Double-chamber micro-fluidic organ chip, preparation method and application

Abstract

The invention discloses a double-chamber micro-fluidic organ chip, a preparation method and application, and belongs to the technical field of biological materials. The double-chamber micro-fluidic organ chip sequentially comprises a top layer, an upper layer, a middle layer and a lower layer from top to bottom, cavities are formed in the upper layer and the lower layer, and micropore arrays for accommodating biological samples are arranged in the cavities; the chip further comprises a plurality of circulation channels which can be respectively communicated with the upper layer chamber and the lower layer chamber, so that a biological sample enters and exits from the micropore array; and the middle layer is a semi-permeable porous membrane. The micro-fluidic organ chip can be used for establishing a metabolic disease model, promoting interaction between organs and realizing space-time dynamic change analysis of tissues and organs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and specifically relates to a double-chamber microfluidic organ chip, a preparation method and an application thereof. Background Art

[0002] Metabolic dysfunction-associated fatty liver disease (MASLD) / non-alcoholic fatty liver disease (NAFLD) is a glycolipid disorder disease accompanied by stress-induced liver injury and insulin resistance. Its pathogenesis ranges from simple fatty liver to non-alcoholic steatohepatitis (NASH) with necrotic inflammation and fibrosis or even cirrhosis. With the rapid changes in modern lifestyles, MASLD has now become the largest chronic liver disease globally, which is closely related to metabolic syndrome and type 2 diabetes. There are multiple hypotheses about the pathogenesis of MASLD, such as genetics, metabolism, oxidative stress and immunity. However, due to the lack of a human biomimetic MASLD model, the understanding of its underlying mechanisms is insufficient. In addition, the efficacy of current MASLD treatment methods is also limited because these treatments mainly focus on alleviating symptoms related to metabolic disorders. Therefore, there is an urgent need to develop a biomimetic MASLD model and corresponding therapeutic interventions.

[0003] For this reason, a double-chamber microfluidic organ chip, a preparation method and an application thereof are proposed. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a double-chamber microfluidic organ chip, a preparation method and an application thereof, which solve the problems in the prior art.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] A double-chamber microfluidic organ chip, which successively includes from top to bottom: a top layer, an upper layer, a middle layer and a lower layer; chambers are provided in both the upper layer and the lower layer, and a microporous array for accommodating biological samples is provided in the chambers; the chip further includes a plurality of flow channels, and can be respectively communicated with the chambers of the upper and lower layers to enable biological samples to enter and exit the microporous array;

[0007] The middle layer is a semi-permeable porous membrane.

[0008] Further, the material of the semi-permeable porous membrane is polycarbonate, fluorinated ethylene polymer, polytetrafluoroethylene, polyvinyl chloride or polyvinyl alcohol.

[0009] Further, the materials of the upper layer and the lower layer are polydimethylsiloxane, polymethyl methacrylate, hydrogel, collagen, polystyrene or styrene-ethylene (butene)-styrene copolymer.

[0010] