Respiratory tract organoid culture system and method for constructing bocavirus infection model
A culture method and technology of the culture system, applied in the direction of virus, culture process, tissue culture, etc., can solve the problem of evaluating the infectivity of Boca virus
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Embodiment 1
[0093] Embodiment 1 human lung organoid culture
[0094] (1) Human tissue collection
[0095] Human lung tissue is the normal lung tissue removed from the hospital through bronchoscopy or surgery. For the resection of normal tissues next to proliferative lesions (including nodules, benign and malignant tumors, etc.), a distance of more than 3 cm should be kept from the proliferative lesions. Rinse normal bronchial mucosal tissue and alveolar tissue. Tissue was cut into pieces approximately 5 mm in diameter and further rinsed with cold PBS. For endoscopic biopsy specimens, at least 500 biopsy samples were collected.
[0096] (2) Tissue separation and cell dissociation
[0097] Tissue fragments and 2mmol / L EDTA cold chelation buffer (5.6mmol / L Na in deionized water 2 HPO 4 ,8.0 mmol / L KH 2 PO 4 , 96.2mmol / L NaCl, 1.6mmol / L KCl, 43.4mmol / L sucrose, 54.9mmol / L D-sorbitol, 0.5mmol / LDL-dithiothreitol) and incubated on ice for 30min. After removing the EDTA buffer, the tissu...
Embodiment 2
[0110] Embodiment 2 organoid infection model
[0111] For human lung organoids, clinical Boca virus-positive samples were used for infection, and real-time fluorescent quantitative PCR technology and Western blotting technology were used for virus infection identification.
[0112] Virus challenge experiment
[0113] Experiments related to Boca virus infection were all carried out in a biosafety level 2 laboratory (BSL-2). Positive samples of Boca virus were selected, and 2ml of prepared aliquoted and preserved samples were taken to incubate organoids for 2 hours. Shake the culture plate to facilitate virus adsorption to host cells. Then the virus liquid was discarded, the cells were washed 3 times with 1×PBS, and the respiratory organoid medium containing 2% FBS was added to continue the culture. Collect organoid culture supernatant and organoid lysate at different times.
[0114] Real-time quantitative PCR and whole genome sequence detection of bocavirus genome in organoi...
Embodiment 3
[0143] Example 3 Detection of Changes in Inflammatory Factors
[0144] After the infection of the organoid Boca virus infection model, the changes of inflammatory factors were detected to determine the inflammatory response of virus infection and the interaction with cells
[0145] Preparation of tissue culture supernatant:
[0146] Centrifuge samples to immediately remove debris and store at -20°C. Avoid multiple (>2) freeze / thaw cycles.
[0147] Tissue culture supernatants may need to be diluted, and tissue / cell extractions should be performed in neutral buffers containing reagents and conditions that do not affect assay performance. Detergents, salts, denaturant concentrations that are too high, pH values that are too high or too low, etc. can negatively affect the assay. Organic solvents should be avoided. Tissue / cell extraction samples should be free of particles such as cells or tissue debris.
[0148] (1) Add 200 μl of washing buffer to each well. Seal and stir ...
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