Organ chip and photosensitive protein activation method based on optogenetics

Abstract

The invention relates to the technical field of biomedicine, in particular to an organ chip based on optogenetics and a photosensitive protein activation method, the organ chip comprises an optical function chip and a near-infrared light source; the optical function chip is light-transmitting, at least two cell cavities for accommodating different organ modules are formed in the optical function chip, a light insulation layer is arranged on the side wall of each cell cavity, and a light converter is arranged in the inner side space of each light insulation layer; the light insulation layer prevents visible light generated by the light converter in the cell cavity from being transmitted to other cell cavities; the preparation material of the light converter comprises an up-conversion material; the near-infrared light source emits near-infrared light into the target cell cavity to excite the light converter in the target cell cavity to emit visible light with the target wavelength, the visible light with the target wavelength activates target photosensitive protein contained in the organ module in the target cell cavity, and the physiological reaction of the organ module is specifically regulated and controlled. According to the invention, accurate regulation and control of physiological reactions of different organ modules in the same organ chip can be realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an organ chip based on optogenetics and a method for activating a photosensitive protein. Background Art

[0002] Microfluidic chips that mimic the physiological function characteristics of a certain tissue or organ are called "organ-on-a-chip". Its goal is to build a human organoid model on a chip that can simulate the physiological or pathological characteristics of human tissues and organs through the cross-integration of technologies such as stem cells, organoids, biomaterials, tissue engineering, microfluidics and microfabrication. As a new model independent of two-dimensional cells and animals, it is used for new drug development and basic medical research. Compared with traditional models, organ chips have the advantages of high throughput, low cost, short cycle, and closer to the real physiological response of the human body. In particular, the organ chip model of multi-organ interaction relies on the microfluidic system to connect different cells with organ functions through circulating fluids. It can simulate the interaction between multiple organs / cells in vitro, while realizing multi-mode biological signal acquisition and more precise environmental regulation, showing unique advantages in simulating physiological processes, drug screening, disease modeling, etc.

[0003] Optogenetics combines optical technology with gene modification to precisely regulate the physiological responses of specific cells through illumination. It has high specificity and time resolution, is easy to operate, and can achieve electrophysiological regulation of cardiomyocytes and regulation of neurons, providing new tools and methods for the study of cardiovascular diseases and the nervous system. Traditional optogenetics requires inserting optical fibers into the animal body to conduct light stimulation in the vicinity of specific cells. The wavelength of visible light transmitted by the optical fiber is optional, and different wavelengths of visible light can activate different photosensitive proteins, correspondingly regulating the physiological responses of cells (excitation or inhibition).

[0004] The combination of these two emerging technologies, organ chips and optogenetics, has attractive application potential in the fields of basic medical research and drug development, but the integration of the two technologies is not simple. The three-dimensional cells or organ modules in the organ chip are extremely small tissues (micrometer level), and the position, intensity and range of light illumination are required to be more precise. Especially in multi-organ chips, how to use visible light to accurately stimulate one of the organs without affecting other organs, there is currently no feasible experimental plan. Secondly, as the research deepens, the experiment will not be satisfied with the application of a single photosensitive protein. Different photosensitive proteins are usually set on multiple organ modules on an organ chip, corresponding to the activation of light with different wavelengths. This requires optical technology to be able to meet the requirements of different wavelengths of light sources to accurately irradiate the position of cells expressing the corresponding photosensitive proteins without affecting other cells. There are also methods in the prior art that use complex optical systems and miniaturized optical equipment to achieve precise regulation of physiological responses of organ modules, but the cost of these instruments and equipment is extremely high and the technical requirements are also high. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides an organ chip based on optogenetics and a method for activating photosensitive proteins, which solves the technical problem that the prior art is difficult to accurately control the position and range of visible light illumination, has high technical barriers and costs, and therefore cannot accurately and differentially (specifically) regulate the physiological responses of organ modules, thus affecting the experimental results.

[0007] (II) Technical solution

[0008] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0009] In a first aspect, an embodiment of the present invention provides an organ chip based on optogenetics, the organ chip comprising: an optical function chip and a near-infrared light source;

[0010] The optical function chip is light-transmissive, and is provided with at least two cell cavities for accommodating different organ modules respectively. A light-isolating layer is provided on the side wall of each cell cavity, and a light converter is provided in the inner space of the light-isolating layer; the light-isolating layer is used to prevent the visible light generated by the light converter in the cell cavity from being transmitted to other cell cavities;

[0011] The near-infrared light source is used to emit near-infrared light of a preset wavelength into the target cell cavity, stimulating the light converter in the target cell cavity to emit visible light of a target wavelength. The visible light of the target wavelength is used to activate the target photosensitive protein contained in the organ module in the target cell cavity, thereby specifically regulating the physiological response of the organ module.

[0012] Optionally, the optical converter is:

[0013] A buffer solution containing upconversion material particles is pre-injected into the cell cavity; and / or,

[0014] A luminescent layer is pre-embedded inside the light-isolating layer of the cell cavity; the luminescent layer contains up-conversion material particles.

