A lung-brain SARS-CoV-2 infection model and its application

Abstract

The present invention discloses a lung-brain SARS-CoV-2 infection model and application, which belongs to the technical field of organ chip models. The lung-brain chip SARS-CoV-2 infection model includes a lung chip and a BBB chip. The upper channel of the lung chip is inoculated with alveolar epithelial cells, and the lower channel is inoculated with pulmonary microvascular endothelial cells. The upper channel of the BBB chip is inoculated with brain microvascular endothelial cells, the back of the porous membrane of the lower channel is inoculated with astrocytes, and the bottom surface of the lower channel is inoculated with microglia. The upper channel of the lung chip is infected with the SARS-CoV-2 virus, and the infected culture medium is collected and mixed with fresh ECM culture medium, and then perfused into the upper channel of the BBB chip. The chip can explore the indirect effects of SARS-CoV-2 infection on the brain at the multi-organ level, and analyze whether the damage of SARS-CoV-2 is caused by the virus directly crossing the BBB into the brain, or by systemic inflammation caused by lung infection. In addition, the system can also monitor multiple cell states and functional disorders in real time after SARS-CoV-2 infection, as well as monitor the response of peripheral immune cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of organ chip models, and specifically relates to a lung-brain SARS-CoV-2 infection model and its application. Background Art

[0002] Novel coronavirus infection (COVID-19) is a systemic disease affecting multiple organs. Although COVID-19 symptoms are primarily concentrated in the respiratory system, clinical data show that 30-40% of COVID-19 patients experience significant neurological symptoms, such as headaches, confusion, loss of smell and taste, cerebrovascular damage, seizures, and encephalitis. To date, the medical community remains undetermined regarding the causes of these neurological symptoms associated with novel coronavirus infection. It remains unclear whether these symptoms are caused by direct invasion of the brain by SARS-CoV-2 or by systemic inflammation triggered by viral infection of the lungs.

[0003] The human brain has a highly selective barrier system, the blood-brain barrier (BBB), which regulates the transport of nutrients and metabolites between the blood and the nervous system. Furthermore, the BBB acts as a protective barrier, preventing toxins and pathogens (including viruses) from entering the brain parenchyma. In some infectious brain diseases, neurotropic viruses, such as HSV-1 and Zika virus, can infect and destroy the BBB and further invade the human brain through the bloodstream. However, whether SARS-CoV-2 can penetrate the brain through a similar pathway across the BBB remains unknown.

[0004] To date, research on brain damage caused by SARS-CoV-2 infection has primarily come from case studies of brain samples collected from deceased individuals infected with the novel coronavirus, animal models, and brain organoids. However, all three types of samples have varying degrees of limitations. For deceased brain samples, the source is very limited, and individual variability is significant. Animal models exhibit different symptoms and pathological changes after SARS-CoV-2 infection due to differences in physiology and genetic background. In recent years, stem cell-derived brain organoids have been increasingly used in neuroscience research, including studies of SARS-CoV-2 infection. However, currently, brain organoid models lack vascular networks, barrier systems, and immune cells, making them inadequate for investigating viral entry pathways into the brain and the immune response during viral infection. Furthermore, given that novel coronavirus infection is a systemic disease affecting multiple organs, no multi-organ humanized models have been developed to study SARS-CoV-2 infection.

[0005] Organ-on-a-chip—a rapidly developing science and technology, formed by the intersection of multidisciplinary approaches—has demonstrated unique advantages in the biomedical field. Based primarily on microfluidic chips, this technology integrates technologies from microfabrication, cell biology, materials, and tissue engineering to construct in vitro biomimetic 3D human organ models, encompassing a variety of living cells, functional tissue interfaces, and biofluids. These models exhibit physiological functions close to those of the human body while enabling precise control of multiple system parameters. This allows researchers to more intuitively study organ behavior and predict or replicate pathological and pharmacological responses in disease. These technologies hold broad application prospects in life science research, disease simulation, new drug development, and precision medicine.

