An assemblable multi-cell co-culture brain organ chip and application thereof

Abstract

This invention belongs to the field of microfluidic organ-on-a-chip technology, and relates to an assemblable multi-cell co-culture brain organ-on-a-chip and its applications. The multi-cell co-culture brain organ-on-a-chip consists of a lower substrate, a middle chip, and an upper chip. The upper chip has one cell culture chamber and eight inlet / outlet ports, while the middle chip has seven cell culture chambers. The six cell culture chambers are arranged in a hexagonal pattern around the central chamber, and adjacent chambers are interconnected through a micro-forestation structure. The lower substrate serves as a supporting base. Compared with existing brain organ-on-a-chips, this chip device can effectively induce and stably generate Lewy bodies, and can reproduce typical pathological features of Parkinson's disease that are difficult to achieve with traditional chips. It is particularly suitable for simulating the blood-brain barrier and co-culture systems of brain parenchymal cells, providing novel device support for the study of Parkinson's disease mechanisms and in vitro drug screening, and has significant application value in the fields of neurological disease model construction and drug development.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic organ-on-a-chip technology, and relates to an assemblable multi-cell co-culture brain organ-on-a-chip and its applications. Background Technology

[0002] Organ-on-a-chip technology is an emerging technology that combines miniaturized models of human organs with microfluidic platforms that simulate physiological environments to facilitate the study of organ structure and function, as well as the understanding of the mechanisms of many diseases. Organ-on-a-chips can be used for drug screening and toxicological evaluation, reducing failed drug development cycles and thus improving drug safety and efficacy. In addition, organ-on-a-chips can be used to replace animal experiments, exhibiting high biosimilarity even in simulated environments, and can provide long-term monitoring and analysis of the physiological and pathological processes under study, thereby offering more effective methods for disease treatment.

[0003] Brain organ-on-a-chip, as an important branch of organ-on-a-chip, is a core in vitro platform for studying neurological diseases (especially Parkinson's disease). The core pathological features of Parkinson's disease are damage to dopaminergic neurons, abnormal aggregation of α-synuclein to form Lewy bodies, accompanied by immune activation of microglia and astrocytes and impaired blood-brain barrier function. The synchronous reproduction of the above multidimensional pathological features is the key to constructing in vitro models of Parkinson's disease and is also a bottleneck that is difficult to overcome with existing technologies.

[0004] Currently, existing brain organ-on-a-chip technologies suffer from two major technological defects that severely limit their application in Parkinson's disease research: First, most existing brain organ-on-a-chips are monolithic fixed structures, focusing primarily on single-cell culture or simple co-culture. The cell culture units are indivisible, making it impossible to pre-treat, precisely seed, and pre-culture different nerve-related cells individually, hindering the orderly co-culture of multiple cells and precise signal regulation between cells. Second, existing chips cannot simultaneously integrate blood-brain barrier units with multiple types of nerve cells (dopaminergic neurons, astrocytes, microglia, etc.), and cannot reproduce the complete pathological chain of Parkinson's disease—"α-synuclein deposition (Lewy body formation) + immune activation + blood-brain barrier damage"—through reasonable structural design and culture strategies. In particular, they cannot stably produce Lewy bodies, a Parkinson's disease-specific pathological marker, resulting in significant deviations between the Parkinson's models constructed by existing chips and the in vivo physiological and pathological states, failing to meet the precise requirements for pathological mechanism research and drug screening. Summary of the Invention

[0005] To address the technical shortcomings of existing brain organ-on-a-chip structures, such as inconvenient operation, inability to reproduce complete pathological features of Parkinson's disease (especially the inability to generate Lewy bodies), and poor multi-cell co-culture effects, the core objective of this invention is to provide an assemblable multi-cell co-culture brain organ-on-a-chip and its applications. Through a unique hierarchical structural design and cell culture strategy, it achieves stable reproduction of Parkinson's disease-specific pathological markers (Lewy bodies) and multi-dimensional pathological features, providing a reliable in vitro tool for the study of neurological diseases (especially Parkinson's disease), while protecting the structural innovation of the chip device itself.

