CN109576155B - Universal nervous system chip - Google Patents

Abstract

The invention provides a universal nervous system chip, which mainly comprises six independent neurovascular units, wherein each neurovascular unit comprises an upper blood brain barrier chamber, a lower nerve chamber, a porous membrane, an upper sample inlet, an upper sample outlet, a lower sample inlet and a lower sample outlet. The invention has the capacity of multi-cell co-culture, can realize the information communication among multiple cells through the design of the porous membrane and the micro-groove, and simulates the whole body nervous system of a human body; information acquisition and monitoring of related research and application can be widely realized through integration of different micro-nano devices.

Description

Universal nervous system chip

Technical Field

The invention belongs to the technical field of microfluidic chips, and relates to a universal nervous system chip based on a microfluidic chip.

Background

Diseases of the nervous system have seriously endangered human life and health, and are attracting more and more attention. Although a large number of researchers are working on the development of drugs for the diseases, the effect achieved by the drugs for the above diseases in clinical treatment is not ideal. The main reason for this dilemma is due to the complexity of the nervous system. On the one hand, the complexity of the structure and, on the other hand, the complexity of the transmission and regulation of the neural signals. Any function of the nervous system (autonomic and involuntary), such as consciousness, cognition, sensation, movement, visual and auditory perception, autonomic nervous function, etc., is related to the transmission, feedback and regulation of nerve signals, which link the cerebral cortex to peripheral receptors or the motor system (skeletal muscles). Although many nerve conduction pathways are known, it is unclear how these signals are transmitted in the conduction pathways, regulated by what factors, and resolved in the cortex; meanwhile, the nervous system is composed of a large variety of cells (neurons, astrocytes, oligodendrocytes, microglia and the like), the transmission of the nerve signals is under the combined action of various cells, the interaction among the cells is already existed, the intricate and complex action forms a neural network, and the function of one cell or a plurality of cells can be summarized by knowing absolutely. In addition, the nervous system is protected by the blood brain barrier, and not all substances, neurotransmitters or small molecule substances capable of serving as signals can pass through, so that the interaction of nerve cells must be considered and the protection and screening effects of the blood brain barrier are simulated to fully understand the functions of the nervous system and provide a repair measure for the damage of the nervous function caused by diseases, and the network construction and the functions of the nervous system can be comprehensively understood only by the obtained neural signals on the basis, so that a foundation is provided for the damage repair. The existing cell model can not simulate the complex interaction between cells in vivo, so a new cell culture model which is closer to the in vivo environment needs to be established. The general neural system chip concept is also based on the above-described reasons.

Mono, neuro-immune-endocrine networks and regulation thereof

The nerve cells form a complicated small network, but the final aim is to serve the whole nervous system and form a large regulation network, so that the function of the nervous system is realized. The nerve-immunity-endocrine network is the most important regulation network for the nervous system to play a role, forms an internal regulation system, has antagonistic mechanisms such as sympathetic nerves, parasympathetic nerves, excitability and inhibitivity, and Th1 and Th2, and is the basis for various nerve signal transmission and nerve function generation.

Hypothalamic and neuroendocrine systems and their central status

Throughout the neuro-immune-endocrine system, the hypothalamus is located in the core. The functions of the device are as follows: 1. an afferent system for receiving the internal and external environment changes; 2. a decision center nucleus with sensing and stress internal and external environment changes, particularly a regulation center which is formed by the nucleus and the pituitary; 3. there is an egress system (shown in fig. 5) that issues instructions to the target mechanism. The hypothalamus integrates three major regulatory pathways: neuro-, endocrine-, immune-responses and thus the central regulatory nervous system, are also the core of the neuro-immune-endocrine network. On the basis of this, various functions of the nervous system or functions of individual nerve cells can be exerted.

(II) neuroendocrine modulation of the hypothalamus

The hypothalamus is composed of normal neurons, secretory neurons and projection neurons. All can secrete hormone, and abnormal cells (tumor cells) from other sources can synthesize and secrete the hormone to act on specific receptors of target organs so as to play the functions of the hormone. Hormones chemically include peptides, proteins, steroids, and the like. Neurons and neuroendocrine cells secrete hormones, acting in an endocrine or paracrine manner.

Secretory neurons of the hypothalamus include three types: 1. large cell neurons secrete Arginine Vasopressin (AVP) and Oxytocin (OXY). 2. Small cell neurons secrete thyroid stimulating hormone (TRH), Corticotropin Releasing Hormone (CRH), somatostatin, GRh, gonadotropin releasing hormone (GnRH) dopamine, etc., and promote pituitary secretion of adrenocorticotropic hormone (ACTh), thyroid stimulating hormone (TSh), Growth Hormone (GH), follicle stimulating hormone (LH), luteinizing hormone (FSH), Prolactin (PRL). 3. The projection neurons, which are mainly located in the lateral subthalamic zone, project to multiple sites, multiple segments and multiple regions of the central nervous system, and the projected nerve fibers finally synapse chemically to release neurotransmitters (such as GABA, glutamic acid and the like), thereby regulating and controlling the activity of target neurons.

