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CN-122012237-A - Organ chip for simulating airway-alveolus continuous barrier function and application of organ chip in COPD model construction and drug evaluation

CN122012237ACN 122012237 ACN122012237 ACN 122012237ACN-122012237-A

Abstract

The invention discloses a three-chamber integrated microfluidic chip for simulating airway-alveolus continuous barrier function and application thereof in COPD drug evaluation. The chip comprises an airway chamber, an alveolus chamber, a blood vessel chamber and a porous PET film, and the continuous space level of the airway-alveolus-qi-blood barrier-blood vessel is rebuilt through a three-chamber integrated bionic structure. The invention respectively establishes an optimized culture system for airway epithelial cells and AT2 cells of COPD patients, obtains airway organoids and alveolar organoids with pathological characteristics, and realizes co-culture in a chip. The chip is provided with a micro sampling port, and can collect effluent liquid at different time points for carrying out inflammatory factor dynamic monitoring. The multi-dimensional detection such as dead and alive staining, ELISA, immunofluorescence and the like is combined to construct an omnibearing evaluation system from morphology to molecules and from inside to outside of cells. The application of erdosteine intervention verification shows that the chip can effectively simulate the airway-alveolus double-part damage characteristics (including morphological shrinkage, cell death increase, inflammatory factor IL-6 increase, mucus factor MUC5B high secretion and the like) of COPD patients and can reliably evaluate the curative effect of the medicine. The invention provides an in-vitro platform with high bionic degree and high stability for COPD mechanism research, drug screening and personalized medicine, and has wide industrialized application prospect in the fields of respiratory disease drug research and development, toxicity evaluation and precise medicine.

Inventors

  • LI QIULING
  • Zhu Zhuoyi
  • Tao Chengxu
  • CHEN XIAOXUE

Assignees

  • 安徽大学

Dates

Publication Date
20260512
Application Date
20260302

Claims (9)

  1. 1. The three-chamber integrated microfluidic chip for simulating the continuous barrier function of the airway and the alveoli is characterized by comprising an airway chamber, a microfluidic chip and a microfluidic chip, wherein the airway chamber is used for culturing airway organoids, and the top of the airway chamber is opened to form a gas-liquid interface; an alveolar chamber for culturing an alveolar organoid in physical communication with the airway chamber sidewall forming a continuous epithelial barrier structure; the vascular chamber is positioned on the upper layer, is separated from the airway chamber and the alveolus chamber on the lower layer through a porous membrane, and is paved with endothelial cells to simulate blood vessels; the porous membrane is positioned between the vascular chamber and the lower culture chamber and is used for realizing signal and substance exchange and constructing an air-blood barrier; a microsampling port in fluid communication with the vascular chamber for collecting effluent.
  2. 2. The chip of claim 1, wherein the airway chamber and alveolar chamber share the same vascular chamber and porous membrane for parallel culture and collaborative analysis of airways and alveolar organoids.
  3. 3. An amplification culture medium for culturing airway organoids of COPD patients is characterized in that the culture medium comprises a basal medium ADVANCED DMEM/F-12, 10% FBS and the following components of an Epidermal Growth Factor (EGF) 25-50 ng/mL, insulin (Insulin) 10-50 μg/mL, transferrin (Transferrin) 5-25 μg/mL, cholera Toxin (Cholera Toxin) 0.1-0.5 μg/mL, retinoic acid （Retinoic Acid）50-100 nM; Noggin 20-50 μg/mL;R-Spondin 1 200-800 ng/mL;A8301 500-1000 nM;CHIR99021 3-10 μM;Y-27632 5-10 μM.
  4. 4. A differentiation medium for culturing alveolar organoids derived from patients with COPD is characterized by comprising a basal medium and a composition comprising 10-50 ng/mL of fibroblast growth factor 10 (FGF 10), 25-50 ng/mL of fibroblast growth factor 7 (FGF 7), 5-20 ng/mL of Surfactant Protein C (SPC), and SB431542 10-50 mu M.
  5. 5. A method of constructing a COPD airway organoid based on the expansion medium of claim 3 comprising the steps of: Obtaining airway tissues of a patient suffering from COPD, and obtaining airway epithelial cells through enzymatic digestion; mixing the cells with matrigel for 3D embedding culture, and adding the amplification culture medium of claim 3; cultured until an airway organoid is formed that is characterized by ciliated cells, secretory cells and basal cells.
  6. 6. A method of constructing COPD alveolar organoids based on the differentiation medium as claimed in claim 4, comprising the steps of: obtaining lung tissues of a patient suffering from COPD, and obtaining a cell suspension through enzyme digestion; Removing fibroblasts to obtain AT2 cells; mixing AT2 cells with matrigel for 3D embedding culture, and adding the differentiation medium according to claim 4; culturing until an alveolar organoid having AT2 cell characteristics and expressing HOPX/Abca3 is formed.
  7. 7. An in vitro model of COPD constructed on the basis of any one of claims 1 to 6, comprising: COPD patients co-cultured in the chip are derived from airway organoids, alveolar organoids and vascular endothelial layers; the chip operates under dynamic perfusion and gas-liquid interface conditions to form an in vitro disease model with a genetic background of COPD patients.
  8. 8. A method for drug screening and efficacy evaluation using the COPD in vitro disease model of claim 7, comprising the steps of: drug intervention, namely applying candidate drugs into the chip for culture; dynamic monitoring of effluent, namely collecting effluent to detect dynamic changes of inflammatory factors and mucus secretion factors; Cell viability evaluation, namely carrying out dead and alive staining on organoids in a chip, and quantitatively analyzing the cell viability; gene expression analysis, namely collecting organoids in a chip to detect gene expression; And (3) evaluating the efficacy of the candidate drug by comparing the index differences of the control group, the COPD model group and the drug treatment group.
  9. 9. Use of the chip of claim 1 or 2, the culture medium of claim 3 or 4, the culture method of claim 5 or 6, the COPD in vitro disease model of claim 7, or the drug evaluation method of claim 8 in the preparation of a kit or device for chronic airway disease research or drug screening.