[0015] Optionally, when the photoconverter is a buffer solution containing upconversion material particles, the concentration of the upconversion material particles in the culture solution in the cell cavity is [10 μg / mL, 300 μg / mL], and the upconversion material particles are nanoparticles or polymer microspheres;

[0016] The nanoparticles are obtained by surface-modifying an upconversion material with a modifier; the modifier includes one or more of silicon dioxide, polyethylene glycol and sodium alginate, and the particle size range of the nanoparticles is [200nm, 500nm];

[0017] The polymer particles are obtained by coating the upconversion material with a polymer; the polymer includes one or more of polyethylene glycol, sodium alginate, dextran or chitosan, and the particle size range of the polymer particles is [10 μm, 50 μm];

[0018] When the light converter is a light-emitting layer, the particle size range of the up-conversion material particles in the light-emitting layer is [30 μm, 80 μm].

[0019] Optionally, the side wall of the cell cavity is vertically arranged, and the light-isolating layer is a circular fence arranged on the inner side wall of the cell cavity.

[0020] Optionally, an interaction channel for exchanging physiological signals between organ modules is provided between two cell chambers of the optical functional chip.

[0021] Optionally, the bottom of the first cell cavity of the optical functional chip is used to accommodate a first organ module, and the bottom of the second cell cavity of the optical functional chip is used to accommodate a second organ module. A straight-plate-shaped light-isolating layer is inserted between the first cell cavity and the second cell cavity. A plurality of first grooves penetrating the light-isolating layer are arranged on the side of the light-isolating layer close to the bottom of the first cell cavity and the second cell cavity to form an interaction channel connecting the first cell cavity and the second cell cavity, and the height of the first groove is in the micron level.

[0022] Optionally, the bottom of the first cell cavity of the optical functional chip is used to inoculate the first organ module, and the bottom of the second cell cavity of the optical functional chip is used to inoculate the second organ module;

[0023] An annular light-isolating layer is arranged on the side walls of the first cell cavity and the second cell cavity, and a second groove connecting the first cell cavity and the second cell cavity is arranged between the first cell cavity and the second cell cavity as an interaction channel.

[0024] Optionally, the optical functional chip is made of a material with high light transmittance; the light isolation layer is made of a material with high light transmittance;

[0025] The highly light-transmitting material comprises: one or more of polydimethylsiloxane, polymethyl methacrylate and quartz glass;

[0026] The light-isolating material includes: one or more of silicon carbide, graphene, carbon nanotubes and carbon powder.

[0027] In a second aspect, an embodiment of the present invention provides a method for activating a light-sensitive protein, the activation method comprising:

[0028] S10, inoculating different organ modules into at least two cell cavities of the organ chip respectively; a light-isolating layer is provided on the side wall of each cell cavity, and a light converter is provided in the inner space of the light-isolating layer; the light-isolating layer is used to prevent the visible light generated by the light converter in the cell cavity from being transmitted to other cell cavities;

[0029] S20. Use a near-infrared light source to emit near-infrared light of a preset wavelength into the target cell cavity, thereby stimulating the light converter in the target cell cavity to emit visible light of a target wavelength. The visible light of the target wavelength is used to activate the target photosensitive protein contained in the organ module in the target cell cavity, thereby specifically regulating the physiological response of the organ module.

[0030] Optionally, the optical functional chip is the optogenetics-based organ chip described in the first aspect.

[0031] (III) Beneficial effects

[0032] The organ chip based on optogenetics proposed in the present invention comprises an optical functional chip and a near-infrared light source; the optical functional chip is light-transmissive, and is provided with at least two cell cavities for accommodating different organ modules respectively, and a light-isolating layer is provided on the side wall of each cell cavity, and a light converter is provided in the inner space of the light-isolating layer; the light-isolating layer is used to prevent the visible light generated in the cell cavity from being transmitted to other cell cavities; the near-infrared light source is used to emit near-infrared light of a preset wavelength into the target cell cavity, and stimulate the light converter in the target cell cavity to emit visible light of a target wavelength, and the visible light of the target wavelength is used to activate the target photosensitive protein contained in the organ module in the target cell cavity, thereby specifically regulating the physiological response of the organ module.

[0033] The present invention combines optogenetics with organ chips, and prevents the visible light generated in the cell cavity from being transmitted to other cell cavities by setting a light-isolating layer on the side wall of each cell cavity, controlling the luminous position and range of visible light, so that the visible light generated by the photoconverter in different cell cavities does not affect each other, thereby achieving the differentiated and precise regulation of the physiological responses of different organ modules in the same organ chip. The present invention uses miniaturized and miniaturized photoconverters (upconversion materials) as light sources for activating photosensitive proteins to realize optogenetic regulation, without supporting complex optical systems and miniaturized optical equipment, through simple near-infrared light sources, remote emission of near-infrared light with strong penetration and low impact on cells and little damage can start the photoconverter, this photosensitive protein activation method is simple and easy, and the technical cost and technical difficulty are correspondingly reduced. The present invention provides an organ chip improved based on optogenetics technology, which is particularly suitable for experimental applications of multi-organ interaction chips, and can provide a new platform for more in-depth and complex biomedical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 A schematic diagram of the structure of an optogenetics-cardiac organ-on-a-chip provided in an embodiment;

[0035] Figure 2 A schematic diagram of the structure of an optogenetics-neuralized heart chip provided in the embodiments;

[0036] Figure 3 for Figure 2 JJ cross section;

[0037] Figure 4 for Figure 2 LL section diagram;

[0038] Figure 5 for Figure 2 A schematic diagram of the structure of the light-isolating layer in FIG.

