Intestinal organ chip analysis method and device based on endogenous autofluorescence parameters

Abstract

The application relates to an intestinal organ chip analysis method and device based on an endogenous autofluorescence parameter, and applies to the technical field of material analysis. The method comprises the following steps: determining autofluorescence images corresponding to multiple preset time points after intestinal organ chip inoculation; for each preset time point, calculating a fluorescence coverage value and a relative fluorescence intensity value corresponding to the preset time point; based on the relative fluorescence intensity values and the fluorescence coverage values of the multiple preset time points, determining fluorescence intensity change trends and fluorescence coverage change trends corresponding to the multiple preset time points respectively; based on a preset correlation rule set, a relative fluorescence intensity value, a fluorescence coverage value, a fluorescence intensity change trend and a fluorescence coverage change trend corresponding to a target preset time point, determining a state analysis result of the intestinal organ chip at the target preset time point; the method can realize non-destructive and dynamic analysis of various states of the intestinal organ chip, thereby reducing the analysis cost and improving the analysis accuracy.

Description

Technical Field

[0001] This application relates to the field of materials analysis technology, and in particular to a method and apparatus for analyzing intestinal organoid microarrays based on endogenous autofluorescence parameters. Background Technology

[0002] With the development of biotechnology, intestinal organoid microarray technology has emerged. This technology relies on microfluidic platforms to construct a monolayer of intestinal epithelium with physiological polarity and barrier activity, which can replicate the cascaded physiological functions of the intestine, such as substance absorption, mucus secretion, and epithelial barrier regulation. It is widely used in drug absorption, toxicity evaluation, nutrient metabolism, and disease model research. The effectiveness of intestinal organoid microarrays is highly dependent on the state of the microarray, such as its maturity.

[0003] In related technologies, the state of intestinal organoid microarrays is usually analyzed by manual judgment or by immunofluorescence staining or gene expression analysis. However, the analysis process can damage the intestinal organoid microarray, making it impossible to achieve dynamic analysis of the state of the intestinal organoid microarray, resulting in high analysis costs and low accuracy. Summary of the Invention

[0004] Therefore, it is necessary to provide an intestinal organoid microarray analysis method and apparatus based on endogenous autofluorescence parameters that can reduce the analysis cost of intestinal organoid microarrays through non-destructive analysis and improve the accuracy of intestinal organoid microarray state assessment, in order to address the above-mentioned technical problems.

[0005] In a first aspect, this application provides a method for analyzing intestinal organoid microarrays based on endogenous autofluorescence parameters, including: At multiple preset time points after intestinal organoid microarray seeding, the intestinal organoid microarray is irradiated with a laser of a specific wavelength and its endogenous autofluorescence signal is collected to obtain autofluorescence images corresponding to each of the multiple preset time points. For each preset time point, the area ratio of the fluorescent positive region in the target region in the corresponding autofluorescence image is calculated as the fluorescence coverage value of the preset time point; and, taking the autofluorescence image acquired at the first preset time point as a reference, the relative fluorescence intensity value of the target region in the corresponding autofluorescence image is calculated to obtain the relative fluorescence intensity value of the preset time point; the target region is the intestinal epithelial region in the intestinal organoid chip; Based on the relative fluorescence intensity values ​​and fluorescence coverage values ​​at multiple preset times, determine the fluorescence intensity change trend and fluorescence coverage change trend corresponding to each of the multiple preset times; Based on a preset association rule set, the relative fluorescence intensity value, fluorescence coverage value, fluorescence intensity change trend, and fluorescence coverage change trend corresponding to a target preset time, the state analysis results of the intestinal organoid microarray at the target preset time are determined; wherein, the preset association rule set includes at least a variety of rules used to indicate the association relationship between the fluorescence intensity value, the fluorescence coverage value, the fluorescence intensity change trend, and the fluorescence coverage change trend, so as to determine different microarray development or abnormal states; the target preset time is any one of the multiple preset times.

[0006] Secondly, this application also provides an intestinal organoid microarray analysis device based on endogenous autofluorescence parameters, comprising: The acquisition module is used to irradiate the intestinal organoid chip with a laser of a specific wavelength at multiple preset time points after inoculation and collect its endogenous autofluorescence signal to obtain autofluorescence images corresponding to each of the multiple preset time points. The calculation module is used to calculate the area ratio of the fluorescent positive region in the target region of the corresponding autofluorescence image for each preset time, as the fluorescence coverage value of the preset time; and, with the autofluorescence image acquired at the first preset time as a reference, calculate the relative fluorescence intensity value of the target region in the corresponding autofluorescence image, to obtain the relative fluorescence intensity value of the preset time; the target region is the intestinal epithelial region in the intestinal organoid chip; The timing processing module is used to determine the fluorescence intensity change trend and the fluorescence coverage change trend corresponding to each of the multiple preset times based on the relative fluorescence intensity value and the fluorescence coverage value at multiple preset times. The analysis module is used to determine the state analysis results of the intestinal organoid microarray at the target preset time based on a preset association rule set, the relative fluorescence intensity value, fluorescence coverage value, fluorescence intensity change trend, and fluorescence coverage change trend corresponding to the target preset time. The preset association rule set includes at least a variety of rules used to indicate the association between the fluorescence intensity value, the fluorescence coverage value, the fluorescence intensity change trend, and the fluorescence coverage change trend, in order to determine different microarray developmental or abnormal states. The target preset time is any one of the multiple preset times.

[0007] The aforementioned method and apparatus for analyzing intestinal organoid microarrays based on endogenous autofluorescence parameters acquires endogenous autofluorescence images of intestinal organoid microarrays at different culture time periods, calculates the relative fluorescence intensity and fluorescence coverage at each preset time point, and then determines the fluorescence intensity and fluorescence coverage trends corresponding to each preset time point based on the relative fluorescence intensity and fluorescence coverage at multiple preset time points. Finally, association analysis is performed using a preset association rule set to obtain the state analysis results of the intestinal organoid microarray at each preset time point. Throughout the analysis process, no additional fluorescent markers are required, achieving non-invasive analysis of intestinal organoid microarrays and reducing analysis costs. Furthermore, by quantifying different states of the intestinal organoid microarray through fluorescence intensity values ​​and fluorescence coverage, dynamic analysis of the intestinal organoid microarray can be achieved, effectively improving the analysis efficiency and accuracy. Attached Figure Description

[0008] To more clearly illustrate the technical solutions in the embodiments of this application or related technologies, the drawings used in the description of the embodiments of this application or related technologies will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0009] Figure 1 This is a flowchart illustrating an intestinal organoid microarray analysis method based on endogenous autofluorescence parameters in one embodiment. Figure 2 This is a flowchart illustrating step 202 in one embodiment; Figure 3 This is a schematic diagram of the application process of intestinal organoid microarray maturity analysis in one embodiment; Figure 4 These are bright-field images and autofluorescence images acquired at different times in one embodiment; Figure 5 This is a schematic diagram of verification data for permeability verification of an intestinal organoid chip involved in one embodiment; Figure 6 This is a schematic diagram of staining results for performance verification of a mature intestinal organoid microarray in one embodiment. Figure 7 This is a schematic diagram of abnormal state staining for an intestinal organoid microarray in one embodiment; Figure 8 This is a structural block diagram of an intestinal organoid microarray analysis device based on endogenous autofluorescence parameters in one embodiment. Detailed Implementation

[0010] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0011] It should be noted that the terms "first," "second," etc., used in this application can be used to describe various elements, but these elements are not limited by these terms. These terms are only used to distinguish the first element from the second element. The terms "comprising" and "having," and any variations thereof, used in this application, are intended to cover non-exclusive inclusion. The term "multiple" used in this application refers to two or more. The term "and / or" used in this application refers to one of the embodiments, or any combination of multiple embodiments.

[0012] In one exemplary embodiment, such as Figure 1 As shown, a method for analyzing intestinal organoid microarrays based on endogenous autofluorescence parameters is provided. Taking a server application as an example, the method includes steps 201 to 204. Wherein: Step 201: At multiple preset time points after intestinal organoid microarray seeding, the intestinal organoid microarray is irradiated with a laser of a specific wavelength and its endogenous autofluorescence signal is collected to obtain autofluorescence images corresponding to each preset time point.