[0006] In the existing technology, there is still a technical gap in the organ chip model for the pathological response of SARS-CoV-2 infection to multiple organs, which needs to be developed and studied by researchers. Summary of the Invention

[0007] This invention provides a new method based on organ-on-a-chip technology to explore the effects of SARS-CoV-2 infection on the brain at the multi-organ level. This invention innovatively constructs a tandem lung-brain organ-on-a-chip model system. Based on this organ-on-a-chip model system, it can simulate the pathological damage caused by SARS-CoV-2 infecting the lungs and subsequently affecting the brain. This system reflects the complex multi-organ pathological responses during SARS-CoV-2 infection and explores the interaction mechanisms between multiple organs during the disease process, filling a gap in this research field.

[0008] The present invention provides a lung-brain SARS-CoV-2 infection model, which is composed of two membrane chips connected in series; the membrane chip includes an upper substrate, a lower substrate and a porous membrane, the upper substrate includes an upper channel, the lower substrate includes a lower channel, and the porous membrane is located between the upper channel and the lower channel;

[0009] The two series-connected membrane chips are respectively a lung chip and a BBB chip;

[0010] The lung chip is inoculated with pulmonary microvascular endothelial cells on the porous membrane on the lower channel side of the sandwich chip, and alveolar epithelial cells on the porous membrane on the upper channel side of the sandwich chip; the culture medium of the upper channel of the lung chip is alveolar epithelial cell culture medium containing SARS-CoV-2, and the culture medium of the lower channel of the lung chip is endothelial cell culture medium containing human peripheral blood mononuclear cells;

[0011] The BBB chip is inoculated with astrocytes on the porous membrane on the lower channel side of the membrane chip, a microglia culture area is inoculated on the bottom surface of the lower channel of the membrane chip, and brain microvascular endothelial cells are inoculated on the porous membrane on the upper channel side of the membrane chip; the culture medium of the upper channel of the BBB chip is the lung chip endothelial culture medium infected with SARS-CoV-2 virus obtained from the lower channel of the lung chip mixed with fresh endothelial culture medium according to a certain ratio.

[0012] Furthermore, in the above technical solution, the porous membrane includes a PET porous membrane and a PDMS porous membrane.

[0013] Furthermore, in the above technical solution, the pore size of the porous membrane is 2-4 μm.

[0014] Furthermore, in the above technical solution, the material of the upper substrate and the lower substrate includes PDMS.

[0015] Furthermore, in the above technical solution, in the alveolar epithelial cell culture medium containing SARS-CoV-2, the SARS-CoV-2 multiplicity of infection MOI = 0.1-10.

[0016] Preferably, the SARS-CoV-2 multiplicity of infection MOI=1.

[0017] Furthermore, in the above technical solution, the upper cell inlet and the upper cell outlet are respectively provided at both ends of the lower channel, and the lower cell inlet and the lower cell outlet are respectively provided at both ends of the lower channel; the lower cell outlet of the lung chip is connected with the upper cell inlet of the BBB chip; and the lung chip is upstream of the BBB chip.

[0018] Furthermore, in the above technical solution, the dimensions of the upper channel and the lower channel are: 1.5 mm wide and 0.2 mm high.

[0019] Furthermore, in the above technical solution, the culture medium of the lower channel of the BBB chip is a mixture of astrocyte culture medium and microglia culture medium in equal volumes.

[0020] The present invention also provides the lung-brain SARS-CoV-2 infection model as a model for exploring the effects of SARS-CoV-2 infection on the lungs and brain at the multi-organ level.

[0021] The present invention also provides the use of the lung-brain SARS-CoV-2 infection model for real-time monitoring of multiple cell states and dysfunctions after SARS-CoV-2 infection, as well as for monitoring the response of peripheral immune cells.

[0022] The present invention also provides a new method for evaluating brain damage caused by SARS-CoV-2 infection based on an organ chip. The specific process is as follows:

[0023] (1) Lung Chip Construction

[0024] Pulmonary microvascular endothelial cells were prepared into a cell suspension of 2,000 cells / μL. 25 μL of the cell suspension was injected into the lower channel of the chip, and the chip was placed upside down in a 37°C incubator and cultured for 2 hours.

[0025] Alveolar epithelial cells were prepared into a cell suspension of 4,000 cells / μL, 25 μL of the cell suspension was injected into the upper channel of the chip, and the chip was placed in a 37°C incubator for overnight culture.