[0039] Figure 6 for Figure 2 KK section view;

[0040] Figure 7 A schematic diagram of the structure of an optogenetics-heart-liver interaction chip provided in the embodiments;

[0041] Figure 8 for Figure 7 MM cross-section diagram in. DETAILED DESCRIPTION

[0042] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0043] In order to better understand the above technical solution, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0044] Embodiment 1

[0045] The embodiment of the present invention provides an organ chip based on optogenetics, which is used to specifically regulate the physiological response of at least two organ modules contained therein. The organ module refers to a cell tissue with organ function.

[0046] The organ chip includes: an optical function chip and a near-infrared light source.

[0047] The optical functional chip is light-transmissive and is provided with at least two cell cavities for accommodating different organ modules respectively. A light-isolating layer is provided on the side wall of each cell cavity, and a light converter is provided in the inner space of the light-isolating layer; the light-isolating layer is used to prevent the visible light generated by the light converter in the cell cavity from being transmitted to other cell cavities.

[0048] Specifically, the preparation material of the optical functional chip includes a high light-transmittance material; the preparation material of the light-isolating layer includes a light-isolating material; the high light-transmittance material includes: one or more of polydimethylsiloxane, polymethyl methacrylate and quartz glass; the light-isolating material includes: one or more of silicon carbide, graphene, carbon nanotubes and carbon powder.

[0049] The near-infrared light source is used to emit near-infrared light of a preset wavelength into the target cell cavity, stimulating the light converter in the target cell cavity to emit visible light of a target wavelength. The visible light of the target wavelength is used to activate the target photosensitive protein contained in the organ module in the target cell cavity, thereby specifically regulating the physiological response of the organ module.

[0050] Specifically, the optical converter may be configured as:

[0051] A buffer solution containing upconversion material particles is pre-injected into the cell cavity; and / or a luminescent layer is pre-embedded inside the light-isolating layer of the cell cavity; the luminescent layer contains upconversion material particles.

[0052] When the light converter is a buffer solution containing upconversion material particles, the concentration of the upconversion material particles in the culture solution in the cell cavity is [10μg / mL, 300μg / mL], and the upconversion material particles are nanoparticles or polymer microspheres. More preferably, the concentration of the upconversion material particles is [100μg / mL, 300μg / mL], so that the visible light power of the stimulated emission of the upconversion material is large enough to better meet the experimental requirements.

[0053] The nanoparticles are obtained by surface modification of the upconversion material with a modifier; the modifier includes one or more of silicon dioxide, polyethylene glycol and sodium alginate, and the particle size of the nanoparticles is in the range of [200nm, 500nm]. The upconversion material particles in the form of nanoparticles can be evenly dispersed around the cells of the organ module without entering the cells.

[0054] The polymer particles are obtained by coating the upconversion material with a polymer; the polymer includes one or more of polyethylene glycol, sodium alginate, dextran or chitosan, and the particle size of the polymer particles ranges from [10 μm to 50 μm]. The polymer particles have good biocompatibility.

[0055] In actual use, a buffer solution prepared by the above-mentioned nanoparticles or polymer particles can be selected according to specific experimental requirements. The buffer solution can be directly injected into the cell cavity of the organ chip, so that the upconversion material particles are suspended in the culture medium and directly contact the cells of the organ module. This method can be called the "injection method".

[0056] When the light converter is a light-emitting layer, the particle size range of the upconversion material particles in the light-emitting layer is [30 μm, 80 μm], and the upconversion material in this particle size range can ensure that it is evenly dispersed in the medium. The light-emitting layer can be made into a specific shape according to the shape of the cell cavity and embedded in the corresponding cell cavity when making the organ chip. This method can be called the "integration method".

[0057] In addition, an interaction channel for exchanging physiological signals between organ modules is provided between two cell chambers of the optical functional chip, so as to study the relationship between organs, such as the heart-brain axis, the gut-brain axis, etc. Specifically, the physiological signal can be an electrical signal transmitted through physical contact or a chemical signal transmitted through liquid. More specifically, the electrical signal can be an electrocardiogram signal, an electroencephalogram signal, an electromyogram signal, an electrodermal signal, etc.; the chemical signal can be a signal molecule secreted by the organ module, specifically, a substance such as hormones and neurotransmitters.