[0013] Among them, intestinal organoid chips are microfluidic culture devices that combine microfluidic chips, culture chambers and other structures for in vitro culture of intestinal organs. Intestinal organoid chips can simulate the intestinal microenvironment, support the growth, differentiation and morphological development of intestinal organs, and serve as an in vitro model carrier for conducting intestinal-related physiological, pathological and drug evaluation studies.

[0014] In this embodiment, the multiple preset time points after inoculation are set with reference to the start of complete culture medium perfusion; for example, the multiple preset time points can be the 3rd, 5th, 7th, 10th, 12th, and 15th day after the start of culture. Of course, the interval between each preset time point can be set according to actual needs.

[0015] Autofluorescence imaging is an optical image obtained by irradiating an intestinal organoid microarray with excitation light of a specific wavelength, causing the intestinal organoid's own biological components to generate endogenous fluorescence upon stimulation, and then acquiring the image through an imaging device. It is understood that autofluorescence does not require additional fluorescent dye labeling, thus it will not cause damage to the intestinal organoid microarray.

[0016] It should be noted that various endogenous fluorescent molecules exist in intestinal organoids, among which flavin coenzymes (including flavin adenine dinucleotide, FAD) are one of the important sources of endogenous fluorescence. Under specific excitation conditions (e.g., 488 nm excitation), these endogenous fluorescent molecules can produce characteristic fluorescence emission signals. The autofluorescence signal detected in this invention reflects, to a certain extent, the changes in endogenous fluorescent components of intestinal epithelial cells and their dynamic changes related to cellular functional status, and can serve as an important indicator for evaluating organoid functional status and injury response under exogenous labeling conditions.

[0017] Step 202: For each preset time, calculate the area ratio of the fluorescent positive region in the target region in the corresponding autofluorescence image as the fluorescence coverage value of the preset time; and, with the autofluorescence image acquired at the first preset time as a reference, calculate the relative fluorescence intensity value of the target region in the corresponding autofluorescence image to obtain the relative fluorescence intensity value of the preset time; the target region is the intestinal epithelial region in the intestinal organoid chip.

[0018] In some embodiments, the average fluorescence intensity of the target region can be calculated for the autofluorescence image corresponding to each preset time, and used as the fluorescence intensity value of the target region; further, the average fluorescence intensity of the target region can be obtained by calculating the average gray value of all pixels in the autofluorescence image, and then the fluorescence intensity value of the target region in the autofluorescence image acquired at the first preset time is used as the reference fluorescence intensity, and the ratio of the average fluorescence intensity of the target region to the reference fluorescence intensity is used as the relative fluorescence intensity value at the preset time.

[0019] In other embodiments, for the autofluorescence image corresponding to each preset time point, the autofluorescence image is first divided into multiple independent single-cell regions using a single-cell segmentation algorithm, the average gray value of each cell is calculated, and then the overall mean of the average gray values ​​of all cells is taken. The overall mean or the normalized value of the overall mean is used as the relative fluorescence intensity value of the target region. The normalized value of the overall mean can be the ratio of the overall mean to the gray value of a reference single cell, and the gray value of the reference single cell can be the average gray value of all cells in the autofluorescence image corresponding to the first preset time point.

[0020] Single-cell segmentation can be achieved using image processing methods such as threshold segmentation, edge detection, machine learning, or instance segmentation.

[0021] Fluorescence coverage (C_coverage) characterizes the spatial proportion of autofluorescent positive regions within the target area; a value closer to 1 indicates a higher degree of coverage. Since the autofluorescence signal in intestinal organoid microarrays primarily originates from epithelial cell populations in metabolically relevant states, the dynamic changes in fluorescence coverage can, to some extent, reflect the spatial expansion, continuity, and barrier structure status of the intestinal epithelial cell population. As organoids mature, epithelial cells gradually proliferate, migrate, and form continuous coverage structures, leading to an increasing trend in C_coverage. Conversely, during drug-induced damage or functional decline, the reduction, breakage, or detachment of fluorescent positive regions can cause a decrease in C_coverage. Therefore, C_coverage can serve as an important label-free indicator for evaluating the integrity and functional changes of the epithelial structure in intestinal organoid microarrays.

[0022] In some embodiments, the process of calculating fluorescence coverage includes: performing background subtraction on the autofluorescence image, for example, by using the rolling ball method to subtract the background; then, using the big rule method to perform automatic global thresholding to obtain a binary image; identifying all fluorescent positive regions within the target area using a connected component labeling algorithm; and calculating the proportion of all fluorescent positive regions to the entire target area to obtain the fluorescence coverage.

[0023] In some embodiments, before identifying all fluorescent positive regions using a connected component labeling algorithm, isolated noise points with an area smaller than a preset threshold (e.g., 100 pixels) can be removed, thereby improving the accuracy of fluorescence coverage.

[0024] Step 203: Based on the relative fluorescence intensity values ​​and fluorescence coverage values ​​at multiple preset times, determine the fluorescence intensity change trend and fluorescence coverage change trend corresponding to each of the multiple preset times.

[0025] Among them, the fluorescence intensity change trend is used to describe the dynamic evolution of the relative fluorescence intensity of intestinal organoid microarrays over time.

[0026] It is understandable that multiple preset times have a temporal sequence. Therefore, by combining the fluorescence intensity values ​​corresponding to each preset time and the temporal sequence of the multiple preset times, the fluorescence intensity change trend at any preset time can be obtained.

[0027] In some embodiments, the relative fluorescence intensity values ​​corresponding to each preset time point can be arranged sequentially according to the time axis, and the rise, fall, stabilization or fluctuation of the relative fluorescence intensity can be quantified by means of curve fitting, difference comparison, slope calculation, etc., so as to obtain the fluorescence intensity change trend at each preset time point.

[0028] In other embodiments, based on the obtained fluorescence intensity change trend, a fluorescence intensity threshold can also be set, so that the fluorescence intensity change trend and the fluorescence intensity threshold can be combined to further determine whether the fluorescence intensity at each time point is abnormal; for example, if the fluorescence intensity change trend is rising but does not exceed the threshold, it is considered to be in the process of growth.

[0029] Among them, the fluorescence coverage change trend is used to describe the dynamic evolution of the fluorescence coverage value of intestinal organoid microarray over time.

[0030] It is understandable that multiple preset times have a temporal sequence. Therefore, by combining the fluorescence coverage values ​​corresponding to each preset time and the temporal sequence of the preset times, the trend of fluorescence coverage change can be obtained.

[0031] In some embodiments, the fluorescence coverage values ​​corresponding to each preset time point can be arranged sequentially according to the time axis, and the rising, falling, stable or fluctuating state of fluorescence coverage can be quantified by curve fitting, difference comparison, slope calculation and other methods, so as to obtain the trend of fluorescence coverage change.

[0032] In other embodiments, based on the obtained fluorescence coverage change trend, a fluorescence coverage threshold can also be set, so that the fluorescence coverage change trend and the fluorescence coverage threshold can be combined to further determine whether the fluorescence coverage at each time point is abnormal.

[0033] Step 204: Based on the preset association rule set, the relative fluorescence intensity value, fluorescence coverage value, fluorescence intensity change trend, and fluorescence coverage change trend corresponding to the target preset time, determine the state analysis results of the intestinal organoid microarray at the target preset time; wherein, the preset association rule set contains at least a variety of rules used to indicate the association between fluorescence intensity value, fluorescence coverage value, fluorescence intensity change trend, and fluorescence coverage change trend, so as to determine different microarray development or abnormal states; the target preset time is any one of multiple preset times.

[0034] The preset association rule set contains a variety of association rules, which are used to indicate the relationship between light intensity value, fluorescence coverage value, fluorescence intensity change trend and fluorescence coverage change trend.

[0035] In some embodiments, the association rules in the preset association rule set are pre-established. For example, under the same culture conditions, multiple reference intestinal organoid chips can be continuously acquired with autofluorescence images until the multiple reference intestinal organoid chips can pass maturity verification, such as FITC-dextran (Fluorescein Isothiocyanate-dextran) permeability detection verification, i.e., they are fully mature, thereby obtaining autofluorescence images corresponding to multiple time points. Then, the fluorescence state of the autofluorescence images corresponding to multiple time points is statistically analyzed. The fluorescence state includes at least fluorescence intensity and fluorescence coverage, and the mean and standard deviation are calculated to construct a fluorescence state reference distribution model. Based on the fluorescence state reference distribution model, a variety of association rules are abstracted.