[0026] The syringe pump was connected to the inlet of the upper and lower channels of the chip at a flow rate of 100 μL / h, and perfusion culture was carried out in a 37°C incubator for 3 days.

[0027] (2) BBB chip construction

[0028] Astrocytes were prepared into a cell suspension of 2,000 cells / μL. 25 μL of the cell suspension was injected into the lower channel of the chip. The chip was then placed upside down in a 37°C incubator and cultured for 2 hours.

[0029] Prepare a microglial cell suspension at 2,000 cells / μL. Aspirate any remaining astrocyte culture medium from the chip channels and inject 25 μL of the microglial cell suspension into the lower channel of the chip. Prepare a brain microvascular endothelial cell suspension at 4,000 cells / μL and inject 25 μL of the cell suspension into the upper channel of the chip. Incubate the chip in a 37°C incubator overnight.

[0030] The syringe pump was connected to the inlet of the upper and lower channels of the chip at a flow rate of 100 μL / h, and perfusion culture was carried out in a 37°C incubator for 3 days.

[0031] (3) Chip function testing

[0032] The integrity of the lung chip barrier was assessed by immunofluorescence staining for VE-cadherin and ZO-1, and alveolar epithelial cell specificity was assessed by expression of type II pneumocyte-specific markers (SPC and HTII-280).

[0033] The integrity of the BBB chip was assessed by immunofluorescence staining for VE-cadherin and ZO-1. FITC-dextran was used to monitor BBB chip permeability. A transmembrane resistance meter was used to measure the transmembrane resistance of the BBB chip.

[0034] (4) Lung Chip Infection with SARS-CoV-2

[0035] After three days of perfusion culture, the upper channel, seeded with alveolar epithelial cells, was inoculated with alveolar epithelial cell culture medium containing SARS-CoV-2 (at a multiplicity of infection (MOI) of 1). The lower channel was inoculated with a mixture of astrocyte culture medium (ScienCell, no. 1801) and microglia culture medium (Procell, no. MP150410) (1:1 volume ratio). After one hour, the upper channel was rinsed twice with fresh culture medium and then infused with 100 μL of fresh virus-free alveolar epithelial culture medium.

[0036] Aspirate the culture medium in the lower channel and add 100 μL of endothelial cell culture medium containing 100,000 human peripheral blood mononuclear cells to simulate immune cells circulating in the body.

[0037] Lay the chip flat and transfer it to a 37°C incubator. Collect the endothelial cell culture medium every two days and store it at -80°C for use as conditioned medium in subsequent experiments. Replace the culture medium with fresh medium and inject 100,000 human peripheral blood mononuclear cells into the lower channel. Continue incubating for six days.

[0038] (5) Infected lung chip endothelial culture medium to treat BBB chip

[0039] The collected virus-infected lung chip endothelial culture medium was mixed with fresh endothelial culture medium at a volume ratio of 1:2 and injected into the upper microvascular channel of the BBB chip. The cells were cultured at 37°C for 4 days, with the medium replaced every 2 days.

[0040] (6) After the BBB chip was perfused with infected lung chip endothelial culture medium, BBB damage detection was performed.

[0041] After perfusion of the BBB chip with infected lung chip endothelial culture medium, changes in BBB integrity can be assessed by permeation with FITC-labeled dextran. Changes in brain microvascular endothelial integrity can be assessed by immunofluorescence staining for extracellular endothelial junctions (e.g., ZO-1, Occludin, Claudin-5, and VE-cadherin). Astrocyte-specific markers (GFAP, S100β) and microglia-specific markers (IBA1, CD11b) can be used to assess the activation status of both glial cell types. Cytokine levels in the culture medium of the upper and lower channels can be measured using a cytokine array kit.

[0042] The present invention establishes a model method for exploring the effects of SARS-CoV-2 infection on brain damage. The model system consists of an upstream lung chip and a downstream BBB chip. SARS-CoV-2 is added to the alveolar epithelial channel of the lung chip, and peripheral blood mononuclear cells are added to the pulmonary microvascular endothelial channel to simulate the peripheral blood immune cells in the circulating blood in vivo, forming the lung injury pathological microenvironment of SARS-CoV-2 pneumonia. By collecting the culture medium of the pulmonary microvascular endothelial channel, mixing it with fresh endothelial culture medium, and injecting it into the brain microvascular endothelial channel of the BBB chip, a brain damage model caused by SARS-CoV-2 infection is constructed.

