An organ-on-a-chip

Abstract

The application provides an organ chip, which comprises, from top to bottom, a liquid collecting layer (1), a pressing layer (2), a glue injection layer (3), a tissue layer (4), a film layer (5) and an oxygen control layer (6). The application provides a novel organ chip, which comprises a film layer and an oxygen control layer, is used for making oxygen pass through the film layer and be eliminated by an oxygen absorbent in the oxygen control layer, liquid cannot pass through the film layer, so that the liquid circulates or is supplemented in the microfluidic channel, and the oxygen concentration in the culture solution is made to be closer to the real physiological environment in the human body based on the cavity and the through structure formed by laminating and sealing of the layers. In addition, a plurality of molds are arranged, so that a plurality of cell models can be simultaneously cultured, and a high-throughput culture effect is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical engineering, and particularly relates to an organ chip. BACKGROUND

[0002] The liver is the most important organ for carrying out metabolic functions in the body, and it is responsible for many important physiological functions such as metabolic detoxification, immune coagulation, and protein synthesis. The liver lobule is considered to be the smallest functional unit of the liver, and its shape is a hexagon. Each liver lobule is composed of hepatocytes radiating from a central vein and is separated by vascular endothelial cells. In the center of each hexagon, there is a central vein. The periphery of the hexagon contains a hepatic portal vein and a hepatic artery, and blood flows into the liver from the hepatic portal vein and the hepatic artery and then flows out through the central vein. Therefore, constructing a blood flow model of the liver lobule in vitro is of great significance for drug development, drug safety testing, and liver disease research.

[0003] Traditional two-dimensional cell culture is difficult to construct complex three-dimensional structures, and animal models also have the problems of high cost and ethics. In recent years, organ chips, as a technology for three-dimensional culture of cells outside the human body to simulate the function of organs in the human body, have developed rapidly. Organ chips have good application prospects in the fields of drug research and development, disease model evaluation, and personalized medicine.

[0004] The basic structure of an organ chip based on a perfusion mode mainly includes a tissue chamber filled with cell hydrogel in the middle and microfluidic channels for cell culture fluid flow on both sides, which can promote the continuous supply of nutrients and oxygen and the removal of metabolic products, thereby facilitating the long-term culture of cells or tissues. However, at the same time, liver lobule chips made using this structure need to avoid the leakage of cell-containing hydrogel from the tissue chamber to the microfluidic channel, which would cause flow obstacles in the cell culture fluid.

[0005] Currently, constructing a physical barrier such as a microcolumn array in the tissue chamber and the culture fluid channel is a main method to solve the above-mentioned problems, and the effect is determined by the surface tension related to the microstructure parameters and the interfacial wettability, as well as the external pressure applied during the loading of the cell-containing hydrogel. However, the presence of these physical barriers also reduces the effective contact area between the cell culture fluid and the extracellular matrix, thereby affecting the uniform stimulation of the cultured cells or tissues. Therefore, a new strategy is needed to construct a temporary barrier to enable the cells or tissues cultured in the liver lobule tissue chamber to receive more uniform and sufficient stimulation. SUMMARY

[0006] In view of the above-mentioned disadvantages of the prior art, the present application aims to provide an organ chip which not only makes the oxygen concentration in the culture solution closer to the real physiological environment in the human body by regulation, but also realizes the culture of multiple cell models at the same time.

[0007] Another object of the present application is to solve the problem of the small effective contact area and insufficient contact between the cell culture solution and the extracellular matrix in the tissue chamber caused by the temporary barrier such as the micro-pillar array in the existing organ chip.

[0008] To achieve the above-mentioned objects and other related objects, the present application is implemented by including the following technical solutions.

[0009] The present application provides an organ chip, which comprises, from top to bottom, a liquid collecting layer, a pressing layer, a glue injection layer, a tissue layer, a film layer and an oxygen control layer.

[0010] According to the above-mentioned technical solutions of the present application, in use, the liquid collecting layer and the pressing layer are fixed relative to each other, and the glue injection layer, the tissue layer, the film layer and the oxygen control layer are fixed relative to each other.

[0011] According to the above-mentioned technical solutions of the present application, in use, the liquid collecting layer and the pressing layer are sealed by being attached to each other.

