Human ad organoid microfluidic screening platform integrating patient plasma / immune components

Abstract

The application provides a human AD organoid microfluidic drug screening platform integrating patient plasma / immune components, retains the complete genetic background of the patient through patient iPSC-derived neural organoids, and can truly reflect individual differences of sporadic AD; through the partition design and independent perfusion control of the neural organoid culture cavity, immune cell circulation cavity and plasma perfusion cavity in the microfluidic chip, the systemic integration and coupling co-culture of the neural organoid, patient-derived microglia-like cells and patient plasma / exosomes in the dynamic microenvironment are realized; through the multi-modal endpoint detection module, five kinds of endpoint data are synchronously collected, and three progressive algorithms of multi-modal pathological load index, individualized drug efficacy prediction index and comprehensive drug efficacy decision score are combined for quantitative evaluation, so that the precise classification of candidate drugs is realized from three levels of pathological load improvement, neural network function recovery and comprehensive drug efficacy decision.

Description

Technical Field

[0001] This invention relates to the fields of stem cell engineering, organ-on-a-chip, disease model construction, and drug screening platform technology, and in particular to a microfluidic drug screening platform for human AD organoids that integrates patient plasma / immune components. Background Technology

[0002] The pathological features of Alzheimer's disease include β-amyloid plaque deposition, neurofibrillary tangles formed by Tau protein hyperphosphorylation, neuroinflammatory responses, and synaptic and neuronal loss. Despite decades of research, effective disease-modifying therapies are still lacking, and most AD drug candidates fail in clinical trials. One important reason for this is the significant difference between preclinical models and the real pathological environment in humans.

[0003] Existing AD drug screening models can be broadly categorized as follows: Traditional two-dimensional cell culture models, while simple to operate and high-throughput, lack three-dimensional tissue structure and intercellular space, failing to reproduce the key pathological feature of AD—Aβ extracellular deposition; transgenic rodent models, although expressing human AD-related mutant genes, only account for about 5% of all AD cases, with sporadic AD, which accounts for over 95%, not involving such highly penetrating genetic mutations. Furthermore, significant species differences exist between humans and rodents, leading to the failure of many drugs effective in animal models in clinical trials, resulting in extremely low conversion rates. In recent years, significant progress has been made in three-dimensional brain organoid technology derived from human induced pluripotent stem cells. These organoids can self-organize in vitro to form layered neural tissue and reproduce core AD pathologies such as Aβ accumulation, Tau hyperphosphorylation, neuroinflammation, and neuronal degeneration. Patient-derived iPSC organoids can preserve individual genetic background and have the potential to capture disease heterogeneity, providing a new tool for personalized drug screening. The combination of microfluidic chip technology and organoids has further promoted the development of this field. Researchers have developed multi-cell type vascularized neuroimmune organoid models that include neurons, microglia, astrocytes and vascular structures, and have realized the simulation of the interaction between different cell types and organs through a microfluidic compartmentalized co-culture system. However, existing AD organoid models still have the following problems: First, most models only focus on the pathological reproduction of nerve cells themselves, failing to introduce peripheral humoral factors from patients into the culture system. Numerous studies have shown that peripheral immunity and metabolic status play a crucial regulatory role in the pathological process of the central nervous system. Neglecting this aspect makes it difficult for models to reflect individual differences in the role of peripheral immunity, plasma factors, and exosomes in the progression of pathology in sporadic AD. Second, existing microfluidic organoid platforms are mostly single static cultures or simple co-cultures, lacking the systematic integration of the three elements of "neural organoids-immune cells-plasma perfusion" in a dynamic microenvironment, and thus unable to simulate the continuous interaction process of neuro-immune-peripheral inflammation in vivo. Third, the efficacy evaluation of existing models often relies on a single endpoint indicator, lacking a multimodal simultaneous detection and quantitative evaluation system for Aβ, pTau, synaptic markers, inflammatory factors, and electrophysiological activity. A single endpoint easily misses the comprehensive effects of drugs on the recovery of neural network function and the regulation of the immune microenvironment, leading to discrepancies between drug screening results and actual clinical efficacy. Due to the aforementioned issues, there is currently a lack of an AD drug screening platform that can truly achieve "patient individualization" and simultaneously assess multi-dimensional drug efficacy.