The hormone secreted by hypothalamus mainly acts on target organs (pituitary), promotes the pituitary to secrete and release or inhibit the release of the hormone, and then acts on peripheral organs such as thyroid gland, gonad, adrenal gland and the like; meanwhile, hormones secreted from the glands of peripheral organs such as mineralocorticoids and glucocorticoids secreted from the adrenal glands, thyroid hormones secreted from the thyroid glands, cytokines secreted from peripheral sensory terminals or visual and auditory receptors, neurotransmitters, and even photoacoustic stimuli all have a feedback regulating (promoting or inhibiting) effect on the hypothalamus or pituitary. This forms a complex set of hypothalamic-pituitary-peripheral glandular circuits that can antagonize (antagonize) each other, regulate the function of the nervous system and peripheral vital organs together, and form an antagonistic network of central → central, central → peripheral, peripheral → central. Therefore, the human cells with the structures are co-cultured, the living environment similar to blood brain barrier simulated nerve cells is constructed, and network signals of the cells, which are co-cultured and interacted with each other, are collected and recorded, so that the essence of regulation and control of a nervous system can be really known. This is also of great significance for the present invention.

In addition to the pituitary, the hypothalamus can regulate the brain stem network via noradrenergic neurons, dopaminergic neurons, 5-HT neurons, which underlies the development of consciousness. While the direct regulation of the amygdala and the hippocampus regulates and controls cognition, emotion and memory. The hypothalamus can receive signals from the retina and project directly to the visual aids; and the auditory signals of the cochlear hair cells are received and directly projected to an auditory auxiliary area, so that the vision and the auditory sense are generated and correspondingly regulated. Even the sense of smell is transmitted to the cortex through hypothalamic metaplasia.

The association between the hypothalamus and the spinal cord is also very tight. The hypothalamus can receive the sensory afferent of the body through the spinothalamic tract, including the sensory afferent of all human receptors and the skin, control the movement of the body through the corticospinal tract after being integrated by the brain, receive the afferent and efferent signals of internal organs, regulate sympathetic/parasympathetic nerves, and enable the pupil to be enlarged, the blood pressure to be increased, the tachycardia to be accelerated and the skeletal muscle vessel to be expanded through the lower inner sympathetic center of the hypothalamus; the parasympathetic center of the hypothalamic anterior zone may elicit a response of coupled vagal bipolar neurons: such as miosis, blood pressure drop, bradycardia, and skeletal muscle vasoconstriction.

Therefore, the hypothalamus, which is the core of the hypothalamus, forms a complex neural network with a plurality of central nervous system components, other human systems, various receptors and peripheral organs, is closely related to consciousness, audio-visual, olfactory, cognitive, emotion, movement, sensation (including skin sensation) and visceral sensorimotor nerve functions, maintains the normal occurrence of each nerve function while regulating the organism through external glandular organs, and the hypothalamus and the receptors or functions are mutually connected to form a mutually regulated network, and the neural networks among different functions are densely overlapped, so that the analysis and comparison of the formed networks or the high-flux detection and the collection of cell potential activities are helpful for understanding the functions of the hypothalamus and the formed networks thereof, and have important significance.

(III) nervous System and immune System

The nervous system can affect immune function through two pathways, one is through the release of neurotransmitters and the other is through the alteration of endocrine activity, which in turn affects immune function. The immune organs such as bone marrow, thymus gland and lymph node enter autonomic nerves, and transmitters (norepinephrine, acetylcholine and peptides) released from the periphery act on immune cells through diffusion. Noradrenaline can inhibit immune reaction, acetylcholine can enhance immune reaction, enkephalin can enhance immune reaction, and beta-endorphin has various effects. Corresponding receptors are present on immune cells.

Meanwhile, various nerve cells can secrete immune regulatory factors to directly enter blood to act on an immune system to play a physiological role. Therefore, a network relation of mutual coordination and control is established between the nervous system and the immune system, the nervous system can conveniently regulate and control the immune system, and the change of the immune system can timely give corresponding feedback to the nervous system.

(IV) endocrine and immune systems

The endocrine system is strictly regulated and controlled by the nervous system, and the endocrine system also has a definite regulation and control effect on the immune system. For example, the pituitary gland-regulated adrenocortical hormone has a wide range of inhibitory effects on various immune responses. The inhibitory effect occurs immediately upon inflammation or stress, which is an important mechanism by which adrenocortical hormones exert an anti-inflammatory effect, and can be considered as a typical paradigm for neuro-immunomodulation.

Growth hormone deficient mice have been shown to develop thymus atrophy, lymphoid tissue degeneration and defective T-lymphocyte function. The above changes returned to normal after supplemental treatment with growth hormone. The reduced immune function that occurs with age may be associated with reduced secretion of growth hormone with age.