[0012] According to the above-mentioned technical solutions of the present application, in use, the liquid collecting layer and the pressing layer are in fluid communication.

[0013] According to the above-mentioned technical solutions of the present application, in use, the glue injection layer, the tissue layer, the film layer and the oxygen control layer are laminated and attached to each other to be sealed.

[0014] According to the above-mentioned technical solutions of the present application, in use, the glue injection layer and the tissue layer are in fluid communication.

[0015] According to the above-mentioned technical solutions of the present application, the lower surface of the liquid collecting layer is formed with a liquid collecting groove, and the groove bottom of the liquid collecting groove is further provided with a liquid collecting hole penetrating through the liquid collecting layer.

[0016] According to the above-mentioned technical solutions of the present application, the lower surface of the pressing layer is formed with a protruding mold.

[0017] In a preferred embodiment, the mold comprises a plurality of mold grooves.

[0018] According to the above-mentioned technical solutions of the present application, the mold groove is further provided with a liquid flow column perpendicular to the pressing layer, and the liquid flow column is provided with a first flow guide hole penetrating through the pressing layer; in use, the liquid collecting groove covers the first flow guide hole.

[0019] According to the above-mentioned technical solutions of the present application, the cross section of the mold groove is a regular hexagon.

[0020] According to the above technical solution of the present application, the mold grooves are arranged in a continuous array.

[0021] According to the above technical solution of the present application, the diameter of the first flow guide hole is less than 100 μm.

[0022] According to the above technical solution of the present application, the glue injection layer is formed with a through mold hole.

[0023] According to the above technical solution of the present application, the glue injection layer is further formed with a plurality of through second flow guide holes and / or a plurality of third flow guide holes.

[0024] According to the above technical solution of the present application, the tissue layer is formed with a through tissue hole, and a plurality of first flow channel grooves are further formed on the upper surface of the tissue layer, which are in fluid communication with the tissue hole.

[0025] According to the above technical solution of the present application, the outer contour of the mold matches the mold hole; in use, the mold penetrates the mold hole and the tissue hole in sequence, and its lower surface is flush with the tissue layer.

[0026] According to the above technical solution of the present application, the second flow guide hole is arranged corresponding to the first flow channel groove, and in use, the second flow guide hole and the corresponding first flow channel groove can flow.

[0027] According to the above technical solution of the present application, a plurality of fourth flow guide holes corresponding to the third flow guide holes are formed on the tissue layer.

[0028] According to the above technical solution of the present application, the second flow guide hole and the third flow guide hole are exposed on the outer surface of the organ chip.

[0029] According to the above technical solution of the present application, the film layer is further provided with a plurality of fifth flow guide holes corresponding to the fourth flow guide holes.

[0030] According to the above technical solution of the present application, the upper surface of the oxygen control layer is formed with a plurality of second flow channel grooves; in use, one end of the second flow channel groove is in communication with the corresponding fifth flow guide hole.

[0031] According to the above technical solution of the present application, in use, the second flow channel groove is filled with an oxygen eliminating agent.

[0032] According to the above technical solution of the present application, the distribution of the second flow channel groove and / or the oxygen eliminating agent matches the physiological metabolic characteristics of the organs in the tissue layer.

[0033] According to the above technical solution of this application, it also includes a backing plate, the thickness of which is the same as the thickness of the tissue layer; in use, the backing plate is disposed between the imprinting layer and the injection layer to release the imprinting state of the mold.

[0034] According to the above technical solution of this application, the thickness of the tissue layer is 1-2 mm.

[0035] According to the above technical solution of this application, the thickness of the adhesive layer is 1-2 mm.

[0036] According to the above technical solution of this application, the thickness of the thin film layer is 30-200 μm.

[0037] According to the above technical solution of this application, the thin film layer is a thin film that allows gas to pass through but not liquid.