[0004] Therefore, there is an urgent need in this field for an organoid drug screening platform for Alzheimer's disease (AD) that can integrate patient plasma / immune components, achieve multi-element coupling and co-culture of neuro-immune-humoral components, and have the ability to simultaneously detect multimodal endpoints and quantitatively evaluate drug efficacy. Summary of the Invention

[0005] The purpose of this invention is to provide a human AD organoid microfluidic drug screening platform that integrates patient plasma / immune components to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a human AD organoid microfluidic drug screening platform integrating patient plasma / immune components, comprising: The patient-derived iPSC neural organoid preparation module is used to reprogram induced pluripotent stem cells from peripheral blood mononuclear cells of patients, and then direct their differentiation into three-dimensional neural organoids containing neurons, astrocytes and microglia. The core module of the microfluidic chip is a multilayer bonded polydimethylsiloxane microfluidic chip, comprising a neural organoid culture chamber, an immune cell circulation chamber, a plasma perfusion chamber, a concentration gradient generation unit, and a waste collection unit. The neural organoid culture chamber, located in the central region of the chip, is equipped with an array of organoid fixation micropillars to accommodate and fix the neural organoids output from the neural organoid preparation module. The immune cell circulation chamber is connected to the neural organoid culture chamber via microchannels to introduce microglial-like cells induced to differentiate from patient PBMCs, achieving neural-immune co-culture. The plasma perfusion chamber is separated from the neural organoid culture chamber by a selectively permeable membrane and is used to introduce patient plasma or plasma exosomes to achieve continuous perfusion stimulation of the organoids by humoral factors. The concentration gradient generation unit is connected to the chip inlet to generate a series of concentration gradients for the drug to be screened. The waste collection unit is connected to the chip outlet to collect culture waste. The multimodal endpoint detection module, integrated downstream of the microfluidic chip or into an external detection system, includes a bright-field / fluorescence microscopy imaging unit, a microelectrode array detection unit, and a microfluidic tandem mass spectrometry interface, which are used to acquire imaging data, electrophysiological data, and inflammatory factor and exosome data of organoids, respectively. The intelligent data analysis and efficacy rating module receives the output data from the multimodal endpoint detection module, and performs data processing, feature fusion, and efficacy rating through the multimodal pathological load index algorithm, the individualized efficacy prediction index algorithm, and the comprehensive efficacy decision scoring algorithm, and outputs the response rating results of the candidate drugs.

[0007] Preferably, the neural organoids are obtained from patient iPSCs through sequential neural induction, neural progenitor cell expansion, and three-dimensional organoid differentiation. Astrocyte precursors are introduced on day 14 of differentiation, and microglia precursors are introduced on day 21. The organoids are cultured until day 42 to obtain three-dimensional neural organoids containing neurons, astrocytes, and microglia.

[0008] Preferably, in the core module of the microfluidic chip, the ratio of glial-like cells in the immune cell circulation chamber to neurons in the neural organoid culture chamber is 1:5; the selectively permeable membrane of the plasma perfusion chamber has a molecular weight cutoff of 100 kDa; the plasma perfusion rate is 0.5-2 μL / min; the drug perfusion rate is 1 μL / min; the drug administration duration is 48-72 hours; and the concentration gradient generation unit generates 5-8 concentration gradients with a concentration range of 0.1 × 10⁻⁸ CJ / mL. 50 Up to 10×IC 50 .

[0009] Preferably, in the multimodal endpoint detection module, the imaging data includes Aβ plaque fluorescence intensity, pTau protein immunofluorescence intensity, and synaptophysin immunofluorescence intensity; the electrophysiological data includes organoid burst firing frequency and network burst rate; the inflammatory factor data includes the concentrations of TNF-α, IL-1β, IL-6, and IFN-γ in the culture medium; and the exosome data includes Aβ exosomes in the waste liquid. 1-42 and pTau 181 Load.

[0010] Preferably, the intelligent data analysis and efficacy rating module uses the Multimodal Pathological Burden Index (MPLI) to quantitatively assess the degree of pathological improvement in organoids after drug intervention. The calculation formula for MPLI is as follows: ; in, and The fluorescence intensity of Aβ plaques in organoids before and after drug administration is shown in two figures, respectively. and The immunofluorescence intensities of pTau protein in organoids before and after drug administration are shown, respectively. and The values ​​represent the synaptophysin immunofluorescence intensity of organoids before and after drug administration, respectively. and The figures represent the combined inflammatory factor index of organoid culture medium before and after drug administration. ,in The concentration of the i-th inflammatory factor, TNF-α, IL-1β, IL-6, or IFN-γ. The baseline concentrations of the corresponding inflammatory factors in the untreated control organoid culture medium are α, β, γ, and δ, which are weighting coefficients that satisfy α+β+γ+δ=1 and are determined by minimizing the prediction error using training set data.

