A pump-free intestinal chip system capable of measuring transmembrane resistance value and a measuring method thereof

Abstract

The present application relates to the field of biomedical engineering, and particularly relates to a pump-free intestinal chip system capable of measuring transmembrane resistance value and a measuring method thereof.The system comprises transmembrane resistance measuring electrodes, an intestinal chip and a swing-type shaker; the intestinal chip is composed of a plurality of chip units arranged in parallel, and each chip unit comprises, from top to bottom, a culture layer, a porous membrane and a perfusion layer; the culture layer is provided with a first end hole, an intermediate hole and a second end hole in sequence, both end holes are communicated with the cavity of the perfusion layer to form a fluid channel, the intermediate hole is used for culturing cells, and the intermediate hole separates the culture layer and the perfusion layer and exchanges materials through the porous membrane; the swing-type shaker is used for providing periodic gravity flow to simulate intestinal peristalsis; the transmembrane resistance measuring electrodes are connected with a resistance meter, and the transmembrane resistance value is obtained through current-voltage measurement.The present application has better simulation of intestinal barrier characteristics, can measure the transmembrane resistance value of the intestinal chip, and is convenient to operate and short in time consumption.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering, and to model construction and characterization methods for organ-on-a-chip technology. Specifically, it provides a pump-free intestinal chip system and its measurement method that can measure transmembrane resistance. Background Technology

[0002] The intestinal barrier is a crucial defense line for maintaining homeostasis in the human body, and its dysfunction is closely related to inflammatory bowel disease and drug-induced intestinal toxicity. Traditional in vitro models, such as the Transwell static culture system, can characterize the integrity and permeability of the intestinal epithelial cell monolayer through transmembrane electrical resistance, but they cannot simulate the fluid shear microenvironment and rhythmic peristalsis of the in vivo intestine, resulting in a significant discrepancy between the in vitro assessment of barrier function and the actual in vivo state.

[0003] In recent years, organ-on-a-chip technology has provided a new approach to constructing intestinal models that more closely resemble physiological models. Intestinal-on-a-chip systems can simulate the dynamic microenvironment of the intestine through microfluidic perfusion and mechanical stress loading. However, most existing intestinal-on-a-chip systems are based on PDMS microfluidic chips, which, due to their narrow channels, make it difficult to measure transmembrane resistance. Currently available intestinal-on-a-chip systems for measuring transmembrane resistance often require complex sensors, making it difficult to monitor changes in intestinal barrier function during dynamic culture. Furthermore, the use of pumps and valves makes the chip system overly complex, reducing experimental success rates and limiting its application in drug screening and disease mechanism research.

[0004] Therefore, developing a pump-free intestinal on-chip system that combines characterization capabilities with rhythmic peristalsis simulation has become an important research direction in the current field of organ-on-a-chip. Summary of the Invention

[0005] The purpose of this invention is to provide a pump-free intestinal chip system and its measurement method that can measure transmembrane resistance, in order to solve the problems that traditional intestinal chip models are difficult to characterize transmembrane resistance and require the introduction of pumps and valves to provide shear force and rhythmic peristalsis, which leads to operational difficulties.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: The present invention provides a pump-free gut chip system for measuring transmembrane resistance, the system comprising a transmembrane resistance measuring electrode, a gut chip, and a oscillating shaker; The intestinal microarray consists of multiple parallel-arranged chip units; each chip unit comprises, from top to bottom, a culture layer, a porous membrane, and a perfusion layer; the culture layer is provided with a first end pore, a middle pore, and a second end pore; the perfusion layer is provided with a cavity for containing culture medium; the porous membrane is provided with a first through pore and a second through pore; the first end pore and the second end pore are respectively connected to the cavity of the perfusion layer through the first through pore and the second through pore to form a fluid channel; the middle pore is used for culturing cells, and the middle pore separates the culture layer and the perfusion layer and facilitates substance exchange through the porous membrane; The swing-type shaker is used to provide a periodic flow of gravity to simulate intestinal peristalsis; The transmembrane resistance measuring electrode includes four platinum wires, arranged in pairs. In each group, one wire is inserted into the middle hole, and the other wire is inserted into the cavity of the perfusion layer through the same end hole. Each group of electrodes is connected to the resistance meter via a wire.