[0058] It should be noted that the up-conversion material contained in the light converter set in each cell cavity can be set according to the wavelength band of visible light that needs to be excited in the cell cavity. For example, if a cell cavity needs to convert near-infrared light into red light during an experiment, the up-conversion material contained in the up-conversion material particles contained in the light converter in the cell cavity can be configured as Y 0.78 VO 4 :0.2Yb, 0.02Ho. This upconversion material can emit red light with a wavelength range of 640 to 660nm under the irradiation of near-infrared light with a wavelength of 980nm. Similarly, other cell cavities of the organ chip can be set with upconversion material particles of different components according to experimental requirements.

[0059] In addition, it should be noted that the organ modules contained in the organ chip usually need to be genetically modified in advance. The currently mature adenovirus / lentivirus transfection or stem cell gene editing method can be used to achieve stable expression of photosensitive proteins in specific types of cells. Such genetically modified organ modules or cell tissues can be purchased as mature products through commercial channels. This embodiment does not involve improvements to the organ modules themselves.

[0060] This embodiment combines optogenetics with organ chips, and sets a photoconverter in the target cell cavity that can emit visible light in a specific band under near-infrared light irradiation, and sets a light-isolating layer on the side wall of the cell cavity to prevent the visible light emitted by the photoconverter in the target cell cavity from transmitting to other cell cavities, thereby controlling the luminous position and range of the visible light, so that the visible light generated by the photoconverters in different cell cavities does not affect each other, thereby achieving specific regulation of the physiological responses of different organ modules in the same organ chip.

[0061] This embodiment uses a miniaturized, micro-scale photoconverter (upconversion material) as a light source for activating photosensitive proteins to achieve optogenetic regulation. It does not require a complex optical system or miniaturized optical equipment. It uses a simple near-infrared light source and a non-contact method to remotely emit near-infrared light with strong penetration and harmless to cells from the outside of the organ chip to start the photoconverter. This remote and specific activation method of photosensitive proteins is simple and easy, and does not require the use of a precise optical instrument to directly irradiate the micron-level organ module. The technical cost and technical difficulty are reduced accordingly. The organ chip improved based on optogenetic technology provided in this embodiment is particularly suitable for experimental applications of multi-organ interaction chips, and can provide a new platform for more in-depth and complex biomedical research.

[0062] Embodiment 2

[0063] Based on the organ chip provided in Example 1, this example takes the optogenetics-cardiac organ chip as an example to explain in detail the specific structure, preparation process and corresponding photosensitive protein activation process of the organ chip.

[0064] The first step is to prepare the optical functional chip.

[0065] like Figure 1 As shown, the optogenetics-heart organoid chip is a three-layer structure, the first layer 1 is a highly light-transmitting glass sheet, and the third layer 3 is a highly light-transmitting glass sheet. The second layer 2 contains three cylindrical cell cavities A, cell cavity B, and cell cavity C. The side walls of the three cell cavities are vertical, and the light-isolating layer 4 is arranged on the inner side walls of the cell cavity to form a circular fence. Each cell cavity is provided with an independent inlet channel 5 and outlet channel 6, and the diameters of the inlet channel 5 and the outlet channel 6 are both [50μm, 100μm].

[0066] Specifically, the manufacturing process of the above optical functional chip is as follows:

[0067] Weigh the polydimethylsiloxane (PDMS) reagent and curing agent, mix them thoroughly in a ratio of 10:1, place the mixed PDMS prepolymer in a vacuum drying pot, and slowly evacuate until the small bubbles in the mixed liquid are completely discharged. Then pour the treated PDMS prepolymer into the first mold. The mold is based on the structure of the second layer structure, including three cell cavities with independent inlet and outlet channels.

[0068] At the same time, a light-shielding layer is prepared. In this embodiment, silicon carbide is used as the light-shielding material, and a PDMS reagent is used as the dispersion medium of silicon carbide. The PDMS reagent, curing agent and silicon carbide particles are mixed, and fully mixed and dispersed by ultrasound to form a PDMS prepolymer with a light-shielding function. The treated PDMS prepolymer with a light-shielding function is then poured into the second mold. The second mold is a circular ring that matches the inner wall of the cylindrical cell cavity according to the description of the light-shielding layer above.

[0069] A small amount of bubbles may be generated during the casting process of the first mold and the second mold, and vacuum treatment is required again. After the bubbles disappear completely, place the mold in a 75°C oven and let it stand for 3 hours to solidify the PDMS prepolymer. After solidification, separate the second layer and the light-isolating layer from the mold respectively. Clean the second layer, the light-isolating layer and the glass sheet B in an oxygen plasma cleaner and bond them together to complete the bonding. Finally, place the PDMS chip in anhydrous ethanol for ultrasonic cleaning for 10 minutes, and then dry it.

[0070] The second step is to seed the cells.