[0011] Preferably, typical values of the weight coefficients α, β, γ, δ are α=0.30, β=0.30, γ=0.20, δ=0.20; a lower MPLI value indicates a better improvement effect of the drug on AD pathology, MPLI≤0.4 is determined as high-efficiency response, 0.4<MPLI≤0.7 is determined as medium-efficiency response, and MPLI>0.7 is determined as low-efficiency response.

[0012] Preferably, the intelligent data analysis and drug efficacy rating module further adopts the individualized drug efficacy prediction index PEPI to integrate electrophysiological activity and exosome markers to predict the long-term therapeutic potential of the drug, and the calculation formula of PEPI is: ; wherein, and are respectively the spike firing frequency of the organoid after drug administration and before drug administration; and are respectively the network burst rate of the organoid after drug administration and before drug administration; and are respectively the loading amount of Aβ in exosomes of the culture medium after drug administration and before drug administration; 1-42 λ, μ, ν are weight coefficients, satisfying λ+μ+ν=1, with typical values of λ=0.35, μ=0.35, ν=0.30; a higher PEPI value indicates a greater potential of the drug in restoring neural network function and removing exosomal pathological load, and PEPI≥0.6 suggests good long-term therapeutic potential.

[0013] Preferably, the intelligent data analysis and drug efficacy rating module adopts the comprehensive drug efficacy decision score CDS to fuse MPLI and PEPI to output the final drug response classification, and the calculation formula of CDS is: ; wherein, and are fusion weights, satisfying + =1, with typical values of =0.55, =0.45; CDS≥0.70 is determined as high-efficiency response, 0.40≤CDS<0.70 is determined as medium-efficiency response, and CDS<0.40 is determined as low-efficiency response.

[0014] Preferably, independent or combined perfusion control between the three functional chambers of the core module of the microfluidic chip is realized through microchannels and a valve control system.

[0015] Preferably, the drug efficacy screening method of the platform comprises the following steps: Step 1: The neural organoids are seeded into the neural organoid culture chamber and fixed using a microcolumn array; microglia-like cells induced to differentiate from PBMCs of the same patient are introduced into the immune cell circulation chamber and co-cultured in a microglia-neuron ratio of 1:5; patient plasma or plasma exosomes are introduced into the plasma perfusion chamber and exchange substances with the neural organoid culture chamber through a selectively permeable membrane, with the perfusion rate controlled at 0.5-2 μL / min; Step 2: The drug to be screened is generated into 5-8 concentration gradients through the concentration gradient generation unit and perfused into the neural organoid culture chamber at a flow rate of 1 μL / min. The drug is continuously administered for 48-72 hours, while maintaining continuous perfusion in the plasma perfusion chamber and the immune cell circulation chamber. Step 3: After drug administration, imaging data, electrophysiological data, and inflammatory factor and exosome data are collected simultaneously before and after drug administration using the multimodal endpoint detection module; Step four: The intelligent data analysis and efficacy rating module calculates MPLI, PEPI and CDS based on the collected data, classifies candidate drugs into high-efficiency response, medium-efficiency response or low-efficiency response according to the CDS value, and outputs efficacy rating report.

[0016] The present invention achieves the following beneficial technical effects compared to the prior art: This invention provides a microfluidic drug screening platform for human AD organoids that integrate patient plasma / immune components. The neural organoids derived from patient iPSCs preserve the patient's complete genetic background, accurately reflecting individual differences in sporadic AD. Through the partitioned design and independent perfusion control of the neural organoid culture chamber, immune cell circulation chamber, and plasma perfusion chamber within the microfluidic chip, the platform achieves systematic integration and coupled co-culture of neural organoids, patient-derived glial-like cells, and patient plasma / exosomes in a dynamic microenvironment, overcoming the limitation of traditional models that cannot simultaneously reproduce the neuro-immune-peripheral inflammatory interactions. The platform simultaneously collects five types of endpoints—Aβ plaques, pTau protein, synaptophysin, inflammatory factors, and electrophysiological activity—through a multimodal endpoint detection module. This invention utilizes point data and combines it with three progressive algorithms—multimodal pathological burden index, personalized drug efficacy prediction index, and comprehensive drug efficacy decision score—for quantitative evaluation. It achieves precise grading of candidate drugs across three levels: improvement in pathological burden, recovery of neural network function, and comprehensive drug efficacy decision. MPLI overcomes the limitations of single-indicator evaluation by weighted integration of the change rates of four pathological indicators: Aβ, pTau, synaptophysin, and inflammatory factors. PEPI compensates for the insufficient predictive ability of static pathological indicators for long-term efficacy by introducing electrophysiological activity and exosome pathological load. CDS achieves a balanced consideration of pathological improvement and functional recovery by fusing the outputs of MPLI and PEPI, providing a quantitative basis for preclinical drug screening and personalized medication decisions. This invention effectively solves the technical problems of traditional AD drug screening models, such as insufficient humanization, inability to reflect individual differences, inability to simultaneously observe the neuro-glial-peripheral inflammation coupling effect, and a single dimension of drug efficacy evaluation. It provides a more clinically valuable technical platform for drug screening and personalized treatment evaluation in sporadic AD. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This invention provides an architecture diagram of a human AD organoid microfluidic drug screening platform that integrates patient plasma / immune components. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] The purpose of this invention is to provide a human AD organoid microfluidic drug screening platform that integrates patient plasma / immune components to solve the problems existing in the prior art.

