CN116144494A - Sepsis-related encephalopathy model and application thereof - Google Patents

Sepsis-related encephalopathy model and application thereof Download PDF

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CN116144494A
CN116144494A CN202211589390.1A CN202211589390A CN116144494A CN 116144494 A CN116144494 A CN 116144494A CN 202211589390 A CN202211589390 A CN 202211589390A CN 116144494 A CN116144494 A CN 116144494A
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sepsis
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porous membrane
membrane
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秦建华
朱丹丹
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Dalian Institute of Chemical Physics of CAS
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    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening

Abstract

The invention discloses a sepsis-related encephalopathy model and application thereof, and belongs to the technical field of organ chip models. The invention comprises a membrane-sandwiched chip, wherein the membrane-sandwiched chip comprises an upper substrate, a porous membrane and a lower substrate which are sequentially arranged from top to bottom, the upper substrate comprises an upper channel, and the lower substrate comprises a lower channel; the membrane-sandwiched chip is a BBB chip; the BBB chip is characterized in that astrocytes and pericytes are inoculated on one side of the porous membrane close to the lower-layer channel, and microglial cells are inoculated on the bottom surface of the lower-layer channel; inoculating human brain microvascular endothelial cells on one side of the porous membrane close to the upper layer channel; the culture medium of the upper layer channel is serum of a sepsis related encephalopathy patient. The invention not only detects the functional damage of the brain barrier in real time, but also detects and researches the effects of the endothelial cells, the pericytes and the glial cells of the brain microvasculature in vitro in real time.

