Multi-organ chip integrating oxygen concentration control and metabolism dynamic monitoring

Abstract

The utility model belongs to the field of organ-like chips, and particularly discloses a multi-organ-like chip integrating oxygen concentration control and metabolism dynamic monitoring. The multi-organoid chip is composed of an upper-layer chip, a PDMS film and a lower-layer chip, wherein a cell culture medium fluid channel, three organoid culture areas and three electrode detection areas are arranged on the lower surface of the upper-layer chip; a Na2SO3 solution fluid channel and an electrode sensing area are arranged on the upper surface of the lower-layer chip; and the lower surface of the upper-layer chip and the upper surface of the lower-layer chip are separated by a polydimethylsiloxane (PDMS) film with the thickness of 100 microns. According to the multi-organ co-culture device, co-culture of multiple organs can be achieved, an oxygen concentration environment similar to human physiology is provided for organoid culture, changes of multiple substances in the organoid microenvironment can be monitored in situ in real time, and the multi-organ co-culture device has wide application value in clinical drug screening and disease model construction.

Description

Technical Field

[0001] The utility model relates to the field of microfluidic chips, and in particular to a multiple organoid chip integrating oxygen concentration control and metabolic dynamic monitoring. Background Art

[0002] Organ-on-a-chip is an emerging cutting-edge cross-disciplinary technology. By integrating organoids and organ chips, it can construct an organ model system with high physiological relevance in vitro. It has important application potential in the fields of tissue and organ development, disease research, drug screening, and regenerative medicine.

[0003] A controllable cellular chemical microenvironment helps to build organoid chips with higher fidelity. This view is particularly reflected in the dissolved oxygen concentration, glucose concentration, lactate concentration and pH in the cell culture medium. In order to predict the response of cells or tissues in vivo as accurately as possible in vitro, various microenvironmental information such as oxygen content, pH value and temperature need to be monitored in the system. Based on the above situation, organoid chips need a universal system to help form a controllable cellular chemical microenvironment.

[0004] However, two problems and challenges currently exist. First, the oxygen concentration environment within organoid chips differs significantly from human physiology. A representative example is the organoid chip published in Nature Communication in 2024 by Xavier Gidol's group at Grenoble Alpes University in France. The research team achieved complete vascularization of organoids on a microfluidic chip for the first time, with the intravascular culture fluid flow rate similar to that of blood. The researchers observed improved organoid growth. However, the dissolved oxygen concentration in the intravascular culture fluid differs significantly from that in blood, remaining in a non-physiological hyperoxic state. This means that the physiological functions of organoids remain unequal to those in the human body. Second, most current organoid chips still require extensive manual sampling to test metabolite changes in the organoid culture medium. This is an invasive process that requires opening the device and then sampling. However, this process is discontinuous, and the ability to effectively and continuously monitor metabolite changes is crucial for long-term organoid culture. To address this, the present invention designs a multi-functional organoid chip that integrates oxygen concentration control and dynamic metabolic monitoring. Utility Model Content

[0005] The purpose of the present invention is to address the shortcomings of the existing technology and to propose a multi-organ-on-a-chip that integrates oxygen concentration control and metabolic dynamic monitoring.

[0006] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A multi-organoid chip that integrates oxygen concentration control and dynamic metabolic monitoring, including an upper chip, a PDMS membrane and a lower chip: wherein, the lower surface of the upper chip is provided with a cell culture medium fluid channel, three organoid culture areas and three electrode detection areas, and the above-mentioned channels are connected to the areas; the lower surface of the upper chip and the upper surface of the lower chip are separated by a 100μm thick PDMS membrane, and the upper surface of the lower chip is provided with a Na2SO3 solution fluid channel and an electrode sensing area.

[0008] Furthermore, the cell culture medium flows in from the cell culture medium perfusion inlet on the lower surface of the upper chip, and then flows out from the cell culture medium perfusion outlet. The Na2SO3 solution flows in from the Na2SO3 solution inlet, flows through the PDMS membrane perforations into the Na2SO3 solution channel on the upper surface of the lower chip, and finally flows upward and out from the Na2SO3 solution outlet.

[0009] Furthermore, the organoid culture area is perfused with a plurality of matrix gel microspheres containing organoid suspension (the diameter of a single microsphere is greater than 500 μm).

[0010] Furthermore, the cell culture medium fluid channel on the lower surface of the upper chip and the Na2SO3 solution fluid channel on the upper surface of the lower chip are symmetrical. The cell culture medium and the Na2SO3 solution flow in the same direction in the channel. The PDMS membrane between the two is breathable. Oxygen in the cell culture medium can pass through the PDMS membrane into the Na2SO3 solution of the lower chip, thereby generating a redox reaction to consume oxygen in the cell culture medium.

