Construction based on epidermal organoid bacterial infection model and drug screening method
By constructing a 3D epidermal organoid infection model and comparing cell viability, the problems of inaccurate and high cost of antibacterial drug screening in the prior art are solved, and more accurate drug screening and clinical treatment strategies are achieved.
Patent Information
- Application Number
- CN202310698332.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-13
- Publication Date
- 2024-12-13
AI Technical Summary
The existing 2D skin cell model and traditional mammalian model have limitations in antibacterial drug screening, which cannot effectively simulate human skin infection and physiological structure, resulting in inaccurate screening results and high cost, limiting the application of high-throughput drug screening. .
A bacterial infection model was constructed based on the 3D epidermal organoid model, and by co-incubating it with bacteria, comparing cell viability with the presence or absence of the drug to be tested, drugs with antibacterial effects were screened out. The specific steps include releasing and digesting 3D organoid cells from matrix gel, counting and mixing with bacteria, setting up experimental groups and control groups, and measuring cell viability to judge the inhibitory effect of the drug.
More accurate drug screening data is achieved, new clinical treatment strategies are provided, high-throughput antibacterial drug screening and drug target development, and improved the research capabilities of disease pathogenic mechanism and drug defense mechanism.
Smart Images
Figure CN119120641A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine and cell engineering technology, and specifically relates to the construction of an epidermal organoid bacterial infection model, and the establishment of a drug screening platform for the model. Background Art
[0002] The skin is one of the largest organs in the mammalian body. Its major functions include protection against fluid and electrolyte loss as well as physical, chemical, and biological insults. Skin and soft-tissue infections (SSTIs) are a significant cause of morbidity and mortality in hospitalized patients and a major treatment challenge for clinicians.
[0003] Early diagnosis, selection of appropriate antimicrobial drugs and timely surgical intervention are the keys to successful treatment. Therefore, it is urgent to construct a suitable high-throughput antimicrobial drug screening model. In the past few decades, 2D (two-dimensional, 2D) primary epidermal cells have been used as a valuable tool for antimicrobial drug screening. However, epidermal cells expanded in 2D form cannot summarize the in vivo cell composition and physiological structure of the human epidermis, and the antimicrobial drug screening results are far from those in vivo. Subsequently, the multi-layer epidermis reconstructed by the air-liquid culture method provided a 3D (three-dimensional, 3D) skin model for the prediction of skin infection drugs. However, the epidermis reconstructed by this model has a limited lifespan, a long modeling cycle and a low success rate, which limits its large-scale application in the direction of high-throughput drug screening. In addition, mouse and other mammalian modeling is a classic antimicrobial drug screening model, but the modeling cycle is long and the cost is high, which limits its accuracy and high throughput.
[0004] Organoids are small clusters of cells that grow in a three-dimensional environment in vitro. They are derived from stem cells and self-organize and differentiate into functional cell types, thus summarizing the structure and function of organs in vivo. The three-dimensional structure, heterogeneity and cell function of organoids are more physiologically relevant in modeling human diseases and predicting drug responses, so they can serve as a link between cell lines and in vivo models. In addition, organoids can be expanded in large quantities in vitro and can be frozen like ordinary cells. At the same time, organoids support gene editing operations, and the ECM culture method also provides a way to explore the effects of cell microenvironment on organoid growth in vitro. These unique advantages have made organoids widely used in academic research and industrial technology. By obtaining a small amount of organ tissue, a large number of organoids can be rapidly expanded in vitro. Currently, they can be used in drug development, disease modeling (genetic diseases, infectious diseases, cancer, viral microbial infections, etc.), developmental biology, regenerative medicine, toxicology and other research. In addition, a variety of organoid biobank platforms have been built at home and abroad for disease typing, which is conducive to the treatment of precision medicine.
[0005] Based on the characteristics of the organoid model, this paper uses epidermal organoids to construct a bacterial infection model for the first time and proposes a suitable drug screening method for this model. This model can be used for high-throughput screening of antibacterial drugs and development of drug targets, and can also be used for the study of disease pathogenesis and drug defense mechanisms, which has important social, economic and scientific significance. Summary of the invention
[0006] In view of this, the present invention aims to provide a drug antibacterial evaluation method based on an infected epidermal organoid model to obtain more accurate drug screening data, and provide a new strategy for the clinical treatment of infected patients.