[0071] The cardiac organoids expressing the light-sensitive protein ChR2 were placed in cell chamber A for suspension culture. The light-sensitive protein ChR2 can be activated by blue light to control the excitation of cardiomyocytes in the cardiac organoids. The cardiac organoids expressing the light-sensitive protein NpHR were placed in cell chamber B for suspension culture. The light-sensitive protein NpHR can be activated by green light to control the inhibition of cardiomyocytes in the cardiac organoids. The cardiac organoids that do not express any light-sensitive proteins were inoculated in cell chamber C.

[0072] In the third step, a buffer solution containing upconversion material particles is injected into the cell cavity.

[0073] Select NaYF with a particle size of about 200 μm 4 :Yb,Tm@SiO 2 and NaYF 4 :Yb,Er@SiO 2 Upconversion material particles dissolved in phosphate buffer solution as photoconverters. NaYF 4 :Yb,Tm@SiO 2 Solution, the concentration of photoconverter in the culture medium reached [100μg / mL, 300μg / mL]. At the same time, NaYF was injected into the cell cavity B. 4 :Yb,Er@SiO 2 The photoconverter concentration in the culture medium reaches [100 μg / mL, 300 μg / mL]. The second layer structure is bonded and sealed to the glass sheet B to obtain an optogenetic organ chip.

[0074] Before gluing and sealing, holes need to be punched at the corresponding positions of glass sheet B to reserve the corresponding inlet and outlet channels of the cell cavity. Use near-infrared light with a wavelength of 980nm to irradiate the cell cavity A from the top or bottom. The upconversion material NaYF contained in the light converter in the cell cavity A 4 :Yb,Tm@SiO 2 The near-infrared light is converted into blue light, which specifically regulates the cardiomyocytes of the cardiac organoid in the cell cavity A and excites them. At the same time, near-infrared light with a wavelength of 980nm is irradiated into the cell cavity B from the top or bottom of the cell cavity B, and the photoconverter NaYF 4 :Yb,Er@SiO 2Convert near-infrared light into green light, and specifically regulate the cardiomyocytes of the cardiac organoids in the cell cavity B to inhibit them. There is no light converter in the cell cavity C, and the light-isolating layer arranged therein ensures that the cardiac organoids in the cell cavity C are not affected by external visible light, nor are they specifically controlled by near-infrared light. In this way, it is possible to achieve guided and precise regulation of the three physiological reactions (excitement, inhibition, and normal) of the cardiomyocytes in the cardiac organoids under the same environmental conditions, which can be used for comparative verification of biomedical experiments. Compared with the single function of exciting specific cells in the prior art, the organ chip of the present invention can achieve bidirectional physiological reaction regulation of excitation and inhibition of organ modules.

[0075] It should be noted that the organ chip provided in this embodiment can be specifically regulated by a near-infrared light source under a natural light environment. The visible light contained in the natural light of the environment has a low light intensity and will not have a significant impact on the organ module in the cell cavity.

[0076] Embodiment 3

[0077] Based on the organ chip provided in Example 1, this example takes the optogenetics-neuralized heart chip as an example to explain in detail the specific structure of the organ chip, its preparation process and the corresponding photosensitive protein activation process.

[0078] The first step is to prepare the upconversion materials.

[0079] Select laboratory synthesized compound Y 0.78 VO 4 :0.2Yb,0.02Ho as light converter, Y 0.78 VO 4 :0.2Yb,0.02Ho can be excited by a 980nm near-infrared light source and emit red light with a wavelength range of 640 to 660nm. 2 O 3 Sc 2 O 3 , Yb 2 O 3 、Ho 2 O 3 NH 3 VO 4 , according to the chemical formula Y 0.78 VO 4 :0.2Yb,0.02Ho calculate the weighing ratio of each raw material and weigh the raw materials, mix the raw materials evenly in a mortar, and then transfer them into a corundum crucible. Put the corundum crucible containing the mixed raw materials into a muffle furnace and calcine at 1000 degrees for 12 hours, then cool down naturally and take it out. The sample taken out is fully ball-milled in a ball mill, and after ball-milling, it is sieved through a 200-mesh standard sieve to obtain a Y0.78VO4:0.2Yb,0.02Ho powder sample with a particle size of less than 75 microns for use.

[0080] The second step is to prepare the optical functional chip.

[0081] like Figures 2 to 4 As shown, the optogenetics-neuralized heart chip includes a stacked upper chip 7 and a lower chip 8, the lower chip 8 includes a cell cavity C and a cell cavity D, the cell cavity C and the cell cavity D are both pools with inverted trapezoidal cross sections, and the bottom area of ​​the pool is smaller than the top area. A straight plate-shaped light-isolating layer 4 is inserted between the cell cavity C and the cell cavity D, as shown in FIG. Figure 5 and 6 As shown, the side of the light-isolating layer 4 close to the bottom of the cell cavity C and the cell cavity D is provided with a plurality of first grooves 9 penetrating the light-isolating layer to form an interaction channel connecting the cell cavity C and the cell cavity D. The height of the first groove 9 is micrometer level. The light-emitting layer 10 can be arranged at any position in the cell cavity C. Preferably, as Figure 2 As shown, the light-emitting layer 10 is a straight plate of similar size to the light-isolating layer 4, and is arranged in parallel in the cell cavity C at a position away from the light-isolating layer 4, so as to further reduce the influence of the visible light generated by the light-emitting layer 10 on the cell cavity D. Slots for inserting the light-isolating layer 4 and the light-emitting layer 10 are also reserved on the upper chip 7 and the lower chip 8, and each cell cavity is also independently provided with a liquid inlet channel 5 and a liquid outlet channel 6.