[0036] For example, based on the fluorescence state reference distribution model, multiple types of association rules can be abstracted, such as time-series correspondence rules, peak correspondence rules, and trend correspondence rules. Among them, time-series correspondence rules are used to define the standard reference range of various fluorescence indicators under different culture durations; peak correspondence rules are used to calibrate the time range corresponding to the peak; and trend correspondence rules are used to define the time range in which various trends appear.

[0037] For example, multiple preset times include five times, namely the first time to the fifth time. If the above analysis is performed based on the five times, the state analysis result of the fifth time can be obtained; if the above analysis is performed based on the first four times (the first time to the fourth time), the state analysis result of the fourth time can be obtained, and so on, the state analysis results of each time can be obtained.

[0038] In other embodiments, a time window of a preset time length closest to the target preset time can be determined. The fluorescence intensity change trend and fluorescence coverage change trend are then clipped using this time window to obtain the target fluorescence intensity change trend and target fluorescence coverage change trend corresponding to the target preset time. Based on a preset association rule set, the relative fluorescence intensity value, fluorescence coverage value, target fluorescence intensity change trend, and target fluorescence coverage change trend corresponding to the target preset time are then used to determine the state analysis result of the intestinal organoid microarray at the target preset time. This process utilizes the target fluorescence intensity change trend and target fluorescence coverage change trend, which have the highest reference value for the target preset time, to determine the state analysis result of the intestinal organoid microarray at the target preset time, further improving the reliability of the state analysis result.

[0039] The aforementioned label-free dynamic analysis method for intestinal organoid microarrays based on autofluorescence parameters involves acquiring endogenous autofluorescence images of the intestinal organoid microarray at different culture time periods, calculating the relative fluorescence intensity and fluorescence coverage at each preset time point, and then determining the fluorescence intensity and fluorescence coverage trends corresponding to each preset time point based on the relative fluorescence intensity and fluorescence coverage at multiple preset time points. Finally, association analysis is performed using a preset association rule set to obtain the state analysis results of the intestinal organoid microarray at each preset time point. Throughout this analysis process, no additional fluorescent markers are required, achieving non-invasive analysis of the intestinal organoid microarray and reducing analysis costs. Furthermore, by quantifying different states of the intestinal organoid microarray through fluorescence intensity values ​​and fluorescence coverage, dynamic analysis of the intestinal organoid microarray can be achieved, effectively improving the analysis efficiency and accuracy.

[0040] In one exemplary embodiment, such as Figure 2 As shown, Figure 2 A flowchart illustrating step 202 in an embodiment of this application is shown. Step 202 includes steps 301 to 303. Wherein: Step 301: Sort multiple preset times in chronological order to obtain a time sequence.

[0041] For example, multiple preset times are sorted in chronological order to obtain a time sequence.

[0042] Step 302: For the k-th preset time in the time series, the ratio of the average fluorescence intensity of the target region in the autofluorescence image corresponding to the k-th preset time to the average fluorescence intensity of the target region in the autofluorescence image corresponding to the first preset time is taken as the relative fluorescence intensity value of the k-th preset time, where k is a positive integer; wherein, the target region includes the intestinal epithelial region in the intestinal organoid chip.

[0043] It should be noted that for intestinal organoid microarrays, the intestinal epithelial region is the key area for representing the maturity of intestinal organoids. Therefore, by identifying the intestinal epithelial region as the target region, interference from other regions can be effectively avoided.

[0044] For example, for the k-th preset time, the average fluorescence intensity I_sample of the target area is determined by the average gray value of all pixels in the target area; for the first preset time, the average fluorescence intensity I_ref of the target area is determined by the average gray value of all pixels in the target area, and the relative fluorescence intensity value I_norm at the k-th preset time is I_sample / I_ref.

[0045] Step 303: For each preset time, the ratio of the total area of ​​the fluorescent positive region in the autofluorescence image corresponding to the preset time to the total area of ​​the autofluorescence image is used as the fluorescence coverage value corresponding to the preset time.

[0046] The fluorescent positive region refers to the region in the autofluorescence image that shows an effective fluorescence signal.

[0047] In some embodiments, fluorescent positive regions can be determined by a connected component labeling algorithm. For example, the autofluorescence image is first processed by background subtraction, threshold segmentation and noise removal to obtain a binary image. Then, the 8-connected component labeling algorithm is used to traverse the image to identify and divide all interconnected fluorescent signal regions, which are the fluorescent positive regions.

[0048] In other embodiments, the fluorescence coverage value at each preset time can be the ratio of the total area of ​​the fluorescent positive region in the autofluorescence image to the total area of ​​the target region in the autofluorescence image at each preset time.

[0049] For example, the fluorescence coverage value corresponding to the preset time can be expressed as C_coverage=ΣA_positive / A_ROI, where ΣA_positive represents the sum of the areas of all fluorescent positive regions at the preset time, and A_ROI represents the total area of ​​the target region.

[0050] In this embodiment, the preset time points are first sorted in chronological order to ensure the consistency of the time logic of data analysis; the relative fluorescence intensity is obtained by using the fluorescence intensity of the intestinal epithelial region at the initial time as a reference, thereby reducing the influence of interference factors such as equipment parameters and sample background fluorescence, and making the fluorescence intensity at different time points comparable in the horizontal direction; at the same time, the fluorescence coverage rate is calculated by the ratio of the area of ​​the regions, ensuring the accuracy of the fluorescence intensity value and the fluorescence coverage rate value.

[0051] In some embodiments, based on a preset association rule set, the relative fluorescence intensity value, fluorescence coverage value, fluorescence intensity change trend, and fluorescence coverage change trend corresponding to a target preset time, the state analysis results of the intestinal organoid microarray at the target preset time are determined, including: Correlation analysis is performed based on the relative fluorescence intensity value, fluorescence coverage value, fluorescence intensity change trend, and fluorescence coverage change trend at the target preset time to determine the actual correlation analysis results at the target preset time.

[0052] The correlation analysis can include trend synchronization analysis, numerical correlation analysis at the same time, peak correlation analysis, etc. For example, trend synchronization analysis is used to describe the consistency of fluorescence intensity and fluorescence coverage over time; numerical correlation analysis at the same time is used to describe the numerical relationship between fluorescence intensity and fluorescence coverage at the same time. For example, a higher fluorescence intensity and a lower fluorescence coverage at the same time may indicate a possible decoupling between metabolism and structure; peak correlation analysis is used to describe the peak relationship between fluorescence intensity and fluorescence coverage.

[0053] Actual correlation analysis results may include whether the changes in fluorescence intensity and fluorescence coverage are synchronized, whether they enter a specified numerical range, the magnitude of the change, etc., and may also include the relationship between the relative fluorescence intensity value or fluorescence coverage value and the corresponding threshold.

[0054] The maturity of intestinal organoid microarrays is used to describe the degree of intestinal epithelial tissue culture within the microarray. Understandably, only by using a mature intestinal organoid microarray can relatively accurate test results be obtained. Therefore, it is necessary to objectively detect and evaluate the maturity of intestinal organoid microarrays.

[0055] Based on the actual association analysis results at the target preset time, rule matching is performed in the preset association rule set to determine the target association rule that matches the actual association analysis results in the preset association rule set.

[0056] In some embodiments, multiple association rules in the preset association rule set are mutually exclusive. Based on this, the preset association rule set can be traversed and matched according to the actual association analysis results to determine the target association rule that matches the actual association analysis results.

[0057] In other embodiments, multiple association rules in the preset association rule set have priorities. Based on this, multiple association rules can be matched sequentially according to their priorities. After a target association rule that matches the actual association analysis result is matched, subsequent matching stops.

[0058] Based on the target association rules, the state analysis results of the intestinal organoid microarray at the target preset time are obtained; the preset association rule set contains the association rules corresponding to different microarray states.

[0059] For example, if the target association rule is the first rule, then the maturity result corresponding to the first rule is used as the state analysis result of the intestinal organoid microarray.