Prolactin (PRL) secreted by the pituitary gland can act to stimulate an immune response. PRL membrane receptors are present on both human T, B lymphocytes and lymphoma cells; after hypophysis is removed, the mice with low immune function can partially recover normal immune function to a certain extent by supplementing PRL; neutralization of PRL with antibodies has been found to inhibit lymphocyte proliferation in a variety of cell lines.

Sex hormones are also sensitive to the immune system, and data indicate that there is a significant gender difference in the immune response between sexes due to differences in sex hormone levels.

(V) immune and endocrine systems

The immune system secretes cytokines, regulating the levels and functions of endocrine hormones, as shown in the table below.

In addition, the immune system can secrete a variety of hormones and neuropeptides like the classical endocrine organs. Has an important influence on the function of the neuroendocrine system. It has now been found that immune cells synthesize up to 10 or more neurotransmitters and hormones (see table below).

Note: hCG, human chorionic gonadotropin; NCDV is Newcastle disease virus; LPS: a lipopolysaccharide; EBV is EB virus; IL-1: interleukin-1; MLR: mixed lymphocyte reaction.

It can be seen that the cell surface of the neuro-immune-endocrine system demonstrates that the relevant receptor receives various information from the other. The central nervous system transmits information to cells of organs, receptors and motor systems through nerve fibers of the central nervous system, sends out instructions and controls the activities of organisms; but the information can be realized by auxiliary treatment and processing through a neuro-immune-endocrine network taking hypothalamus as a core; meanwhile, the functions of the nervous system are further exerted and regulated by processing and diffusing cell information through the wandering endocrine and immune cells and generating new information. Hormones, neurotransmitters, cytokines are common languages for the neuro-immune-endocrine network. The generation of nervous system lesions is necessarily related to the neural network.

Therefore, the universal nervous system chip is beneficial to simulating the nerve cells in a blood brain barrier in vivo, exploring the functions of the central nerve cells → the central nerve cells, the central nerve cells → the peripheral nerve cells, the peripheral nerve cells → the central nerve cells, the central nerve cells → the endocrine cells, the endocrine cells → the central nerve cells, the central nerve cells → the immune cells, the immune cells → the central nerve cells, and constructing a neural network, so that the physiological mechanisms of generation, transmission, function formation and regulation of various information of the nervous system can be known, the functions in the pathological process can be explored, clues and bases are provided for diagnosis and treatment and prognosis judgment of diseases of the nervous system, and new treatment strategies and targets are formed, thereby having great scientific significance and clinical application value.

Second, the concept of the invention

The growth mode and state of cells in the microstructure of human tissue and organs are different, for example, capillary endothelial cells are composed of a single layer of endothelial cells, a thin layer of basement membrane is wrapped outside the cells, and nerve cells grow under the three-dimensional condition, and the cells are closely connected with each other to form a three-dimensional whole. The traditional in vitro culture model can only form a cell growth form of only two dimensions or only three dimensions, and can not meet the requirements of in vitro two-dimensional and three-dimensional cross co-culture.

Organ microfluidic chips (organ on a chips) are a subset of microfluidic chips, and have been recognized by the industry as the mainstream platform for precise control of mammalian cells and their microenvironment. The micro-fluidic chip has the following advantages in constructing the simulated organ in vitro: 1. the micro-nano size of the unit component in the micro-fluidic chip can simultaneously accommodate molecules, cells, tissues and even organs; 2. the chip has a special fluid accurate control system, so that the chip can measure physical quantity, chemical quantity and biomass simultaneously and has the characteristics of small volume, low consumption, high flux and the like; 3. the chip can simultaneously culture a plurality of cells contained in the organ, and the spatial arrangement of the cells can imitate the physiological structure of the organ; 4. it can reconstruct the physiological environment of the organ in vivo, such as fluid shear force and signal molecule concentration gradient. The organ chip can simulate organs from three aspects of composition, structure and environment, and has high simulation degree. At present, the work of simulating various organs by applying a microfluidic technology is carried out, but the work of assembling a plurality of neurovascular units, simulating a nervous system and detecting the system in real time is still in a blank stage. The invention firstly simulates the nervous system in vitro and has great application prospect in basic biological research and medicine research and development centers.

Application of the invention (Universal nervous System chip)

The universal nervous system chip can simulate the real in-vivo conditions of various nerve cells, immune cells and endocrine cells, record potential current change, understand and control mechanisms, and can be used for research, research and development of neurophysiology, neuropsychology, pathology and pharmacology, and related food, materials and devices, and transformation and application customization services of clinical and healthy precise medicine. The diseases specifically involved are as follows:

the most common, critical and difficult cerebral diseases such as cerebral and spinal cord ischemia

Stroke is currently the most major disease endangering human health. Stroke is most commonly associated with cerebral ischemia-reperfusion injury. Can lead to consciousness, cognitive dysfunction, hemiplegia, language disorder and the like of a patient. Seriously affecting the quality of life of the patient. The function and damage mechanism of nerve cells in the infarct area and the penumbra area after cerebral ischemia are not completely clear, the ischemia process in vivo can be simulated through the nervous system chip, different cell types and the regulation and control effect of hypothalamus-pituitary on cerebral ischemia damage are analyzed simultaneously, the pathogenic mechanism and the passage related to cerebral ischemia are searched, the potential target point of treatment is found on the basis, and new medicaments are designed or developed and new treatment strategies are provided. Similarly, the most common, critical and distressed brain diseases also include smog, atretic syndrome, cerebral hemorrhage, coma, botanic people, epilepsy, dementia, diabetic encephalopathy, insomnia, anxiety, depression, schizophrenia, and the like.