[0082] Similar to the preparation of the second layer structure in Example 2, weigh the PDMS reagent and curing agent, mix them thoroughly in a ratio of 10:1, place the mixed PDMS prepolymer in a vacuum drying pot, and slowly evacuate until the small bubbles in the mixed liquid are completely discharged. After that, pour the treated PDMS prepolymer into the mold. The molds correspond to the preparation of the upper chip (independent inlet and outlet channels and cell cavities C, D), the lower chip (cell cavities C, D) and the slots for inserting the light-isolating layer and the light-emitting layer. A small amount of bubbles may be generated during the casting process of the mold, and it is necessary to vacuumize again. After the bubbles disappear completely, place the mold in a 75°C oven and let it stand for 3 hours to cure the PDMS prepolymer. After curing, separate the upper chip and the lower chip from the mold. The upper chip and the lower chip are ultrasonically cleaned in anhydrous ethanol for 10 minutes, and then dried.

[0083] Specifically, the light-shielding layer of this embodiment is a PDMS sheet with multiple gaps (first grooves) with a height of 0.5 to 2 nm at the bottom. When preparing, silicon carbide particles need to be added before the PDMS reagent and curing agent are mixed, and then fully mixed and dispersed by ultrasound to form a PDMS prepolymer with light-shielding function, which forms a cell contact channel barrier after curing. Similarly, when preparing the luminous layer, Y particles with a particle size of less than 75 microns need to be added before the PDMS reagent and curing agent are mixed. 0.78 VO 4: 0.2Yb, 0.02Ho powder (upconversion material), fully mixed and dispersed by ultrasound, become PDMS prepolymer with luminescent function, and form a luminescent layer after curing. Figure 2 , embed the light-isolating layer and the light-emitting layer into the slots reserved for the lower chip according to the positional relationship mentioned above, and can also be directly glued to the corresponding positions when necessary.

[0084] The third step is to seed the cells in the cell chamber.

[0085] Dopaminergic neuron cells expressing the photosensitive protein ChrimsonR were inoculated at the bottom of cavity C, and cardiomyocytes (iPS-CMs) differentiated from human induced pluripotent stem cells were inoculated in cavity D. Dopaminergic neuron cells can be activated by red light to induce the excitation of neuron cells. After inoculation, dopaminergic neuron cells can adhere to the wall and grow tree-like axons that cover the bottom of cavity C, and extend to the interaction channel and even cavity D. Similarly, cardiomyocytes can grow adherently after inoculation in cavity D, and they are short cylindrical with branches that cover the bottom of cavity D. Some cell branches contact the axons of dopaminergic neuron cells in the gap (first groove) of the interaction channel, forming a physical connection between the two.

[0086] After inoculating the cells, the upper chip and the lower chip inoculated with cells are bonded together to obtain an optogenetic-neuralized heart chip. By irradiating the cells with near-infrared light (wavelength of 980nm) that does not damage the cells, the light converter in the light-emitting layer on one side of the cell cavity C can be activated to convert the near-infrared light into red light, specifically regulating the dopaminergic neurons in the cell cavity C to make them excited. The light-isolating layer has a light-shielding function, and the red light in the cell cavity C cannot enter the cell cavity D, and the near-infrared light will not specifically activate the cardiomyocytes (iPS-CMs) in the cell cavity D. The cardiomyocytes in the cell cavity D will only be affected by the dopaminergic neurons that are physically connected to them. In this way, the purpose of regulating iPS-CMs can be achieved by controlling the excitation of dopaminergic neurons through light stimulation.

[0087] Embodiment 4

[0088] Based on the organ chip provided in Example 1, this example takes the optogenetics-heart-liver interaction chip as an example to explain in detail the specific structure of the organ chip, its preparation process and the corresponding photosensitive protein activation process.

[0089] The first step is to prepare the optical functional chip.