[0007] In a first aspect of the present invention, a non-diagnostic and non-therapeutic drug screening method based on an organoid bacterial infection model is provided, comprising the steps of:
[0008] (I) releasing the 3D organoid from the matrix gel and performing a digestion process to obtain cells from the 3D organoid;
[0009] (II) counting the cells from the 3D organoid, taking a predetermined number of cells from the 3D organoid and mixing them with bacteria, and co-incubating them to obtain a first co-incubation mixture; wherein the mixed co-incubation includes an experimental group and a negative control group; wherein the experimental group is incubated in the presence of a test drug, and the negative control group is incubated in the absence of a test drug, and the other conditions are the same as those of the experimental group;
[0010] (III) Determine the cell viability A1 of the experimental group and the cell viability A0 of the negative control group, and compare them. If the cell viability A0 of the negative control group is significantly lower than the cell viability A1, it indicates that the drug to be tested is an antibacterial agent that has an inhibitory effect on the bacteria.
[0011] In another preferred embodiment, the significantly lower than refers to A0 / A1≤0.75, preferably A0 / A1≤0.5, more preferably A0 / A1≤0.25 or ≤1 / 5.
[0012] In some embodiments, the 3D organoid in step (I) is a freshly obtained 3D cultured organoid.
[0013] In another preferred embodiment, the 3D organoids are selected from the following group: human organs, mouse organs, dog organs, and monkey organs.
[0014] In another preferred embodiment, the 3D organoids are skin organoids, and the skin organoids include epithelial organoids.
[0015] In some embodiments, the bacterial infection solution in step (II) is a solution obtained by diluting bacteria with organoid culture medium, and the bacteria are selected from the following group: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa; the bacteria can be antibacterial or non-antibacterial.
[0016] In some embodiments, releasing the 3D organoids from the matrix gel in step (I) refers to destroying the matrix gel components by enzymatic hydrolysis without destroying the 3D structure of the organoids, thereby obtaining an organoid suspension suspended in the culture medium.
[0017] In another preferred embodiment, the enzymatic hydrolysis uses dispase at a concentration of 0.5-2 U / mL, and the digestion conditions are 35-40° C. for 0.5-2 h. Preferably, the dispase is Tryple.
[0018] In some embodiments, in step (II), the mixed co-incubation further includes a positive control group; the positive control group is incubated in the presence of a known drug, and the other conditions are the same as those of the experimental group and the negative control group.
[0019] In a second aspect of the present invention, a method for constructing a non-diagnostic and non-therapeutic organoid bacterial infection model is provided, comprising the steps of:
[0020] (I) releasing the 3D organoid from the matrix gel and performing a digestion process to obtain cells from the 3D organoid;
[0021] (II) Counting the cells from the 3D organoids, taking a predetermined number of cells from the 3D organoids and mixing them with bacteria, and co-incubating them to obtain a first co-incubation mixture; wherein the mixed co-incubation includes an experimental group and a negative control group; wherein the experimental group is incubated in the presence of a test drug, and the negative control group is incubated in the absence of a test drug, and the other conditions are the same as those of the experimental group.
[0022] In some embodiments, the 3D organoids in step (I) are freshly obtained 3D cultured organoids.
[0023] In another preferred embodiment, the 3D organoids are selected from the following group: human organs, mouse organs, dog organs, and monkey organs.
[0024] In another preferred embodiment, the 3D organoids are skin organoids, and the skin organoids include epithelial organoids.
[0025] In some embodiments, the bacterial infection solution in step (II) is a solution obtained by diluting bacteria with organoid culture medium, and the bacteria are selected from the following group: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa; the bacteria can be antibacterial or non-antibacterial.
[0026] In some embodiments, releasing the 3D organoids from the matrix gel refers to destroying the matrix gel components by enzymatic hydrolysis without destroying the 3D structure of the organoids, thereby obtaining an organoid suspension suspended in the culture medium.