Description

Sepsis-related encephalopathy model and application thereof
Technical Field
The invention belongs to the technical field of organ chip models, and particularly relates to a sepsis-related encephalopathy model and application thereof.
Background
Sepsis-associated encephalopathy (sepis-associated encephalopathy, SAE), also known as sepsis encephalopathy, is a diffuse brain dysfunction caused by sepsis due to infections in other parts than the brain. In recent years, the incidence of sepsis has increased. Studies have shown that over 50% of patients with sepsis have combined SAE and that mortality increases significantly once sepsis has combined SAE, mortality can increase to 70% with increasing SAE severity. Currently, SAE has become an important health threat, affecting public health of social development, and needs to be solved. SAE is multifactorial, multiple pathological mechanisms are involved in parallel, such as blood brain barrier destruction, astrocyte activation, microglial activation, etc., which can lead to neuronal damage and thus cognitive dysfunction. However, the pathophysiology and underlying molecular mechanisms are still not completely understood so far.
At present, great difficulty still exists in developing SAE pathogenesis research, and the outstanding difficulty is in building an SAE model. At present, a mouse model and a 2D cell culture model are most commonly used. However, the mouse model has a certain difference from the human body in terms of genetic background, physiological structure and the like; the SAE pathogenesis is a series of pathophysiological processes caused by peripheral inflammatory reaction entering brain parenchyma through broken blood brain barrier, and the 2D cell culture model lacks organ pathophysiological micro-system, so a humanized bionic model with the pathophysiological micro-system needs to be established. The body organ chip (organs-on-chips) is an organ physiological micro-system constructed on the chip, and can simulate the near-physiological tissue cell microenvironment in vitro by realizing three-dimensional cell culture and accurate fluid control in a micro-channel, so that the defect that the traditional 2D cell culture and animal model are difficult to embody the complex physiological functions of human tissues and organs is overcome to a certain extent.
Organ chips, which are a rapidly developing scientific technology, are formed by intersecting and converging multidisciplinary technologies, and have demonstrated their unique advantages in the biomedical field. The technology is mainly based on micro-fluidic chips, integrates micro-processing, cell biology, materials, biological tissue engineering and other discipline technologies, constructs a bionic 3D human organ model in vitro, and comprises a plurality of living cells, functional tissue interfaces, biological fluids and the like. The model has physiological functions approaching the human level, while also being able to accurately control a plurality of system parameters. Researchers can more intuitively study the behaviors of the organism, predict or reproduce pathological reactions and pharmacological reactions in diseases. Has wide application prospect in the fields of life science research, disease simulation, new medicine research and development, accurate medical treatment and the like.
The microfluidic chip has several advantages as a cell culture carrier: firstly, different channel sizes can be designed according to practical application by means of a microfluidic chip technology, a certain space limitation, namely physical factor control is provided, and the three-dimensional growth state of cells is maintained. Second, fluid control helps to promote the exchange of nutrients and oxygen, providing a good living environment for cell culture. Finally, PDMS is used as a manufacturing material of the microfluidic chip, has good light transmission and air permeability, can monitor and observe cells in real time, is favorable for fully utilizing oxygen by the cells, and maintains the growth state of the cells. However, the optimization of the in vitro SAE model and operation is still blank by combining the microfluidic technology with the humanized SAE in vitro model.
In the prior art, a humanized three-dimensional sepsis related encephalopathy micro-fluidic bionic model with organ pathophysiology micro-system characteristics is established for a double-channel membrane-sandwiched chip, and the model also belongs to the technical blank, and is developed and researched by researchers.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a sepsis-related encephalopathy model and application thereof, and a novel method for exploring the influence of sepsis on the brain in vitro in three-dimensional level. The invention constructs an in vitro humanized three-dimensional sepsis related encephalopathy bionic model system, can simulate the pathological damage process of a body inflammatory reaction to brain when in sepsis based on the organ chip model system, explores the occurrence mechanism of brain damage in the sepsis process, and fills the blank of the research field.
The invention provides a sepsis-related encephalopathy model, which comprises a membrane-clamping chip, wherein the membrane-clamping chip comprises an upper substrate, a porous membrane and a lower substrate which are sequentially arranged from top to bottom, the upper substrate comprises an upper channel, the lower substrate comprises a lower channel, and the porous membrane is positioned between the upper channel and the lower channel;
the membrane-sandwiched chip is a BBB chip;
the BBB chip is characterized in that astrocytes and pericytes are inoculated on one side of the porous membrane close to the lower-layer channel, and microglial cells are inoculated on the bottom surface of the lower-layer channel; and inoculating human brain microvascular endothelial cells on one side of the porous membrane close to the upper layer channel.
Further, the porous membrane comprises a PET porous membrane or a PDMS porous membrane, etc.
Further, the pore diameter of the porous membrane is 1-10 μm.
Further, the materials of the upper substrate and the lower substrate comprise PDMS.
Further, two ends of the upper layer channel are respectively provided with an upper layer cell inlet and an upper layer cell outlet, and two ends of the lower layer channel are respectively provided with a lower layer cell inlet and a lower layer cell outlet. The sizes of the upper layer channel and the lower layer channel are as follows: 1.5mm wide and 0.2mm high.