[0043] The SARS-CoV-2 infection brain injury model provided by the present invention not only simulates the connection between the lungs and the brain during viral infection and performs real-time detection of functional damage to the lung-brain barrier, but also can study the role of peripheral immune cells and glial cells in brain damage caused by SARS-CoV-2 infection.

[0044] The SARS-CoV-2 infection brain injury model provided by the present invention can use commonly used biological cell detection methods to detect BBB barrier damage, various cell states and inflammatory factor levels, including FITC-dextran permeability detection, cell viability detection, cell immunofluorescence staining, qRT-PCR, cytokine array detection, etc.

[0045] This invention utilizes organ-on-a-chip technology, utilizing biocompatible and light-transmitting PDMS and PET porous membranes to facilitate cell imaging and observation. The device is suitable for monitoring changes in barrier tissues (such as the alveolar barrier and the blood-brain barrier) following viral infection, as well as various cellular behaviors and changes, such as changes in mRNA, protein expression, cytokine secretion, and cell death. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the lung-brain SARS-CoV-2 infection model constructed by the lung chip and BBB chip in series of the present invention.

[0047] Figure 2 Schematic diagram of the structure and functional divisions of the microfluidic chip of the present invention.

[0048] Among them, A is a lung chip: 1. PET porous membrane, 2. alveolar epithelial cell culture area, 3. pulmonary microvascular endothelial cell culture area.

[0049] B is the BBB chip: 4, PET porous membrane, 5, brain microvascular endothelial cell culture area, 6, astrocyte culture area, 7, microglia culture area.

[0050] Figure 3This is the viral infection status of the alveolar chip under the action of viruses (side view of the lung chip);

[0051] Figure 4 Various cell changes in the BBB chip under the action of viruses. DETAILED DESCRIPTION

[0052] The present invention will be further described in the following examples, but are not intended to limit the present invention.

[0053] Example 1

[0054] The lung chip and BBB chip were constructed using the membrane chip.

[0055] like Figure 1-2 As shown, the sandwich chip is formed by gluing and sealing an upper substrate and a lower substrate, with a PET porous membrane provided between the upper and lower substrates. An upper channel is provided on the upper substrate, and a lower channel is provided on the lower substrate. An upper cell inlet and an upper cell outlet are provided on the upper channel, and a lower cell inlet and a lower cell outlet are provided on the lower channel. When the sandwich chip is assembled, the upper channel and the lower channel are in an "x" shape. The dimensions of the upper channel and the lower channel are both 1.5 mm wide and 0.2 mm high. The pore size of the PET porous membrane is 2 μm. The material of the upper and lower substrates is PDMS.

[0056] The lung chip is inoculated with pulmonary microvascular endothelial cells on the PET porous membrane on the lower channel side of the sandwich chip, and alveolar epithelial cells on the PET porous membrane on the upper channel side of the sandwich chip. Alveolar epithelial cell culture medium containing SARS-CoV-2 (SARS-CoV-2 multiplicity of infection MOI = 1) is added to the upper channel through the upper cell inlet, and endothelial cell culture medium containing human peripheral blood mononuclear cells is added to the lower channel through the lower cell inlet to simulate the immune cells circulating in the body.

[0057] The BBB chip is inoculated with astrocytes on the PET porous membrane on the lower channel side of the sandwich chip, a microglial cell culture area is inoculated on the bottom surface of the lower channel of the sandwich chip, and brain microvascular endothelial cells are inoculated on the PET porous membrane on the upper channel side of the sandwich chip.

[0058] The lung chip is upstream and the BBB chip is downstream.

[0059] The lung chip endothelial culture medium infected with SARS-CoV-2 virus obtained from the lower channel of the lung chip was mixed with fresh endothelial culture medium (ScienCell, no. 1001) in a volume ratio of 1:2 as conditioned medium and injected into the upper channel of the BBB chip; the culture medium of the lower channel was a mixed culture medium of astrocyte culture medium (ScienCell, no. 1801) and microglia culture medium (Procell, no. MP150410) (volume ratio of 1:1) to establish a lung-brain SARS-CoV-2 infection model.

