CN218465834U

CN218465834U - Organ chip for on-line light transparency and on-line observation

Info

Publication number: CN218465834U
Application number: CN202221796100.6U
Authority: CN
Inventors: 朱; 俞婷婷; 钟响
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2022-07-06
Filing date: 2022-07-06
Publication date: 2023-02-10
Anticipated expiration: 2032-07-06

Abstract

The utility model belongs to organ chip, biomedical engineering, tissue optics field, more specifically relates to an organ chip that is used for transparent and the on-line observation of online light. This organ chip stacks up from bottom to top and is provided with transparent stratum basale, first pattern layer, second pattern layer and transparent apron layer, wherein the second through-hole in the second pattern layer uses as the observation hole, first through-hole in the first pattern layer uses as the cultivation hole of artifical micro-organ, first through-hole and second through-hole position one-to-one and be linked together, the separation of cultivateing hole and observation hole has been realized in the cooperation each other, be favorable to reducing the medium complexity between camera lens and sample in order to improve observation quality. The organ chip can acquire fine structure information of the organ chip and the artificial micro-organ with high precision and high flux, can be suitable for online optical transparency and online observation of the organ chip, and does not influence the survival state of the artificial micro-organ.

Description

Organ chip for on-line light transparency and on-line observation

Technical Field

The utility model belongs to organ chip, biomedical engineering, tissue optics field, more specifically relates to an organ chip that is used for on-line light transparent and on-line observation.

Background

The organ chip is a new scientific technology which realizes the simulation of organism organ functions by culturing cells in an in vitro chip. By means of ingenious bionic design, the organ chip can simulate the form and functions of a real organ to a certain extent, can provide a model similar to a real organism for medical, pharmaceutical and life science researches, and provides data for substituting animals and clinical experiments for researches such as drug research and screening, disease model construction and the like. Various organ chips have been developed and applied to specific clinical studies.

The research and development of the organ chip can not carry out accurate three-dimensional measurement on the artificial micro-organs in the chip. Traditional medical imaging techniques such as CT, MRI, etc. can achieve three-dimensional imaging measurement of real organs, but these methods have too low resolution to be applied to imaging measurement on miniaturized chips. The optical microscopic imaging technology provides an important means for acquiring structural information of tissues and organs with high resolution. In order to acquire three-dimensional structural information of biological tissues, a traditional method mostly adopts a tissue section mode, a sample is firstly cut into slices of several micrometers to dozens of micrometers, and then an image with cell and even subcellular resolution is acquired through a fluorescence microscope. And the structure of a large amount of non-biological tissues exists in a chip of partial organs, the slicing method is obviously not suitable. Another method is to use an optical sectioning microscope, such as a confocal laser scanning microscope, a two-photon microscope, a fluorescence-illuminated sheet microscope, etc., or other tomographic imaging techniques, such as OCT, etc., to obtain fine structural information of the target.

However, the organ chip has a multi-layer complex structure, and the artificial micro-organ cultured therein often has a certain thickness, and due to the mismatch of refractive indexes between the components, the propagation path of light in the sample is shifted towards various directions, and a strong scattering tendency is shown, so that the imaging depth of the optical imaging system is limited, further the imaging quality of the tissue is gradually reduced along with the increase of the depth, and the difficulty of high-quality imaging of the organ chip is increased. In addition, the existing organ chip usually washes the micro-organs in the microfluidic channel in one direction, and the shearing force of the fluid can influence the physiological activity of the artificial micro-organs.

When the existing organ chip is used for high-resolution and high-flux observation, a plurality of problems exist, and the problems need to be solved urgently.

SUMMERY OF THE UTILITY MODEL

To prior art's defect, the utility model aims to provide an organ chip for online optical transparency and online observation to solve the technical problem that the formation of image quality is not good enough, micro-organ physiological activity that prior art organ chip exists when online optical transparency and observe artifical micro-organ can not guarantee etc.