[0060] In some embodiments, the state analysis results of intestinal organoid microarrays may include, in addition to maturity results, target association rules corresponding to the maturity results, as well as raw analysis data of autofluorescence images, thereby facilitating traceability by analysis and testing personnel.

[0061] In the above embodiments, the maturity of intestinal organoid microarrays is automatically determined by combining two types of time-series change characteristics: fluorescence intensity and fluorescence coverage. This avoids the subjectivity of manual judgment, realizes the quantitative analysis of intestinal organoid microarray maturity, and ensures the reliability of the maturity determination.

[0062] In some embodiments, the preset association rule set includes the following state determination rules determined based on fluorescence intensity value, fluorescence coverage value, fluorescence intensity change trend, and fluorescence coverage change trend: If the decrease in fluorescence coverage exceeds a preset threshold, the intestinal organoid microarray is determined to be in an abnormal state.

[0063] Understandably, if the decrease in fluorescence coverage exceeds the preset threshold, it indicates that the integrity of the intestinal epithelial tissue structure has changed drastically in a short period of time. Therefore, the state of the intestinal organoid chip at that preset time is determined to be abnormal.

[0064] If the intestinal organoid microarray is not in an abnormal state, and the fluorescence coverage value is greater than the first preset threshold, the relative fluorescence intensity value is greater than the second preset threshold, and the trends of fluorescence intensity change and fluorescence coverage change are both in a stable state, then the intestinal organoid microarray is determined to be in a mature state.

[0065] Among them, the stable state refers to the fluctuation range of the fluorescence intensity change trend and the fluorescence coverage change trend within the preset range.

[0066] For example, at a preset time, if the fluorescence coverage value is greater than a first preset threshold (coverage threshold), the fluorescence intensity value is greater than a second preset threshold (intensity threshold), and the trends of fluorescence intensity change and fluorescence coverage change are both in a stable state (i.e., maintaining high stability), the state of the intestinal organoid chip at that preset time is determined to be mature.

[0067] It is understandable that fluorescence intensity can reflect the state of cell metabolism, and fluorescence coverage can reflect the state of intestinal epithelial tissue structure. When the fluorescence intensity trend remains stable or increases relative to the fluorescence intensity reference benchmark, and the fluorescence coverage increases relative to the fluorescence coverage reference benchmark, it means that the cell metabolism-related state of the intestinal organoid chip is developing normally in sync with the intestinal epithelial tissue structure. When the fluorescence coverage value at the preset time is greater than the coverage threshold and the fluorescence intensity value is greater than the fluorescence intensity threshold, it means that the integrity of the intestinal epithelial structure and the proportion of functional regions have reached the maturity standard. Therefore, the state of the intestinal organoid chip at the preset time is determined to be the mature state.

[0068] If the intestinal organoid microarray is not in an abnormal state, but the relative fluorescence intensity value does not reach the second preset threshold, the fluorescence coverage value does not reach the first preset threshold, and the trends of fluorescence intensity change and fluorescence coverage change do not show synchronous increase, then the intestinal organoid microarray is determined to be in an immature state.

[0069] Understandably, the stable trends in fluorescence intensity and fluorescence coverage indicate that there has been no significant developmental progress in the metabolic state and tissue structure of intestinal epithelial cells. At the same time, neither of these indicators exceeded the corresponding judgment threshold, indicating insufficient cell metabolism, a low proportion of epithelial functional areas, and that the tissue has not yet completed differentiation and development, thus it is judged to be in an immature state.

[0070] When the intestinal organoid microarray is not in an abnormal state, both the fluorescence intensity and fluorescence coverage trends show a synchronous increase, and the fluorescence coverage value does not exceed the first preset threshold, thus confirming that the intestinal organoid microarray is in a maturing state.

[0071] It is understandable that the fluorescence intensity and fluorescence coverage trends both increased synchronously, indicating that the cell metabolism-related state of the intestinal organoid chip was developing normally in sync with the intestinal epithelial tissue structure. Furthermore, the fluorescence coverage value at the preset time was greater than the coverage threshold, indicating that the intestinal epithelial structure had not yet formed a continuous monolayer and that the epithelial polarity and barrier function were insufficient. Therefore, the state of the intestinal organoid chip at the preset time was determined to be in a maturing state.

[0072] When the state of the intestinal organoid microarray is not abnormal, the fluorescence coverage shows an upward trend followed by a downward trend, and the fluctuation of the fluorescence intensity does not exceed the preset amplitude threshold, thus determining that the state of the intestinal organoid microarray is a structural remodeling state.

[0073] It is understandable that the trends of fluorescence intensity and fluorescence coverage both increase first and then decrease, indicating the existence of differentiation processes such as villization. This reflects the transformation of intestinal epithelium from a two-dimensional fused monolayer to a mature epithelium with a three-dimensional villous structure. Therefore, the state of the intestinal organoid chip at this preset time is a state of structural remodeling.

[0074] In the above embodiments, by using the temporal variation characteristics of fluorescence intensity and fluorescence coverage, the synchronicity of indicators, and threshold judgment conditions, multiple states such as mature, maturing, structural remodeling, immature, and abnormal are distinguished. Thus, different developmental stages and abnormal states of intestinal organoid chips can be comprehensively distinguished according to the preset state mapping relationship, effectively avoiding the subjectivity of manual judgment.

[0075] In some embodiments, if the decrease in fluorescence coverage exceeds a preset decrease threshold, the state of the intestinal organoid microarray is determined to be abnormal, including: If the decrease in fluorescence coverage exceeds the preset decrease threshold, and the fluorescence intensity shows an upward trend, the abnormal state of the intestinal organoid microarray is determined to be the decoupling of functional and structural states.

[0076] It is understandable that the decrease in fluorescence coverage exceeds the preset threshold, and the increase in fluorescence intensity indicates structural damage and stress response in the intestinal epithelial tissue. Therefore, the abnormal state is determined to be the decoupling of functional and structural states.

[0077] If the decrease in both the fluorescence intensity and fluorescence coverage trends exceeds the preset decrease threshold, the abnormal state of the intestinal organoid microarray is determined to be comprehensive metabolic and structural inhibition.

[0078] Understandably, the decrease in both fluorescence intensity and fluorescence coverage exceeded the preset threshold, indicating that the metabolic activity of the intestinal epithelial tissue and the epithelial structure collapsed synchronously. Therefore, the abnormal state was determined to be a comprehensive inhibition of both function and structure.

[0079] In the above embodiments, the abnormal states corresponding to the decrease in fluorescence coverage are further subdivided. By combining the differences in fluorescence intensity and fluorescence coverage, two different abnormal types are accurately distinguished: the decoupling of functional state and structural state, and the comprehensive inhibition of metabolism and structure. This refines the judgment dimensions of abnormal states and makes the maturity results more accurate.

[0080] In some embodiments, acquiring autofluorescence images of the intestinal organoid microarray at multiple preset time points after inoculation includes: At multiple preset time points after intestinal organoid microarray seeding, the intestinal organoid microarray is irradiated with a laser of the target wavelength and images are acquired to obtain autofluorescence images of the intestinal organoid microarray at each preset time point after seeding.

[0081] Among them, lasers of the target wavelength are used to excite the intestinal organoid chip to produce endogenous autofluorescence.

[0082] For example, an intestinal organoid chip is excited with a laser with a wavelength of 480nm to 500nm to produce endogenous autofluorescence; the emission light acquisition wavelength is set to 500nm to 550nm to complete the acquisition of autofluorescence images of the intestinal epithelial region. Throughout the acquisition process, the excitation light intensity and exposure time remain constant to ensure the reliability of the autofluorescence images.

[0083] For a better understanding of the above embodiments, please refer to [link / reference]. Figure 3 , Figure 3 The following is a schematic diagram of the application process involved in the embodiments of this application, including steps S1 to S7, wherein: S1, Intestinal organoid chip construction. For example, intestinal organoids derived from human induced pluripotent stem cells or human tissue-derived intestinal organoids are seeded onto a chip with a specified structure to obtain an intestinal organoid chip.

[0084] S2, Acquire autofluorescence images. For example, the intestinal organoid chip is irradiated with laser at multiple preset times to induce autofluorescence, and autofluorescence images at each time point are acquired.