Further, the culture medium of the upper layer channel is serum of a sepsis-related encephalopathy patient.
Further, the culture medium of the lower channel is an astrocyte culture medium, a pericyte culture medium and a microglial culture medium which are mixed in equal volumes.
The invention also provides application of the sepsis-related encephalopathy model in-vitro humanization and three-dimensional horizontal exploration of the influence of sepsis-related encephalopathy on the brain.
The invention also provides application of the sepsis-related encephalopathy model in monitoring various cell states and dysfunctions in the brain in real time and monitoring responsiveness to personalized treatment when the sepsis-related encephalopathy is performed.
The invention also provides an evaluation method of the sepsis-related encephalopathy model, which comprises the following specific processes:
(1) BBB chip construction
Astrocytes and pericytes were prepared as a cell suspension of 2,000 cells/. Mu.L at a 2:1 addition ratio, 20. Mu.L of the cell suspension was injected into the lower layer channel of the chip, and the chip was placed in an incubator at 37℃and cultured for 2 hours with adherence.
Microglial cells were prepared as a cell suspension of 2,000 cells/. Mu.L. The remaining astrocyte culture medium in the chip channels was blotted dry and 20 μl of microglial suspension was injected into the chip lower channels. Human brain microvascular endothelial cells were made into 2X 10 7 Mu.l of the cell suspension was injected into the upper layer channel of the chip. The chips were placed in an incubator at 37℃overnight for incubation.
The syringe pump is communicated with the inlets of the upper and lower channels of the chip, the flow rate is 100 mu L/h, and the chips are subjected to perfusion culture in an incubator at 37 ℃ for 3 days.
(2) Chip function detection
The barrier integrity of the BBB chip was examined by immunofluorescent staining of vascular endothelial cell cadherin VE-cadherin and zonulin ZO-1. BBB chip permeability was monitored using FITC-labeled dextran (FITC-dextran). And monitoring the transmembrane resistance value of the BBB chip by using a transmembrane resistor.
(3) Peripheral blood collection for sepsis-associated encephalopathy patients
Collecting peripheral blood of patients with Sepsis meeting diagnosis standard of Sepsis-3.0 and SAE patients meeting diagnosis standard of Sepsis related encephalopathy, collecting the supernatant after taking 3000 rpm/min, and preserving at-80deg.C.
And simultaneously, carrying out disease severity assessment (APACHE II and SOFA scores) and cognitive dysfunction assessment (MMSE and MoCA) on the patient to prepare for individual treatment of the patient.
(4) BBB chip for treating serum of sepsis related encephalopathy patient
The collected sepsis-associated brain disease patient serum was injected into the upper layer microvascular channels of the BBB chip. Culturing at 37deg.C for 4 days, and changing liquid every 2 days.
(5) BBB injury detection after serum perfusion of BBB chip of sepsis related encephalopathy patient
After the BBB chip was perfused with serum from sepsis-related encephalopathy patients, BBB integrity changes could be assessed by the permeability of FITC-labeled dextran. Brain microvascular endothelial integrity changes were assessed by immunofluorescent staining for endothelial extracellular junctions (e.g., ZO-1, occludin, claudin-5, VE-cadherin). The activation states of both glial cells were detected by astrocyte-specific markers (GFAP, s100deg.beta) and microglial-specific markers (IBA 1, CD11 b). Inflammatory factor levels in the up-and-down channel media were detected by cytokine array kit.
The beneficial effects are that:
the invention establishes a model method for exploring the influence of sepsis on brain injury. The model system consists of humanized brain microvascular endothelial cells, pericytes, astrocytes, microglia and a membrane-sandwiched chip. And (3) constructing a sepsis-induced brain injury model by collecting serum of a sepsis-related encephalopathy patient and injecting the serum into a brain microvascular endothelial channel of the BBB chip.
The sepsis-related encephalopathy bionic model provided by the invention not only simulates the sepsis-related encephalopathy pathogenesis, detects the functional damage of the brain barrier in real time, but also can detect and research the effects of brain microvascular endothelial cells, pericytes and glial cells in the brain damage caused by sepsis in real time and dynamically in vitro.
The sepsis-related encephalopathy bionic model provided by the invention can detect BBB barrier damage, various cell states and inflammatory factor levels by adopting a cell detection means commonly used in biology, and comprises FITC-dextran permeability detection, cell viability detection, cell immunofluorescence staining, qRT-PCR, cytokine array detection and the like.
The invention utilizes organ chip technology, and the PDMS and PET porous membrane with good biocompatibility and light transmittance provide great convenience for imaging and observing cells. The device is suitable for detecting changes in barrier tissue levels (blood brain barrier) and behaviors and changes of various cells in the brain after virus infection, such as mRNA changes, protein expression changes, cytokine secretion, cell death and the like.
Drawings
Fig. 1 is a schematic diagram of the structure of a capsular chip according to the present invention.
Wherein 1 is an upper layer channel, 2 is a lower layer channel, and 3 is a PET porous membrane.
Fig. 2 is a schematic diagram of the structural and functional partitioning of a sepsis-associated brain model of the present invention.
Fig. 3 is a 3D stereoscopic image of a sepsis associated brain disease model of the present invention.
FIG. 4 is an observation of various cell states in a sepsis-associated brain disease model according to the present invention.
Figure 5 shows various cellular changes in sepsis-associated brain disease models in response to serum from sepsis patients.
Detailed Description
The following examples further illustrate the invention, but are not intended to limit it.
Example 1
And constructing the BBB chip by using the membrane-sandwiched chip.