(II) other representative important problems

1. Trauma of brain and spinal cord

Cerebrospinal trauma is after a primary injury, and secondary nerve damage is an important factor in exacerbating the condition. At the moment, the blood brain barrier of the injured part is opened, the whole body is in a stress state, and the immune system is inhibited to different degrees according to the severity and time of injury. Thus the neuro-immune-endocrine network is completely different from that in the normal physiological state. The current research on cerebrospinal trauma, especially the research on molecular mechanism, cannot completely simulate the complex environment, including blood brain barrier opening, endocrine system activation, immunosuppression and the like, but the animal experimental research is obviously greatly different from the human condition. The general nervous system chip can well simulate the cell environment, explore the change rule between nerve cells and a neural network under the condition of trauma, and provide a promising scheme for acute-phase treatment and long-term repair of nervous system injury. Meanwhile, the physiological change and the psychological demand of trauma patients can be paid more attention to, corresponding medicines or other auxiliary treatment means can be developed, and comprehensive and systematic treatment is provided for the trauma patients.

2. Tumor of brain and spinal cord

Cerebrospinal tumors, especially malignant tumors such as gliomas and metastases, are still a problem of neurological diseases at present. It has poor therapeutic effect. High-grade glioma or metastatic tumor is easy to relapse or disseminate no matter the surgical excision degree, and becomes a difficult problem in the field of neuroscience. The growth environment and biological characteristics of the tumor are obviously not comprehensive by simply culturing cells or simulating the growth environment and biological characteristics by animal experiments. Tumor cells grow in the complex environment of the nervous system, interact with normal nerve cells and are subjected to multiple regulation, so that the invasion, metastasis and growth capacities of the tumor cells are also related to multiple factors, and the tumor cells need to be observed and explored from the perspective of a neural network. The universal nervous system chip is applied, so that the real growth mode of tumor cells can be simulated, and the pathogenic key factors of the tumor cells can be searched; meanwhile, the kit helps to judge the sensibility of tumor recurrence, metastasis and dissemination, prognosis and radiotherapy and chemotherapy and also helps to develop novel chemotherapeutic drugs.

3. Brain and spinal cord function prediction and rehabilitation

The characteristics of the central nervous system diseases can affect the functions of the nervous system, and the vulnerability and difficult recovery of the functions of the nervous system bring great difficulties to the treatment of the diseases, and also become a challenge in the field of neuroscience. Meanwhile, the loss of nervous system functions, such as consciousness, cognition, movement, sensation and the like, causes great dysfunction to patients, reduces the quality of life, and brings heavy burden to the patients in treatment. How to evaluate and predict brain and spinal cord function? How effectively do rehabilitation? All the methods are established on the premise of knowing the specific mechanism of the neural network, are obtained by large data image collection and clinical follow-up and combining the general neural system chip and the molecular mechanism of the neural network, and form a basic and clinical dual network on the basis. The system needs to be analyzed, and together with the cooperation with rehabilitation medicine, the treatment is carried out aiming at the rehabilitation target, or corresponding medicines or auxiliary appliances are developed, relevant psychological excitation and guidance are developed, the dysfunction of the brain and the spinal cord caused by various reasons is improved, and the diagnosis and treatment level of the brain and the spinal cord diseases is improved.

4. Exploration of general anesthesia mechanism

The general anesthesia mechanism is one of the fields which are not known by human at present, and the exploration of the general anesthesia mechanism is a hot point for many years at home and abroad. The general anesthesia mechanism has important significance for accurately regulating and controlling the general anesthesia induction, the intraoperative maintenance and the awakening process of a patient in clinical anesthesia, and the oxygen supply of the intraoperative blood pressure, heartbeat, respiration, important organs of the whole body, particularly the oxygen supply of brain tissues and the prevention and treatment of the problem of postoperative cognitive dysfunction. The current adopted research means is mainly simple cell and animal experiments and meets the bottleneck, the universal nervous system chip can simulate the interaction relationship of human complex neural networks, so that the research on the general anesthesia molecular mechanism enters a brand new stage, a brand new thought is provided for finally overcoming the difficulty, and the development of novel general anesthesia medicaments is facilitated.

Disclosure of Invention

The invention provides a universal nervous system chip based on a microfluidic chip technology, which is used for carrying out in-vitro simulation on a nervous system for the first time and has great application prospect in basic biological research and medicine research and development centers.

