CN116904545A - High-content lung injury evaluation system based on human lung organoids - Google Patents

High-content lung injury evaluation system based on human lung organoids Download PDF

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CN116904545A
CN116904545A CN202310889941.4A CN202310889941A CN116904545A CN 116904545 A CN116904545 A CN 116904545A CN 202310889941 A CN202310889941 A CN 202310889941A CN 116904545 A CN116904545 A CN 116904545A
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evaluation system
lung
organoids
compound
cells
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李晓波
孟庆涛
王晶
许朵
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Capital Medical University
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079Mitochondria

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Abstract

The application discloses a high-content lung injury evaluation system based on a human lung organoid, and belongs to the technical field of cell biology. The application provides a rapid evaluation system for compound-induced lung injury by using lung organoids, and constructs a rapid, effective and reliable screening and evaluation system for finding early lung injury and clarifying lung injury mechanism, medicine and therapy. The screening system established by the application can realize rapid screening of the compound for lung organoids by utilizing human tissue culture, and can rapidly discover toxicity of the compound and early damage indexes.

Description

High-content lung injury evaluation system based on human lung organoids
Technical Field
The application relates to the technical field of cell biology, in particular to a high-content lung injury evaluation system based on a human lung organoid.
Background
The integrity of the lung tissue ensures that the lung performs a gas exchange function and an immune defence function. Lung epithelial cells include ciliated cells, rod-like cells, goblet cells and undifferentiated basal cells. The small bronchial epithelial cell types include ciliated cells, rod-like cells and neuroendocrine cells. Alveolar epithelial cells mainly include type I alveolar epithelial cells and type II alveolar epithelial cells. The different cell types of lung tissue communicate with each other and interact to maintain pulmonary homeostasis, function and promote repair of lung lesions.
The application of the planar culture cell model is a classical research method in the biomedical field, and the model has the advantages of economy, rapidness, simple culture and the like. However, since the composition of the planar cultured cells is single, there is a clear difference from the in vivo real situation, and the research result obtained based on the cell experiment alone is often insufficient to represent the physiological and pathological processes actually occurring in the living body. Furthermore, animal models have been widely used to elucidate human lung development, physiology, and pathogenesis, however, animal to human itself differences do not accurately reproduce interactions between human lung environment and cells. In addition, based on the 3R principle, the use of animal experiments should be reduced as much as possible.
The 3D lung organoids are derived from human tissue and contain a variety of cell types that reproduce the characteristics of the human airway. These properties make the lung organoids a model for studying lung injury, repair/regeneration mechanisms of lung injury, and phenotypic changes in lung disease, while the lung organoids show great potential for use in toxicity assessment or drug testing related research fields.
The prior art method utilizes normal human lung tissue to construct a lung organoid. These lung organoids can be passaged multiple times and cryopreserved. However, existing evaluation systems have multiple uses for planar cells or animal models. Planar cells are single in cell type and cannot achieve interactions between cells. The animal model has long test period, and the animal and human body have certain difference before, so the animal model cannot be directly applied to the human body.
Disclosure of Invention
The application aims to provide a high-content lung injury evaluation system based on human lung organoids, which aims to solve the problems in the prior art.
In order to achieve the above object, the present application provides the following solutions:
the application provides a lung organoid toxicity evaluation system based on high content analysis, which utilizes a compound to infect a lung organoid and evaluates lung injury caused by the compound according to morphological changes and physiological indexes of the lung organoid.
Preferably, the compounds include small molecule compounds (gut flora metabolites, HY-L078 derived from MCE) and particulate matter (PM 2.5 Carbon black, perfluorooctanoic acid, alumina, etc.).
Preferably, the physiological indicators include cell viability, mitochondrial and golgi damage.
Preferably, for cell viability and golgi and mitochondrial staining, probes are directly added to the culture wells for staining; for antibody staining, organoids were isolated using cell recovery solution, fixed and immunofluorescent stained.
Preferably, the cell viability, mitochondria and golgi apparatus are detected with high connotation, and golgi apparatus and mitochondria are stained with golgi apparatus and mitochondrial staining probes to detect changes in golgi apparatus and mitochondria; the cell viability is detected by using a cell viability detection kit.
Preferably, the mitochondrial detection kit is M7510 of Thermo Fisher; the golgi detection kit is C1034 of bi yun tian.
Preferably, the physiological index further comprises antibody detection, wherein different kinds of cells in the organoid are stained by using antibodies of different kinds of cells, and the influence on the different cells after the infection is explored.