And manufacturing a PDMS channel chip: after the chip template is molded, parameters of a template channel are measured and silanization modification is carried out on the chip. And (3) preparing a Sylgard184 monomer and a PDMS initiator according to a volume ratio of 10:1, pouring the mixture onto a surrounding chip template, carrying out vacuum degassing for 20min, drying at 80 ℃ for 30min in a drying oven, curing the PDMS, and taking out. Naturally cooling, peeling the solidified chip from the template by using a blade, then dividing the PDMS chip into proper size, wrapping the PDMS chip by using a preservative film, and punching out an inlet and an outlet by using a puncher.
And (3) assembling a film-clamping chip: the 2 μm pore size PET porous membrane was cut to the appropriate size and attached to the upper channel chip. And putting the lower layer channel and the upper layer channel attached with the porous membrane into a plasma sealing machine for oxygen injection and the like, and aligning and pasting the upper layer channel and the lower layer channel together. And placing the adhered film-sandwiched chip into a 60 ℃ oven for 30min, so that the film-sandwiched chip is fully and firmly adhered.
As shown in fig. 1-2, the membrane-sandwiched chip is formed by bonding and sealing an upper substrate and a lower substrate, and a PET porous membrane is arranged between the upper substrate and the lower substrate. The upper layer substrate is provided with an upper layer channel, and the lower layer substrate is provided with a lower layer channel. The upper layer channel is provided with an upper layer cell inlet and an upper layer cell outlet, and the lower layer channel is provided with a lower layer cell inlet and a lower layer cell outlet. When the laminated chip is assembled, the upper layer channel and the lower layer channel are in an 'x' shape. The dimensions of the upper layer channel and the lower layer channel are 1.5mm wide and 0.2mm high. The pore diameter of the PET porous membrane is 2 mu m. The upper substrate and the lower substrate are made of PDMS.
The BBB chip is characterized in that astrocytes and pericytes are inoculated on a PET porous membrane positioned on the side of a lower channel of the membrane-clamping chip, microglial cells are inoculated on the bottom surface of the lower channel of the membrane-clamping chip, and human brain microvascular endothelial cells are inoculated on the PET porous membrane positioned on the side of an upper channel of the membrane-clamping chip.
Example 2
A method for exploring the influence of sepsis on brain in vitro and three-dimensional level based on organ chip technology. The specific process is as follows:
(1) Sterilizing and coating film chip
The membrane-sandwiched chip was placed in a 6cm petri dish, and the petri dish was left open and placed in an ultra-clean bench for ultraviolet irradiation overnight.
The upper and lower channels of the sandwich chip were each filled with 50. Mu.g/mL Fibronectin solution and placed in an incubator at 37℃overnight. Before seeding the cells, the fibronectin solution was blotted and washed three times with PBS.
(2) Construction of BBB chip:
astrocytes and pericytes were prepared as a cell suspension of 2,000 cells/. Mu.L in a volume ratio of 2:1, 20. Mu.L of the cell suspension was injected into a chip-on-chip lower layer channel, and the membrane-on-chip was placed in a 37℃incubator upside down and cultured for 2 hours with adherence, so that the astrocytes and pericytes adhered to a PET porous membrane on the side of the lower layer channel. Microglial cells were prepared as a cell suspension of 2,000 cells/. Mu.L. Sucking the residual astrocyte and pericyte culture medium in the lower channel of the sandwiched membrane chip to dry, and injecting 20 mu L of microglial suspension into the lower channel of the sandwiched membrane chip to adhere microglial cells to the lower layerThe bottom surface of the track. Human brain microvascular endothelial cells were made into 2X 10 7 Mu.l of the cell suspension was injected into the upper layer channel of the membrane-sandwiched chip. The membrane-sandwiched chip was placed in an incubator at 37℃overnight to allow the brain microvascular endothelial cells to adhere to the PET porous membrane on the side of the upper channel. The injection pump is communicated with an upper cell inlet and a lower cell inlet of the membrane-clamping chip, and an upper cell outlet and a lower cell outlet are connected with the collecting device. The culture medium of the upper layer channel is serum of a sepsis related encephalopathy patient. The culture medium of the lower layer channel is an astrocyte culture medium, a pericyte culture medium and a microglial culture medium which are mixed in equal volumes. The culture medium was cultured by perfusion in an incubator at 37℃for 3 days at a flow rate of 100. Mu.L/h.
(3) Chip function detection
The integrity of the BBB chip barrier was examined by immunofluorescent staining of vascular endothelial cell cadherin VE-cadherin and zona claudin ZO-1, and immunofluorescent staining of astrocytes (GFAP, S100deg.P, AQP 4), pericytes (HBVP-GFAP), microglial cells (Iba 1) were performed, respectively, to observe that the on-chip nerve cells were intact (see FIG. 4). BBB chip permeability was monitored using FITC-labeled dextran (FITC-dextran). And monitoring the transmembrane resistance value of the BBB chip by using a transmembrane resistor.
(4) Constructing a sepsis related encephalopathy bionic model:
because the SAE onset process is a series of reactions caused by peripheral blood inflammatory factors entering the brain parenchyma through the blood brain barrier, blood serum of SAE patients is used for perfusion of brain microvascular endothelial channels for 24 hours respectively after 3 days of perfusion, and the construction of a sepsis-related encephalopathy bionic model is completed.
On the fourth day, four cells on the BBB chip were detected separately by immunofluorescence, and it was found that the brain microvascular endothelial extracellular junctions (ZO-1 indicated tight junctions, VE-cadherein indicated adhesive junctions) were significantly attenuated in the SAE group; significant decrease in pericyte coverage (α -SMA indicates pericytes) astrocytes are significantly activated (GFAP is significantly up-regulated); the obvious activation of microglia (significant upregulation of IBA 1) suggests that systemic inflammatory response caused by sepsis may lead to damage to the nervous system of the patient, which in turn may lead to clinically occurring neurological symptoms. The results are shown in FIG. 5.
The foregoing disclosure is illustrative of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims.