In order to achieve the purpose, the invention adopts the technical scheme that:

a universal nervous system chip mainly comprises six independent neurovascular units which are closely related and connected with each other and are open to the outside, and the six independent neurovascular units can realize wide system coupling of nerve-immunity-endocrine-microecology.

The six independent neurovascular units comprise two annular units A, B and four rectangular units A, B, C, D, the two annular units A, B are symmetrically arranged, two rectangular units A, B are arranged between the ends of the two annular units, namely, the two annular units A, B and the two rectangular units A, B form an ellipse-like structure, and the center of the two annular units is provided with another two rectangular units C, D. The lower chip cavity of annular unit A, B and the lower chip cavity of rectangle unit A, B between connect through annular funnel microgroove, connect through rectangle funnel microgroove between the lower chip cavity of rectangle unit A, B, C, D, wherein, annular funnel microgroove distributes in annular unit lower chamber bottom surface, rectangle funnel microgroove distributes under two rectangle units between the chamber, funnel microgroove is used for the physical separation and the connection of neuron axon, makes to carry out information transfer between the neuron. The lower chips of the six independent units are communicated through the rectangular funnel micro-grooves and the annular funnel micro-grooves, and the porous membrane and the funnel micro-groove structure enable the six independent neurovascular units to be communicated with each other, so that functional continuity among multiple networks in a nervous system is simulated. The rectangular funnel micro-groove has the length of 50-100 μm, the width of 3-10 μm and the height of 3 μm; the micro-groove of the ring-shaped funnel has the length of 500-.

Each neurovascular unit comprises an upper layer chip, a lower layer chip and a middle porous membrane, and metabolites generated by cells cultured in the upper layer chip permeate into the lower layer chip through the middle porous membrane, so that cell non-contact information communication of an upper layer cell culture system and a lower layer cell culture system is realized. The upper chip comprises an upper inlet, an upper cavity and an upper outlet; the upper layer inlet and the upper layer outlet are communicated through the upper layer cavity. The upper layer cavity has a height of 50-100 μm, a length of 300-600 μm and a width of 100-300 μm. The lower chip comprises a lower inlet, a lower chamber and a lower outlet; the lower layer inlet and the lower layer outlet are communicated through the lower layer chamber. The lower chip chamber has a height of 100-. The upper chip chamber and the lower chip chamber of the six independent units are separated by an intermediate porous membrane. The porous film has a thickness of 5-15 μm, and pore diameters of 0.4 μm, 1 μm, 3 μm, and 5 μm. After the chip is integrally sealed, the upper layer chip cavity and the lower layer chip cavity are aligned in the center, and the upper layer cavity is slightly larger than the lower layer cavity.

The upper liquid level of the upper chip cavity of the six independent units is integrated with a TEER microelectrode array and an optical/acoustic signal input and output device, a micro-nano monitoring device such as molecular oxygen/glucose/pH is integrated between liquids, and the bottom surface of the upper chip cavity is integrated with a micro-nano monitoring device such as molecular oxygen/glucose/pH, a hydrogen peroxide sensor microelectrode array and an electric potential sensor microelectrode array; the TEER microelectrode array is used for detecting whether a blood brain barrier is complete, the optical/acoustic signal input and output device is used for performing photoelectric stimulation and signal detection on cells, the micro-nano monitoring devices such as molecular oxygen/glucose/pH and the like are used for detecting the concentration of molecular oxygen/glucose generated by cell metabolism and the pH in a culture solution, the hydrogen peroxide sensor microelectrode array is used for detecting hydrogen peroxide generated by cell metabolism and further monitoring the fate and state of the cells, and the potential sensor microelectrode array is used for detecting action potential generated by neurons and electroencephalogram-like field potential generated by neuron groups and further extracting information transmitted by the neuron network potential. A TEER microelectrode array, a molecular oxygen/glucose/pH micro-nano monitoring device and the like are integrated on the liquid surface of the lower chip cavity, a TEER microelectrode array, a molecular oxygen/glucose/pH micro-nano monitoring device and the like are integrated between liquids, and a hydrogen peroxide sensor microelectrode array and a potential sensor microelectrode array are integrated on the bottom surface of the lower chip cavity; the TEER microelectrode array is used for detecting whether a blood brain barrier is complete, the micro-nano monitoring devices such as molecular oxygen/glucose/pH and the like are used for detecting the concentration of molecular oxygen/glucose generated by cell metabolism and the pH in a culture solution, the hydrogen peroxide sensor microelectrode array is used for detecting hydrogen peroxide generated by cell metabolism and further monitoring the fate and state of cells, and the potential sensor microelectrode array is used for detecting action potentials generated by neurons and electroencephalogram-like field potentials generated by neuron groups and further extracting information transmitted by the neuron network potentials.

A universal nervous system chip, six neurovascular units can realize the wide association, inspection or contrast research, evaluation and prediction (including precise medical individualized clinical service and the like) of consciousness, sensation, cognition, physical and/or visceral movement and organ system nervous-immune-endocrine-microecology physiology, pathology, pharmacology, materials (including research and development materials of medicines, health products, foods and the like) and/or devices (including extensible instruments and equipment research and development).