[0006] The technical solution of the present invention: An assemblable multi-cell co-culture brain organ-on-a-chip adopts a three-layer detachable and assemblable structure, including a lower substrate, a middle chip and an upper chip; The upper chip is the blood-brain barrier unit culture layer, equipped with one independent cell culture chamber and eight symmetrically distributed inlet / outlet ports. The cell culture chamber is 8-12 mm long and 2-5 mm wide, with inlet / outlet radii of 0.8-1.2 mm. The cell culture chamber is used to culture blood-brain barrier-related cells. The inlet / outlet ports are used for aeration of the cell culture chamber on the lower middle chip, for culture medium perfusion, sample addition and collection, or for connecting to an external perfusion system, providing a dynamic biomimetic culture environment for the blood-brain barrier unit cells, simulating the state of blood flow in vivo. Blood-brain barrier-related cells include astrocytes, human brain microvascular endothelial cells, and pericytes, among others. The upper-layer chip and the middle-layer chip are separated by a single-layer or double-layer polycarbonate porous membrane with a pore size of 8 μm; The middle layer of the chip is the core layer for brain parenchymal cell culture, with seven independent cell culture chambers. The central cell culture chamber is a regular hexagon, and the other six cell culture chambers are evenly arranged in a regular hexagonal pattern around the central cell culture chamber. Adjacent cell culture chambers are interconnected by micro-fence structures. The central cell culture chamber is used to culture dopaminergic neurons, and the six surrounding cell culture chambers are used to seed different neural cells related to constructing the human brain microenvironment, including glial cells. The spacing between the micro-fence structures is 0.5-0.8 mm, which allows cytokines, small molecule nutrients, and signaling molecules to diffuse freely, while effectively preventing cell migration across chambers and avoiding cross-contamination.

[0007] The upper and middle layers of the chip are 2-10mm in height, which can meet the needs of two-dimensional or three-dimensional cell culture. For two-dimensional cell culture on the chip, Geltrex basement membrane matrix can be used to pre-coat the cell culture chamber. For three-dimensional cell culture on the chip, one or more of silica gel, agarose, chitosan, sodium alginate, and Matrigel matrix gel can be selected to pre-coat the cell culture chamber.

[0008] The lower substrate is 1mm high and is a flat support base plate used to support the middle and upper chips.

[0009] The upper chip, middle chip and lower substrate are fixedly connected by screws and nuts. The contact surface of adjacent layers is sealed with a polytetrafluoroethylene hydrophobic tape layer, which can effectively compact the contact surface, prevent leakage between layers and ensure the stability of long-term cell culture.

[0010] The upper-layer chip, middle-layer chip, and lower-layer substrate can be made of plastic, resin, glass, quartz, silicon, ceramic, or metal. The manufacturing process is simple and can be mass-produced using standard soft lithography technology, making it highly practical.

[0011] The chip of this invention has a wide range of applications. It can be used to construct a normal blood-brain barrier-neural chip model for blood-brain barrier function research and neuropharmaceutical permeability screening. It can also be used to construct a brain chip model of Parkinson's disease, which can reproduce the key pathological features of Parkinson's disease through a dual-attack strategy of α-synuclein pre-prepared fibrils and immune stimulation.

[0012] During the model construction process, the inoculated cells can be flexibly selected, including one or more of the following: human or animal brain microvascular endothelial cells, astrocytes, microglia, neuronal cell lines, human or animal brain nerve-related primary cells, and brain nerve-related functional cells induced from human pluripotent stem cells.