[0090] like Figure 7As shown, the optogenetics-heart-liver interaction chip has a three-layer structure, including a first layer 11, a second layer 12 and a third layer 13 stacked in sequence, the first layer 11 is a glass sheet, the second layer 12 is provided with a cell cavity E, a cell cavity F, an independent liquid inlet channel 5, and an independent liquid outlet channel, and the second layer 12 and the third layer 13 are provided with a second groove 14 connecting the cell cavity E and the cell cavity F. The cell cavity E and the cell cavity F are both cylindrical, and the bottoms of the cell cavity E and the cell cavity F are connected through the interaction channel formed by the second groove 14 on the second layer 12 and the third layer 13. The inner wall of the cell cavity E is sequentially provided with an annular light-emitting layer 10 and a light-isolating layer 4 from the inside to the outside, and the inner wall of the cell cavity F is provided with a light-isolating layer 4. A sampling port 15 can also be led out on the interaction channel connecting the cell cavity E and the cell cavity F. Based on this setting, the culture media in the cell cavity E and the cell cavity F are connected, and the secretions of the organ modules in the cell cavity E and the cell cavity F are communicated through the culture media, that is, the organ modules in the cell cavity E and the cell cavity F influence each other through chemical and biological factors, and the culture media samples of both can be obtained through the sampling port 15.

[0091] In the same manner as in Example 2, the second chip, the third chip, the light-shielding layer and the light-emitting layer are prepared according to the shapes described in this example. The difference from Example 2 is that the up-conversion material particles used in the light-emitting layer of this example are composed of NaYF 4 :Yb,Tm@SiO 2 (Photoconverter) that can be excited by a 980nm laser to emit light.

[0092] The cleaned and dried light-isolating layer and the light-emitting layer are sequentially embedded in the cell cavity E, with the light-isolating layer being located outside the light-emitting layer. Only the light-isolating layer is embedded on the inner wall of the cell cavity F. Then the second layer and the third layer are aligned and bonded.

[0093] In the second step, cells are seeded in the cell chamber.

[0094] iPS-CMs expressing the photosensitive protein ChR2 are inoculated in the cell cavity E. The photosensitive protein ChR2 can be activated by blue light, inducing the excitation of iPS-CMs cells. Hepatocytes differentiated from human induced pluripotent stem cells are inoculated in the cell cavity F. After inoculating the cells, the second and first layers of glass slides are bonded together to obtain an optogenetics-heart-liver interaction chip. Before bonding the glass slides, holes need to be punched at the locations of the independent inlet and outlet channels and the sampling ports of the interaction channels.

[0095] By irradiating iPS-CMs with near-infrared light without damaging them, the photoconverter in the luminous fence A on one side of the cell cavity A is activated, converting the near-infrared light into blue light, specifically regulating the iPS-CMs in the cell cavity A to make them excited. The biological and chemical factors secreted by the excited iPS-CMs affect the liver cells in the cell cavity B through the interaction channel. In this way, the purpose of regulating liver cells can be achieved by controlling the excitement of iPS-CMs through light stimulation.

[0096] Embodiment 5

[0097] The embodiment of the present invention provides a method for activating a photosensitive protein, which is used to regulate the physiological response of an organ module containing the photosensitive protein. The optical functional chip is the organ chip based on optogenetics as described in embodiments 1 to 4. The activation method comprises:

[0098] S10, inoculating different organ modules into at least two cell cavities of the organ chip respectively; a light-isolating layer is provided on the side wall of each cell cavity, and a light converter is provided in the inner space of the light-isolating layer; the light-isolating layer is used to prevent the visible light generated by the light converter in the cell cavity from transmitting to other cell cavities.

[0099] S20. Use a near-infrared light source to emit near-infrared light of a preset wavelength into the cell cavity, thereby stimulating the light converter in the cell cavity to emit visible light of a target wavelength. The visible light of the target wavelength is used to activate the target photosensitive protein contained in the organ module in the cell cavity, thereby specifically regulating the physiological response of the organ module.

[0100] The activation method of the photosensitive protein provided in this embodiment is that the organ chip used is provided with a photoconverter in the target cell cavity that can emit visible light of a specific band under remote near-infrared light irradiation, and a light-isolating layer is provided on the side wall of the cell cavity to prevent the visible light emitted by the photoconverter in the target cell cavity from transmitting to other cell cavities, thereby controlling the luminous position and range of the visible light, so that the visible light generated by the photoconverters in different cell cavities does not affect each other, thereby achieving specific regulation of the physiological responses of different organ modules in the same organ chip. This remote and specific activation method of the photosensitive protein is simple and easy to implement, and does not require the use of a precise optical instrument to directly irradiate the micron-level organ module, and the technical cost and technical difficulty are correspondingly reduced. It is particularly suitable for the experimental application of multi-organ interaction chips, and can provide a new platform for more in-depth and complex biomedical research.

[0101] The present invention is described with reference to the flowcharts and / or block diagrams of the methods, devices (systems) and computer program products according to the embodiments of the present invention. It should be understood that each process and / or box in the flowchart and / or block diagram, as well as the combination of the processes and / or boxes in the flowchart and / or block diagram, can be implemented by computer program instructions.

[0102] It should be noted that in the claims, any reference numerals placed between brackets shall not be construed as limiting the claims. The word "comprising" does not exclude the presence of components or steps not listed in the claims. The word "a" or "an" preceding a component does not exclude the presence of a plurality of such components. The invention may be implemented by means of hardware comprising several different components and by means of a suitably programmed computer. In the claims enumerating several means, several of these means may be embodied by the same hardware. The use of the words first, second, third, etc., is for convenience of expression only and does not indicate any order. These words may be understood as part of the component name.