[0085] S3, Two-parameter extraction. For example, for the autofluorescence image at each time step, normalized fluorescence intensity and fluorescence coverage are calculated.

[0086] S4, Maturity Assessment. For example, this is determined based on relative fluorescence intensity and fluorescence coverage.

[0087] For the mature state, the fluorescence intensity and fluorescence coverage trends show a synchronous increase or remain stable at a high level, and the fluorescence coverage value is greater than the coverage threshold (e.g., 0.1), while the relative fluorescence intensity value is greater than the intensity threshold (e.g., 1.5).

[0088] For the immature state, the judgment conditions are that the trends of fluorescence intensity change and fluorescence coverage change show a stable trend, the fluorescence coverage value is not greater than the coverage threshold, and the fluorescence intensity value is not greater than the intensity threshold.

[0089] For a product in a mature state, the criteria for judgment are that both the fluorescence intensity change trend and the fluorescence coverage change trend increase synchronously, and the fluorescence coverage value is not greater than the coverage threshold.

[0090] For the structural remodeling state, the criteria for judgment are that both the fluorescence intensity change trend and the fluorescence coverage change trend are first increasing and then decreasing.

[0091] For abnormal states, the judgment condition is that the decrease in fluorescence coverage exceeds the magnitude threshold.

[0092] Furthermore, for the metabolic and structural decoupling state in the abnormal state, the judgment condition is that the decrease in fluorescence coverage exceeds the amplitude threshold, and the fluorescence intensity shows an upward trend.

[0093] For the state of complete inhibition of metabolism and structure in the abnormal state, the judgment condition is that the decrease in both the fluorescence intensity change trend and the fluorescence coverage change trend exceeds the amplitude threshold.

[0094] S5, Functional Verification. This step is optional; it allows for functional verification of intestinal organoid microarrays that are determined to be in a mature state to ensure the functional integrity of the microarray. For example, functional verification can be FITC-dextran permeability verification.

[0095] S6, Chip Application. This step is optional; for example, an intestinal organoid chip determined to be mature can be used for drug toxicity evaluation.

[0096] S7, Output Results. Output the state analysis results of the intestinal organoid microarray.

[0097] For example, the microfluidic chip can be a bilayer chip with a polycarbonate porous membrane (pore size 0.45 μm, membrane area 0.33 cm²). Before seeding, 200 μg / mL of Collagen IV and 100 μg / mL of Matrigel are pre-infused into the channels of the microfluidic chip, and it is incubated overnight at 4°C for pretreatment. Simultaneously, it is coated at 37°C for 1 hour before seeding. Then, intestinal organoids differentiated from human induced pluripotent stem cells (iPSCs) or intestinal organoids derived from human tissue are digested into single cells or small cell clusters (approximately 30 μm to 50 μm in diameter) and seeded at a preset density (e.g., 2.5 × 10⁻⁶). 5 cells / cm² up to 5×10 5 Intestinal organoid microarrays were obtained by inoculating the microarray with cells / cm², allowing it to stand for 2-3 days, and then perfusing it with complete culture medium at a flow rate of 10 μL / h. The complete culture medium is a pre-prepared composite cell culture medium made by adding serum, growth factors and other additives to the basal culture medium, which provides conditions for the in vitro culture of intestinal organoids.

[0098] Subsequently, autofluorescence imaging was performed on days 3, 5, 7, 10, 12, and 15 of culture. Specifically, an inverted fluorescence microscope (excitation: 488nm laser, emission: 525 / 550nm bandpass filter), 10× objective lens, same exposure time (1000ms) and excitation intensity (10%) was used to obtain autofluorescence images. Simultaneously, bright-field images for the corresponding days were also acquired as raw data, such as... Figure 4 As shown, Figure 4 Bright-field and autofluorescence images acquired at different times are shown.

[0099] according to Figure 4 The autofluorescence images at different time points are shown. After background subtraction using image processing software, the epithelial region is selected as the target region, and the fluorescence intensity value I_norm = I_sample / I_ref is calculated, where I_sample is the mean of the average fluorescence intensity of all single cells in the target region, and I_ref is the average fluorescence intensity of all single cells in the target region of the autofluorescence image on day 3. Simultaneously, a large-scale threshold segmentation method is used to remove isolated noise points smaller than 100 pixels. An 8-connected region labeling algorithm is used to calculate the area of ​​the fluorescently positive region. The ratio of the area of ​​the fluorescently positive region to the total area of ​​the target region is then used as the fluorescence coverage rate. Based on the fluorescence intensity values, fluorescence coverage rate values, fluorescence intensity trends, and fluorescence coverage rate trends corresponding to multiple preset time points, the state analysis results of the intestinal organoid microarray are determined. The state analysis results are shown in Table 1 below. Table 1 Day 3 1.00±0.36 (reference value) 0.006±0.004 / immature state Day 5 1.39±0.23 0.058±0.029 <![CDATA[8.6×10 -6 ]]> In the process of maturing Day 7 1.56±0.15 0.219±0.064 <![CDATA[6.7×10⁻ 6 ]]> Mature state Day 10 1.54±0.17 0.211±0.048 <![CDATA[6.9×10⁻ 6 ]]> Mature state Day 12 1.48±0.13 0.123±0.038 <![CDATA[7.8×10⁻ 6 ]]> Structural remodeling status Day 15 1.29±0.1 0.121±0.025 <![CDATA[8.6×10⁻ 6 ]]> Structural remodeling status The apparent permeability coefficient was determined by FITC-dextran permeability testing. The apparent permeability coefficient is an indicator used to quantify the function of the intestinal epithelial barrier. The lower the apparent permeability coefficient, the denser and more complete the intestinal epithelial barrier is. In this embodiment, the monolayer of intestinal organoids on day 3 is usually not fully covered by biofilm, so FITC-dextran permeability testing was not performed on day 3, and the data was empty.

[0100] In this embodiment, a 4,000 Dalton (kDa) FITC-dextran was used to perform permeability testing on the intestinal organoid microarray from day 5 to day 15 to verify the integrity and functional effect of the microarray epithelial barrier.

[0101] The specific operation is as follows: Replace the original culture medium in the upper channel of the intestinal organoid microarray with 4 kDa FITC-dextran HBSS (Hank's Balanced Salt Solution), at a concentration of 0.5 mg / mL to 1.0 mg / mL. Add blank HBSS solution without fluorescent dye to the lower channel of the microarray. Place the intestinal organoid microarray in a swing perfusion apparatus and incubate at 37°C for 30 to 60 minutes. After incubation, aspirate 100 μL of the lower channel liquid and detect it using a fluorescence microplate reader. Set the excitation wavelength to 485 nm and the emission wavelength to 535 nm, and measure the fluorescence intensity of the sample. Calculate the FITC-dextran concentration in the lower channel using a pre-plotted standard curve, and further calculate the apparent permeability coefficient. The validation data are as follows: Figure 5 As shown.

[0102] As shown in Table 1, on day 3, the relative fluorescence intensity was 1.00 (reference value), and the fluorescence coverage was 0.006 ± 0.004. At this point, both indicators were at extremely low levels, with the fluorescence coverage far below the coverage threshold of 0.1 (wherein, the coverage threshold of 0.1 was determined based on observations from continuous culture experiments under the same culture conditions. In these continuous culture experiments, it was found that when the coverage threshold first exceeded approximately 0.10, the intestinal organoid microarray simultaneously exhibited stable FITC-dextran low permeability, continuous ZO-1 tight junctions, and mature small intestinal epithelial morphology; therefore, the coverage threshold was determined to be 0.1; of course, this threshold can be adjusted based on experimental data for culture systems under different conditions), indicating an immature state; simultaneously, combined with... Figure 4 The image states in the images can indirectly prove this conclusion, namely, the cells are distributed in an island-like pattern in the bright field image and do not form a continuous monolayer; no significant autofluorescence is observed in the autofluorescence image.