[0013] The beneficial effects of this invention are as follows: The chip adopts a layered, assemblable structure, which solves the problem of inconvenient operation of existing integrated chips, allowing for individual pretreatment and culture of different cells before assembly; the hexagonal surrounding chamber combined with the micro-fence structure enables multi-cell co-culture and inter-cell signal exchange, completely reproducing the function of the neurovascular unit; the dual-strike strategy can accurately construct a pathological model of Parkinson's disease, and the model highly matches the in vivo physiological and pathological state; the chip is simple to prepare and highly stable, and can be widely used in the fields of neuropathological research and drug screening, providing an ideal in vitro experimental platform for related research. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the overall structure of the assemblable multi-cell co-culture brain organ-on-a-chip in Embodiment 1 of the present invention; Figure 2 The following are top views of the upper chip, middle chip, and lower substrate in Embodiment 1 of the present invention; wherein (a) is a top view of the upper chip, clearly showing the distribution structure of a single cell culture chamber and eight inlet / outlet ports; (b) is a top view of the middle chip, clearly showing the hexagonal arrangement of the seven cell culture chambers and the micro-fence structure between adjacent chambers; and (c) is a top view of the lower substrate. Figure 3These are microscopic images of the morphology of various cells in the brain chip after seeding in Example 2 of the present invention, which visually present the growth status of various cells after seeding; (a) are dopaminergic neurons, (b) are astrocytes, (c) are microglia, and (d) are human brain microvascular endothelial cells. Figure 4 The following is a graph showing the results of blood-brain barrier function verification in Example 2 of the present invention; (a) is a fluorescence characterization graph of functional proteins of astrocytes (GFAP, green), (b) is a fluorescence characterization graph of functional proteins of human brain microvascular endothelial cells (ZO-1, green), and (c) is a statistical result graph of the penetration rate of detection chips for fluorescent tracers of different molecular weights. Figure 5 The image shows the results of detecting Parkinson's pathological features of dopaminergic neurons on the brain chip in Embodiment 3 of the present invention. It is a fluorescence image of the formation of PFF aggregates (red) on dopaminergic neurons, which visually shows the formation of Lewy bodies (PFF aggregates), a pathological marker of Parkinson's disease. Figure 6 The image shows the results of detecting Parkinson's pathological features of astrocytes and microglia on a brain chip in Example 3 of the present invention. It is a statistical graph of the changes in fluorescence intensity of astrocytes (expressing GFAP characteristic protein) and microglia (expressing CD68 characteristic protein), which visually shows that astrocytes and microglia have been activated. Figure 7 This is a statistical comparison chart of the permeability of sodium fluorescein to the blood-brain barrier before and after the construction of the brain chip Parkinson's model in Embodiment 3 of the present invention, which visually shows the extent of barrier function impairment.

[0015] In the figure: 1. Cell culture chamber in the middle of the middle chip; 2. Cell culture chamber around the middle chip; 3. Micro-fence structure; 4. Cell culture chamber of the upper chip; 5. Inlet / outlet; 6. Polycarbonate porous membrane; 7. Screws and nuts; 8. Polytetrafluoroethylene hydrophobic tape sealing layer. Detailed Implementation

[0016] The specific embodiments of the present invention will be further described in detail below with reference to the technical solution and accompanying drawings.

[0017] Example 1 The steps for preparing an assemblable multi-cell co-culture brain organochip are as follows: Using soft lithography, upper and middle layers of chips are fabricated using thermoplastic polymer PMMA (polymethyl methacrylate) as the substrate, and precisely processed to form cell culture chambers and microfence structures of preset sizes. The upper-layer chip has one independent cell culture chamber 4 and eight symmetrically distributed inlet / outlet ports 5. The cell culture chamber is 10mm long and 3mm wide, and the inlet / outlet ports have a radius of 1mm. The middle-layer chip has seven independent cell culture chambers. The middle chamber 1 is a regular hexagon with a side length of 5mm, and the surrounding six cell culture chambers 2 are a combination of circles and regular hexagons arranged in a ring. Micro-fence structures 3 are set between adjacent cell culture chambers, with a spacing of 0.5-0.8mm between the micro-fence structures. The chip single-layer height is 2-10mm. Polished borosilicate glass is used as the lower-layer substrate, and its dimensions are precisely matched with the upper-layer and middle-layer chips. M3 stainless steel screws and nuts 7 are used as interlayer fasteners, and a polytetrafluoroethylene (PTFE) hydrophobic tape sealing layer is used as the interlayer sealing structure. The PTFE hydrophobic tape sealing layer is sized to match the chip contact surface to achieve reliable sealing and prevent leakage. The overall structure of the multi-cell co-culture brain organ chip is as follows: Figure 1 As shown.