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] Example 1: like Figure 1 As shown in this embodiment, the human Alzheimer's disease organoid microfluidic drug screening platform integrating patient plasma and immune components mainly comprises four functional modules: a patient-derived induced pluripotent stem cell neural organoid preparation module, a microfluidic chip core module, a multimodal endpoint detection module, and an intelligent data analysis and efficacy rating module. The platform first isolates peripheral blood mononuclear cells from the peripheral blood of Alzheimer's patients. Through non-integrative reprogramming technology, these peripheral blood mononuclear cells are reprogrammed into induced pluripotent stem cells, which then differentiate into three-dimensional neural organoids containing neurons, astrocytes, and microglia. The prepared neural organoids are seeded into the neural organoid culture chamber of the microfluidic chip. Simultaneously, microglia-like cells induced from peripheral blood mononuclear cells of the same patient are introduced into the immune cell circulation chamber, and patient plasma or plasma exosomes are introduced into the plasma perfusion chamber, achieving systematic integration and coupled co-culture of neural organoids, immune cells, and patient humoral factors in a dynamic microenvironment. The drug to be screened is perfused into the neural organoid culture chamber after generating a series of concentration gradients through a concentration gradient generation unit. After drug administration, imaging data, electrophysiological data, inflammatory factors and exosome data are collected simultaneously through the multimodal endpoint detection module. Finally, the intelligent data analysis and efficacy rating module calculates the multimodal pathological burden index, individualized efficacy prediction index and comprehensive efficacy decision score based on the collected data, and outputs the response grading results of the candidate drugs.

[0023] In the patient-derived induced pluripotent stem cell (iPSC) neural organoid preparation module, peripheral blood was first collected from Alzheimer's patients, and peripheral blood mononuclear cells were separated using density gradient centrifugation. The separated peripheral blood mononuclear cells were transduced into a non-integrating Sendai virus vector containing four reprogramming factors: Oct4, Sox2, Klf4, and c-Myc. After transduction, they were seeded into Matrigel-coated culture dishes and continuously cultured in iPSC medium until embryonic stem cell-like clones appeared. Clones with typical morphology were selected for expansion and identification. The pluripotency of iPSCs was verified by alkaline phosphatase staining, immunofluorescence detection of pluripotency markers, and in vitro tri-germ layer differentiation experiments. After obtaining qualified iPSCs, they underwent neural induction differentiation: iPSCs were cultured in a neural induction medium, and neural organoids were obtained sequentially through neural induction, neural progenitor cell expansion, and three-dimensional organoid differentiation. Specifically, neural induction was performed from day 0 to day 7 of differentiation, neural progenitor cell expansion was carried out from day 7 to day 14, astrocyte precursors were introduced on day 14, and microglia precursors were introduced on day 21. The cells were co-cultured until day 42 to obtain three-dimensional neural organoids containing neurons, astrocytes, and microglia. The culture medium was changed every two days during differentiation, and morphological changes in the organoids were observed under an inverted microscope. The obtained neural organoids had a diameter of approximately 300-500 micrometers and exhibited a typical cortical-like layered structure, suitable for subsequent microfluidic chip seeding.