[0103] On day 5, the relative fluorescence intensity was 1.39 ± 0.23, and the fluorescence coverage was 0.058 ± 0.029. Compared to day 3, both the fluorescence intensity and fluorescence coverage showed an upward trend, but the fluorescence coverage of 0.058 ± 0.029 was still less than the coverage threshold of 0.1, indicating that it was in a maturing state; simultaneously, combined with Figure 4 The image states in the images indirectly support this conclusion: in the bright-field images, cell proliferation has completely covered the culture layer, forming a continuous monolayer; in the autofluorescence images, autofluorescence is distributed in a dotted pattern; and the apparent permeability of FITC-dextran is verified to be 8.6 × 10⁻⁻⁻⁶. 6 The speed of cm / s indicates that the intestinal epithelial barrier is initially formed at this time, but it is not yet dense and has high permeability.

[0104] On day 7, the relative fluorescence intensity was 1.56 ± 0.15, and the fluorescence coverage was 0.219 ± 0.064. Compared to day 5, both the fluorescence intensity and fluorescence coverage showed a continuous upward trend, and the fluorescence coverage of 0.219 ± 0.064 was greater than the coverage threshold of 0.1. The relative fluorescence intensity was greater than the preset fluorescence intensity threshold of 1.5 (wherein, the preset fluorescence intensity threshold of 1.5 was determined based on continuous culture experiments under the same culture conditions. In the continuous culture experiments, it was found that when the relative fluorescence intensity first exceeded approximately 1.5, the intestinal organoid microarray simultaneously exhibited stable FITC-dextran low permeability, continuous ZO-1 tight junctions, and mature small intestinal epithelial morphology. Therefore, the preset fluorescence intensity threshold was determined to be 1.5; of course, for culture systems under different conditions, this threshold can be adjusted according to experimental data), indicating a mature state; simultaneously, combined with Figure 4 The image states in the images indirectly support this conclusion: in the bright-field images, the cells have completely covered the culture layer, forming a continuous and dense monolayer; in the autofluorescence images, autofluorescence is significantly increased and distributed in a sheet-like pattern; and FITC-dextran permeability testing shows an apparent permeability coefficient of 6.7 × 10⁻⁶. -6 cm / s, which is a further decrease compared to day 5, indicates that the intestinal epithelial barrier function is becoming more intact.

[0105] On day 10, the relative fluorescence intensity was 1.54 ± 0.17, and the fluorescence coverage was 0.211 ± 0.048. Compared to day 7, both fluorescence intensity and fluorescence coverage showed a continuous upward trend and remained at a high level. The fluorescence coverage was still greater than the coverage threshold of 0.1, indicating a mature state. Simultaneously, combined with... Figure 4 The image states in the images indirectly support this conclusion: in the bright-field images, cells have formed a continuous and dense epithelial layer with cellular protrusions; in the autofluorescence images, the autofluorescence signal still shows a regional distribution, locally concentrated in the areas of structural protrusions; and FITC-dextran permeability verification shows an apparent permeability coefficient of 6.9 × 10⁻⁶. -6 The value of cm / s remained basically the same as on day 7, still at a low level, indicating that the intestinal epithelial barrier function remained intact.

[0106] On day 12, the fluorescence intensity was 1.48 ± 0.13, and the fluorescence coverage was 0.123 ± 0.038. Looking at the entire time series, the fluorescence intensity began to decline from its peak on day 7 (1.56), and the fluorescence coverage also began to decrease from its peak on day 7 (0.219), showing an overall trend of first rising and then falling. However, the fluctuation in fluorescence intensity did not exceed the preset amplitude threshold (30%), which was considered a state of structural remodeling. Simultaneously, combined with... Figure 4The image states in the images indirectly support this conclusion: continuous epithelial cells are visible in the bright-field images, but there are signs of cell clump detachment; the autofluorescence signal in the autofluorescence images is attenuated compared to the mature stage; and FITC-dextran permeability verification shows an apparent permeability coefficient of 7.8 × 10⁻⁶. -6 cm / s, which begins to rise in the more mature stage, indicates that the intestinal epithelial barrier function is weakened along with the differentiation and remodeling of villous structures.

[0107] On day 15, the fluorescence intensity was 1.29 ± 0.10, and the fluorescence coverage was 0.121 ± 0.025. Both indicators decreased slowly compared to day 12, continuing the overall trend of first increasing and then decreasing. Furthermore, the fluctuation in fluorescence intensity did not exceed the preset threshold (30%), indicating a structural remodeling state. Simultaneously, combined with... Figure 4 The image states in the images indirectly support this conclusion: continuous epithelial cells are still visible in the bright-field images, and cell clumps continue to detach; the autofluorescence signal remains at a low level in the autofluorescence images; and FITC-dextran permeability verification shows an apparent permeability coefficient of 8.6 × 10⁻⁶. -6 cm / s, the apparent permeability coefficient continued to rise, indicating that the differentiation and remodeling of villous structure continued and the intestinal epithelial barrier function was further weakened.

[0108] In some embodiments, to further verify the effectiveness of the analytical method provided in this application, the performance of the intestinal organoid microarray on day 10 was further verified. Based on the analytical method provided in this application, it was determined that the intestinal organoid microarray on day 10 was in a mature state. Therefore, ZO-1 immunofluorescence staining and villous structure immunofluorescence identification were performed on the intestinal organoid microarray on day 10, and the results are as follows: Figure 6 As shown, by Figure 6 As can be seen, the immunofluorescence staining images of ZO-1 (a protein) and ZO-1+DAPI (a fluorescent dye for cell nuclei) confirm that the intestinal organoid microarray on day 10 formed a continuous, clear, tightly connected ring structure, indicating mature intestinal epithelial barrier function. The immunofluorescence staining images of villi (where red represents fluorescent markers of epithelial cell adhesion molecules, blue represents fluorescent markers of cell nuclei, and green represents fluorescent markers of villi) confirm that the intestinal organoid microarray on day 10 had differentiated into small intestinal villi structures, and the epithelium exhibited a polar arrangement, indicating structural maturity and approximation to physiological structures. Therefore, the analytical method provided in this application can accurately determine whether an intestinal organoid microarray is mature.

[0109] In other embodiments, to verify the accuracy of abnormal state determination, different batches of intestinal organoid microarrays were used. Intestinal organoid microarrays that had reached maturity on day 10 were continuously treated with hydrogen peroxide (H2O2, 500 μM) added to the perfusion fluid of the upper channel for 48 hours. Oxidative stress was simulated before treatment (0 h), and at 12 h, 24 h, and 48 h after treatment. Autofluorescence images were acquired after treatment, such as... Figure 7 As shown in Table 2 below, the fluorescence intensity and fluorescence coverage were calculated. Table 2 0h 1.752±0.004 0.247±0.030 Mature state 12h 1.045±0.062 0.157±0.015 Abnormal state (total suppression) 24h 1.359±0.034 0.222±0.025 Abnormal state (stress rebound) 48h 1.252±0.053 0.072±0.014 Abnormal state (continuously deteriorating) As shown in Table 2, in the early stages of H2O2 stimulation (0 h to 12 h), both fluorescence intensity and fluorescence coverage exhibited a sharp decline, with decreases of approximately 40% and 36%, respectively, exceeding the preset decline threshold (30%), indicating an abnormal state (comprehensive inhibition of metabolism and structure). Between 12 h and 24 h, fluorescence intensity briefly rebounded to 1.359, and fluorescence coverage also rebounded to 0.222 (close to the initial maturation level), exhibiting stress rebound characteristics, still indicating an abnormal state. Between 24 h and 48 h, fluorescence coverage again dropped sharply to 0.072, and fluorescence intensity fell back to 1.252, indicating a continued deterioration in cell activity and structural integrity. These results demonstrate that this method can effectively identify the dynamic evolution of oxidative stress injury from acute inhibition, stress rebound, to continuous deterioration.