[0038] As described above, the organ-on-a-chip of the present invention has the following beneficial effects:

[0039] This application provides a novel organ-on-a-chip, comprising a thin film layer and an oxygen control layer. Oxygen is allowed to pass through the thin film layer and be eliminated by an oxygen absorber in the oxygen control layer, while liquid cannot pass through the thin film layer. This allows liquid to circulate or replenish within microfluidic channels. Based on the cavity and through-structure formed by the stacked and sealed layers, the oxygen concentration distribution in the culture medium within the tissue layer more closely resembles the actual physiological environment in the human body. Furthermore, multiple molds are provided, enabling simultaneous culture of multiple cell models and achieving high-throughput culture. Attached Figure Description

[0040] Figure 1 The diagram shown is an exploded view of the organ-on-a-chip according to the present invention.

[0041] Figure 2 The diagram shown is a schematic representation of the fluid collection layer of the organ-on-a-chip according to the present invention.

[0042] Figure 3 The diagram shown is a schematic representation of the imprinted layer of the organ-on-a-chip according to the present invention.

[0043] Figure 4 The diagram shown is a schematic representation of the adhesive layer of the organ-on-a-chip according to the present invention.

[0044] Figure 5 The diagram shown is a structural schematic of the tissue layer of the organ-on-a-chip according to the present invention.

[0045] Figure 6 The diagram shown is a schematic representation of the thin film layer of the organ-on-a-chip according to the present invention.

[0046] Figure 7 The diagram shown is a structural schematic of the oxygen control layer of the organ-on-a-chip according to the present invention.

[0047] Figure 8 A schematic diagram showing the overall structure of the organ chip of the present application.

[0048] Figure 9 A schematic diagram showing the structure of the solidified hydrogel formed in the tissue layer in the organ chip of the present application.

[0049] Figures 1-9 Reference numerals are as follows:

[0050] 1 collection layer

[0051] 11 collection groove

[0052] 12 collection hole

[0053] 2 embossing layer

[0054] 21 mold

[0055] 22 first flow guide hole

[0056] 3 glue injection layer

[0057] 31 glue injection groove

[0058] 32, 33 second flow guide hole

[0059] 34, 35, 36, 37 third flow guide hole

[0060] 4 tissue layer

[0061] 41, 42 first flow channel groove

[0062] 43 tissue hole

[0063] 5 film layer

[0064] 6 oxygen control layer

[0065] 61, 62 second flow channel groove

[0066] 7 pad DETAILED DESCRIPTION

[0067] The embodiments of the present application will be described in detail by the following specific examples, and other advantages and effects of the present application can be easily understood by those skilled in the art from the contents disclosed in the specification.

[0068] Reference is made to Figures 1-9It is to be understood that the structures, proportions, sizes, etc. shown in the drawings accompanying the present specification are merely intended to facilitate the understanding of the content disclosed by the present specification for those skilled in the art to understand and read, and are not intended to limit the conditions under which the present application can be implemented, and therefore do not have technical significance. Any modification of the structure, change of the proportional relationship or adjustment of the size, without affecting the effects that can be achieved by the present application and the purposes that can be achieved, should still fall within the scope of the technology disclosed by the present application.

[0069] As shown in Figure 1 , the present application provides an organ chip, which comprises, from top to bottom, a liquid collecting layer 1, a pressing layer 2, a glue injection layer 3, a tissue layer 4, a film layer 5 and an oxygen control layer 6.

[0070] In the present application, through the superposition and cooperation between the layers, the oxygen control layer 6 can act on the cell culture space through the film layer 5, so as to eliminate the oxygen in this space, thereby better simulating the oxygen environment inside the human body.

[0071] In a specific embodiment as shown in Figure 1 and Figure 2 , the lower surface of the liquid collecting layer 1 is formed with a liquid collecting groove 11, and the groove bottom of the liquid collecting groove 11 is further provided with a liquid collecting hole 12 penetrating through the liquid collecting layer 1.

[0072] In a specific embodiment as shown in Figure 1 and Figure 3 , the lower surface of the pressing layer 2 is formed with a protruding mold 21; the mold 21 comprises a plurality of mold grooves, and a liquid flow column perpendicular to the pressing layer 2 is arranged in the mold groove, and a first flow guide hole 22 penetrating through the pressing layer 2 is arranged on the liquid flow column.

[0073] In a specific embodiment as shown in Figure 1 , Figure 2 and Figure 3 , in use, the liquid collecting groove 11 covers the first flow guide hole 22.