[0024] The core module of the microfluidic chip is a multilayer bonded polydimethylsiloxane microfluidic chip, which includes a neural organoid culture chamber, an immune cell circulation chamber, a plasma perfusion chamber, a concentration gradient generation unit, and a waste collection unit. The neural organoid culture chamber is located in the central region of the chip. At the bottom of the chamber is an array of organoid fixation micropillars, each 100 micrometers high and 50 micrometers in diameter, with a spacing of 150 micrometers between adjacent micropillars. These micropillars are used to contain and fix the neural organoids output from the neural organoid preparation module, preventing displacement during perfusion. The immune cell circulation chamber is connected to the neural organoid culture chamber via a microchannel 50 micrometers wide and 30 micrometers deep, used to introduce microglial-like cells induced from peripheral blood mononuclear cells in patients, achieving neural-immune co-culture. The plasma perfusion chamber is separated from the neural organoid culture chamber by a selectively permeable membrane with a molecular weight cutoff of 100 kDa. This membrane allows small proteins, exosomes, and metabolites in the plasma to pass through while preventing the passage of microorganisms and cells. It is used to introduce patient plasma or plasma exosomes, enabling continuous perfusion stimulation of organoids by humoral factors. The concentration gradient generation unit, connected to the chip inlet, employs a classic Christmas tree-shaped microchannel network structure. Through branching and mixing channels, it serially dilutes the single-concentration drug solution to be screened with blank culture medium, generating 5-8 concentration gradients of drug solutions. The concentration range is set from 0.1 times the half-maximal inhibitory concentration (ICD) to 10 times the ICD, allowing for the parallel application of the drug to multiple organoids after generating a series of concentration gradients. The waste collection unit, connected to the chip outlet via a capillary tube, is used to collect culture waste for subsequent exosome extraction and detection. The three functional chambers—neural organoid culture chamber, immune cell circulation chamber, and plasma perfusion chamber—are interconnected by a microchannel and a valve control system to achieve independent or combined perfusion control. The valve control system consists of pneumatic microvalves integrated on the chip, which control the opening and closing of each channel through external air pressure, thereby achieving precise regulation of the perfusion timing of different chambers.

[0025] In the organoid inoculation and co-culture stage, the neural organoids prepared in step one were carefully aspirated using a pipette and inoculated into the neural organoid culture chamber of the microfluidic chip. The organoids were then fixed in the central region of the chamber using a microcolumn array. Peripheral blood mononuclear cells from the same patient were cultured for 7 days in an induction medium containing macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor to induce differentiation into microglial-like cells. These microglial-like cells were introduced into the immune cell circulation chamber at a ratio of 1:5 to neurons, and circulation perfusion was initiated for neural immune co-culture. Peripheral blood from the patient was collected and centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant plasma was separated, or plasma exosomes were further extracted by ultracentrifugation. The patient plasma or plasma exosomes were introduced into the plasma perfusion chamber, where they exchanged substances with the neural organoid culture chamber through a selectively permeable membrane with a molecular weight cutoff of 100 kDa. The plasma perfusion rate was controlled at 0.5-2 μL per minute. The entire co-culture system was maintained in an incubator at 37 degrees Celsius with 5% carbon dioxide by volume. The culture medium was a neural basal medium supplemented with B27 additive, N2 additive, glutamine, and penicillin-streptomycin antibiotics.

[0026] During the drug gradient application and dynamic perfusion phase, the drug to be screened was dissolved in dimethyl sulfoxide and diluted to the stock concentration with blank culture medium. The drug solution was then injected into the inlet of the concentration gradient generation unit via a syringe pump. The concentration gradient generation unit used a series of branching and mixing channels to gradient mix the drug solution with the blank culture medium, generating drug solutions with 5-8 concentration gradients, ranging from 0.1 times the half-maximal inhibitory concentration (ICD) to 10 times the ICD. Each gradient drug solution was perfused into the corresponding organoid in the neural organoid culture chamber at a flow rate of 1 μL per minute, with continuous administration for 48-72 hours. During administration, continuous perfusion of plasma or exosomes in the plasma perfusion chamber and circulation perfusion of microglial-like cells in the immune cell circulation chamber were maintained simultaneously to simulate continuous immune surveillance and humoral factor exposure in vivo. Morphological changes and growth status of the organoids were observed and recorded under an inverted microscope every 12 hours during the administration process.