[0027] In another preferred embodiment, the enzymatic hydrolysis uses dispase at a concentration of 0.5-2 U / mL, and the digestion conditions are 35-40° C. for 0.5-2 h. Preferably, the dispase is Tryple.
[0028] In some embodiments, in step (II), the mixed co-incubation further includes a positive control group; the positive control group is incubated in the presence of a known drug, and the other conditions are the same as those of the experimental group and the negative control group.
[0029] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 Bright field photograph of isolated and cultured mouse epidermal organoids.
[0031] Figure 2 This is a transmission electron micrograph of the mouse epidermal organoid structure. (Note: basal cell (SB); spinous layer (SP); granular layer (GR); keratin granules (KG); stratum corneum (SC)).
[0032] Figure 3 The statistical results of the number of methicillin-resistant Staphylococcus aureus USA300 colonized in mouse epidermal organoids at different time points; the number of colonization gradually increased within 24 hours.
[0033] Figure 4 The results of flow cytometry detection of the number of dead cells (7-AAD+) in mouse epidermal organoids at different time points after infection with USA300.
[0034] Figure 5 These are the results of detecting cell viability in the mouse epidermal organoid infection model after treatment with methicillin and vancomycin.
[0035] Figure 6 The results of cell viability detection in the mouse epidermal organoid infection model after the infected mouse epidermal organoids were treated with the "Matrigel re-embedding" method and treated with methicillin and vancomycin.
[0036] Figure 7 Schematic diagram of the drug screening method of the present invention. DETAILED DESCRIPTION
[0037] After extensive and in-depth research, the inventor unexpectedly discovered a drug screening method based on an organoid bacterial infection model, on which the present invention was completed.
[0038] the term
[0039] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and molecular biology, cell and tissue culture, microbiology related terms and laboratory procedures used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.
[0040] The term "3D organoids" used in this article refers to small clusters of cells grown in a 3D environment in vitro. They are derived from stem cells and self-organize and differentiate into functional cell types, which can recapitulate the structure and function of organs in vivo.
[0041] The term "MOI" used herein is an abbreviation of multiplicity of infection, which is the ratio of the number of bacterial particles present in a confined space to the number of target cells.
[0042] The term “co-incubation” as used herein refers to the co-culture of organoids with bacteria at a fixed MOI in the absence of Matrigel encapsulation.
[0043] The term "tissue" refers to biopsy material or explant taken from a patient or animal or tissue produced in vitro.
[0044] The term "culture medium" refers to a growth-supporting liquid of nutrients and substances used to culture cells. Examples of suitable culture media include DMEM, Ham's, and RPMI.
[0045] In one aspect, the present invention provides a method for constructing an epidermal organoid infection model. In one embodiment, taking mouse epidermal organoids as an example, the colonization of methicillin-resistant Staphylococcus aureus USA300 in mouse epidermal organoids peaked at 24 hours, and the reduction in cell viability reached a maximum at 24 hours.
[0046] In one aspect, the present invention provides 3D organoids obtained by the aforementioned method for constructing an organoid viral infection model.
[0047] In one aspect, the present invention provides a method for constructing the aforementioned organoid infection model or the use of 3D organoids in infection research.
[0048] In one aspect, the present invention provides a method for identifying an organoid infection model. In an experimental case, taking mouse epidermal organoids infected with USA300 as an example, the organoid lysate after 2, 4, 8, and 24 hours of infection was plated on LB agar plates to quantify the number of bacterial colonization; in an experimental case, the organoid single cell lysate was analyzed by flow cytometry to quantify the proportion of dead cells in the organoid.
[0049] In one aspect, the present invention provides a method for drug sensitivity screening of infected organoids. In an experimental case, using primary mouse skin organoids infected with USA300 as an example, vancomycin concentration-dependently reversed the reduction of organoid cell viability caused by infection and inhibited the amount of USA300 internalized in the organoid.
[0050] In one aspect, the present invention provides a method for drug sensitivity screening of the aforementioned infection organoid model.