[0074] Through the above structure and cooperation, in use, the liquid flowing out of the second flow guide hole 22 in the pressing layer 2 can be collected through the liquid collecting groove 11 on the liquid collecting layer 1, and then flow out of the chip through the liquid collecting hole 12.

[0075] In a specific embodiment as shown in Figure 1 and Figure 4In the specific embodiment shown, the glue injection layer 3 is formed with a mold hole 31.

[0076] In one specific embodiment as shown in Figure 1 and Figure 4 In the specific embodiment shown, the glue injection layer 3 is formed with a plurality of second flow holes 32, 33. Figure 4 In the specific embodiment shown, the second flow holes are two.

[0077] In one specific embodiment as shown in Figure 1 and Figure 4 In the specific embodiment shown, the glue injection layer 3 is formed with a plurality of third flow holes 34, 35, 36, 37. Figure 4 In the specific embodiment shown, the third flow holes are four.

[0078] In one specific embodiment as shown in Figure 1 and Figure 5 In the specific embodiment shown, the tissue layer 4 is formed with a tissue hole 43, and a plurality of first flow channels 41, 42 are formed on the upper surface of the tissue layer 4, which are in fluid communication with the tissue hole 43; the second flow holes are arranged corresponding to the first flow channels, and in use, the second flow holes are in fluid communication with the corresponding first flow channels.

[0079] In one specific embodiment as shown in Figure 1 and Figure 5 In the specific embodiment shown, the tissue layer 4 is formed with a fourth flow hole corresponding to the third flow hole.

[0080] In one specific embodiment as shown in Figure 1 and Figure 8 In the specific embodiment shown, the outer contour of the mold 21 matches the mold hole 31; in use, the mold 21 penetrates the mold hole 31 and the tissue hole 43 in turn, and its lower surface is flush with the tissue layer 4.

[0081] In one specific embodiment as shown in Figure 1 and Figure 6 In the specific embodiment shown, the film layer 5 is further provided with a fifth flow hole corresponding to the fourth flow hole.

[0082] In one specific embodiment as shown in Figure 1 and Figure 7 In the specific embodiment shown, the upper surface of the oxygen control layer 6 is formed with a plurality of second flow channels, specifically two, as shown in 61, 62; in use, one end of the second flow channel is in communication with the corresponding fifth flow hole.

[0083] In one specific embodiment as shown in Figure 8In the specific embodiment shown, the organ-on-a-chip further includes a pad 7, the thickness of which is the same as the thickness of the tissue layer 4. Figure 8 As shown, during use, the pad 7 is disposed between the imprinting layer 2 and the injection layer 3 to release the imprinting state of the mold 21. The imprinting state of the mold 21 refers to the hydrogel containing cells imprinted by the mold 21 into the tissue pores 43.

[0084] In a like Figure 3 In the specific embodiment shown, the cross-section of the mold groove is a regular hexagon. This allows for the accurate construction of a blood flow model of a real liver lobule in vivo.

[0085] In a like Figure 3 In the specific embodiment shown, several mold slots are arranged in a continuous array, preferably, as shown in the example. Figure 3 As shown, several of the mold slots have a honeycomb structure.

[0086] In one specific embodiment, the diameter of the first flow-guiding hole 22 is less than 100 μm. Therefore, when the organ-on-a-chip of this application is subjected to the surface tension of the hydrogel, the hydrogel will not clog the first flow-guiding hole 22 during the imprinting process.

[0087] In one specific embodiment, the thickness of the tissue layer 4 is 1 to 2 mm. For example, it can be 1 mm, 1.5 mm, or 2 mm.

[0088] In a preferred embodiment, the thickness of the adhesive layer 3 is 1-2 mm. For example, it can be 1 mm, 1.5 mm, or 2 mm.

[0089] In a preferred embodiment, the thickness of the thin film layer 5 is 50–200 μm. For example, it can be 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, or 200 μm.

[0090] In a preferred embodiment, the thin film layer 5 is a film that allows gas to pass through but not liquids. In a preferred embodiment, the thin film layer 5 may be a PDMS film. PDMS film is a fully transparent, high-precision film material made by cross-linking and curing PDMS silicone rubber. It is hydrophobic, biocompatible, and breathable; water forms droplets on the film and slides off, but water vapor and oxygen can pass through the film.