[0027] During the multimodal endpoint data acquisition phase, after drug administration, the following four types of endpoint data were simultaneously collected using the multimodal endpoint detection module. The first type was imaging data, which included Aβ plaque fluorescence intensity, pTau protein immunofluorescence intensity, and synaptophysin immunofluorescence intensity of organoids acquired via confocal microscopy. Specifically, after drug administration, organoids were fixed with 4% paraformaldehyde for 30 minutes, washed three times with phosphate buffer, and stained with thiosulfate S for Aβ plaques, AT8 antibody for pTau protein immunofluorescence, and anti-synaptophysin antibody for synaptophysin immunofluorescence. Images were acquired at 400x magnification using a confocal microscope after staining, and ImageJ software was used to analyze the images and calculate the Aβ plaque fluorescence intensity, pTau protein immunofluorescence intensity, and synaptophysin immunofluorescence intensity. All fluorescence intensity values ​​were normalized to organoid volume. The second category is electrophysiological data. A microelectrode array detection unit integrated into the bottom of the neural organoid culture chamber records the spike firing frequency and network burst rate of the organoids in real time. The recording time is 10 minutes, and the sampling frequency is 25 kHz. A threshold detection method is used to identify spike firing events, with the spike firing detection threshold set to 5 times the standard deviation of the baseline signal. A network burst is defined as an event in which at least 5 electrodes simultaneously detect spike firing within a 100-millisecond time window. The third category is inflammatory factor data. Culture medium from the outlet of the neural organoid culture chamber is collected, and the concentrations of tumor necrosis factor α, interleukin-1β, interleukin-6, and interferon-γ in the culture medium are detected using a microfluidic tandem mass spectrometry interface or enzyme-linked immunosorbent assay (ELISA). The fourth category is exosome data. Culture waste liquid from the waste collection unit is collected, and exosomes are extracted using ultracentrifugation. The morphology of exosomes is identified by transmission electron microscopy, the number of exosome particles is determined by nanoparticle tracking analysis, and Aβ in exosomes is detected using ELISA. 1-42 and pTau 181 The loading amount was normalized by exosome particle count. Baseline data prior to drug administration were collected using the same method before drug administration.

[0028] In the intelligent data analysis and efficacy rating stage, the intelligent data analysis and efficacy rating module receives pre- and post-drug administration data output from the multimodal endpoint detection module and first calculates the Multimodal Pathological Burden Index (MPLI). The formula for calculating MPLI is: ; in, and The fluorescence intensity of Aβ plaques in organoids before and after drug administration is shown in two figures, respectively. and The immunofluorescence intensities of pTau protein in organoids before and after drug administration are shown, respectively. and The values ​​represent the synaptophysin immunofluorescence intensity of organoids before and after drug administration, respectively. and The figures represent the combined inflammatory factor index of organoid culture medium before and after drug administration. ,in The concentration of the i-th inflammatory factor, TNF-α, IL-1β, IL-6, or IFN-γ. The baseline concentration of the corresponding inflammatory factor in the untreated control organoid culture medium; α, β, γ, and δ are weighting coefficients, satisfying α+β+γ+δ=1, determined by minimizing prediction error using training set data. A lower MPLI value indicates a better improvement in the pathology of Alzheimer's disease; an MPLI less than or equal to 0.4 is considered a highly effective response, an MPLI greater than 0.4 and less than or equal to 0.7 is considered a moderately effective response, and an MPLI greater than 0.7 is considered a poorly effective response.

[0029] The intelligent data analysis and drug efficacy rating module further employs the Personalized Drug Efficacy Prediction Index (PEPI) to integrate electrophysiological activity and exosome biomarkers to predict the long-term efficacy potential of drugs. The formula for calculating PEPI is: ; in, and The spike firing frequencies of organoids were measured before and after drug administration, respectively. and Network burst rates of organoids were defined before and after drug administration, respectively. and Aβ levels in culture medium exosomes before and after drug administration were respectively. 1-42 The loading capacity; λ, μ, and ν are weighting coefficients that satisfy λ+μ+ν=1, with typical values ​​of λ=0.35, μ=0.35, and ν=0.30; the higher the PEPI value, the greater the potential of the drug in restoring neural network function and clearing exosome pathological loads, and PEPI≥0.6 indicates good long-term efficacy potential.

[0030] The intelligent data analysis and drug efficacy rating module ultimately uses a comprehensive drug efficacy decision score (CDS) that integrates MPLI and PEPI to output the final drug response grade. The calculation formula for CDS is: ; in, and To integrate weights, satisfy + =1, typical value is =0.55, =0.45; CDS ≥ 0.70 is considered a high-efficiency response, 0.40 ≤ CDS < 0.70 is considered a medium-efficiency response, and CDS < 0.40 is considered a low-efficiency response. The intelligent data analysis and efficacy rating module classifies candidate drugs into three levels—high-efficiency response, medium-efficiency response, or low-efficiency response—according to the above criteria, and outputs an efficacy rating report, providing quantitative basis for preclinical drug screening and personalized medication decisions.

[0031] This embodiment achieves the systematic integration of patient-derived neural organoids, patient-derived microglial cells, patient plasma, and exosomes in a microfluidic dynamic environment through the above-mentioned technical solution. It simultaneously collects five types of endpoint data through a multimodal endpoint detection module and realizes quantitative evaluation from pathological burden improvement and neural network function recovery to comprehensive drug efficacy decision through three progressive algorithms: MPLI, PEPI, and CDS. This effectively solves the technical problems of traditional Alzheimer's drug screening models, such as insufficient humanization, inability to reflect individual differences, lack of a multi-factor coupling environment of neuroimmune peripheral inflammation, and single dimension of drug efficacy evaluation.