In order to achieve the above object, the present invention provides an organ chip for online optical transparency and online observation, which is stacked from bottom to top with a transparent substrate layer, a first pattern layer, a second pattern layer and a transparent cover plate layer; wherein:

the first pattern layer contains a chip pattern of the organ chip, and the chip pattern comprises a micro-fluidic main channel and a plurality of first through holes which are dispersedly arranged on the micro-fluidic main channel and communicated with the micro-fluidic main channel;

the pattern contained in the second pattern layer comprises a plurality of second through holes, and the plurality of second through holes in the second pattern layer are in one-to-one correspondence with and communicated with the plurality of first through holes in the first pattern layer;

the bottom of the second pattern layer and the top of the transparent substrate layer are respectively connected with the top and the bottom of the microfluidic main channel, so that the microfluidic main channel becomes a closed channel;

and two ends of the second pattern layer are arranged in a protruding manner relative to the transparent cover plate layer, and a main channel liquid inlet hole and a main channel liquid outlet hole which are communicated with the microfluidic main channel are respectively arranged at two ends of the second pattern layer.

Preferably, a middle channel is further arranged between every two first through holes on the first pattern layer; the protruding parts at the two ends of the second pattern layer are also provided with a middle channel liquid inlet hole and a middle channel liquid outlet hole.

Preferably, the material of the first pattern layer and the second pattern layer is PDMS.

Preferably, the first through hole and the second through hole have the same cross-sectional shape, are circular or polygonal through holes, and have a U-shaped bottom at the bottom.

Preferably, a connection layer is further disposed between the transparent substrate layer and the first pattern layer, the connection layer is made of PDMS, and the hardness of the PDMS material used in the connection layer is less than that of the PDMS material used in the first pattern layer; the bottom of the second pattern layer and the top of the connecting layer are respectively connected with the top and the bottom of the micro-fluidic main channel, so that the micro-fluidic main channel becomes a closed channel.

Preferably, the thickness of the connection layer is in the range of 5-20 μm.

Preferably, the thickness of the first pattern layer is 1.5 ± 0.1mm, and the thickness of the second pattern layer is 100 ± 10 μm.

Preferably, the transparent substrate layer is a glass slide or a cover slip; the transparent cover plate layer is a glass slide or a cover glass.

Preferably, the main channel liquid inlet hole, the main channel liquid outlet hole, the middle channel liquid inlet hole and the middle channel liquid outlet hole are all arranged in a boss mode.

Generally, through the utility model discloses above technical scheme who conceives compares with prior art, has following beneficial effect:

(1) The utility model provides a pair of organ chip, range upon range of transparent stratum basale from bottom to top, first pattern layer, second pattern layer and transparent apron layer of being provided with, wherein the second through-hole in the second pattern layer uses as the observation hole, first through-hole in the first pattern layer uses as the cultivation hole of artifical micro-organ, first through-hole and second through-hole position one-to-one and be linked together, mutually support the separation that has realized cultivateing hole and observation hole, be favorable to reducing the medium complexity between camera lens and sample in order to improve the observation quality. The organ chip can acquire the fine structure information of the organ chip and the artificial micro-organ with high precision and high flux, can be suitable for online transparent and online observation of the organ chip, and does not influence the survival state of the artificial micro-organ.

(2) The utility model provides a pair of organ chip seals through setting up of just putting opening, ultra-thin PDMS and slide, has realized high resolution's optical imaging. In the preferred embodiment, the U-shaped bottom surface is formed by surface tension to help the cells aggregate into spheres. Therefore, the method can acquire the fine structure information of the organ chip and the artificial micro-organ with high precision and high flux, is suitable for the online transparent and online observation of the organ chip, and does not influence the living state of the artificial micro-organ. Meanwhile, a transparent special intermediate channel is arranged in the chip structure, the transparent reagent is circularly introduced into the intermediate channel, the transparent reagent in the intermediate channel slowly enters the culture chamber in the chip by utilizing the diffusion effect, and the influence of the fluid shearing force on the artificial micro-organ in the culture chamber can be reduced.

(3) The utility model discloses the organ chip that provides in some embodiments adopts PDMS to form micro-fluidic channel, cultivates the hole and observes the hole for chip subject material, utilizes the soft lithography mode, utilizes surface tension to form U type culture hole, adopts the design of cultivateing the hole and observing the hole separation simultaneously, has improved observation quality effectively.