[0091] In a like Figure 4In the specific embodiment shown, the second flow holes 32, 33 of the glue injection layer 3 and the third flow holes 34, 35, 36, 37 are exposed to the outer surface of the organ chip.

[0092] In a preferred embodiment, the distribution of the second flow channel and / or the oxygen scavenger is matched to the physiological metabolic characteristics of the organ in the tissue layer. This is because different organs require different oxygen content distributions, and this is a biomimetic structural design, so that the actual human metabolic environment can be better simulated. In a specific embodiment, the size and arrangement of the second flow channel can be matched according to the control of the oxygen concentration in the actual process, and in order to better eliminate oxygen in the organ chip culture system, it can be arranged in a targeted manner. Arranged as Figure 7 As shown, a more specific arrangement of the second flow channel is provided, which has two flow channels 61 and 62, and the two flow channels are staggered and densely arranged in an S shape.

[0093] In use, the culture solution can enter from the second flow holes 32 and 33 and exit from the collection hole 12, so as to simulate the actual blood flow of the liver lobule model in the body. Alternatively, in use, the culture solution enters from the collection hole 12 and exits from the second flow holes 32 and 33. Alternatively, in use, the culture solution enters from one of the second flow holes such as 32 and exits from the collection hole 12 or the other second flow hole 33.

[0094] In a specific embodiment, in use, the second flow channel is filled with an oxygen scavenger.

[0095] In a specific embodiment, in use, the cell-containing hydrogel is injected from the second flow hole and fills the tissue holes 43 in the tissue layer, and then the mold 21 of the imprinting layer 2 is lowered to complete the imprinting; then the culture solution is injected from the second flow hole to expel the excess hydrogel from the organ chip; after the hydrogel solidifies, the imprinting of the imprinting layer is removed and the culture solution is perfused from the second flow hole and / or the collection hole to culture the cells; and the oxygen scavenger is perfused from the third flow hole to eliminate and control the oxygen concentration in the organ chip during culture.

[0096] The culture solution and the oxygen scavenger Scavenger can be driven by gravity or pumped externally, and the present application does not make specific limitations thereon. The oxygen scavenger can be a liquid Scavenger for controlling the oxygen concentration.

[0097] In a more specific embodiment, the cell culture process using the organ chip in the present application is as follows:

[0098] Before injecting the hydrogel containing cells, the spacer 7 is used to separate the imprinting layer 2 and the injection layer 3, the hydrogel containing cells is injected from the second flow hole 32, and the spacer 7 is removed immediately after the hydrogel fills the tissue hole 43, so that the bottom end of the imprinting layer 2 is tightly attached to the film layer 5 to complete the imprinting; then the culture solution is injected from one of the second flow holes 32 to make the excess hydrogel flow along the mold 21 and the tissue hole 43 and be discharged from the other second flow hole 33; after the hydrogel solidifies, the imprinting layer 2 is lifted by a certain distance and separated from the injection layer 3 by the spacer 7, and the culture solution is continuously injected from the second flow holes 32 and 33 or the liquid collecting hole 12 to culture the cells; the liquid scavenger is injected from the third flow holes 34 and 35 to absorb oxygen to control the oxygen concentration of the surrounding environment. Finally, the shape of the solidified hydrogel 8 formed in the tissue hole 43 is shown in the shadow part of Figure 9 .

[0099] Compared with the prior art, the beneficial effects of the present application are:

[0100] (1) The simultaneous large-scale culture of multiple cell models is realized, and the high-throughput characteristics are achieved;

[0101] (2) The setting of the oxygen control layer can make the oxygen concentration distribution in the culture solution closer to the physiological environment of the human body;

[0102] (3) The setting of the mold and other temporary barriers with special structures can make the culture solution fully and uniformly stimulate cell growth;

[0103] (4) The design of the inlet and outlet of multiple culture solutions and the construction of the regular hexagonal structure of the tissue hole can well construct the blood flow model of the real liver lobule in vivo and adjust the inlet and outlet of the liquid according to the experimental requirements.

[0104] Therefore, the present application effectively overcomes the various shortcomings in the prior art and has high industrial utilization value.