When the universal nervous system chip is used, the middle layer porous membrane and the upper layer chip are sealed by irreversible oxygen plasma, and the chip is placed in an oven with the temperature of 60-100 ℃ to be heated for 10-30 minutes; then the lower chip 9 is directly sealed on the glass slide by plasma; and finally, coating a thin layer of PDMS polymer on the upper surface of the lower chip after plasma treatment, and directly aligning, bonding and curing the two layers of chips to form a closed two-layer co-culture chip. When the cells are planted, the brain capillary endothelial cells are planted on the upper surface of the porous membrane, and complete culture solution for the brain capillary endothelial cells is arranged above and below the porous membrane; when the endothelial cells of the cerebral capillaries grow over the upper surface of the porous membrane and form tight connection, inverting the device, and then planting the glial cell suspension on the lower surface of the porous membrane; when the brain capillary endothelial cells and the glial cells are co-cultured until the glial cells basically grow over the lower surface of the porous membrane, the stably cultured neurons are implanted into the lower cavity of the chip, so that an indirect co-culture system is formed.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention combines culture units of various cells by utilizing a method of integrating multilayer microfluidic chips, assembles various chip models on the same chip according to the structure of a human brain nervous system, establishes connection through a ring/rectangular funnel micro-groove, can simply, conveniently and quickly realize wide association, inspection or comparative research, evaluation and prediction (including precise medical individualized clinical service and the like) of consciousness, sensation, cognition, body and/or visceral movement whole body organ system nerve-immunity-endocrine-microecology physiology, pathology, pharmacology, materials (including research and development materials of medicines, health products, foods and the like) and/or devices (including extensible instruments, equipment research and development) and each independent unit can be effectively divided so as to be convenient for respective control and observation.

(2) The invention has independent cell culture chambers, and information acquisition and monitoring of related research and application can be widely realized through integration of different micro-nano devices in the chambers.

Drawings

FIG. 1 is a schematic diagram of the overall structure and a schematic diagram of a single unit structure of the present invention;

FIG. 2 is a top view of the overall structure of the present invention;

FIG. 3 is a schematic view of the structure of a microelectrode array of the present invention;

FIG. 4 is a side view of a partial structure of the present invention;

fig. 5 is a diagram of a prior art efferent system with instruction to a target mechanism.

In the figure: 1 upper chip entrance; 2, an upper chip outlet; 3 an upper chamber; 4, an upper chip; 5 middle porous membrane; 6 lower chip inlet; 7, a lower chip outlet; 8 a lower chamber; 9 a lower chip;

10 a ring unit A; 11 a ring unit B; 12 rectangular units A; 13 rectangular units B; 14 rectangular units C; 15 rectangular units D; 16 ring-shaped funnel micro-grooves; 17 rectangular funnel micro-grooves;

18 upper liquid level of the upper chamber; 19 the lower bottom surface of the upper chamber; 20 upper TEER microelectrode array; 21. 22, 23 an upper hydrogen peroxide sensor microelectrode array; 24. 25, 26 an upper layer electric potential sensor microelectrode array;

27 lower bottom surface of the lower chamber; 28 lower TEER microelectrode array; 29. 30, 31 a microelectrode array of a lower hydrogen peroxide sensor; 32. 33, 34 a microelectrode array of a lower layer potential sensor;

35. 36 rectangular unit middle layer porous membrane, annular unit middle layer porous membrane; 37. 38 inlet and outlet of upper chip of rectangular unit; 39. 40 rectangular unit lower chip inlet and outlet; 41. 42 inlets/outlets of upper and lower chips of the ring unit; 43. 44 brain capillary endothelial cells on the upper surface of the rectangular unit middle porous membrane and brain capillary endothelial cells on the upper surface of the annular unit middle porous membrane; 45. 46, 47 projection neurons, gray matter astrocytes and intermediate neurons in the lower chip of the rectangular unit; 48. 49 oligodendrocytes, white matter astrocytes in the lower chip of the annular unit; 50 side view of a toroidal funnel micro-trough connecting a rectangular unit with a toroidal unit.

C/N in FIG. 3 represents a counter electrode and a negative electrode; and R represents a reference electrode.

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and description, but not for the purpose of limiting the invention.

Example 1

A universal nervous system chip, as shown in fig. 1: a universal nervous system chip mainly comprises six independent neurovascular units, each unit has a similar structure and comprises an upper chip 4, a middle porous membrane 5 and a lower chip 9; the middle porous membrane 5 is provided with micropores, metabolites generated by cells cultured in the upper chip can permeate into the lower chip through the micropores, and cell non-contact information communication and mutual influence of the upper and lower cell culture systems can be realized.

The upper-layer chips of the six independent units respectively comprise an upper-layer chip inlet 1, an outlet 2 and an upper-layer cavity 3, and the upper-layer cavity 3 is used as a cell cavity; the inlet 1 and outlet 2 communicate through the cell chamber.