[0018] The upper chip is the blood-brain barrier unit culture layer. The size of its cell culture chamber can be adjusted within the range of 8-12 mm in length and 2-5 mm in width, and the radius of the inlet and outlet can be adjusted within the range of 0.8-1.2 mm. This cell culture chamber is used for co-culturing astrocytes and human brain microvascular endothelial cells. The inlet and outlet can be used for ventilation of the middle chamber, culture medium perfusion, sample addition and collection, and can also be connected to an external perfusion system to provide a dynamic biomimetic culture environment for endothelial cells, simulating the state of blood flow in vivo. The upper chip and the middle chip are separated by a single or double layer of polycarbonate porous membrane with a pore size of 8 μm.

[0019] The middle layer of the chip serves as the core layer for culturing brain parenchymal cells. Six surrounding cell culture chambers are arranged in a uniform hexagonal pattern around the central chamber, with adjacent chambers interconnected by a micro-barrier structure. The spacing between the micro-barriers is controlled at 0.5-0.8 mm, allowing the free diffusion of cytokines, small molecule nutrients, and signaling molecules while effectively preventing cell migration across chambers and avoiding cross-contamination. The central cell culture chamber is used to culture dopaminergic neurons, while the surrounding chambers are used to seed human brain microvascular endothelial cells, astrocytes, microglia, and other neuronal cells.

[0020] The lower substrate is a flat support plate with a height of 1mm, used to support the middle and upper layer chips.

[0021] The fabrication process of an assemblable multi-cell co-culture brain organ-on-a-chip includes PMMA substrate pretreatment, laser cutting, pre-assembly pretreatment, cleaning, and assembly, as follows: PMMA substrate treatment: Remove the protective film from the surface of the substrate and wipe it with a lint-free cloth dampened with alcohol to remove dust; if hydrophobic and leak-proof measures are required, apply acrylic double-sided tape to the surface and remove air bubbles, then apply polytetrafluoroethylene hydrophobic tape.

[0022] Laser cutting: Place the PMMA substrate on a laser engraving machine, adjust the height of the laser head, and start the cutting program to obtain the chip's layer structure; ultra-thin small-sized parts can be trimmed with scissors.

[0023] Pre-assembly treatment: After wiping each layer of chips with alcohol, ultrasonically clean them three times with ultrapure water for 15 minutes each time, and let them air dry naturally in a fume hood; glue the lower structure to the base plate with acrylic glue and let it stand overnight at room temperature; then soak the chips in benzalkonium chloride disinfectant for 2 hours to sterilize them, rinse them three times with sterilizing water, air dry them in a clean bench, and then sterilize them overnight with ultraviolet light.

[0024] After the chip fabrication was completed, cell seeding was performed: immature dopaminergic neurons differentiated to day 8 using human induced pluripotent stem cells (iPSCs). Simultaneously, human brain microvascular endothelial cells, human astrocytes, and human microglia in the logarithmic growth phase were harvested, digested with trypsin, and resuspended for later use. The immature dopaminergic neurons were then seeded at a rate of 1×10⁻⁶ cells / year. 5 Cells / mL were seeded in the cell culture chamber in the middle of the mid-layer chip; human brain microvascular endothelial cells, astrocytes, and microglia were seeded at a density of 1×10⁻⁶ cells / mL. 5 Cells / mL were seeded into six cell culture chambers surrounding the middle layer of the chip; simultaneously, human brain microvascular endothelial cells were seeded at a density of 5 × 10⁶ cells / mL. 4 Cells were seeded at a density of [number] cells / mL on the upper surface of an 8 μm porous membrane, and astrocytes were seeded at the same density on the lower surface of the porous membrane. After seeding, fresh culture medium was added to each cell culture chamber, and the cells were incubated at 37°C with 5% CO2 for 48 hours to ensure full cell adhesion. The cell culture chamber arrangement and microfence structure of the middle layer chip are shown below. Figure 2 As shown in (b), the cell culture chamber and inlet / outlet structure of the upper chip are as follows: Figure 2 As shown in (a), the lower substrate is as follows Figure 2 (c)