Preferably, the organoids are isolated using a cell recovery solution, immobilized and immunofluorescent stained.
Preferably, the cell recovery solution is 354253 by Corning.
Preferably, the fixation is fixation with paraformaldehyde.
Preferably, the immunofluorescent staining comprises whole body immunofluorescent staining or preparing frozen sections for immunofluorescent staining.
The application discloses the following technical effects:
the application provides a compound toxicity evaluation system based on high content utilization of lung organoids, and constructs a rapid, effective and reliable screening and evaluation system for finding early lung injury and clarifying lung injury mechanism, medicine and therapy. The screening system established by the application can realize rapid screening of the compound for lung organoids by utilizing human tissue culture, and can rapidly discover toxicity of the compound and early damage indexes.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the high content of Calcein/PI cell activity and cytotoxicity assays of normal human lung organoid compounds following contamination and analysis of lung organoid morphology such as organoid area, organoid diameter, etc. using image J;
FIG. 2 is a graph showing the ultrastructural screening of high content of small molecular compounds of normal human lung organoids after contamination.
Detailed Description
Various exemplary embodiments of the application will now be described in detail, which should not be considered as limiting the application, but rather as more detailed descriptions of certain aspects, features and embodiments of the application.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the application. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The technical scheme of the application is conventional in the field, and the reagents or raw materials are purchased from commercial sources or are disclosed.
Example 1 cultivation of lung organoids:
1. after obtaining the normal tissue at the far end of the lung, the normal tissue is placed in tissue preservation solution MACS (Meitianni) 130-100-008, and the tissue preservation solution can preserve the tissue for 24-48 hours. Tissue activity is affected over time and should be treated as soon as possible, in this example stored for 12 hours.
2. Tissues were washed 5 times in ice-cold PBS (without calciferous).
3. Shearing lung tissue to a size of less than 2mm 3 The pellet was placed in a 15mL tube, the tissue was washed with 10mL of ice in PBS, the tissue was allowed to settle by gravity for 10s, and the supernatant was aspirated off.
4. The above steps were repeated 10 times.
5. 5mL of a mild cell dissociation reagent, stemcell, 100-0485, was added and the tube incubated at 37℃on a 35rpm shaker for 60min.
6. 400g after the incubation was completed, centrifuged at 4℃for 5min, and the gentle cell dissociation reagent was pipetted off.
7. 10mL of 4 ℃ PBS+1% FBS is added, the mixture is vigorously blown for 20 times, gravity sedimentation is carried out for 10s, the supernatant is sucked and transferred to a new 15mL centrifuge tube through a 70 mu m cell screen, and a filtrate containing cancer stem cell clusters is obtained.
8. 10mL of 4℃PBS was added after each filtration, and the mixture was re-filtered, and the total filtration was performed three times.
9. The filtrate was centrifuged at 300g at 4℃for 5min.
10. 10mL 4℃PBS+1% FBS was used for resuspension.
11. The pipette tip was pre-wetted and 10. Mu.L of the selected filtrate was aspirated and placed on a glass slide or cytometer for counting, approximately 500-1000 stem cell clusters per culture well.
12. Again at 300g,4℃for 5min.
13. Add 25. Mu.L of basal lung organoid culture medium Stemcell:05060 per culture well, resuspend pellet, again according to 1:1 adding an equal volume of Matrigel matrix gel, and after fully mixing, paving 50 mu L of Matrigel matrix gel in the middle of a 24-well plate for each well to avoid generating bubbles.
14. The 24-well plate was incubated in an incubator at 37℃for 10min to allow Matrigel to solidify.
15. After gel coagulation, 500 μl of basal lung organoid medium was added to each well and cultured in an incubator at 37 ℃. The basal lung organoid medium was changed every 3 days, ensuring that fresh basal lung organoid medium was pre-warmed prior to changing fluid.
Example 2 method of contamination of lung organoid-based compounds
1 Small molecule Compound contamination
1.1 the mature lung organoids were removed from the incubator and the medium was discarded.
1.2 intestinal flora metabolites at a concentration of 10nM are added to the lung organoid basal medium and 500. Mu.L per well is added to the lung organoid, taking care not to destroy the matrigel, and small molecule compounds are able to penetrate the matrigel.
1.3 after all additions, 24 well plates were placed in 5% CO for cultivation of lung organoids 2 Culturing in a 37 ℃ incubator until the lung organoids develop and mature.
2 contamination of particulate matter
2.1 dissociation of the lung organoids to be passaged with mild cell dissociation reagents on a shaker for 15 minutes.
2.2 organoid pellet was resuspended by adding organoid growth medium, and the pellet was thoroughly mixed.
2.3 aspiration of 150. Mu.L of organoid-containing Medium according to the number of infected wells, addition of 15. Mu.L of infectious agent, PM with 10. Mu.g/mL of infectious agent 2.5 After thoroughly mixing, 165 μl of 2.5% Matrigel was added rapidly.
2.4 sucking the mixed culture medium of organoids, particles and matrigel according to the size of the pore plate, adding the mixed culture medium into the pore plate, fully spreading the bottom of the plate, and placing the mixture into 37 ℃ and 5% CO 2 Culturing in an incubator.