[0007] In the above technical solution, the transmembrane resistance measuring electrode and the intestinal chip are further positioned by an electrode support; The electrode support includes two parallel side plates and a top plate connected to the top of the two side plates, the side plates and the top plate forming a downward-facing groove. The top plate is provided with four limiting posts and four platinum wire insertion holes; The limiting posts are distributed around the central hole, and their diameter is 0.5 mm-3 mm; The diameter of the platinum wire insertion hole is 0.5 mm-3 mm.

[0008] In the above technical solution, the culture layer is further made of polystyrene (PS) or polymethyl methacrylate (PMMA) and has a height of 5 mm to 5 cm. The perfusion layer is made of polystyrene (PS) or polymethyl methacrylate (PMMA), and the cavity depth of the perfusion layer is 0.1 mm to 1 cm. The porous membrane is made of polycarbonate (PC), polyester (PET) or polytetrafluoroethylene (PTFE) and has a thickness of 0.1~20μm; the pore size of the first through hole and the second through hole is 0.1~20 μm.

[0009] In the above technical solution, the swing angle of the swinging rocker is 0~60° and not 0, and the swing speed is 0~100 rpm and not 0.

[0010] In the above technical solution, the opening width of the electrode support groove is 1mm to 1cm larger than the width of the intestinal chip, and the width of the intestinal chip is 1cm to 5cm.

[0011] In the above technical solution, the bottom of the platinum wire inserted into the middle hole is 0.1 mm to 1 cm away from the porous membrane, the two platinum wires inserted into the same hole are spaced 0.1 mm to 0.5 cm apart, and the diameter of the platinum wire is 0.5 mm to 2 mm.

[0012] In the above technical solution, the culture layer, porous membrane, and perfusion layer are further bonded together by double-sided tape, glue, or hot pressing.

[0013] In the above technical solution, the intestinal chip is further integrated into a single structure by combining multiple chip units.

[0014] Another aspect of the present invention provides a method for measuring transmembrane resistance using the above-described pumpless intestinal chip system, comprising the following steps: (1) Sterilize or disinfect the intestinal chip and the transmembrane resistance measuring electrode; (2) Intestinal cells were seeded into the central well of the intestinal microarray and cultured statically after seeding; (3) The intestinal microarray was placed on a swing-type shaker in an incubator for incubation; (4) Connect the transmembrane resistance measuring electrode to the resistor meter, provide current through the resistor meter and measure voltage to obtain the transmembrane resistance value.

[0015] The beneficial effects of this invention are as follows: 1. The pump-free intestinal chip system provided by this invention separates the culture layer and the perfusion layer through a porous membrane to achieve material exchange, and a oscillating shaker provides periodic gravity flow to simulate intestinal peristalsis, thereby achieving pump-free perfusion. Compared with traditional models, this system better simulates the intestinal barrier characteristics and does not involve the large number of pumps and valves required in traditional intestinal chips, making it convenient to operate and time-saving.

[0016] 2. This invention provides a transmembrane resistance measuring electrode that matches a pump-free intestinal chip, which can measure the transmembrane resistance value of the intestinal chip, solving the problem that traditional intestinal chips cannot measure the transmembrane resistance value.

[0017] 3. The pump-free intestinal chip system provided by this invention avoids the use of PDMS material commonly used in traditional intestinal chips, which can reduce the adsorption of small molecule drugs by the intestinal chip and is beneficial for the development of drug-related applications.