[0051] In one aspect, the present invention provides a method for detecting the number of internalized bacteria in the aforementioned infection organoid model after drug treatment.
[0052] The present invention is further described below by specific embodiments, but the present invention is not limited to the following examples. In addition, the reagents and materials used in the following examples are also all commercially available, and if not clearly stated, the methods and conditions used are also all processed according to known methods and conditions.
[0053] Example 1
[0054] This embodiment provides a construction of a methicillin-resistant Staphylococcus aureus USA300 infection model based on mouse epidermal organoids, comprising the following steps:
[0055] 1. Establishment of mouse epidermal organoid model:
[0056] 1) Sample cleaning: After soaking the mouse skin tissue in 75% alcohol for 3 minutes, wash the skin several times with PBS containing 1% double antibody to remove obvious fat tissue.
[0057] 2) Sample digestion and shearing: ① Place the tissue in Advanced DMEM / F12 diluted to 2.5U / mL dispase, digest at 37℃ for 1h, and peel off the epidermal tissue with tweezers; ② Cut the epidermal tissue into about 0.5mm 3 Add 0.25% trypsin to small pieces, digest at 37℃ for 20-30min, add medium containing 10% FBS to stop digestion, let it stand and precipitate, and collect the supernatant.
[0058] 3) Filtration: The supernatant collected in step 2) was filtered through a 70 μm filter, the filtrate was collected, the cell density was counted, and the cell pellet was collected by centrifugation at 300×g for 10 min at room temperature.
[0059] 4) Gel preparation: Dilute Matrigel with Advanced DMEM / F12 at a ratio of 1:1, resuspend the cell pellet at a density of 2500 cells / 10 μL, and drop each drop into a six-well culture plate preheated in an incubator, about 40 μL, and place in an incubator at 37°C with 5% CO2 for 30 minutes to allow the gel to solidify.
[0060] 5) After sufficient solidification, add mouse epidermal organoid culture medium, the composition of the culture medium is: Glutamax, 1×; HEPES, 10mM; B27 without Vitamin-A, 1×; BSA, 0.1%; N-acetylcysteine, 1mM; Forskolin, 10μM; EGF, 50ng / mL; Wnt3a, 100ng / mL; Y-27632, 10μM; Penicillin-streptomycin mixture, 1×. The above components are dissolved in Advanced DMEM / F12 culture medium and cultured at 37°C and 5% CO2 for 7 days. From the second day, A-83-01, 1μM, is added to the culture medium components, and the culture medium is replaced every other day. The morphological structure of the organoid under an optical microscope is as follows: Figure 1 As shown, a 200-300μm structure with a cornified inner core is generated. Transmission electron microscopy results show that the organoids have a multi-layered phenotype. Including the basal layer (Stratum basale, SB): a layer of short columnar basal cells; spinous layer (Stratum spinosum, SP): 4 to 10 layers of polygonal, larger spinous cells, and multiple oval, membrane-bounded lamellar granules can also be seen; granular layer (Stratum granulosum, GR): 3-5 layers of flat spindle-shaped cells, with a large number of transparent keratin granules (keratohyalin granules, KG) in the cytoplasm; stratum corneum (Stratum corneum, SC): multiple layers of flat keratinocytes, the cells are completely cornified, without nuclei and cytoplasm. The basal layer (BC) is located on the outside of the organoid; while the spinous layer (SP), granular layer (GR) and stratum corneum (SC) are located in the center of the organoid. (Results see Figure 2 )
[0061] 2. Establishment of mouse epidermal organoid USA300 infection model:
[0062] 1) Remove the culture medium from the epidermal organoids and gently rinse once with PBS. Dilute dispase to 1U / mL with Advanced DMEM / F12 medium. Add 1.5mL of 1U / mL dispase to each well and use a Pasteur pipette to blow away the solidified Matrigel structure. Place in the incubator for 1 hour to digest the Matrigel and release the organoids.
[0063] 2) Collect all released organoids, mix thoroughly with a Pasteur pipette, take out 1 mL of organoid suspension, centrifuge at 300×g for 5 min, discard the supernatant, add 2 mL of Tryple and digest at 37°C for 15 min, pipette up and down with a 1 mL pipette for dozens of times to digest the organoids into single cells, and count the number of cells.