2.5 changes in organoid morphology were observed microscopically every six hours.
Example 3 evaluation System for toxicity of Lung organoids based on high content analysis
1. If the activity and cytotoxicity of Calcein/PI cells or the detection of probes such as mitochondria and Golgi apparatus are used, the culture medium is directly discarded, and the diluted probes are added into the hole containing the lung organoid along the hole wall (to cover matrigel) and the matrigel is not destroyed. Incubation for a defined period of time allows for detection with high connotation.
2. For example, when incubating with an antibody that binds to the fluorescent secondary antibody, it is necessary to separate the organoids from the matrigel using cornin Cell Recovery Solution (cell recovery solution), and then to perform whole immunofluorescent staining after fixation with paraformaldehyde or to make frozen sections for immunofluorescent staining. The method mainly comprises the following steps:
2.1 the medium in each well was discarded (for organoids cultured in suspension, organoids were placed in EP tubes together with the medium, 300g was added after centrifugation at 4℃for 5min, cell Recovery Solution) and 1mL Cell Recovery Solution was added to each well and placed on ice for 20-30min.
2.2 wetting the tip with ice PBS in advance, adding to the well of Cell Recovery Solution, gently blowing with the tip 5 times (without mainly destroying the structure of the lung organoids), collecting in a 1.5mL EP tube or 15mL tube by volume, centrifuging at 300g at 4℃for 5min.
2.3 after centrifugation, the supernatant was discarded, 4% paraformaldehyde was added for fixation for 2 hours, again 400g, and centrifugation was performed at 4℃for 5min, and the supernatant was discarded. The lung organoids of this step can be used for direct staining or staining after the preparation of frozen sections.
2.4 for direct staining, the fixed organoids were washed once with PBS, centrifuged at 300g at 4℃for 5min, and the supernatant was discarded. 5% BSA was added and the mixture was blocked for 1 hour on a 20rpm rotating disk.
After 2.5, the supernatant was discarded after centrifugation at 300g for 5min at 4 ℃. PBS was added to wash three times for 5 minutes each.
2.6 after washing, the supernatant was discarded after centrifugation at 300g for 5min at 4 ℃. Primary antibody was added and incubated overnight at 4 ℃ on a 20rpm turntable.
After the incubation of 2.7, 300g was centrifuged at 4℃for 5min, and the primary antibody was recovered. PBS was added for 3 washes of 10min each.
2.8 after washing, the secondary antibody was recovered by centrifugation at 300g,4℃g and 4℃for 5min. PBS was added for 3 washes of 10min each.
2.10 after washing, the supernatant was discarded after centrifugation at 300g for 5min at 4 ℃. DAPI was added and incubated on a 20rpm turntable for 30min.
After 2.11 incubation, 300g was centrifuged at 4℃for 5min, the organoid was resuspended by adding an anti-fluorescence-attenuation capper and spread on a slide, and finally a cover slip was placed for observation.
2.12 if the frozen sections are used for staining, the sections at the temperature of-80 ℃ are taken out and placed in a section rack for temperature return for 5-10min.
2.13 sections were washed twice in PBS for 5min each.
2.14 washing with tissue grade xylene for 15min, repeated 2 times. Washing with 100% ethanol for 5min was repeated 2 times. Washing with 90% ethanol for 5min. Washing with 70% ethanol for 5min. Using dH 2 O was washed for 5min.
2.15 placing the slices into a slice box, and heating the citrate by using a microwave oven in a buffer way until the citrate boils, and then heating the citrate for 10 minutes with low fire.
2.16 sections were removed from sodium citrate buffer, cooled and washed with PBS buffer for 2 minutes.
2.17 air-drying the sections and marking out the portions of organoid sections using an immunohistochemical pen.
2.18 blocking for 1h with 5% BSA.
2.19 washing with PBS three times, each for 5min. The primary antibody was added for incubation overnight.
2.20 washed 33 times with PBS for 5min each.
2.21 adding secondary antibody, and incubating for 1h at room temperature in dark place.
2.22 washed 33 times with PBS for 5min each.
2.23 DAPI was added and incubated at room temperature for 30min in the dark.
2.24 washed 33 times with PBS, and after 5min each time, DAPI-containing caplets were added and observed under a confocal microscope.
The detection results are shown in fig. 1-2, the detection results are shown in fig. 1, the cell viability screening is carried out after the HY-L078 part of intestinal flora metabolites of the MCE are infected, the ultrastructural screening is carried out after the HY-L078 part of intestinal flora metabolites of the MCE are infected, and the screening and evaluation system for quickly, effectively and reliably finding early lung injury and clarifying lung injury mechanism, medicine and therapy is constructed based on the high content analysis. The screening system established by the application can realize rapid screening of the compound for lung organoids by utilizing human tissue culture, and can rapidly discover toxicity of the compound and early damage indexes.
The above embodiments are only illustrative of the preferred embodiments of the present application and are not intended to limit the scope of the present application, and various modifications and improvements made by those skilled in the art to the technical solutions of the present application should fall within the protection scope defined by the claims of the present application without departing from the design spirit of the present application.