[0011] Furthermore, the cell culture medium on the lower surface of the upper chip passes through the PDMS membrane through the pH detection inlet, the glucose detection inlet, and the lactic acid detection inlet to the electrode sheet sensing area of the lower chip, and undergoes electrochemical reaction therewith.

[0012] Furthermore, the electrode sheet adopts a two-dimensional MXene conductive material.

[0013] After the upper chip, PDMS membrane, and lower chip are fixed and formed, three organoid culture areas are formed. Each of the three culture areas contains multiple Matrigel microspheres (the diameter of a single microsphere is greater than 500μm) with organoid suspension. Cell culture medium penetrates through the gaps between the microspheres, and seed cells can grow into organoids through self-assembly in the Matrigel.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] This chip can simultaneously meet the co-culture and free combination of three different organoids. It can be widely used to simulate systemic diseases involving multiple organs (such as cancer, metabolism, inflammatory infection, etc.) and multi-organ drug effects and toxicity assessments that are difficult to simulate in vitro experiments. It fills a gap in the industry and has broad clinical application value.

[0016] The design of the PDMS membrane and the Na2SO3 solution channel in the lower chip generates a redox reaction that consumes oxygen in the cell culture medium, providing an oxygen concentration environment similar to that of the human body for organoid culture, which is beneficial to the formation of the physiological microstructure of organoids, the maintenance of physiological metabolic levels, and long-term culture.

[0017] The addition of electrochemical sensors enables real-time monitoring of changes in multiple substances in the organoid microenvironment, dynamically and quantitatively reflecting the activity status of the organoid, which is beneficial for organoid metabolic assessment and drug toxicity evaluation in disease modeling and drug testing.

[0018] This chip has two major advantages. One is that it provides an oxygen concentration environment similar to that of human physiology for organoid culture; the other is that it can dynamically monitor changes in pH, glucose and lactate concentrations in real time without sampling. At the same time, this chip uses a peristaltic pump and liquid storage bottle, which can facilitate liquid replacement and sampling at any time, and can be adapted to the detection methods of conventional microfluidic chips and microscopy observation.

[0019] This chip is processed using organic glass plates (PMMA) and PDMS as matrices, can be mass-produced, and has low chip production costs and high yield, and has great commercial potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the overall structure of a multi-organ-on-a-chip proposed in the present invention that integrates oxygen concentration control and metabolic dynamic monitoring.

[0021] Figure 2 This is a schematic diagram of the structure of the upper chip in a multi-organoid chip that integrates oxygen concentration control and metabolic dynamic monitoring proposed in the present invention.

[0022] Figure 3 This is a schematic diagram of the structure of a multi-organ-on-a-chip PDMS membrane that integrates oxygen concentration control and metabolic dynamic monitoring proposed in the present invention.

[0023] Figure 4 This is a schematic diagram of the structure of the lower chip in a multiple organoid chip that integrates oxygen concentration control and metabolic dynamic monitoring proposed in the present invention.

[0024] Figure 5 This is a schematic diagram of the overall structure of the assembled multiple organoid chips proposed in the present invention, which integrates oxygen concentration control and metabolic dynamic monitoring.

[0025] Figure 6 This is a schematic diagram of cell culture in the organoid culture area of a multiple organoid chip that integrates oxygen concentration control and metabolic dynamic monitoring proposed in the present invention.

[0026] Figure 7 This is a schematic diagram of the multiple organoid chip working system proposed in the present invention, which integrates oxygen concentration control and metabolic dynamic monitoring.