[0064] 3) Prepare a standard growth curve of drug-resistant Staphylococcus aureus USA300 to determine the CFU value corresponding to the OD value. USA300 in the logarithmic growth phase was added to the liquid culture medium at a ratio of 1:100. The culture was shaken at 37°C and 220 rpm / min. 1 mL of bacterial solution was taken every 1 hour, and the CFU was counted on an agar plate and the OD value at the emission wavelength of 600 nm was measured at the same time.
[0065] 4) Select OD between 0.35-0.41, which corresponds to USA300 of 1.2×10 7 CFU / mL.
[0066] 5) Resuspend 1×10 6 USA300 bacteria, and resuspend 1 × 10 in 500 μL of culture medium. 5 Organoids, the two components were evenly mixed and transferred to a 24-well plate, and cultured in a 37°C, 5% CO2 incubator to complete the infection process and obtain USA300-infected epidermal organoid cultures.
[0067] Example 2
[0068] This embodiment provides a method for detecting the number of bacteria internalized and cell viability in epidermal organoids infected with USA300, comprising the following steps:
[0069] 1. Quantitative determination of the number of bacteria internalized in USA300-infected epidermal organoids.
[0070] 1) Cultivate the mouse epidermal organoids infected with USA300 obtained in Example 1, and remove the culture plates after infection is completed (0 h), culturing for 2 h, 4 h, 8 h, and 24 h, respectively.
[0071] 2) Let stand for 3 minutes to allow the organoids to settle to the bottom of the well. Discard 800 μL of the supernatant and be gentle to avoid sucking away the organoids.
[0072] 3) Add 800 μL PBS and repeat step 2).
[0073] 4) Fill the volume to 1 mL with epidermal organoid culture medium, add gentamicin to a final concentration of 500 μg / mL, and incubate in an incubator for 1 h to kill extracellular bacteria.
[0074] 5) Repeat steps 2) and 3).
[0075] 6) Add 2 mL of Tryple, incubate at 37°C for 15 min, and pipette up and down several dozen times with a 1 mL pipette tip to lyse the organoids.
[0076] 7) Collect the lysate, centrifuge at 3000×g for 5 min, discard the supernatant, and evenly suspend the precipitate with 500 μL PBS.
[0077] 8) Take 10 μL of the dilution and spread it on LB agar plate, place it in a 37°C, 5% CO2 incubator overnight, and count the CFU value. (Results are shown in Figure 3 )
[0078] 2. Determination of cell viability of epidermal organoids infected with USA300.
[0079] 1) Cultivate the mouse epidermal organoids infected with USA300 obtained in Example 1, and remove the culture plates after infection is completed (0 h), culturing for 2 h, 4 h, 8 h, and 24 h, respectively.
[0080] 2) Collect organoids from five infection time points, centrifuge at 300×g for 5 min, and discard the supernatant.
[0081] 3) Resuspend the organoid pellet with 2 mL of Tryple and incubate at 37°C for 15 min. Use a 1 mL pipette to pipette up and down a dozen times until the organoids are digested into single cells.
[0082] 4) Add culture medium containing 10% FBS to stop digestion and centrifuge at 300×g for 10 min.
[0083] 5) Discard the supernatant and resuspend in 100 μL PBS. Add 5 μL 7-ADD dye and incubate at room temperature in the dark for 15 min.
[0084] 6) Sieve with a 40μm mesh and run the flow cytometer to detect the percentage of 7-ADD positive cells (i.e. dead cells). (Results are shown in Figure 4 )
[0085] Example 3
[0086] This embodiment provides a drug screening method based on the epidermal organoid USA300 infection model, comprising the following steps:
[0087] 1) Remove the culture medium from the epidermal organoids and gently rinse once with PBS. Dilute dispase to 1U / mL with Advanced DMEM / F12 medium. Add 1.5mL of 1U / mL dispase to each well and use a Pasteur pipette to blow away the solidified Matrigel structure. Place in the incubator for 1 hour to digest the Matrigel and release the organoids.