The lower-layer chip of the six independent units comprises a lower-layer inlet 6, an outlet 7 and a lower-layer chamber 8, and the lower-layer chamber 8 is used as a cell chamber; the inlet 6 and outlet 7 communicate through the cell chamber.

As shown in fig. 2: the six independent units comprise two annular units A10 and B11 and four rectangular units A12, B13, C14 and D15. The annular units A10 and B11 and the two rectangular units A12 and B13 form an ellipse-like shape, and the center of the ellipse-like shape is provided with the other two rectangular units C14 and D15. The lower chip chambers of the annular units A10 and B11 are connected with the lower chip chambers of the rectangular units A12 and B13 through an annular funnel micro-groove 17; the lower chip chambers of the rectangular units A12, B13, C14 and D15 are connected through a rectangular funnel micro-groove 18. The ring-shaped funnel micro-groove is positioned at the bottom of the lower chip chamber of the ring-shaped units A10 and B11; the annular funnel micro-groove is positioned between the bottoms of lower-layer chip chambers of rectangular units A12 and C14, between the bottoms of lower-layer chip chambers of rectangular units A12 and D15, between the bottoms of lower-layer chip chambers of rectangular units C14 and D15, between the bottoms of lower-layer chip chambers of rectangular units C14 and B13, and between the bottoms of lower-layer chip chambers of rectangular units D15 and B13.

As shown in fig. 3: the upper liquid level 18 of the upper-layer cavity of the six units is integrated with a TEER microelectrode array 20, and electrodes are arranged in parallel in a strip shape; the lower bottom surface 28 of the upper chamber is integrated with a hydrogen peroxide sensor microelectrode array 29-31 and an electric potential sensor microelectrode array 32-34, and the hydrogen peroxide microelectrode array and the electric potential sensor microelectrode array are arranged in a crossed and parallel mode; the lower bottom surface 19 of the lower layer cavity is integrated with a TEER microelectrode array 21, a hydrogen peroxide sensor microelectrode array 22-24 and an electric potential sensor microelectrode array 25-27, the TEER microelectrode array corresponds to the TEER microelectrode array on the upper liquid level of the upper cavity, and the hydrogen peroxide microelectrode array and the electric potential sensor microelectrode array are arranged in a crossed and parallel mode; C/N represents a counter electrode and a negative electrode; and R represents a reference electrode.

As shown in fig. 4: 51. 52, 53 and 54 are respectively a side view of a rectangular unit upper chip, a side view of a rectangular unit lower chip, a side view of a ring unit upper chip and a side view of a ring unit lower chip in the six units; 35. 36 are a rectangular unit interlayer porous membrane, a circular unit interlayer porous membrane, respectively; 37. 38 are the inlet and outlet of the upper chip of the rectangular unit respectively; 39. 40 are the inlet and outlet of the lower chip of the rectangular unit respectively; 41. 42 are the inlet/outlet of the upper and lower chips of the ring unit, respectively; 43. 44 are cerebral capillary endothelial cells on the upper surface of the rectangular unit intermediate porous membrane and cerebral capillary endothelial cells on the upper surface of the annular unit intermediate porous membrane respectively; 45. 46, 47 are projection neurons, gray matter astrocytes and intermediate neurons in the lower chip of the rectangular unit respectively; 48. 49 are oligodendrocyte and white matter astrocyte in the lower chip of the annular unit respectively; and 50 is a side view of the annular funnel micro-groove connecting the rectangular unit and the annular unit.

When the universal nervous system chip is used, firstly, irreversible oxygen plasma sealing is carried out on the middle porous membrane 5 and the upper chip 4, the chip is heated for 20 minutes in an oven at 80 ℃, then the lower chip 9 is directly plasma-sealed on a glass slide with a proper size, finally, a thin PDMS polymer layer is coated on the upper surface of the lower chip 9 after plasma treatment, and the two chips are directly aligned, bonded and cured to form a closed two-layer co-culture chip. When the cells are planted, the brain capillary endothelial cells are planted on the upper surface of the porous membrane, and complete culture solution for the brain capillary endothelial cells is arranged above and below the porous membrane; when the endothelial cells of the cerebral capillaries grow over the upper surface of the porous membrane and form tight connection, inverting the device, and then planting the glial cell suspension on the lower surface of the porous membrane; when the brain capillary endothelial cells and the glial cells are co-cultured until the glial cells basically grow over the lower surface of the porous membrane, the stably cultured neurons are implanted into the lower cavity of the chip, so that an indirect co-culture system is formed.

The invention establishes non-contact information communication between the upper layer and the lower layer of cells of each neurovascular unit through the middle porous membrane 5, establishes contact information transmission between different neurons in six neurovascular units through the annular funnel micro-groove 16 and the rectangular funnel micro-groove 17, and can be used as a good platform for researching the mutual relation of neural networks, drug screening and toxicity evaluation.