[0027] In the figure: 01 upper chip, 02 PDMS membrane, 03 lower chip, 1 is the cell culture medium perfusion inlet, 2 is the cell culture medium perfusion outlet, 3 is the cell culture medium fluid channel, 4 is the organoid culture area, 5 is the pH detection inlet, 6 is the glucose detection inlet, 7 is the lactic acid detection inlet, 8 is the Na2SO3 solution inlet, 9 is the Na2SO3 solution outlet, 10 is the Na2SO3 solution fluid channel, 11 is the pH detection electrode groove, 12 is the pH detection electrode sheet, 13 is the glucose detection electrode groove, 14 is the glucose detection electrode sheet, 15 is the lactic acid detection electrode groove, and 16 is the lactic acid detection electrode sheet. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0029] like Figure 1-7As shown, a multiple organoid chip that integrates oxygen concentration control and metabolic dynamic monitoring includes an upper chip 01, a PDMS membrane 02 and a lower chip 03. After the upper chip 01, the PDMS membrane 02 and the lower chip 03 are bonded together, they are fixed with screws and nuts. The lower surface of the upper chip 01 is provided with a cell culture medium perfusion inlet 1, a cell culture medium perfusion outlet 2, a cell culture medium fluid channel 3, three organoid culture areas 4, a pH detection inlet 5, a glucose detection inlet 6, a lactate detection inlet 7, a Na2SO3 solution inlet 8 and a Na2SO3 solution outlet 9; the lower surface of the upper chip 01 and the upper surface of the lower chip 03 are separated by a 100μm thick PDMS membrane 02 (polydimethylsiloxane), on which there are perforations corresponding to the pH detection inlet 5, the glucose detection inlet 6, the lactate detection inlet 7, the Na2SO3 solution inlet 8 and the Na2SO3 solution outlet 9. The upper surface of the lower chip 03 is provided with a Na2SO3 solution fluid channel 10, a pH detection electrode groove 11, a pH detection electrode sheet 12, a glucose detection electrode groove 13, a glucose detection electrode sheet 14, a lactic acid detection electrode groove 15 and a lactic acid detection electrode sheet 16, as well as a Na2SO3 solution inlet 8 and a Na2SO3 solution outlet 9 corresponding to the upper chip 01. The Na2SO3 solution fluid channel 10 is 500 μm wide and 1 mm deep. The concentration of the injected Na2SO3 solution can be 2%, 3%, 4%, 5% or 6%. The depth of the Na2SO3 solution inlet 8, the Na2SO3 solution outlet 9, the pH detection electrode groove 11, the glucose detection electrode groove 13 and the lactic acid detection electrode groove 15 are all 1 mm. The thickness of the pH detection electrode sheet, the glucose detection electrode sheet 14 and the lactic acid detection electrode sheet 16 are all 1 mm.

[0030] The cell culture medium fluid channel 3 on the lower surface of the upper chip 01 and the Na2SO3 solution fluid channel 10 on the upper surface of the lower chip 03 are symmetrical. The cell culture medium and the Na2SO3 solution flow in the same direction in the channel. The PDMS membrane 02 between the two is breathable. The oxygen in the cell culture medium can pass through the PDMS membrane 02 and enter the Na2SO3 solution of the lower chip 03, thereby generating a redox reaction to consume the oxygen in the cell culture medium. This process can regulate the dissolved oxygen concentration of the culture medium entering the organoid culture area and simulate the blood oxygen environment in the human body.

[0031] After the upper chip 01, PDMS membrane 02 and lower chip 03 are fixed and formed, three organoid culture areas are formed. Each of the three culture areas contains multiple matrix gel microspheres (the diameter of a single microsphere is greater than 500μm) with organoid suspension. The cell culture medium penetrates through the gaps between the microspheres, and the seed cells can grow into organoids through self-assembly in the matrix gel.

[0032] Cell culture medium on the lower surface of upper chip 01 passes through pH detection port 5, glucose detection port 6, and lactate detection port 7, respectively, through PDMS membrane 02 to the electrode sensing area of lower chip 03, where it undergoes an electrochemical reaction. Changes in the electrochemical signals reflect changes in the concentration of cellular metabolites. The conductive material of all three electrodes is two-dimensional MXeneTi3C2Tx, which enhances the reaction signal strength.

[0033] When using, the steps are:

[0034] 1) Before assembling the chip, place the upper chip with the lower surface facing up and add multiple Matrigel microspheres containing organoid suspension (with a single microsphere diameter greater than 500 μm) to each of the three organoid culture areas until the entire area is filled;

[0035] 2) The upper chip 01 is left to stand until the matrigel solidifies, and then the upper chip 01, PDMS membrane 02, and lower chip 03 are assembled in sequence, and the matrigel further solidifies;

[0036] 3) Cell culture medium perfusion inlet 1, cell culture medium perfusion outlet 2, Na2SO3 solution inlet 8, and Na2SO3 solution outlet 9 are respectively connected to external peristaltic pumps to circulate culture medium. The cells are placed in a cell culture incubator, with the cell culture medium perfusion channel perfusing organoid culture medium and the Na2SO3 solution channel perfusing Na2SO3 solution.

[0037] 4) Organoids self-assemble in the organoid culture area and gradually grow into mature organoids. The electrode sensing area monitors changes in pH, glucose, and lactate concentrations in the cell culture medium in real time.