[0088] 2) Collect all released organoids, mix thoroughly with a Pasteur pipette, take out 1 mL of organoid suspension, centrifuge at 300×g for 5 min, discard the supernatant, add 2 mL of Tryple and digest at 37°C for 15 min, pipette up and down with a 1 mL pipette for dozens of times to digest the organoids into single cells, and count the number of cells.
[0089] 3) Select OD between 0.35-0.41, which corresponds to USA300 of 1.2×10 7 CFU / mL.
[0090] 4) Use epidermal organoid culture medium to adjust the density of mouse epidermal organoids to 2×10 5 cells / mL, and USA300 was adjusted to 2.5×10 6 CFU / mL.
[0091] 5) Dilute the drug to be tested to ten times the specified concentration using epidermal organoid culture medium. If the DMSO content exceeds 0.1%, prepare a DMSO control at the same time.
[0092] 6) In a 96-well plate, the experimental grouping is set as follows:
[0093] (i) Normal mouse epidermal organoid control group: containing 50 μL organoid suspension + 50 μL culture medium;
[0094] (ii) Infection of mouse epidermal organoids experimental group: 50 μL organoid suspension + 40 μL USA300 suspension + 10 μL culture medium; 100 μL system contains 1×10 4 Mouse epidermal organoids and 1×10 5 USA300, MOI=10;
[0095] (iii) DMSO blank control group: containing 50 μL organoid suspension + 40 μL USA300 suspension + 10 μL DMSO diluent;
[0096] (iv) Infected mouse epidermal organoid administration group: containing 50 μL organoid suspension + 40 μL USA300 suspension + 10 μL test drug dilution.
[0097] Incubate in a 37°C, 5% CO2 incubator for 24 h.
[0098] Let the plate stand for 3 minutes, carefully discard 80 μL of the culture medium supernatant in each well with a pipette to avoid sucking away the organoids, and add Advanced DMEM / F12 to a final volume of 100 μL / well.
[0099] Add 100 μL CellTiter-Lumi TM The luminescent cell viability detection reagent was incubated at room temperature with shaking and in the dark for 30 minutes, and then allowed to stand for 30 minutes to allow the luminescent signal to stabilize.
[0100] A multifunctional microplate reader with chemiluminescence detection function was used for chemiluminescence detection, and the relative viability of cells was directly calculated based on the chemiluminescence readings. (Results are shown in Figure 5 )
[0101] Example 4
[0102] This example provides a method for evaluating the degree of bacterial internalization after drug treatment in an epidermal organoid infection model, comprising the following steps:
[0103] 1) Remove the culture medium from the epidermal organoids and gently rinse once with PBS. Dilute dispase to 1U / mL with Advanced DMEM / F12 medium. Add 1.5mL of 1U / mL dispase to each well and use a Pasteur pipette to blow away the solidified Matrigel structure. Place in the incubator for 1 hour to digest the Matrigel and release the organoids.
[0104] 2) Collect all released organoids, mix thoroughly with a Pasteur pipette, take out 1 mL of organoid suspension, centrifuge at 300×g for 5 min, discard the supernatant, add 2 mL of Tryple and digest at 37°C for 15 min, pipette up and down with a 1 mL pipette for dozens of times to digest the organoids into single cells, and count the number of cells.
[0105] 3) Select OD between 0.35-0.41, which corresponds to USA300 of 1.2×10 7 CFU / mL.
[0106] 4) Use epidermal organoid culture medium to adjust the density of mouse epidermal organoids to 2×10 5 / mL, and adjust USA300 to 2.5×10 6 CFU / mL.
[0107] 5) Dilute the drug to be tested to ten times the specified concentration using epidermal organoid culture medium. If the DMSO content exceeds 0.1%, prepare a DMSO control at the same time.