(4) The utility model utilizes the soft lithography technology to obtain the chip mold and uses PDMS to manufacture and assemble the chip; then, introducing a transparentizing reagent by using the chip fluid system; and (3) carrying out imaging detection by using a fluorescence microscope or other tomography technologies to obtain the three-dimensional structure information of the artificial micro-organ. The utility model provides an organ chip design of the online live body light transparent method of adaptation has greatly improved the flux that sample treatment and formation of image were surveyd. Therefore, the utility model discloses effectively combine with multiple imaging technique, provide high-resolution, high flux solution for the three-dimensional structure information who acquires artifical micro-organ.

Drawings

Fig. 1 is a schematic diagram of the layered structure of an organ chip according to some embodiments of the present invention.

Fig. 2 shows a chip pattern of a first pattern layer, i.e., a culture layer, according to some embodiments of the present invention.

Fig. 3 illustrates the pattern of a second patterned layer, i.e., a viewing layer, in some embodiments of the invention.

Fig. 4 is a schematic diagram of the whole structure of the organ chip according to some embodiments of the present invention.

Fig. 5 is a schematic view of a layered structure of an organ chip provided with a connection layer according to some embodiments of the present invention.

Fig. 6 is a comparison of the micro-organ matching the tumor on the chip before and after the organ chip is transparent on line according to some embodiments of the present invention.

The same reference numbers will be used throughout the drawings to refer to the same or like elements or structures, wherein:

1-a transparent substrate; 2-a first pattern layer; 21-a microfluidic main channel; 22-a first via; 23-an intermediate channel; 3-a second pattern layer; 31-a second via; 4-a transparent cover plate layer; 5-a main channel liquid inlet hole; 6-main channel liquid outlet hole; 7-liquid inlet hole of the middle channel; 8-liquid outlet holes of the middle channel; 9-a tie layer; 10-boss.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the invention.

The utility model provides an organ chip suitable for online light is transparent and online observation, through organ chip structural design, let in transparent reagent in this organ chip and can be in order to carry out online transparency to artifical micro-organ, and can directly adopt confocal formation of image, two-photon formation of image or the microscopic formation of image of slide fluorescence come online observation organ chip after transparent, adopt the culture solution to resume this artifical micro-organ after the observation is finished, can drop into operation once more after finishing resuming, can keep artifical micro-organ live state all the time.

As shown in fig. 1 and 4, an organ chip suitable for on-line transparency and on-line observation provided in some embodiments of the present invention is provided with a transparent substrate layer 1, a first pattern layer 2, a second pattern layer 3, and a transparent cover layer 4, which are stacked from bottom to top; wherein:

the first pattern layer 2 contains a chip pattern of the organ chip. In some embodiments, the chip pattern of the first pattern layer 2 is as shown in fig. 2, and the chip pattern includes a microfluidic main channel 21 and a plurality of first through holes 22 dispersedly arranged on the microfluidic main channel 21 and communicated with the microfluidic main channel 21.

The pattern of the second pattern layer 3 comprises a plurality of second through holes 31, and the plurality of second through holes 31 in the second pattern layer 3 are in one-to-one correspondence and communication with the plurality of first through holes 22 in the first pattern layer 2; the pattern of the second patterned layer 3 is shown in fig. 3. The overall structure of the organ chip is schematically shown in FIG. 4. During operation, second through-hole 31 in the second pattern layer 3 uses as the observation hole, and first through-hole 22 in the first pattern layer 2 uses as the cultivation hole of artifical micro-organ, and first through-hole 22 and second through-hole 31 position one-to-one and be linked together, mutually support and have realized the separation of cultivateing hole and observation hole, are favorable to reducing the medium complexity between camera lens and sample in order to improve observation quality.

The bottom of the second pattern layer 3 and the top of the transparent substrate layer 1 are connected to the top and bottom of the microfluidic main channel 21, respectively, for making the microfluidic main channel 21 a closed channel.

Two ends of the second pattern layer 3 are protruded relative to the transparent cover plate layer 4, and the protruded parts at the two ends are respectively provided with a main channel liquid inlet hole 5 and a main channel liquid outlet hole 6 communicated with the microfluidic main channel 21, as shown in fig. 3.

In some embodiments, the first pattern layer 2 and the second pattern layer 3 are PDMS blocks with different patterns prepared by a soft lithography method.