[0018] 4. The pump-free intestinal microarray system provided by this invention can achieve throughput experiments by designing the well spacing of the culture layer to meet the standard 96-well plate specifications, thereby improving experimental repeatability. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the layered structure of the intestinal chip of the present invention; Figure 2 This is a schematic diagram of the electrode support for the transmembrane resistance measuring electrode. a is the front view, b is the left view, c is the top view, and d is the perspective view. Figure 3 A schematic diagram illustrating the principle of measuring transmembrane resistance using electrodes in a chip; Figure 4 The transmembrane resistance measuring electrode was used to measure the resistance-concentration plots of different potassium chloride (KCl) solutions; Figure 5 This is a graph showing the change in transmembrane resistance of the gut microarray as a function of culture days in Example 3. In the diagram: 1-1: Culture layer; 1-2: Porous membrane; 1-3: Perfusion layer; 1-1a: First end hole; 1-1b: Middle hole; 1-1c: Second end hole; 1-2a: First through hole; 1-2b: Second through hole; 1-3a: Cavity; 2-1a, 2-1b, 2-1c and 2-1d: Platinum wire insertion holes; 2-2a, 2-2b, 2-2c and 2-2d: Limiting posts. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0022] The following is a detailed description of a pumpless intestinal chip system and its measurement method for measuring transmembrane resistance, as described in this invention.

[0023] Example 1 A pump-free gut-on-a-chip system for measuring transmembrane resistance includes transmembrane resistance measuring electrodes, a gut chip, and a oscillating shaker; Figure 1As shown, the intestinal microarray consists of multiple parallel-arranged chip units; each chip unit, from top to bottom, includes a culture layer 1-1, a porous membrane 1-2, and a perfusion layer 1-3; the culture layer is provided with a first end hole 1-1a, a middle hole 1-1b, and a second end hole 1-1c; the perfusion layer 1-3 is provided with a cavity 1-3a for containing culture medium; the porous membrane 1-2 is provided with a first through hole 1-2a and a second through hole 1-2b; the first end hole 1-1a and the second end hole 1-1c are respectively connected to the perfusion layer through the first through hole 1-2a and the second through hole 1-2b. The cavities 1-3a of layer 1-3 are connected to form a fluid channel; the intermediate pore 1-1b is used for cell culture, and the intermediate pore 1-1b separates the culture layer 1-1 from the perfusion layer 1-3 and facilitates material exchange through the porous membrane 1-2; the oscillating shaker is used to provide periodic gravity flow to simulate intestinal peristalsis; the transmembrane resistance measuring electrode includes four platinum wires, arranged in groups of two. In each group of electrodes, one wire is inserted into the intermediate pore 1-1b, and the other wire is inserted into the cavity 1-3a of the perfusion layer 1-3 through the same end hole. Each group of electrodes is connected to the resistance meter through a wire.

[0024] like Figure 3 The measurement principle diagram shows that I1 and I2 are one set of platinum wire electrodes, and V1 and V2 are another set of platinum wire electrodes. The resistance value is measured based on Ohm's law.

[0025] Example 2 A pump-free gut-on-a-chip system for measuring transmembrane resistance includes transmembrane resistance measuring electrodes, a gut chip, and a oscillating shaker; Figure 1 As shown, the intestinal microarray consists of multiple parallel-arranged chip units; from top to bottom, each chip unit includes a culture layer 1-1, a porous membrane 1-2, and a perfusion layer 1-3; the culture layer 1-1 is provided with a first end hole 1-1a, a middle hole 1-1b, and a second end hole 1-1c; the perfusion layer 1-3 is provided with a cavity 1-3a for containing culture medium; the porous membrane 1-2 is provided with a first through hole 1-2a and a second through hole 1-2b; the first end hole 1-1a and the second end hole 1-1c are connected to the cavity 1-3a of the perfusion layer 1-3 through the first through hole 1-2a and the second through hole 1-2b, respectively. The system is designed to form a fluid channel; the intermediate pore 1-1b is used for cell culture, and the intermediate pore 1-1b separates the culture layer 1-1 from the perfusion layer 1-3 and facilitates material exchange through the porous membrane 1-2; the oscillating shaker is used to provide periodic gravity flow to simulate intestinal peristalsis; the transmembrane resistance measuring electrode includes four platinum wires, arranged in groups of two. In each group of electrodes, one wire is inserted into the intermediate pore 1-1b, and the other wire is inserted into the cavity 1-3a of the perfusion layer 1-3 through the same end hole. Each group of electrodes is connected to a resistance meter via wires. A commercial resistance meter is used to provide current and measure voltage to obtain the transmembrane resistance value.