Claims (9)

1. The sepsis related encephalopathy model is characterized by comprising a membrane-clamping chip, wherein the membrane-clamping chip comprises an upper substrate, a porous membrane and a lower substrate which are sequentially arranged from top to bottom, the upper substrate comprises an upper channel, the lower substrate comprises a lower channel, and the porous membrane is positioned between the upper channel and the lower channel;
the membrane-sandwiched chip is a BBB chip;
the BBB chip is characterized in that astrocytes and pericytes are inoculated on one side of the porous membrane close to the lower-layer channel, and microglial cells are inoculated on the bottom surface of the lower-layer channel; and inoculating human brain microvascular endothelial cells on one side of the porous membrane close to the upper layer channel.
2. The sepsis-related brain disease model according to claim 1, wherein the porous membrane comprises a PET porous membrane or a PDMS porous membrane.
3. The sepsis-related brain disease model according to claim 1, characterized in that the pore size of the porous membrane is 1-10 μιη.
4. The sepsis-related brain disease model according to claim 1, wherein the materials of the upper and lower substrates comprise PDMS.
5. The sepsis-related brain disease model according to claim 1, wherein both ends of the upper layer channel are provided with an upper layer cell inlet and an upper layer cell outlet, respectively, and both ends of the lower layer channel are provided with a lower layer cell inlet and a lower layer cell outlet, respectively.
6. The sepsis-related brain disease model according to claim 1, wherein the medium of the upper layer channel is sepsis-related brain disease patient serum.
7. The sepsis-related brain disease model according to claim 1, wherein the medium of the lower channel is an equal volume of mixed astrocyte, pericyte and microglial medium.
8. Use of a sepsis-associated brain disease model according to any one of claims 1 to 7 for in vitro humanization, three-dimensional level exploration of the effects of sepsis-associated brain disease on the brain.
9. Use of a sepsis-associated brain disease model according to any one of claims 1 to 7 for real-time monitoring of a plurality of cell states and dysfunctions in the brain and monitoring responsiveness to personalized therapy in sepsis-associated brain disease.
CN202211589390.1A 2022-12-09 2022-12-09 Sepsis-related encephalopathy model and application thereof Pending CN116144494A (en)

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