[0032] This invention has illustrated its principles and implementation methods using specific examples. The descriptions of these embodiments are merely illustrative of the method and its core ideas; furthermore, those skilled in the art will recognize that modifications may be made to the specific implementation methods and application scope based on the principles of this invention. Therefore, the content of this specification should not be construed as limiting the invention.

Claims

1. A human AD organoid microfluidic drug screening platform integrating patient plasma / immune components, characterized in that: comprising: a patient-derived iPSC neural organoid preparation module, configured to reprogram peripheral blood mononuclear cells of a patient to obtain induced pluripotent stem cells, and further directionally differentiate the induced pluripotent stem cells into three-dimensional neural organoids comprising neurons, astrocytes and microglial cells; a microfluidic chip core module, which is a multi-layer bonded polydimethylsiloxane microfluidic chip, comprising a neural organoid culture chamber, an immune cell circulation chamber, a plasma perfusion chamber, a concentration gradient generation unit and a waste liquid collection unit; the neural organoid culture chamber is located in the central area of the chip, and is provided with an organoid fixation microcolumn array, configured to accommodate and fix the neural organoids output by the neural organoid preparation module; the immune cell circulation chamber is connected to the neural organoid culture chamber through a microchannel, configured to introduce microglia-like cells induced and differentiated from patient PBMCs to realize neural-immune co-culture; the plasma perfusion chamber is separated from the neural organoid culture chamber by a selectively permeable membrane, configured to introduce patient plasma or plasma exosomes to realize continuous perfusion stimulation of the organoids by humoral factors; the concentration gradient generation unit is connected to the inlet end of the chip, configured to generate a series of concentration gradients of the drug to be screened; the waste liquid collection unit is connected to the outlet end of the chip, configured to collect culture waste liquid; a multimodal endpoint detection module, integrated downstream of the microfluidic chip or in an external detection system of the chip, comprising a bright-field / fluorescence microscopic imaging unit, a microelectrode array detection unit and a microfluidic tandem mass spectrometry interface, which are respectively configured to acquire imaging data, electrophysiological data, and inflammatory factor and exosome data of the organoids; an intelligent data analysis and drug efficacy rating module, configured to receive the output data of the multimodal endpoint detection module, perform data processing, feature fusion and drug efficacy rating through a multimodal pathological load index algorithm, a personalized drug efficacy prediction index algorithm and a comprehensive drug efficacy decision scoring algorithm, and output a response grading result of the candidate drug.

2. The human AD organoid microfluidic drug screening platform integrating patient plasma / immune components according to claim 1, characterized in that: The neural organoids are obtained by subjecting patient iPSCs to neural induction, neural progenitor cell expansion and three-dimensional organoid differentiation in sequence, wherein astrocyte precursors are introduced on the 14th day of differentiation, microglial cell precursors are introduced on the 21st day of differentiation, and co-culture is carried out until the 42nd day to obtain the three-dimensional neural organoids comprising neurons, astrocytes and microglial cells.

3. The human AD organoid microfluidic drug screening platform integrating patient plasma / immune components according to claim 1, characterized in that: In the core module of the microfluidic chip, the ratio of glial-like cells in the immune cell circulation chamber to neurons in the neural organoid culture chamber is 1:5; the selective permeable membrane of the plasma perfusion chamber has a molecular weight cutoff of 100 kDa; the plasma perfusion rate is 0.5-2 μL / min; the drug perfusion rate is 1 μL / min; the drug administration duration is 48-72 hours; and the concentration gradient generation unit generates 5-8 concentration gradients with a concentration range of 0.1 × 10⁻⁸ CJ / mL. 50 Up to 10×IC 50 .

4. The human AD organoid microfluidic drug screening platform integrating patient plasma / immune components according to claim 1, characterized in that: The multimodal endpoint detection module includes imaging data such as Aβ plaque fluorescence intensity, pTau protein immunofluorescence intensity, and synaptophysin immunofluorescence intensity; electrophysiological data such as organoid burst firing frequency and network burst rate; inflammatory factor data such as the concentrations of TNF-α, IL-1β, IL-6, and IFN-γ in the culture medium; and exosome data such as Aβ exosomes in waste fluid. 1-42 and pTau 181 Load.