[0060] Example 2

[0061] A new method based on organ-on-a-chip technology to explore the impact of SARS-CoV-2 infection on the brain at the multi-organ level. The specific process is as follows:

[0062] (1) Sterilization and coating of film-coated chips

[0063] Place the membrane chip in a 6 cm culture dish, open the culture dish and place it in a clean bench for ultraviolet irradiation overnight.

[0064] The upper and lower channels of the membrane chip were each filled with 50 μg / mL fibronectin solution and placed in a 37°C incubator for 24 hours. Before seeding cells, the fibronectin solution was aspirated and the cells were washed three times with PBS.

[0065] (2) Construction of lung chip:

[0066] A 2,000 cell / μL suspension of pulmonary microvascular endothelial cells was prepared. 25 μL of this suspension was injected into the lower channel of the chip, which was then inverted and placed in a 37°C incubator for 2 hours to allow the cells to adhere to the PET porous membrane on the lower channel. A 4,000 cell / μL suspension of alveolar epithelial cells was prepared. 25 μL of this suspension was injected into the upper channel of the chip, which was then placed in a 37°C incubator for overnight culture to allow the cells to adhere to the PET porous membrane on the upper channel. The upper channel of the Lung Chip was cultured with alveolar epithelial cell culture medium (90% RPMI 1640 basal medium + 10% fetal bovine serum + 1% penicillin / streptomycin); the lower channel was cultured with pulmonary microvascular endothelial cell culture medium (Procell, no. CM-0565). Connect a syringe pump to the upper and lower cell inlets of the membrane chip, and connect the upper and lower cell outlets to a collection device. Culture the cells at a flow rate of 100 μL / h and incubate in a 37°C incubator for 3 days.

[0067] (3) Construction of BBB chip:

[0068] A 2,000 cell / μL astrocyte suspension was prepared. 25 μL of this suspension was injected into the lower channel of the chip. The chip was then placed upside down in a 37°C incubator and cultured for 2 hours to allow the astrocytes to adhere to the PET porous membrane on the lower channel. A 2,000 cell / μL microglia suspension was prepared. Any remaining astrocyte culture medium in the lower channel of the chip was aspirated. 25 μL of the microglia suspension was injected into the lower channel of the chip, allowing the microglia to adhere to the bottom surface of the lower channel. A 4,000 cell / μL brain microvascular endothelial cell suspension was prepared. 25 μL of this suspension was injected into the upper channel of the chip. The chip was then incubated overnight at 37°C to allow the brain microvascular endothelial cells to adhere to the PET porous membrane on the upper channel. Connect a syringe pump to the upper and lower cell inlets of the membrane chip, and connect the upper and lower cell outlets to a collection device. Culture the cells at a flow rate of 100 μL / h and incubate in a 37°C incubator for 3 days.

[0069] (4) Chip function testing

[0070] The integrity of the lung chip barrier was assessed by immunofluorescence staining for VE-cadherin and ZO-1, and alveolar epithelial cell specificity was assessed by expression of type II pneumocyte-specific markers (SPC and HTII-280).

[0071] The integrity of the BBB chip was assessed by immunofluorescence staining for VE-cadherin and ZO-1. FITC-dextran was used to monitor BBB chip permeability. A transmembrane resistance meter was used to measure the transmembrane resistance of the BBB chip.

[0072] (5) Construction of lung-brain SARS-CoV-2 infection model:

[0073] After three days of perfusion culture, the upper channel seeded with alveolar epithelial cells was inoculated with alveolar epithelial cell culture medium containing SARS-CoV-2 (SARS-CoV-2 multiplicity of infection = 1). One hour later, the upper channel was rinsed twice with fresh culture medium and then infused with 100 μL of fresh virus-free alveolar epithelial culture medium. The culture medium in the lower channel was aspirated and then inoculated with 100 μL of endothelial cell culture medium containing 100,000 human peripheral blood mononuclear cells (hPBMCs) to simulate circulating immune cells in vivo. The chip was then placed flat in a 37°C incubator and incubated. Endothelial cell culture medium was collected every two days and stored at -80°C for use as conditioned medium in subsequent experiments.