In some embodiments, the first pattern layer 2 is fabricated as follows: firstly drawing a photoetching drawing of a first pattern layer, manufacturing a corresponding mask, then forming a corresponding male mold on a silicon wafer through soft photoetching, pouring PDMS prepolymer on the silicon wafer, curing in an oven at 80 ℃ for 10-30min, taking down a PDMS block with a formed pattern after the curing is finished, and cutting the PDMS block into a proper size to obtain the first pattern layer. The soft lithography is specifically: firstly, spin-coating photoresist with a preset thickness on a silicon wafer, then carrying out pre-baking, covering a mask on the silicon wafer after the completion of the pre-baking, carrying out alignment exposure in a photoetching machine, and carrying out post-baking after the exposure is completed. And then development and fixing are performed.

In some embodiments, the second pattern layer is formed by the following method: firstly drawing a photoetching drawing of a second pattern layer, manufacturing a corresponding mask, then forming a corresponding male mold on a silicon wafer through soft photoetching, pouring PDMS prepolymer on the silicon wafer, curing in an oven at 80 ℃ for 10-30min, taking down a PDMS block with a formed pattern after the curing is finished, and cutting the PDMS block into a proper size to obtain the second pattern layer.

In some embodiments, the first through holes 22 on the first pattern layer 2 are further provided with intermediate channels 23 between two of them; and the protruding parts at the two ends of the second pattern layer are also provided with a middle channel liquid inlet hole 7 and a middle channel liquid outlet hole 8. The intermediate channel is arranged so that each first through hole 22 not only has the fluid from the microfluidic main channel to pass through, but also has the fluid from the intermediate channel in the other direction to pass through, and the transparent reagent is circulated from the intermediate channel 23, so that the transparent reagent in the intermediate channel slowly enters the culture chamber in the chip by using the diffusion effect, and the influence of the fluid shear force on the artificial micro-organ in the culture chamber can be reduced. In the preferred embodiment, the circulation of the transparent reagent from the central channel to the heart micro-organ can last for 0.5-1h; for tumor micro-organs, the gas can be continuously introduced for 0.5-2h; for the brain-like organ, the infusion can be continued for 6-12h. In a preferred embodiment, the microfluidic main channel and the intermediate channel are perpendicular to each other.

In some embodiments, the main channel liquid inlet hole 5, the main channel liquid outlet hole 6, the middle channel liquid inlet hole 7 and the middle channel liquid outlet hole 8 are all provided with a boss 10, and the main channel liquid inlet hole 5, the main channel liquid outlet hole 6, the middle channel liquid inlet hole 7 and the middle channel liquid outlet hole 8 are led out through the boss 10, so that liquid inlet and liquid outlet are facilitated. The figure is provided with 6 bosses which respectively correspond to a main channel liquid inlet hole 5, a main channel liquid outlet hole 6, two middle channel liquid inlet holes 7 and two middle channel liquid outlet holes 8.

In some embodiments, the cross-sectional shapes of the first through hole 22 and the second through hole 31 are the same, and are circular or polygonal, such as hexagonal or octagonal; the bottom of the first through hole 22 is a U-shaped bottom, and the U-shaped bottom is beneficial to three-dimensional culture of cells. The U-shaped bottom can be prepared by the following method: after the first pattern layer 2 is prepared, 0.005-0.015g of uncured PDMS prepolymer is taken by a liquid transfer machine, dripped into the first through hole 22 of the first pattern layer 2, and after standing for 3-8 minutes, the PDMS prepolymer can spontaneously form a U-shaped bottom surface under the action of surface tension, and then heated and cured to obtain the first pattern layer with the through hole of the U-shaped bottom.

In some embodiments, as shown in fig. 5, a connection layer 9 is further disposed between the glass slide substrate layer 1 and the first pattern layer 2, the connection layer 9 is made of PDMS, and the hardness of the PDMS material used for the connection layer 9 is less than that of the PDMS material used for the first pattern layer 2, which is beneficial to firmly connecting the first pattern layer 2 and the transparent substrate layer 1, and compared with a PDMS-PDMS interface, the PDMS-PDMS interface is more beneficial to spreading the PDMS and is beneficial to forming a U-shaped bottom. And the bottom of the second pattern layer and the top of the connecting layer are respectively connected with the top and the bottom of the microfluidic main channel, so that the microfluidic main channel becomes a closed channel.

In some embodiments, the thickness of the tie layer 9 is in the range of 5-20 μm.