[0026] The intestinal microarray has a hole spacing that conforms to the specifications of a 96-well plate, and the culture layer 1-1, porous membrane 1-2, and perfusion layer 1-3 are firmly bonded together by hot pressing. The culture layer 1-1 and the perfusion layer 1-3 are made of PS, the porous membrane 1-2 is made of PET, the culture layer 1-1 is 3 cm high, the porous membrane 1-2 has a pore size of 2 μm and a thickness of 8 μm, and the cavity 1-3a of the perfusion layer 1-3 has a depth of 5 mm. The transmembrane resistance measuring electrode and the intestinal chip are positioned by an electrode support. The electrode support includes two parallel side plates and a top plate connected to the top of the two side plates. The side plates and the top plate enclose a downward-facing cavity. The opening width of the cavity is 2 mm larger than the width of the intestinal chip. In this embodiment, the width of the intestinal chip is 3 cm. The top plate is provided with four limiting posts and a platinum wire insertion hole. The limiting posts are distributed around the middle hole 1-1b and have a diameter of 1 mm. The diameter of the platinum wire insertion hole is 1 mm. The bottom of the platinum wire inserted into the middle hole 1-1b is 1-21 mm away from the porous membrane, and the bottom of the platinum wire inserted into the end hole is 1 mm away from the bottom of the cavity 1-3a. The two platinum wires inserted into the same hole are spaced 1 mm apart, and the diameter of the platinum wire is 0.8 mm.

[0027] After the electrode holder of the transmembrane resistance measuring electrode is fixed to the platinum wire, it is inserted into KCl solutions of different concentrations. Figure 4 It can be observed that as the concentration increases, the reciprocal of the resistance value of the solution measured using the transmembrane resistance measuring electrode increases linearly, demonstrating the rationality of the design of the transmembrane resistance measuring electrode.

[0028] Example 3 The transmembrane resistance is measured using the pumpless intestinal chip system described in Example 2, which is capable of measuring transmembrane resistance. The steps include: (1) Sterilize or disinfect the intestinal chip and the transmembrane resistance measuring electrode; (2) Intestinal cells were seeded into the middle well of the intestinal chip, with a quantity of 100,000 cells, a medium volume of 100 μL in the middle well, a perfusion layer medium volume of 1 mL, and static culture for 8-36 h after seeding. (3) The intestinal chip was placed on a swing shaker in an incubator for culture. The swing angle of the swing shaker was set to 10° and the swing speed was 10 rpm. The control group was a static culture condition without the introduction of a swing shaker to provide fluid conditions. (4) The intestinal microarray was cultured continuously for 6 days under two conditions, with the culture medium changed daily. The transmembrane resistance of the intestinal microarray under the two conditions was measured using a transmembrane resistance measuring electrode. The change of transmembrane resistance of the intestinal microarray with the number of culture days was obtained. Figure 5 ).

[0029] The results showed that the transmembrane resistance of the intestinal chip in Example 3 was higher than that of the control group within 6 days, indicating that the barrier function of the pump-free intestinal chip system of the present invention is superior.