[0074] Fresh culture medium was replaced and 100,000 human peripheral blood mononuclear cells were injected into the lower channel. Culture was continued for 6 days. During this period, immunofluorescence detection showed that SARS-CoV-2 mainly infects alveolar epithelial cells and replicates in them in large quantities. The results are as follows Figure 3 shown.

[0075] The collected virus-infected lung chip endothelial culture medium (conditioned medium) was mixed with fresh endothelial culture medium at a volume ratio of 1:2 and injected into the upper channel of the BBB chip seeded with endothelial cells. The culture medium of the lower channel was a mixed culture medium of astrocyte culture medium (ScienCell, no. 1801) and microglial culture medium (Procell, no. MP150410) (volume ratio 1:1). Culture was carried out at 37°C for 4 days, and the medium was changed every 2 days. On the fourth day, the three types of cells on the BBB chip were detected by immunofluorescence. It was found that the extracellular junctions of brain microvascular endothelial cells (ZO-1 indicates tight junctions, VE-cadherin indicates adherens junctions) were significantly weakened in the virus-infected group; astrocytes were significantly activated (GFAP and S100β were significantly upregulated); and microglia were significantly activated (IBA1 and CD11b were significantly upregulated), indicating that the neurological symptoms of patients infected with the new coronavirus are caused by systemic inflammation caused by viral infection of the lungs. The results are as follows Figure 4 shown.

Claims

1. A lung-brain SARS-CoV-2 infection model, characterized by: It consists of two membrane chips connected in series; the membrane chip includes an upper substrate, a lower substrate and a porous membrane, the upper substrate includes an upper channel, the lower substrate includes a lower channel, and the porous membrane is located between the upper channel and the lower channel; The two series-connected membrane chips are respectively a lung chip and a BBB chip; The lung chip is inoculated with pulmonary microvascular endothelial cells on the porous membrane on the lower channel side of the sandwich chip, and alveolar epithelial cells on the porous membrane on the upper channel side of the sandwich chip; the culture medium of the upper channel of the lung chip is alveolar epithelial cell culture medium containing SARS-CoV-2, and the culture medium of the lower channel of the lung chip is endothelial cell culture medium containing human peripheral blood mononuclear cells; The BBB chip is inoculated with astrocytes on the porous membrane on the lower channel side of the membrane chip, a microglial cell culture area is inoculated on the bottom surface of the lower channel of the membrane chip, and brain microvascular endothelial cells are inoculated on the porous membrane on the upper channel side of the membrane chip; the culture medium of the upper channel of the BBB chip is the lung chip endothelial culture medium infected with SARS-CoV-2 virus obtained from the lower channel of the lung chip mixed with fresh endothelial culture medium in proportion; The upper channel has an upper cell inlet and an upper cell outlet at both ends, and the lower channel has a lower cell inlet and a lower cell outlet at both ends; the lower cell outlet of the lung chip is connected to the upper cell inlet of the BBB chip; the lung chip is upstream of the BBB chip; The culture medium of the lower channel of the BBB chip is a mixture of astrocyte culture medium and microglia culture medium in equal volumes; In the alveolar epithelial cell culture medium containing SARS-CoV-2, the SARS-CoV-2 multiplicity of infection MOI is 0.1-10.

2. The lung-brain SARS-CoV-2 infection model according to claim 1, characterized in that: The porous membrane includes a PET porous membrane and a PDMS porous membrane.

3. The lung-brain SARS-CoV-2 infection model according to claim 1, characterized in that: The pore size of the porous membrane is 2-4 μm.

4. The lung-brain SARS-CoV-2 infection model according to claim 1, characterized in that: The materials of the upper substrate and the lower substrate include PDMS.

5. Use of the lung-brain SARS-CoV-2 infection model according to any one of claims 1 to 4, characterized in that: It is used as a model to explore the effects of SARS-CoV-2 infection on the lungs and brain at the multi-organ level.

6. Use of any one of claims 1 to 4 in the lung-brain SARS-CoV-2 infection model, characterized in that: Real-time monitoring of multiple cell states and dysfunctions after SARS-CoV-2 infection, as well as applications in monitoring the responses of peripheral immune cells.

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