[0025] Example 2 The blood-brain barrier-neural chip model was constructed using an assemblable multi-cell co-cultured brain organ-on-a-chip, with the following steps: First, the porous membrane surface was coated with a matrix gel diluted 1:100 by volume to promote cell adhesion and growth. Human brain microvascular endothelial cells and astrocytes were seeded into the corresponding cell culture chambers according to the designed structure and cultured in a 37°C, 5% CO2 saturated humidity incubator. After 2-3 days of seeding, the cells adhered and fused. Cell morphology was observed using an inverted phase contrast microscope to determine whether the human brain microvascular endothelial cells exhibited a typical cobblestone morphology and whether the astrocyte protrusions grew normally.

[0026] Immunofluorescence staining was used to characterize cellular functional features: the expression and membrane localization of ZO-1, a marker protein of tight junctions in human brain microvascular endothelial cells, were detected to assess tight junction formation; the expression of GFAP, a characteristic protein of astrocytes, was detected to assess its differentiation and maturation; for chip permeability testing, two fluorescent tracers with different molecular weights, 376 kDa sodium fluorescein and 70 kDa FITC-glucan, were used to compare the permeability differences between cells seeded on one side of a porous membrane and assembling a bilayer membrane structure and cells seeded on both sides of a porous membrane to form a monolayer membrane structure.

[0027] The verification results showed that the human brain microvascular endothelial cells and astrocytes within the chip were in good growth condition, and the cell morphology and functional protein expression were as expected. Figure 4 As shown in (a) and (b); the permeability test results are as follows: Figure 4 As shown in (c), the chip exhibits extremely low permeability to the macromolecular FITC-glucan. The permeability coefficient closely matches in vivo physiological values ​​and the reference range for classic in vitro chips. Furthermore, the permeability of the bilayer membrane structure is significantly lower than that of the single-layer membrane, indicating that the chip maintains its blood-brain barrier function and possesses significant molecular weight selective permeability. The microscopic morphology of various cell types after seeding is shown in [image / image]. Figure 3 As shown in (a)-(d).

[0028] Example 3 The following steps were taken to construct a Parkinson's disease microarray model using an assemblable multi-cell co-culture brain organoid chip: After 48 hours of adherent cell culture, the old culture medium in the middle chamber of the mid-layer chip was discarded, and complete culture medium for dopaminergic neurons containing 1 mg / mL of fluorescently labeled α-synuclein pre-fabricated fibrils (PFF) was added. The surrounding chambers were simultaneously replaced with fresh culture medium for continued culture. Chip assembly was then performed. A porous membrane seeded with astrocytes was placed above the middle cell culture chamber, cell side down. A porous membrane seeded with human brain microvascular endothelial cells was precisely aligned with the membrane, cell side up. After aligning the upper and middle layers, polytetrafluoroethylene hydrophobic tape was placed between the layers and secured with screws and nuts to ensure a leak-proof seal. 80 μL of endothelial cell culture medium was added to the upper chip cell culture chamber. After co-incubating dopaminergic neurons with PFF for 48 hours, the culture medium was replaced with normal neuronal culture medium for another 24 hours. Subsequently, neuronal culture medium containing 0.2 μg / mL of interferon-γ was added for 24 hours, and then the medium was replaced with fresh normal culture medium. The Parkinson's disease chip model was thus completed.

[0029] Model pathological feature verification: During the 7th-9th day of chip culture, fluorescence microscopy was used to observe the PFF aggregation in dopaminergic neurons. The results are as follows: Figure 5 As shown, distinct Lewy body-like PFF aggregates were observed within neurons, consistent with typical pathological markers of Parkinson's disease. Immunofluorescence staining with GFAP and CD68 and fluorescence intensity analysis revealed a significantly increased proportion of activated astrocytes and microglia. Figure 6 As shown, the model indicates the formation of a neuroinflammatory microenvironment. The integrity of the blood-brain barrier was assessed using sodium fluorescein permeability, and the results are as follows: Figure 7 As shown, the permeability of the Parkinson's model group was significantly higher than that of the normal control group, indicating that pathological damage leads to the disruption of the blood-brain barrier integrity, which is highly consistent with the pathological process of Parkinson's disease in vivo.