[0110] In other embodiments, a group was randomly selected from chips that had reached maturity on day 10 of culture. 100 μM 5-fluorouracil (5-FU, dissolved in the culture medium) was added to the upper channel perfusion solution, and the chips were treated continuously for 48 h. Autofluorescence images were acquired before treatment (0 h), and at 12 h, 24 h, and 48 h after treatment, as shown below. Figure 7 As shown in the figure, the fluorescence intensity and fluorescence coverage were then calculated, and the results are shown in Table 3 below: Table 3 0h 1.586±0.012 0.237±0.033 Mature state 12h 1.279±0.029 0.078±0.006 Abnormal state 24h 1.176±0.038 0.035±0.008 Abnormal state (continuously deteriorating) 48h 0.627±0.016 0.032±0.007 Abnormal state (continuously deteriorating) As shown in Table 3, under the action of antimetabolites (5-FU), fluorescence coverage decreased sharply within 12 hours (a decrease of approximately 67%, far exceeding the 30% amplitude threshold), indicating rapid damage to the integrity of the epithelial structure. Fluorescence intensity showed a slow decreasing trend from 12 to 24 hours, with decreases of approximately 19% and 26%, respectively, not exceeding the amplitude threshold, suggesting that the metabolic-related state was still maintained to some extent in the early stages of drug action. By 48 hours, the fluorescence intensity decreased by approximately 60%, and the fluorescence coverage decreased by approximately 87%, both exceeding the 30% amplitude threshold, indicating a continuous deterioration of comprehensive metabolic and structural inhibition. These results demonstrate that this method can effectively identify the time difference between early structural damage and delayed metabolic inhibition caused by antimetabolites, enabling phased monitoring of the dynamic damage process.

[0111] It should be noted that the chips used in Table 3 are from different experimental batches than the chips corresponding to Table 1. The absolute values ​​of chips from different experimental batches may differ, but the rules for judging the trend of change are the same.

[0112] Among related technologies, destructive labeling, such as immunofluorescence staining or gene expression analysis, is mainly used. These methods require fixation, permeabilization, or lysis of the sample, which destroys the sample integrity and cannot be used for subsequent experiments or continuous dynamic monitoring. For example, transepithelial resistance measurement can reflect the overall barrier integrity in real time, but it is not sensitive to local structural changes (such as early cell shedding or microvilli dysplasia) and is easily affected by electrode design and culture medium composition. Although FITC-dextran permeability detection can directly assess the function of macromolecular barriers, it is usually used as an endpoint detection and cannot determine in advance whether the model is ready for testing.

[0113] The method provided in this application requires no additional fluorescent markers throughout the entire process, enabling non-invasive analysis of intestinal organoid chips and reducing analysis costs. Furthermore, by quantifying different states of the intestinal organoid chip through fluorescence intensity and fluorescence coverage, dynamic analysis of the chip can be achieved, effectively improving the analysis efficiency and accuracy. In addition, the method uses a conventional wide-field fluorescence microscope instead of the high-cost FLIM (Fluorescence Lifetime Imaging Microscopy) system, achieving metabolism-related imaging through fluorescence intensity (rather than lifetime) analysis, significantly lowering the equipment barrier. The dual-dimensional time-series analysis method combining FAD single parameter and fluorescence coverage simplifies the process while maintaining high consistency with gold standards (e.g., FITC-dextran permeability). The entire detection process is non-invasive, enabling continuous dynamic monitoring of live cells and tracking the complete developmental cycle from inoculation to functional maturity on the same chip, providing an industrially viable solution for standardized quality control and drug screening of organoid chips.

[0114] It should be understood that although the steps in the flowcharts of the above embodiments are shown sequentially according to the arrows, these steps are not necessarily executed in the order indicated by the arrows. Unless explicitly stated herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least some steps in the flowcharts of the above embodiments may include multiple steps or multiple stages. These steps or stages are not necessarily completed at the same time, but can be executed at different times. The execution order of these steps or stages is not necessarily sequential, but can be performed alternately or in turn with other steps or at least some of the steps or stages in other steps. It is understood that the steps in different embodiments can be freely combined as needed, and all non-contradictory solutions formed by such combinations are within the scope of protection of this application.

[0115] Based on the same inventive concept, this application also provides an intestinal organoid microarray analysis device based on endogenous autofluorescence parameters for implementing the intestinal organoid microarray analysis method based on endogenous autofluorescence parameters described above. The solution provided by this device is similar to the solution described in the above method. Therefore, the specific limitations of one or more intestinal organoid microarray analysis device embodiments based on endogenous autofluorescence parameters provided below can be found in the limitations of the intestinal organoid microarray analysis method based on endogenous autofluorescence parameters described above, and will not be repeated here.

[0116] In one exemplary embodiment, such as Figure 8The diagram illustrates a schematic of an intestinal organoid microarray analysis device based on endogenous autofluorescence parameters. The intestinal organoid microarray analysis device 500 based on endogenous autofluorescence parameters includes: The acquisition module 501 is used to irradiate the intestinal organoid chip with a laser of a specific wavelength at multiple preset time points after inoculation and collect its endogenous autofluorescence signal to obtain autofluorescence images corresponding to each preset time point.

[0117] The calculation module 502 is used to calculate the area ratio of the fluorescent positive region in the target region of the corresponding autofluorescence image for each preset time, as the fluorescence coverage value of the preset time; and, with the autofluorescence image acquired at the first preset time as a reference, calculate the relative fluorescence intensity value of the target region in the corresponding autofluorescence image to obtain the relative fluorescence intensity value of the preset time; the target region is the intestinal epithelial region in the intestinal organoid chip.

[0118] The timing processing module 503 is used to determine the fluorescence intensity change trend and fluorescence coverage change trend corresponding to each of the multiple preset times based on the relative fluorescence intensity values ​​and fluorescence coverage values ​​at multiple preset times.

[0119] Analysis module 504 is used to determine the state analysis results of the intestinal organoid microarray at the target preset time based on a preset association rule set, the relative fluorescence intensity value, fluorescence coverage value, fluorescence intensity change trend, and fluorescence coverage change trend corresponding to the target preset time. The preset association rule set contains at least a variety of rules to indicate the association between fluorescence intensity value, fluorescence coverage value, fluorescence intensity change trend, and fluorescence coverage change trend, so as to determine different microarray development or abnormal states. The target preset time is any one of multiple preset times.

[0120] In some embodiments, the calculation module 502 is used to sort multiple preset times in chronological order to obtain a time sequence; for the k-th preset time in the time sequence, the ratio of the average fluorescence intensity of the target region in the autofluorescence image corresponding to the k-th preset time to the average fluorescence intensity of the target region in the autofluorescence image corresponding to the first preset time is used as the relative fluorescence intensity value of the k-th preset time, where k is a positive integer; for each preset time, the ratio of the total area of ​​the fluorescent positive region in the target region in the autofluorescence image corresponding to the preset time to the total area of ​​the target region in the autofluorescence image is used as the fluorescence coverage value corresponding to the preset time.

[0121] In some embodiments, the analysis module 504 is used to perform correlation analysis based on the relative fluorescence intensity value, fluorescence coverage value, fluorescence intensity change trend, and fluorescence coverage change trend at a target preset time to determine the actual correlation analysis result at the target preset time; perform rule matching in a preset correlation rule set based on the actual correlation analysis result at the target preset time to determine the target correlation rule in the preset correlation rule set that matches the actual correlation analysis result; and obtain the state analysis result of the intestinal organoid chip at the target preset time based on the target correlation rule; the preset correlation rule set contains the correlation rules corresponding to different chip states.

[0122] In some embodiments, the preset association rule set includes the following state determination rules based on fluorescence intensity value, fluorescence coverage value, fluorescence intensity change trend, and fluorescence coverage change trend: if the decrease in fluorescence coverage change trend exceeds a preset decrease threshold, the intestinal organoid chip is determined to be in an abnormal state; if the intestinal organoid chip is not in an abnormal state, the fluorescence coverage value is greater than a first preset threshold, the relative fluorescence intensity value is greater than a second preset threshold, and both the fluorescence intensity change trend and the fluorescence coverage change trend are in a stable state, the intestinal organoid chip is determined to be in a mature state; if the intestinal organoid chip is not in an abnormal state, the relative fluorescence intensity value does not reach the threshold. If the fluorescence intensity and fluorescence coverage values ​​do not reach the second preset threshold and the fluorescence coverage value does not increase synchronously, the intestinal organoid microarray is determined to be in an immature state. If the intestinal organoid microarray is not in an abnormal state, and the fluorescence intensity and fluorescence coverage values ​​both increase synchronously, and the fluorescence coverage value does not exceed the first preset threshold, the intestinal organoid microarray is determined to be in a maturing state. If the intestinal organoid microarray is not in an abnormal state, and the fluorescence coverage value first increases and then decreases, and the fluctuation of the fluorescence intensity value does not exceed the preset amplitude threshold, the intestinal organoid microarray is determined to be in a structural remodeling state.