Description

Organ chip for simulating airway-alveolus continuous barrier function and application of organ chip in COPD model construction and drug evaluation Technical Field The invention belongs to the technical field of microfluidic chips and organoid culture, and particularly relates to a microfluidic organ chip for integrated airway organoid and alveolar organoid co-culture and application thereof in COPD model construction, drug screening and mechanism research. Background Chronic Obstructive Pulmonary Disease (COPD) is a chronic respiratory disease characterized by airflow limitation, airway remodeling and alveolar structural destruction, the pathological mechanisms of which include chronic inflammation, increased mucus secretion, impaired cilia function, and extracellular matrix degradation. The existing research shows that the occurrence of COPD is closely related to long-term inflammatory reaction, mucus hypersecretion, cilia injury and abnormal degradation of extracellular matrix, and the diseases have obvious tissue heterogeneity and inter-patient variability, which brings great challenges for constructing a human-derived in-vitro model with stable pathological manifestations. The current commonly used models of COPD mainly include smoke-exposed mouse models and elastase-induced models, although these animal models can reproduce part of COPD pathological features such as alveolar destruction and inflammatory infiltration, they have difficulty accurately simulating the three-dimensional anatomy of the human airway-alveolar interface and long-term chronic disease course due to species differences and structural complexity limitations. In addition, animal models are difficult to embody individual differences of patients, limit individual drug screening and mechanism research, and the traditional two-dimensional cell culture technology is simple and convenient to operate, but lacks gas-liquid interfaces and three-dimensional cell-extracellular matrix interactions, and cannot simulate complex intercellular communication and barrier functions in COPD. In recent years, the lung organoid technology is gradually applied to the research of lung diseases, and the research shows that the lung organoid function can better reconstruct the polar structure and multicellular composition of lung epithelium, thereby providing a new platform for simulating lung pathology. However, the traditional organoid culture system is static culture, lacks key pathological factors such as vascularization, airflow stress, inflammation microenvironment and the like, and is difficult to completely simulate the chronic progress of COPD. More importantly, existing lung organoid models are typically focused on a single epithelial structure (airway only or alveolar only), are unable to reconstruct both airway and alveolar structures simultaneously, and do not have conditions that mimic the synergy of multiple cell types of vascular endothelium-epithelium. The microfluidic organ chip technology can more truly simulate the micro-environment of in-vivo tissues by integrating physiological related factors such as fluid shear force, gas-liquid interface, chemical gradient and the like. However, both the existing lung chips and organoid cultures have difficulty simulating the multicellular, multi-structural injury characteristics of COPD, lacking an in vitro platform capable of integrating patient-derived airway-alveolar organoids, multichamber co-culture, and vascularized dynamic perfusion simultaneously. Therefore, an in-vitro platform capable of integrating airway-alveolus double structures, vascularization microenvironment and chronic inflammation simulation simultaneously is developed, and has important scientific significance and application value for COPD mechanism research and drug evaluation. Disclosure of Invention The invention aims to solve the technical problems that the existing COPD in-vitro model is difficult to simulate the double-structure damage of an air passage and an air alveolus, lacks the specificity of a patient and vascularization dynamic microenvironment, provides a three-chamber integrated microfluidic chip for simulating the continuous barrier function of the air passage and the air alveolus and application thereof in the construction of the COPD model and the drug evaluation, and can realize the stable maintenance of organoids and the monitoring of indexes such as inflammation, survival rate and the like under the condition of a trace sample, thereby providing a technical means with high bionic degree and high stability for the accurate modeling and clinical drug evaluation of respiratory diseases such as COPD and the like. In order to solve the technical problems, the invention provides a three-chamber integrated microfluidic chip for simulating the continuous barrier function of an airway and an alveolus, which comprises an airway chamber and an alveolus chamber which are physically communicated through side walls, a va