In some embodiments, the first patterning layer 2 has a thickness of 1.5 ± 0.1mm, and the second patterning layer 3 has a thickness of 100 ± 10 μm.

In some embodiments, the transparent substrate layer 1 is a glass slide or cover slip; the transparent cover plate layer 4 is a glass slide or a cover glass.

In some embodiments, the cross-sectional shapes of the first through holes 22 and the second through holes 31 are circular, the respective layers are sequentially bonded by using a Plasma process according to the sequence of the glass slide-the connecting layer-the first pattern layer 2-the second pattern layer 3-the cover glass-the material inlet/outlet boss, the positions of the circular through holes of the first pattern layer 2 and the second pattern layer 3 are ensured to be in one-to-one correspondence, and finally, the corresponding microfluidic facilities are connected, so that the overall structure of the microfluidic organ chip is shown in fig. 5. The utility model provides an above-mentioned adaptation the utility model discloses the organ chip design of transparent reagent and transparent method has solved the disappearance problem that prior art organ chip normal position transparent method required time is longer, produce deformation great and online organ chip transparent method.

The utility model discloses some embodiments utilize above-mentioned organ chip to carry out the transparent method of observing of online live body to artifical micro-organ, including following step:

s1, mixing 60% of medical iodixanol injection (sigma) with a culture solution according to a certain proportion, uniformly mixing by using a liquid transfer gun, and adding one or more of calcium chloride, potassium chloride, zinc chloride, sodium chloride and the like to adjust the ion concentration to obtain a transparent reagent.

S2, transparency: filling the obtained light transparent reagent in a pump, discharging bubbles in the chip, and continuously pouring the light transparent reagent into the chip at a speed of 2-200 μ L/min for half an hour.

S3, observation: after the perfusion is finished, the chip can be moved to a microscope for observation.

S4, recovery: continuously perfusing the culture solution into the chip at a speed of 2-200 μ L/min for 60 min, and then returning to the incubator.

The organ chip provided by the utility model is suitable for various artificial micro-organs, and the micro-organs can be purchased and obtained from the market or cultured and manufactured by self according to the method of the prior art. For culturing three-dimensional artificial micro-organs, such as myocardial ball, tumor ball, brain-like artificial micro-organs and the like, only cell suspension (50-100 ten thousand per milliliter) is required to be introduced into the chip, and then the chip is centrifuged and soaked in culture solution for culture; for heart or brain-like, corresponding cell factors are needed to be added during culture, and the three-dimensional artificial micro-organ can be cultured in a culture hole of an organ chip by referring to a culture method of the three-dimensional artificial micro-organ in the prior art.

In one embodiment, the organ chip of the present invention is used for on-line transparency and detection of tumor micro-organ samples, and specifically comprises the following steps:

(1) Manufacturing an adaptive chip: respectively drawing photoetching drawings of the chip middle layers corresponding to the first pattern layer 2 and the second pattern layer 3 according to the drawings of fig. 2 and fig. 3, manufacturing corresponding masks, forming corresponding male molds on a silicon wafer through soft photoetching, pouring PDMS prepolymer into the silicon wafer, curing for 20min in an oven at 80 ℃, taking off PDMS blocks with patterns after curing, and cutting the PDMS blocks into proper sizes to obtain the first pattern layer 2 and the second pattern layer 3; sequentially bonding each layer of components according to the sequence of the glass slide, the connecting layer, the first pattern layer 2, the second pattern layer 3, the cover glass, the boss of the main channel liquid inlet hole, the boss of the main channel liquid outlet hole, the boss of the middle channel liquid inlet hole and the boss of the middle channel liquid outlet hole, filling 0.01g of PDMS prepolymer into the hole in the first pattern layer 2, centrifuging to form a U-shaped bottom surface, and curing again; after the curing is completed, the corresponding microfluidic devices are connected to obtain the microfluidic chip shown in fig. 4 and 5.

(2) Artificial micro-organ culture: a1 million/mL suspension of tumor cells, hct116 transfected with GFP protein, in rpmi1640 was applied to the chip obtained as described above, and the chip was centrifuged at 400G for 5 minutes, followed by static culture.