[0105] The above embodiments only exemplarily illustrate the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above embodiments without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should be covered by the claims of the present application.

Claims

1. An organ-on-a-chip, characterized in that, The organ chip comprises, from top to bottom, a liquid collecting layer (1), a stamping layer (2), a glue injection layer (3), a tissue layer (4), a membrane layer (5) and an oxygen control layer (6); in use, the liquid collecting layer (1) is fixedly connected with the stamping layer (2), the glue injection layer (3), the tissue layer (4), the membrane layer (5) and the oxygen control layer (6) are fixedly connected, and the liquid collecting layer (1) is in fluid communication with the stamping layer (2); the glue injection layer (3) is in fluid communication with the tissue layer (4); a lower surface of the liquid collecting layer (1) is provided with a liquid collecting groove (11), a groove bottom of the liquid collecting groove (11) is provided with a liquid collecting hole (12) penetrating through the liquid collecting layer (1); a lower surface of the stamping layer (2) is provided with a protruding mold (21); the mold (21) comprises a plurality of mold grooves; the mold grooves are further provided with liquid flow columns perpendicular to the stamping layer (2), and the liquid flow columns are provided with first flow guide holes (22) penetrating through the stamping layer (2); in use, the liquid collecting groove (11) covers the first flow guide holes (22); the glue injection layer (3) is provided with a penetrating mold hole (31); the glue injection layer (3) is further provided with a plurality of penetrating second flow guide holes (32, 33) and / or a plurality of third flow guide holes (34, 35, 36, 37); the membrane layer (5) is a membrane through which gas can pass but liquid cannot pass; the tissue layer (4) is provided with a penetrating tissue hole (43), and a plurality of first flow channel grooves (41, 42) are further formed on an upper surface of the tissue layer (4), and the first flow channel grooves (41, 42) are in fluid communication with the tissue hole (43).

2. The organ-on-chip according to claim 1, characterized in that In use, the liquid collecting layer (1) is attached to the stamping layer (2) to seal; And / or, in use, the glue injection layer (3), the tissue layer (4), the membrane layer (5) and the oxygen control layer (6) are laminated and attached to seal.

3. The organ chip according to claim 1, wherein The cross section of the mold groove is a regular hexagon; And / or, a plurality of the mold grooves are arranged in a continuous array.

4. The organ-chip of claim 3, wherein The diameter of the first flow guide hole (22) is less than 100 μm.

5. The organ-on-chip of claim 1, wherein The outer contour of the mold (21) matches the mold hole (31); in use, the mold (21) penetrates the mold hole (31) and the tissue hole (43) in sequence, and the lower surface of the mold (21) is flush with the tissue layer (4); And / or, the second flow guide hole is arranged corresponding to the first flow channel groove, and, in use, the second flow guide hole and the corresponding first flow channel groove are in fluid communication; And / or, a plurality of fourth flow guide holes corresponding to the third flow guide holes are formed on the tissue layer (4); And / or, the second flow guide hole (32, 33) and the third flow guide hole (34, 35, 36, 37) are exposed on the outer surface of the organ chip.

6. The organ-on-chip according to claim 5, characterized in that The membrane layer (5) is further provided with a fifth flow guide hole penetratingly arranged corresponding to the fourth flow guide hole; And / or, the upper surface of the oxygen control layer (6) is formed with several second flow channel grooves (61, 62); in use, one end of the second flow channel groove is in communication with the corresponding fifth flow guide hole.

7. The organ-on-chip according to claim 6, characterized in that In use, the second flow channel groove is filled with an oxygen elimination agent; And / or, the distribution of the second flow channel groove and / or the oxygen elimination agent matches the physiological metabolic characteristics of the organs in the tissue layer.

8. The organ chip according to claim 1, wherein, Further comprising a backing plate (7) having the same thickness as the tissue layer (4); in use, the backing plate (7) is arranged between the embossing layer (2) and the glue injection layer (3) to release the embossing state of the mold; And / or, the thickness of the tissue layer (4) is 1-2 mm; And / or, the thickness of the glue injection layer (3) is 1-2 mm; And / or, the thickness of the film layer (5) is 30-200 μm.

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