture chamber are filled with a mixed culture medium composed of Dulbecco's modified Eagle's complete medium, fetal bovine serum complete medium, and penicillin-streptomycin mixed liquid complete medium, so that the C2BBe1 intestinal cells are cultured under a gas-liquid exchange interface until the C2BBe1 intestinal cells are reprogrammed to a precursor state with stemness.

6. The method of reprogramming C2BBe1 intestinal cells according to claim 5, wherein, The addition proportions of the Dulbecco's modified Eagle's complete medium, the fetal bovine serum complete medium, and the penicillin-streptomycin mixed liquid complete medium are 90%, 10%, and 1%, respectively.

7. The method of reprogramming C2BBe1 intestinal cells according to claim 1, wherein, The method of inoculating C2BBe1 intestinal cells in a double-layer culture chamber further comprises the following steps: Oxygen is introduced into the gas-liquid exchange interface to make the C2BBe1 intestinal cells in an oxygen-infiltrated state.

8. The method of reprogramming C2BBe1 intestinal cells according to claim 7, wherein, The concentration distribution expression of the oxygen comprises: wherein, is the number of moles of oxygen consumed per unit time, is the oxygen concentration in the CO2incubator at 37 °C, is the hyperbolic cosine function, is the first order rate constant for the oxygen decomposition reaction, L is a constant value, z is the height of the three-dimensional structure of the intestine formed in the gas-liquid exchange interface model.

9. The method of reprogramming C2BBe1 intestinal cells according to claim 8, wherein, The concentration distribution expression of the oxygen is solved by the following method: The following equation is solved by combining the boundary conditions: wherein, D is the diffusion coefficient of oxygen in C2BBe1 intestinal cells.

10. A C2BBe1 intestinal cell reprogramming system, characterized in that, The method comprises the following steps: The verification module is configured to combine the in-vivo intestinal tube model and the in-vitro gas-liquid exchange interface model to verify the mechanical stimulation characteristics specific to different development stages of the intestinal tube, and obtain a verification result. The in-vivo intestinal tube model comprises an early endoderm intestinal tube model corresponding to an early development stage of the intestinal tube and a late endoderm intestinal tube model corresponding to a late development stage of the intestinal tube. In the in-vitro gas-liquid exchange interface model, cells in the early development stage of the intestinal canal form a rectangular structure on the gas-liquid exchange interface, and cells in the late development stage of the intestinal canal construct a three-dimensional crypt-villus structure model on the gas-liquid exchange interface. The inoculation module is configured to inoculate C2BBe1 intestinal cells in a double-layer culture chamber according to the verification result, so that the C2BBe1 intestinal cells are reprogrammed to a precursor state with stemness.

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