[0038] The above is only a preferred specific implementation method of the present invention, but the protection scope of the present invention is not limited to this. Any technician familiar with the technical field within the technical scope disclosed by the present invention can make equivalent replacements or changes based on the technical solution and utility model concept of the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A multi-organoid chip integrating oxygen concentration control and metabolic dynamic monitoring, characterized in that: An upper chip (01), a PDMS membrane (02) and a lower chip (03): wherein the lower surface of the upper chip (01) is provided with a cell culture medium fluid channel, three organoid culture areas and three electrode detection areas, and the above-mentioned channels are connected to the areas; the lower surface of the upper chip (01) and the upper surface of the lower chip (03) are separated by a PDMS membrane (02); a Na2SO3 solution fluid channel and an electrode sensing area are provided on the upper surface of the lower chip (03), and the upper chip (01) is provided with a cell culture medium perfusion inlet (1), a perfusion outlet (2), The cell culture medium fluid channel (3), the organoid culture area (4), the pH detection inlet (5), the glucose detection inlet (6), the lactic acid detection inlet (7), the Na2SO3 solution inlet (8) and the Na2SO3 solution outlet (9); the upper surface of the lower chip (03) is provided with a Na2SO3 solution fluid channel (10), a pH detection electrode groove (11), a pH detection electrode sheet (12), a glucose detection electrode groove (13), a glucose detection electrode sheet (14), a lactic acid detection electrode groove (15) and a lactic acid detection electrode sheet (16).

2. The multi-organ-on-a-chip integrating oxygen concentration control and metabolic dynamic monitoring according to claim 1, characterized in that: The organoid culture area (4) is perfused with a plurality of matrix gel microspheres containing an organoid suspension, and the diameter of a single microsphere is greater than 500 μm. The diameter of the organoid culture area (4) is 4 mm.

3. The multi-organ-on-a-chip integrating oxygen concentration control and metabolic dynamic monitoring according to claim 2, characterized in that: The cell culture medium fluid channel (3) is 500 μm wide and 1 mm deep. The cell culture medium perfusion inlet (1), cell culture medium perfusion outlet (2), organoid culture area (4), pH detection inlet (5), glucose detection inlet (6) and lactate detection inlet (7) are all 1 mm deep. The Na2SO3 solution inlet (8) and Na2SO3 solution outlet (9) are perforated.

4. The multi-organ-on-a-chip integrating oxygen concentration control and metabolic dynamic monitoring according to claim 3, characterized in that: After the upper chip (01), the PDMS membrane (02) and the lower chip (03) are attached, they are fixed with screws and nuts.

5. The multi-organ-on-a-chip integrating oxygen concentration control and metabolic dynamic monitoring according to claim 4, characterized in that: The PDMS membrane (02) is 100 μm thick and is perforated at positions corresponding to the pH detection inlet (5), glucose detection inlet (6), lactic acid detection inlet (7), Na2SO3 solution inlet (8) and Na2SO3 solution outlet (9) of the upper chip (01).

6. The multi-organ-on-a-chip integrating oxygen concentration control and metabolic dynamic monitoring according to claim 5, characterized in that: The Na2SO3 solution fluid channel (10) is 500 μm wide and 1 mm deep. The concentration of the injected Na2SO3 solution can be 2%, 3%, 4%, 5% or 6%. The Na2SO3 solution inlet (8), the Na2SO3 solution outlet (9), the pH detection electrode groove (11), the glucose detection electrode groove (13) and the lactic acid detection electrode groove (15) are all 1 mm deep. The pH detection electrode sheet, the glucose detection electrode sheet (14) and the lactic acid detection electrode sheet (16) are all 1 mm thick.

7. The multi-organ-on-a-chip integrating oxygen concentration control and metabolic dynamic monitoring according to claim 6, characterized in that: The pH detection electrode sheet, the glucose detection electrode sheet (14) and the lactic acid detection electrode sheet (16) are all made of a two-dimensional MXene conductive material.

8. The multi-organ-on-a-chip integrating oxygen concentration control and metabolic dynamic monitoring according to claim 6, characterized in that: The cell culture medium fluid channel and the Na2SO3 solution fluid channel are symmetrical, and the cell culture medium and the Na2SO3 solution flow in the same direction in the two channels. The PDMS membrane (02) between the two is air permeable, and oxygen in the cell culture medium can pass through the PDMS membrane (02) into the Na2SO3 solution of the lower chip (03), thereby generating an oxidation-reduction reaction to consume oxygen in the cell culture medium.

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