[0103] In addition, it should be noted that, in the description of this specification, the description of the terms "one embodiment", "some embodiments", "embodiment", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0104] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments after knowing the basic creative concept. Therefore, the claims should be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0105] Obviously, those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention should also include these modifications and variations.

Claims

1. An organ chip based on optogenetics, characterized in that: The organ chip comprises: an optical function chip and a near-infrared light source; The optical function chip is light-transmissive, and is provided with at least two cell cavities for accommodating different organ modules respectively. A light-isolating layer is provided on the side wall of each cell cavity, and a light converter is provided in the inner space of the light-isolating layer; the light-isolating layer is used to prevent the visible light generated by the light converter in the cell cavity from being transmitted to other cell cavities; the preparation material of the light converter includes an up-conversion material; The near-infrared light source is used to emit near-infrared light of a preset wavelength into the target cell cavity, stimulating the light converter in the target cell cavity to emit visible light of a target wavelength. The visible light of the target wavelength is used to activate the target photosensitive protein contained in the organ module in the target cell cavity, thereby specifically regulating the physiological response of the organ module.

2. The organ chip according to claim 1, characterized in that: The optical converter is: A buffer solution containing upconversion material particles is pre-injected into the cell cavity; and / or, A luminescent layer is pre-embedded inside the light-isolating layer of the cell cavity; the luminescent layer contains up-conversion material particles.

3. The organ chip according to claim 2, characterized in that: When the light converter is a buffer solution containing upconversion material particles, the concentration of the upconversion material particles in the culture solution in the cell cavity is [10 μg / mL, 300 μg / mL], and the upconversion material particles are nanoparticles or polymer microspheres; The nanoparticles are obtained by surface-modifying an upconversion material with a modifier; the modifier includes one or more of silicon dioxide, polyethylene glycol and sodium alginate, and the particle size range of the nanoparticles is [200nm, 500nm]; The polymer particles are obtained by coating the upconversion material with a polymer; the polymer includes one or more of polyethylene glycol, sodium alginate, dextran or chitosan, and the particle size range of the polymer particles is [10 μm, 50 μm]; When the light converter is a light-emitting layer, the particle size range of the up-conversion material particles in the light-emitting layer is [30 μm, 80 μm].

4. The organ chip according to claim 1, characterized in that: The side wall of the cell cavity is vertically arranged, and the light-isolating layer is a circular fence arranged on the inner side wall of the cell cavity.

5. The organ chip according to claim 1, characterized in that: An interaction channel for exchanging physiological signals between organ modules is provided between two cell cavities of the optical functional chip.

6. The organ chip according to claim 5, characterized in that: The bottom of the first cell cavity of the optical functional chip is used to accommodate the first organ module, and the bottom of the second cell cavity of the optical functional chip is used to accommodate the second organ module. A straight-plate-shaped light-isolating layer is inserted between the first cell cavity and the second cell cavity. A plurality of first grooves penetrating the light-isolating layer are arranged on the side of the light-isolating layer close to the bottom of the first cell cavity and the second cell cavity to form an interaction channel connecting the first cell cavity and the second cell cavity, and the height of the first groove is in the micron level.

7. The organ chip according to claim 5, characterized in that: The bottom of the first cell cavity of the optical functional chip is used for inoculating the first organ module, and the bottom of the second cell cavity of the optical functional chip is used for inoculating the second organ module; An annular light-isolating layer is arranged on the side walls of the first cell cavity and the second cell cavity, and a second groove connecting the first cell cavity and the second cell cavity is arranged between the first cell cavity and the second cell cavity as an interaction channel.

8. The organ chip according to claim 1, characterized in that: The preparation material of the optical functional chip includes a highly light-transmitting material; the preparation material of the light-isolating layer includes a light-isolating material; The highly light-transmitting material comprises: one or more of polydimethylsiloxane, polymethyl methacrylate and quartz glass; The light-isolating material comprises: one or more of silicon carbide, graphene, carbon nanotubes and carbon powder.

9. A method for activating a photosensitive protein, characterized in that: The activation method includes: S10, inoculating different organ modules into at least two cell cavities of the organ chip respectively; a light-isolating layer is provided on the side wall of each cell cavity, and a light converter is provided in the inner space of the light-isolating layer; the light-isolating layer is used to prevent the visible light generated by the light converter in the cell cavity from being transmitted to other cell cavities; the preparation material of the light converter includes an upconversion material; S20. Use a near-infrared light source to emit near-infrared light of a preset wavelength into the target cell cavity, thereby stimulating the light converter in the target cell cavity to emit visible light of a target wavelength. The visible light of the target wavelength is used to activate the target photosensitive protein contained in the organ module in the target cell cavity, thereby specifically regulating the physiological response of the organ module.

10. The activation method according to claim 9, characterized in that: The optical functional chip is an organ chip based on optogenetics as described in any one of claims 1 to 8.

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