[0108] 6) In a 24-well plate, the experimental groups are set as follows:
[0109] (i) Infected mouse epidermal organoid control group: 500 μL organoid suspension + 400 μL USA300 suspension + 100 μL culture medium; 1 mL of the system contains 1×10 5 Mouse epidermal organoids and 1×10 6 USA300, MOI=10
[0110] (ii) DMSO blank control group: containing 500 μL organoid suspension + 400 μL USA300 suspension + 100 μL DMSO diluent;
[0111] (iii) Infected mouse epidermal organoid administration group: containing 500 μL organoid suspension + 400 μL USA300 suspension + 100 μL test drug dilution.
[0112] 7) Incubate in a 37°C, 5% CO2 incubator for 24 h.
[0113] 8) Let the plate stand for 3 minutes, and carefully discard the supernatant of the culture medium in each well with a pipette to avoid sucking away the organoids. Add 1 mL of PBS containing 1% P / S to each well, let the plate stand for 3 minutes, and discard the supernatant.
[0114] 9) Add 1 mL of PFA to each well and incubate at room temperature for 1 h.
[0115] 10) Repeat step 8).
[0116] 11) Add 1 mL of OWB (organoid washing buffer, PBS containing 0.1% Triton X-100 and 0.2% BSA) to each well and incubate at 4°C for 15 min.
[0117] 12) After the plate was left to stand for 3 minutes, the supernatant was discarded, and 200 μL of Anti-Staphylococcus aureus antibody diluted in OWB was added and incubated at 4°C overnight.
[0118] 13) Add 1 mL of OWB, shake gently for 10 minutes, let the plate stand for 3 minutes, and carefully discard the supernatant of the culture medium in each well with a pipette to avoid sucking away the organoids. Repeat 3 times and discard the supernatant.
[0119] 14) Add FITC conjugated Goat Anti-Rabbit IgG antibody diluted in 200 μL OWB and incubate at room temperature in the dark for 1 hour.
[0120] 15) Repeat step 13)
[0121] 16) Add 200 μL 1× DAPI staining solution and incubate at room temperature in the dark for 1 h.
[0122] 17) Add 1 mL of OWB, shake gently for 10 minutes, let the plate stand for 3 minutes, and carefully discard the supernatant of the culture medium in each well with a pipette to avoid sucking away the organoids.
[0123] 18) The mouse epidermal organoids were scanned by laser confocal microscopy at 5 μm / layer in the Z-axis, and the maximum fluorescence signal of each layer was superimposed to evaluate the internalization degree of USA300 after drug treatment.
[0124] Comparative Example 1
[0125] This comparative example provides another drug screening method for the epidermal organoid USA300 infection model, which is based on the drug sensitivity screening method of the common tumor organoids and some infected organoids "Matrigel re-embedding", including the following steps:
[0126] 1) Obtain the organoids infected for 24 hours in Example 1, wash them twice with Advanced DMEM / F12, resuspend the organoids with 5% Matrigel, and evenly add 20 μL to each well of a 96-well plate, containing 1×10 4 Organoids were placed in the incubator for 30 minutes to allow them to solidify. At the same time, uninfected normal organoids were set up as controls at the beginning of infection.
[0127] 2) Add 100 μL of culture medium / DMSO dilution / test drug dilution to each group and incubate in a 37°C, 5% CO2 incubator for 24 h.
[0128] 3) Add 100 μL CellTiter-Lumi TM The luminescent cell viability detection reagent was incubated at room temperature with shaking and in the dark for 30 minutes, and then allowed to stand for 30 minutes to allow the luminescent signal to stabilize.
[0129] 4) Use a multifunctional microplate reader with chemiluminescence detection function to perform chemiluminescence detection, and directly calculate the relative viability of the cells based on the chemiluminescence readings. (Results are shown in Figure 6 )
[0130] Compared with implementation case 3 (result 5), there was no statistical difference between the drug treatment group and the infection group in result 6. This indicates that the "Matrigel re-embedding" operation may significantly reduce the viability of epidermal organoids after infection, suggesting that this drug sensitivity method may not be suitable for bacterial infection types that have a greater impact on cell viability.
[0131] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.