[0030] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the implementation. The scope of protection of the present invention should be determined by the scope defined in the claims. Other variations or modifications can be made based on the above description. Obvious variations or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A pump-free intestinal chip system for measuring transmembrane resistance, characterized in that: The system includes transmembrane resistance measuring electrodes, an intestinal chip, and a oscillating shaker; The intestinal microarray consists of multiple parallel-arranged chip units; each chip unit comprises, from top to bottom, a culture layer, a porous membrane, and a perfusion layer; the culture layer is provided with a first end pore, a middle pore, and a second end pore; the perfusion layer is provided with a cavity for containing culture medium; the porous membrane is provided with a first through pore and a second through pore; the first end pore and the second end pore are respectively connected to the cavity of the perfusion layer through the first through pore and the second through pore to form a fluid channel; the middle pore is used for culturing cells, and the middle pore separates the culture layer and the perfusion layer and facilitates substance exchange through the porous membrane; The swing-type shaker is used to provide a periodic flow of gravity to simulate intestinal peristalsis; The transmembrane resistance measuring electrode includes four platinum wires, arranged in pairs. In each group, one wire is inserted into the middle hole, and the other wire is inserted into the cavity of the perfusion layer through the same end hole. Each group of electrodes is connected to the resistance meter via a wire.

2. The pumpless intestinal chip system for measuring transmembrane resistance according to claim 1, characterized in that: The transmembrane resistance measuring electrode and the intestinal chip are positioned by an electrode holder; The electrode support includes two parallel side plates and a top plate connected to the top of the two side plates, the side plates and the top plate forming a downward-facing groove. The top plate is provided with four limiting posts and four platinum wire insertion holes; The limiting posts are distributed around the central hole, and their diameter is 0.5 mm-3 mm; The diameter of the platinum wire insertion hole is 0.5 mm-3 mm.

3. The pumpless intestinal chip system for measuring transmembrane resistance according to claim 1, characterized in that: The culture layer is made of polystyrene or polymethyl methacrylate, and its height is 5 mm to 5 cm. The perfusion layer is made of polystyrene or polymethyl methacrylate, and the cavity depth of the perfusion layer is 0.1 mm to 1 cm. The porous membrane is made of polycarbonate, polyester or polytetrafluoroethylene, and its thickness is 0.1~20μm; the pore diameter of the first through hole and the second through hole is 0.1~20 μm.

4. The pumpless intestinal chip system for measuring transmembrane resistance according to claim 1, characterized in that: The swing angle of the swing-type rocker is 0~60° and not 0, and the swing speed is 0~100 rpm and not 0.

5. The pumpless intestinal chip system for measuring transmembrane resistance according to claim 2, characterized in that: The opening width of the electrode support slot is 1 mm to 1 cm larger than the width of the intestinal chip; The width of the intestinal chip is 1 cm to 5 cm.

6. The pumpless intestinal chip system for measuring transmembrane resistance according to claim 1, characterized in that: The bottom of the platinum wire inserted into the middle hole is 0.1 mm to 1 cm away from the porous membrane. The two platinum wires inserted into the same hole are spaced 0.1 mm to 0.5 cm apart, and the diameter of the platinum wire is 0.5 mm to 2 mm.

7. The pumpless intestinal chip system for measuring transmembrane resistance according to claim 1, characterized in that: The culture layer, porous membrane, and perfusion layer are integrally formed by double-sided tape, glue, or hot pressing.

8. The pumpless intestinal chip system for measuring transmembrane resistance according to claim 1, characterized in that: The intestinal chip is composed of multiple chip units integrated into a single structure.

9. A method for measuring transmembrane resistance using the pump-free intestinal chip system according to any one of claims 1-8, characterized in that, Includes the following steps: (1) Sterilize or disinfect the intestinal chip and the transmembrane resistance measuring electrode; (2) Intestinal cells were seeded into the central well of the intestinal microarray and cultured statically after seeding; (3) The intestinal microarray was placed on a swing-type shaker in an incubator for incubation; (4) Connect the transmembrane resistance measuring electrode to the resistor meter, provide current through the resistor meter and measure voltage to obtain the transmembrane resistance value.

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