[0030] The above results demonstrate that the assemblable multi-cell co-culture brain organ-on-a-chip of the present invention can stably construct blood-brain barrier models and Parkinson's disease models. The pathological characteristics of the models are consistent with the in vivo state, and they can be effectively used for the study of the pathogenesis of Parkinson's disease and the screening of neuro-drugs, fully meeting the experimental and application needs of those skilled in the art.

Claims

1. An assemblable multi-cell co-culture brain organ-on-a-chip, characterized in that, This assemblable multi-cell co-culture brain organ-on-a-chip adopts a three-layer detachable and assemblable structure, including a lower substrate, a middle chip, and an upper chip. The upper chip is the blood-brain barrier unit culture layer, with one independent cell culture chamber and eight symmetrically distributed inlet and outlet ports. The cell culture chamber is 8-12 mm long and 2-5 mm wide, and the inlet and outlet ports have a radius of 0.8-1.2 mm. The cell culture chamber is used to culture blood-brain barrier-related cells, and the inlet and outlet ports are used to ventilate the cell culture chamber on the lower middle chip, for culture medium perfusion, sample addition and collection, or to connect to an external perfusion system, providing a dynamic biomimetic culture environment for the cells of the blood-brain barrier unit, simulating the state of blood flow in vivo. The upper-layer chip and the middle-layer chip are separated by a single-layer or double-layer polycarbonate porous membrane with a pore size of 8 μm; The middle layer of the chip is the core layer for brain parenchymal cell culture, with 7 independent cell culture chambers. The central cell culture chamber is a regular hexagon, and the other 6 cell culture chambers are evenly arranged in a regular hexagon around the central cell culture chamber. Adjacent cell culture chambers are interconnected by a micro-fence structure. The central cell culture chamber is used to culture dopaminergic neurons, and the 6 surrounding cell culture chambers are used to inoculate different neural cells related to constructing the human brain microenvironment. The lower substrate is 1mm high and is a flat support base plate used to support the middle and upper chips.

2. The assemblable multi-cell co-culture brain organ-on-a-chip according to claim 1, characterized in that, The upper-layer chip, the middle-layer chip, and the lower-layer substrate are fixedly connected by screws and nuts, and the contact surfaces of adjacent layers are sealed with a polytetrafluoroethylene hydrophobic tape layer.

3. The assemblable multi-cell co-culture brain organ-on-a-chip according to claim 1, characterized in that, The upper-layer chip, middle-layer chip, and lower-layer substrate can be made of plastic, resin, glass, quartz, silicon, ceramic, or metal.

4. The assemblable multi-cell co-culture brain organ-on-a-chip according to claim 1, characterized in that, Blood-brain barrier-related cells include astrocytes, human brain microvascular endothelial cells, and pericytes.

5. The assemblable multi-cell co-culture brain organ-on-a-chip according to claim 1, characterized in that, The spacing between the micro-fence structures is 0.5-0.8 mm.

6. The assemblable multi-cell co-culture brain organ-on-a-chip according to claim 1, characterized in that, The upper and middle layers of the chip are 2-10 mm in height to meet the requirements of two-dimensional or three-dimensional cell culture. For two-dimensional cell culture on the upper and middle layers, the cell culture chamber is pre-coated with Geltrex basement membrane matrix. For three-dimensional cell culture on the upper and middle layers, one or more of silica gel, agarose, chitosan, sodium alginate, and Matrigel matrix gel are selected to pre-coat the cell culture chamber.

7. An assemblable multi-cell co-culture brain organ-on-a-chip for constructing a normal blood-brain barrier-neural chip model for blood-brain barrier function research and neuropharmaceutical permeability screening.

8. An assemblable multi-cell co-culture brain organ-on-a-chip for constructing a brain chip model of Parkinson's disease, which reproduces the key pathological features of Parkinson's disease through a dual-attack strategy of α-synuclein pre-fabricated fibrils and immune stimulation.

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