Claims
1. A non-diagnostic and non-therapeutic drug screening method based on an organoid bacterial infection model, comprising the steps of: (I) releasing the 3D organoid from the matrix gel and performing a digestion process to obtain cells from the 3D organoid; (II) counting the cells from the 3D organoid, taking a predetermined number of cells from the 3D organoid and mixing them with bacteria, and co-incubating them to obtain a first co-incubation mixture; wherein, The mixed co-incubation includes an experimental group and a negative control group; wherein the experimental group is incubated in the presence of the drug to be tested, while the negative control group is incubated in the absence of the drug to be tested, and the other conditions are the same as those of the experimental group; (III) Determine the cell viability A1 of the experimental group and the cell viability A0 of the negative control group, and compare them. If the cell viability A0 of the negative control group is significantly lower than the cell viability A1, it indicates that the drug to be tested is an antibacterial agent that has an inhibitory effect on the bacteria. In another preferred embodiment, the significantly lower than refers to A0 / A1≤0.75, preferably A0 / A1≤0.5, more preferably A0 / A1≤0.25 or ≤1 / 5.
2. The method according to claim 1, characterized in that The 3D organoids described in step (I) are freshly obtained 3D cultured organoids. In another preferred embodiment, the 3D organoids are selected from the following group: human organs, mouse organs, dog organs, and monkey organs. In another preferred embodiment, the 3D organoids are skin organoids, and the skin organoids include epithelial organoids.
3. The method according to claim 1, characterized in that The bacterial infection solution in step (II) is a solution obtained by diluting bacteria with organoid culture medium, and the bacteria are selected from the following group: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa; the bacteria can be antibacterial or non-antibacterial.
4. The method according to claim 1, characterized in that The releasing of the 3D organoids from the matrix gel in step (I) refers to destroying the matrix gel components by enzymatic hydrolysis without destroying the 3D structure of the organoids, thereby obtaining an organoid suspension suspended in the culture medium. In another preferred embodiment, the enzymatic hydrolysis uses dispase at a concentration of 0.5-2 U / mL, and the digestion conditions are 35-40° C. for 0.5-2 h. Preferably, the dispase is Tryple.
5. The method according to claim 1, characterized in that In step (II), the mixed co-incubation also includes a positive control group; the positive control group is incubated in the presence of a known drug, and the other conditions are the same as those of the experimental group and the negative control group.
6. A method for constructing a non-diagnostic and non-therapeutic organoid bacterial infection model, comprising the steps of: (I) releasing the 3D organoid from the matrix gel and performing a digestion process to obtain cells from the 3D organoid; (II) counting the cells from the 3D organoid, taking a predetermined number of cells from the 3D organoid and mixing them with bacteria, and co-incubating them to obtain a first co-incubation mixture; wherein, The mixed co-incubation includes an experimental group and a negative control group; wherein the experimental group is incubated in the presence of the drug to be tested, while the negative control group is incubated in the absence of the drug to be tested, and the other conditions are the same as those of the experimental group.
7. The method according to claim 6, characterized in that The 3D organoids described in step (I) are freshly obtained 3D cultured organoids. In another preferred embodiment, the 3D organoids are selected from the following group: human organs, mouse organs, dog organs, and monkey organs. In another preferred embodiment, the 3D organoids are skin organoids, and the skin organoids include epithelial organoids.
8. The method according to claim 6, characterized in that The bacterial infection solution in step (II) is a solution obtained by diluting bacteria with organoid culture medium, and the bacteria are selected from the following group: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa; the bacteria can be antibacterial or non-antibacterial.
9. The method according to claim 6, characterized in that The releasing of 3D organoids from matrix gel refers to destroying the matrix gel components by enzymatic hydrolysis without destroying the 3D structure of the organoids, thereby obtaining an organoid suspension suspended in the culture medium. In another preferred embodiment, the enzymatic hydrolysis uses dispase at a concentration of 0.5-2 U / mL, and the digestion conditions are 35-40° C. for 0.5-2 h. Preferably, the dispase is Tryple.
10. The method according to claim 6, characterized in that In step (II), the mixed co-incubation also includes a positive control group; the positive control group is incubated in the presence of a known drug, and the other conditions are the same as those of the experimental group and the negative control group.