5. The human AD organoid microfluidic drug screening platform integrating patient plasma / immune components according to claim 1, characterized in that: The intelligent data analysis and drug efficacy rating module adopts a multimodal pathological load index MPLI to quantitatively evaluate the pathological improvement degree of the organoids after drug intervention, and the calculation formula of MPLI is: ； in, and The fluorescence intensity of Aβ plaques in organoids before and after drug administration is shown in two figures, respectively. and The immunofluorescence intensities of pTau protein in organoids before and after drug administration are shown, respectively. and The values ​​represent the synaptophysin immunofluorescence intensity of organoids before and after drug administration, respectively. and The figures represent the combined inflammatory factor index of organoid culture medium before and after drug administration. ,in The concentration of the i-th inflammatory factor, TNF-α, IL-1β, IL-6, or IFN-γ. The baseline concentrations of the corresponding inflammatory factors in the untreated control organoid culture medium are α, β, γ, and δ, which are weighting coefficients that satisfy α+β+γ+δ=1 and are determined by minimizing the prediction error using training set data.

6. The human AD organoid microfluidic drug screening platform integrating patient plasma / immune components according to claim 5, characterized in that: Typical values of the weight coefficients α, β, γ, δ are α=0.30, β=0.30, γ=0.20, δ=0.20; a lower MPLI value indicates a better improvement effect of the drug on AD pathology, MPLI ≤ 0.4 is determined as high-efficiency response, 0.4 < MPLI ≤ 0.7 is determined as medium-efficiency response, and MPLI > 0.7 is determined as low-efficiency response.

7. The human AD organoid microfluidic drug screening platform integrating patient plasma / immune components according to claim 5, characterized in that: The intelligent data analysis and drug efficacy rating module further adopts a personalized drug efficacy prediction index PEPI to integrate electrophysiological activity and exosome markers to predict the long-term therapeutic potential of the drug, and the calculation formula of PEPI is: ； in, and The spike firing frequencies of organoids were measured before and after drug administration, respectively. and Network burst rates of organoids were defined before and after drug administration, respectively. and Aβ levels in culture medium exosomes before and after drug administration were respectively. 1-42 The loading capacity; λ, μ, and ν are weighting coefficients that satisfy λ+μ+ν=1, with typical values ​​of λ=0.35, μ=0.35, and ν=0.30; the higher the PEPI value, the greater the potential of the drug in restoring neural network function and clearing exosome pathological loads, and PEPI≥0.6 indicates good long-term efficacy potential.

8. The human AD organoid microfluidic drug screening platform integrating patient plasma / immune components according to claim 7, characterized in that: The intelligent data analysis and drug efficacy rating module uses a comprehensive drug efficacy decision score (CDS) that integrates MPLI and PEPI to output the final drug response grade. The calculation formula for CDS is as follows: ； in, and To integrate weights, satisfy + =1, typical value is =0.55, =0.45; CDS≥0.70 is considered a high-efficiency response, 0.40≤CDS<0.70 is considered a medium-efficiency response, and CDS<0.40 is considered an inefficient response.

9. The human AD organoid microfluidic drug screening platform integrating patient plasma / immune components according to claim 1, characterized in that: The three functional chambers of the microfluidic chip core module are connected by microchannels and a valve control system to achieve independent or combined perfusion control.

10. The human AD organoid microfluidic drug screening platform integrating patient plasma / immune components according to claim 1, characterized in that: The efficacy screening method of the platform includes the following steps: Step 1: The neural organoids are seeded into the neural organoid culture chamber and fixed using a microcolumn array; microglia-like cells induced to differentiate from PBMCs of the same patient are introduced into the immune cell circulation chamber and co-cultured in a microglia-neuron ratio of 1:5; patient plasma or plasma exosomes are introduced into the plasma perfusion chamber and exchange substances with the neural organoid culture chamber through a selectively permeable membrane, with the perfusion rate controlled at 0.5-2 μL / min; Step 2: The drug to be screened is generated into 5-8 concentration gradients through the concentration gradient generation unit and perfused into the neural organoid culture chamber at a flow rate of 1 μL / min. The drug is continuously administered for 48-72 hours, while maintaining continuous perfusion in the plasma perfusion chamber and the immune cell circulation chamber. Step 3: After drug administration, imaging data, electrophysiological data, and inflammatory factor and exosome data are collected simultaneously before and after drug administration using the multimodal endpoint detection module; Step four: The intelligent data analysis and efficacy rating module calculates MPLI, PEPI and CDS based on the collected data, classifies candidate drugs into high-efficiency response, medium-efficiency response or low-efficiency response according to the CDS value, and outputs efficacy rating report.

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