(3) Preparing a corresponding transparent reagent: mixing the 1mL of 60% iodixanol injection with a standard DMEM culture solution according to a ratio of 1: the concentration of potassium ions is 1.8mM; the concentration of calcium ion is 0.2mM; the concentration of magnesium ions was 0.2mM and the concentration of sodium ions was 80mM.

(4) And (3) transparency: the obtained light transparent reagent is filled in a microflow pump, bubbles in the chip are exhausted, and then the light transparent reagent is continuously poured into the chip at the speed of 40 mu L/min for half an hour.

(5) And (4) observation: after the completion of the perfusion, the chip is moved to the microscope for observation, as shown in fig. 6, wherein fig. 6 (a) is an image of different depths before the light is transparent, and fig. 6 (b) is an image of different depths after the light is transparent. It can be seen that the tumor micro-organs in the chip become transparent gradually after the light is transparent, and meanwhile, the structure of the chip can still be kept complete, and the imaging depth is obviously improved.

(6) And (3) recovering: the culture solution is continuously perfused into the chip at the speed of 40 muL/min for 60 minutes, and then the chip is put back into the incubator, and the tumor micro-organ is still kept in a living state.

It will be understood by those skilled in the art that the foregoing is merely a preferred embodiment of the present invention, and is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

Claims

1. An organ chip for online light transparency and online observation is characterized in that a transparent substrate layer (1), a first pattern layer (2), a second pattern layer (3) and a transparent cover plate layer (4) are stacked from bottom to top; wherein:

the first pattern layer (2) contains a chip pattern of the organ chip, and the chip pattern comprises a micro-fluidic main channel (21) and a plurality of first through holes (22) which are dispersedly arranged on the micro-fluidic main channel (21) and communicated with the micro-fluidic main channel (21);

the pattern contained in the second pattern layer (3) comprises a plurality of second through holes (31), and the plurality of second through holes (31) in the second pattern layer (3) are in one-to-one correspondence and communication with the plurality of first through holes (22) in the first pattern layer (2);

the bottom of the second pattern layer (3) and the top of the transparent substrate layer (1) are respectively connected with the top and the bottom of the micro-fluidic main channel (21) so as to enable the micro-fluidic main channel (21) to be a closed channel;

and two ends of the second pattern layer (3) are arranged in a protruding way relative to the transparent cover plate layer (4), and a main channel liquid inlet hole (5) and a main channel liquid outlet hole (6) which are communicated with the micro-fluidic main channel (21) are respectively arranged at two ends of the second pattern layer.

2. The organ chip of claim 1, characterized in that the first pattern layer (2) is further provided with intermediate channels (23) between each two of the first through holes (22); the protruding parts at the two ends of the second pattern layer (3) are also provided with a middle channel liquid inlet hole (7) and a middle channel liquid outlet hole (8).

3. The organ chip of claim 1, wherein the material of the first pattern layer (2) and the second pattern layer (3) is PDMS.

4. The organ chip of claim 1, wherein the first through-hole (22) and the second through-hole (31) have the same cross-sectional shape, and are circular or polygonal through-holes, and the bottom of the first through-hole (22) has a U-shaped bottom.

5. The organ chip of claim 1, wherein a connection layer (9) is further disposed between the transparent substrate layer (1) and the first pattern layer (2), the connection layer (9) is made of PDMS, and the hardness of the PDMS material used for the connection layer (9) is less than that of the PDMS material used for the first pattern layer (2); the bottom of the second pattern layer (3) and the top of the connecting layer (9) are respectively connected with the top and the bottom of the micro-fluidic main channel (21) so as to enable the micro-fluidic main channel (21) to be a closed channel.

6. The organ chip of claim 5, characterized in that the thickness of the connection layer (9) is in the range of 5-20 μm.

7. The organ chip according to claim 1, wherein the thickness of the first pattern layer (2) is 1.5 ± 0.1mm, and the thickness of the second pattern layer (3) is 100 ± 10 μm.

8. The organ chip of claim 1, wherein the transparent substrate layer (1) is a glass slide or a cover glass; the transparent cover plate layer (4) is a glass slide or a cover glass.

9. The organ chip of claim 1, wherein the main channel liquid inlet hole (5), the main channel liquid outlet hole (6), the intermediate channel liquid inlet hole (7) and the intermediate channel liquid outlet hole (8) are provided by bosses (10).