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Construction method and application of organoid virus infection model

A construction method and virus infection technology, applied in the fields of biomedicine and cell engineering, can solve the problems of long cycle, unfavorable management, pathogenic microorganism contamination, etc.

Pending Publication Date: 2020-07-28
张高华奥生命科学(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are individual differences between animal models, and the experimental results have large errors. Therefore, batches of animals are required for experiments, which seriously damages animal welfare; it will also cause contamination of pathogenic microorganisms among animals, which is not conducive to management; the success rate of model construction is low and the cycle is long; Moreover, the number of models is small and there is no possibility of high-throughput screening; the most important thing is that the expression of the receptor Ace2 gene of the new coronavirus is different among species, which limits the applicability of the experimental results of model animals

Method used

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  • Construction method and application of organoid virus infection model
  • Construction method and application of organoid virus infection model
  • Construction method and application of organoid virus infection model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Example 1: Isolation and culture of human lung organoids

[0110] 1. Take the lung tissue resected by lung surgery and place it in the pre-cooled basal medium at 4°C. The basal medium components are: Advanced DMEM / F12 medium, penicillin, GlutaMax and HEPES to maintain the tissue cells. active.

[0111] 2. Cut the lung tissue obtained in step 1 into 0.5mm with surgical scissors 3 and transferred to a 15mL centrifuge tube, adding 10mL of washing medium (composition: DMEM medium, 1% fetal bovine serum, 1% penicillin) and repeatedly pipetting to obtain small pieces of lung tissue.

[0112] 3. Let the small pieces of lung tissue obtained in step 2 settle down, and remove the supernatant. Then add 10 mL of washing medium, mix by pipetting, and obtain small pieces of lung tissue.

[0113] 4. Remove the excess washing medium as much as possible from the small pieces of lung tissue obtained in step 3, and add 37°C preheated digestion medium, the composition of which is isolat...

Embodiment 2

[0132] Example 2: Isolation and culture of human liver organoids

[0133] 1. Take the liver tissue resected by liver surgery and place it in the pre-cooled basal medium at 4°C. The basal medium components are: Advanced DMEM / F12 medium, penicillin, GlutaMax and HEPES to maintain the tissue cells. active. , to obtain small pieces of liver tissue

[0134] 2. Cut the liver tissue obtained in step 1 into 0.5mm with surgical scissors 3 and transferred to a 15mL centrifuge tube, adding 10mL washing medium (composition: DMEM medium, 1% fetal bovine serum, 1% penicillin) and blowing repeatedly.

[0135] 3. Let the small pieces of lung tissue obtained in step 2 settle down, and remove the supernatant. Then add 10 mL of washing medium, mix by pipetting, and obtain small pieces of liver tissue.

[0136] 4. Remove as much excess washing medium as possible from the small pieces of lung tissue obtained in step 3, and add 37°C preheated digestion medium, the composition of which is collag...

Embodiment 3

[0152] Example 3: Isolation and culture of human intestinal organoids

[0153] 1. Take the intestinal tissue resected by intestinal surgery and place it in the pre-cooled basal medium at 4°C. The basal medium components are: Advanced DMEM / F12 medium, penicillin, GlutaMax and HEPES to maintain the tissue cell activity.

[0154] 2. Use a scalpel to gently scrape off the fluff on the inner wall of the intestinal tissue obtained in step 1, and then use surgical scissors to cut it into 0.5cm 3 and transfer it to a 15mL centrifuge tube, add 10mL PBS, and pipette repeatedly to obtain small pieces of intestinal tissue.

[0155] 3. Set aside the small pieces of intestinal tissue obtained in step 2 to settle, and remove the supernatant. Add 10 mL of PBS solution containing 5 mM EDTA and let stand at 4° C. for 25 minutes for digestion.

[0156] 4. Transfer the intestinal tissue digested in step 3 to a new Petri dish containing 10 mL of PBS, repeatedly pipet the intestinal tissue about...

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Abstract

The invention discloses a construction method and application of an organoid virus infection model. The construction method of the organoid virus infection model comprises the following steps: (1) mixing a 3D cultured organoid with a virus infection solution, and then carrying out planarization co-incubation to complete an infection process; and (2) after infection is completed, enabling the organoid to be out of the planarization state, and returning to the 3D culture state again, so as to obtain the 3D organoid subjected to isolated culture. Establishment of the model can be used for high-throughput screening and drug research and development of antiviral drugs as well as research on viral disease pathogenesis and defense mechanisms of virus infection caused by natural occurrence or experiments, and a new strategy is provided for clinical virus of treatment infected patients.

Description

technical field [0001] The invention belongs to the technical fields of biomedicine and cell engineering, and specifically relates to a construction method and application of an organoid virus infection model. Background technique [0002] Coronaviruses are a broad family of viruses. Currently, six species (229E, NL63, OC43, HKU1, MERS-CoV, and SARS-CoV) are known to infect humans and cause respiratory diseases. SARS-CoV-2 is the first Seven disease-causing coronaviruses that can cause deadly respiratory disease and are highly contagious and pathogenic. [0003] In the traditional virus research process, cell lines such as African green monkey kidney cells (Vero, AGMK, BGM) and human embryonic lung fibroblasts are used to inoculate the virus, the virus is isolated after amplification and culture, and then inoculated into animal models such as mice , rats, guinea pigs and hamsters for virological research, animal models are widely used in the study of the pathogenesis of vir...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N7/00C12Q1/70C12Q1/02C12R1/93
CPCC12N5/0688C12N5/0671C12N5/0679C12N7/00C12Q1/025G01N33/5014G01N33/5088C12N2513/00C12N2770/20021C12N2503/02G01N2500/10
Inventor 张启生赵冰方敏
Owner 张高华奥生命科学(上海)有限公司
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