Claims (9)

1. A high-content lung injury evaluation system based on human lung organoids is characterized in that the lung organoids are infected by a compound, and the lung injury caused by the compound is evaluated according to the morphological change and the physiological index of the lung organoids.
2. The evaluation system according to claim 1, wherein the compound comprises a small molecule compound and a particulate.
3. The evaluation system of claim 1, wherein the physiological indicators include cell viability, mitochondrial and golgi damage.
4. The evaluation system according to claim 3, wherein the cell viability, mitochondria and golgi apparatus are detected with high connotation, and golgi apparatus and mitochondria are stained with golgi apparatus and mitochondrial staining probes to detect changes in golgi apparatus and mitochondria; the cell viability is detected by using a cell viability detection kit.
5. The evaluation system according to claim 1, wherein the physiological index further comprises antibody detection, wherein different kinds of cells in the organoid are stained with antibodies to the different kinds of cells, and the effect on the different cells after contamination is investigated.
6. The evaluation system according to claim 5, wherein the organoids are isolated by means of cell recovery solutions and fixed and immunofluorescent stained.
7. The evaluation system according to claim 6, wherein the cell recovery solution is Corning 354253.
8. The evaluation system according to claim 6, wherein the immobilization is immobilization with paraformaldehyde.
9. The evaluation system of claim 6, wherein the immunofluorescent staining comprises whole body immunofluorescent staining or frozen section preparation immunofluorescent staining.
CN202310889941.4A 2023-07-19 2023-07-19 High-content lung injury evaluation system based on human lung organoids Pending CN116904545A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117757885A (en) * 2023-12-26 2024-03-26 首都医科大学 Intestinal flora metabolite screening method based on high content

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117757885A (en) * 2023-12-26 2024-03-26 首都医科大学 Intestinal flora metabolite screening method based on high content

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