The above-mentioned embodiments only express the embodiments of the present invention, but not should be understood as the limitation of the scope of the invention patent, it should be noted that, for those skilled in the art, many variations and modifications can be made without departing from the concept of the present invention, and these all fall into the protection scope of the present invention.

Claims (6)

1. A universal nervous system chip is characterized in that the chip mainly comprises six independent neurovascular units which are closely related and connected with each other and are open to the outside, and the six independent neurovascular units can realize wide system coupling of nerve-immunity-endocrine-microecology; the six independent neurovascular units comprise two annular units and four rectangular units, the two annular units A, B are symmetrically arranged, two rectangular units A, B are arranged between the end parts of the two annular units, and the other two rectangular units C, D are arranged in the middle of an area enclosed by the two annular units and the two rectangular units; the lower chip chambers of the annular unit A, B and the rectangular unit A, B are communicated through an annular funnel micro-groove, the lower chip chambers of the rectangular unit A, B, C, D are communicated through a rectangular funnel micro-groove, and the funnel micro-groove is used for physical separation and connection of neuron axons to promote information transmission among the neurons;

each neurovascular unit comprises an upper chip, a lower chip and a middle porous membrane, metabolites generated by cells cultured in the upper chip permeate into the lower chip through the middle porous membrane, and cell non-contact information communication of an upper cell culture system and a lower cell culture system is realized; the upper chip comprises an upper inlet, an upper chamber and an upper outlet, and the upper inlet and the upper outlet are communicated through the upper chamber; the lower chip comprises a lower inlet, a lower cavity and a lower outlet, and the lower inlet is communicated with the lower outlet through the lower cavity; after the chip is integrally sealed, the centers of the upper layer cavity and the lower layer cavity are aligned, and the upper layer cavity is larger than the lower layer cavity;

the upper liquid surface of the upper chip cavity of the six independent neurovascular units is integrated with a TEER microelectrode array and an optical/acoustic signal input and output device, the inter-liquid is integrated with a molecular oxygen/glucose/pH micro-nano monitoring device, and the bottom surface is integrated with a molecular oxygen/glucose/pH micro-nano monitoring device, a hydrogen peroxide sensor microelectrode array and a potential sensor microelectrode array; the upper liquid surface of the lower chip cavity is integrated with a TEER microelectrode array and a molecular oxygen/glucose/pH micro-nano monitoring device, the middle liquid surface is integrated with the TEER microelectrode array and the molecular oxygen/glucose/pH micro-nano monitoring device, and the bottom surface is integrated with a hydrogen peroxide sensor microelectrode array and a potential sensor microelectrode array; the TEER microelectrode array is used for detecting whether a blood brain barrier is complete, the optical/acoustic signal input and output device is used for performing photoelectric stimulation and signal detection on cells, the molecular oxygen/glucose/pH micro-nano monitoring device is used for detecting the concentration of molecular oxygen/glucose generated by cell metabolism and the pH in a culture solution, the hydrogen peroxide sensor microelectrode array is used for detecting hydrogen peroxide generated by cell metabolism so as to monitor the fate and state of the cells, and the potential sensor microelectrode array is used for detecting action potentials generated by neurons and electroencephalogram-like field potentials generated by neuron groups and extracting information transmitted by the neuron network potentials.

2. The universal nervous system chip of claim 1, wherein the rectangular funnel micro-groove has a length of 50-100 μm, a width of 3-10 μm, and a height of 3 μm; the micro-groove of the ring-shaped funnel has the length of 500-.

3. The CNS chip as claimed in claim 1, wherein the upper chamber of the upper chip has a height of 50-100 μm, a length of 300-600 μm, and a width of 100-300 μm.

4. The CNS chip of claim 1, wherein the lower chamber of the lower chip has a height of 100-200 μm, a length of 300-600 μm, and a width of 100-300 μm.

5. The universal nervous system chip according to claim 1, wherein the porous film has a thickness of 5-15 μm and a pore size of 0.4 μm, 1 μm, 3 μm, 5 μm.

6. A method of using the universal nervous system chip of claim 1, comprising:

when in use, the middle porous membrane and the upper chip are sealed by irreversible oxygen plasma, and are heated in an oven at 60-100 ℃ for 10-30 minutes; then directly sealing the lower chip on the glass slide by plasma; finally, coating a layer of PDMS polymer on the upper surface of the lower chip after plasma treatment, aligning, bonding and curing the two chips to form a closed two-layer co-culture chip;

when the cells are planted, firstly, the brain capillary endothelial cells are planted on the upper surface of the porous membrane, and complete culture solution for the brain capillary endothelial cells is arranged on the upper and lower parts of the porous membrane; inverting the device when the endothelial cells of the cerebral capillaries grow over the upper surface of the porous membrane and form tight connection; then planting the glial cell suspension on the lower surface of the porous membrane; when the brain capillary endothelial cells and the glial cells are co-cultured until the glial cells basically grow over the lower surface of the porous membrane, the stably cultured neurons are implanted into the lower cavity of the chip, so that an indirect co-culture system is formed.

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