[0123] In some embodiments, the preset state mapping relationship includes: if the decrease in fluorescence coverage exceeds a preset decrease threshold and the fluorescence intensity is increasing, the abnormal state of the intestinal organoid chip is determined to be the decoupling of functional and structural states; if the decrease in both fluorescence intensity and fluorescence coverage exceeds a preset decrease threshold, the abnormal state of the intestinal organoid chip is determined to be the comprehensive inhibition of metabolism and structure.

[0124] The modules in the aforementioned intestinal organoid microarray analysis device based on endogenous autofluorescence parameters can be implemented entirely or partially through software, hardware, or a combination thereof. These modules can be embedded in or independent of the processor in a computer device, or stored in the memory of a computer device as software, so that the processor can call and execute the corresponding operations of each module.

[0125] It should be noted that the user information (including but not limited to user device information, user personal information, etc.) and data (including but not limited to data used for analysis, data stored, data displayed, etc.) involved in this application are all information and data authorized by the user or fully authorized by all parties, and the collection, use and processing of the relevant data must comply with relevant regulations.

[0126] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this application.

[0127] The above embodiments are merely illustrative of several implementation methods of this application, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.

Claims

1. A method for analyzing intestinal organoid microarrays based on endogenous autofluorescence parameters, characterized in that, The method includes: At multiple preset time points after intestinal organoid microarray seeding, the intestinal organoid microarray is irradiated with a laser of a specific wavelength and its endogenous autofluorescence signal is collected to obtain autofluorescence images corresponding to each of the multiple preset time points. For each preset time point, the area ratio of the fluorescent positive region in the target region in the corresponding autofluorescence image is calculated as the fluorescence coverage value of the preset time point; and, taking the autofluorescence image acquired at the first preset time point as a reference, the relative fluorescence intensity value of the target region in the corresponding autofluorescence image is calculated to obtain the relative fluorescence intensity value of the preset time point; the target region is the intestinal epithelial region in the intestinal organoid chip; Based on the relative fluorescence intensity values ​​and fluorescence coverage values ​​at multiple preset times, determine the fluorescence intensity change trend and fluorescence coverage change trend corresponding to each of the multiple preset times; Based on a preset association rule set, the relative fluorescence intensity value, fluorescence coverage value, fluorescence intensity change trend, and fluorescence coverage change trend corresponding to a target preset time, the state analysis results of the intestinal organoid microarray at the target preset time are determined; wherein, the preset association rule set includes at least a variety of rules used to indicate the association relationship between the fluorescence intensity value, the fluorescence coverage value, the fluorescence intensity change trend, and the fluorescence coverage change trend, so as to determine different microarray development or abnormal states; the target preset time is any one of the multiple preset times.

2. The method according to claim 1, characterized in that, For each preset time point, the area ratio of the fluorescent positive region within the target region in the corresponding autofluorescence image is calculated as the fluorescence coverage value for the preset time point; and, using the autofluorescence image acquired at the first preset time point as a reference, the relative fluorescence intensity value of the target region in the corresponding autofluorescence image is calculated to obtain the relative fluorescence intensity value for the preset time point, including: The multiple preset times are sorted in chronological order to obtain a time sequence; For the k-th preset time in the time sequence, the ratio of the average fluorescence intensity of the target region in the autofluorescence image corresponding to the k-th preset time to the average fluorescence intensity of the target region in the autofluorescence image corresponding to the first preset time is taken as the relative fluorescence intensity value of the k-th preset time, where k is a positive integer; For each preset time, the ratio of the total area of ​​the fluorescent positive region in the target region of the autofluorescence image corresponding to the preset time to the total area of ​​the target region in the autofluorescence image is used as the fluorescence coverage value corresponding to the preset time.

3. The method according to claim 1, characterized in that, The analysis results of determining the state of the intestinal organoid microarray at the target preset time, based on a preset association rule set, the relative fluorescence intensity value, fluorescence coverage value, fluorescence intensity change trend, and fluorescence coverage change trend, include: Based on the relative fluorescence intensity value, fluorescence coverage value, fluorescence intensity change trend and fluorescence coverage change trend at the target preset time, a correlation analysis is performed to determine the actual correlation analysis result at the target preset time. Based on the actual association analysis results at the target preset time, rule matching is performed in the preset association rule set to determine the target association rule in the preset association rule set that matches the actual association analysis results; Based on the target association rules, the state analysis results of the intestinal organoid chip at the target preset time are obtained; the preset association rule set contains the association rules corresponding to different chip states.

4. The method according to claim 3, characterized in that, The preset association rule set includes the following state determination rules determined based on the fluorescence intensity value, the fluorescence coverage value, the fluorescence intensity change trend, and the fluorescence coverage change trend: If the decrease in fluorescence coverage exceeds a preset threshold, the state of the intestinal organoid chip is determined to be abnormal. If the state of the intestinal organoid chip is not abnormal, the fluorescence coverage value is greater than a first preset threshold, the relative fluorescence intensity value is greater than a second preset threshold, and the fluorescence intensity change trend and the fluorescence coverage change trend are both in a stable state, then the state of the intestinal organoid chip is determined to be mature. If the state of the intestinal organoid chip is not abnormal, but the relative fluorescence intensity value does not reach the second preset threshold, the fluorescence coverage value does not reach the first preset threshold, and the fluorescence intensity change trend and the fluorescence coverage change trend do not show a synchronous increase, then the state of the intestinal organoid chip is determined to be immature. If the intestinal organoid chip is not in an abnormal state, and both the fluorescence intensity change trend and the fluorescence coverage change trend show a synchronous increase, and the fluorescence coverage value does not exceed the first preset threshold, then the intestinal organoid chip is determined to be in a maturing state. If the state of the intestinal organoid chip is not abnormal, and the fluorescence coverage changes first increases and then decreases, and the fluctuation of the fluorescence intensity does not exceed the preset amplitude threshold, then the state of the intestinal organoid chip is determined to be a structural remodeling state.

5. The method according to claim 4, characterized in that, If the decrease in fluorescence coverage exceeds a preset threshold, the state of the intestinal organoid chip is determined to be abnormal, including: If the decrease in fluorescence coverage exceeds a preset decrease threshold and the fluorescence intensity is increasing, the abnormal state of the intestinal organoid chip is determined to be the decoupling of functional and structural states. If the decrease in both the fluorescence intensity change trend and the fluorescence coverage change trend exceeds the preset decrease threshold, the abnormal state of the intestinal organoid chip is determined to be comprehensive metabolic and structural inhibition.

6. A microarray analysis device for intestinal organoids based on endogenous autofluorescence parameters, characterized in that, The device includes: The acquisition module is used to irradiate the intestinal organoid chip with a laser of a specific wavelength at multiple preset time points after inoculation and collect its endogenous autofluorescence signal to obtain autofluorescence images corresponding to each of the multiple preset time points. The calculation module is used to calculate the area ratio of the fluorescent positive region in the target region of the corresponding autofluorescence image for each preset time, as the fluorescence coverage value of the preset time; and, with the autofluorescence image acquired at the first preset time as a reference, calculate the relative fluorescence intensity value of the target region in the corresponding autofluorescence image, to obtain the relative fluorescence intensity value of the preset time; the target region is the intestinal epithelial region in the intestinal organoid chip; The timing processing module is used to determine the fluorescence intensity change trend and the fluorescence coverage change trend corresponding to each of the multiple preset times based on the relative fluorescence intensity value and the fluorescence coverage value at multiple preset times. The analysis module is used to determine the state analysis results of the intestinal organoid microarray at the target preset time based on a preset association rule set, the relative fluorescence intensity value, fluorescence coverage value, fluorescence intensity change trend, and fluorescence coverage change trend corresponding to the target preset time. The preset association rule set includes at least a variety of rules used to indicate the association between the fluorescence intensity value, the fluorescence coverage value, the fluorescence intensity change trend, and the fluorescence coverage change trend, in order to determine different microarray developmental or abnormal states. The target preset time is any one of the multiple preset times.

Images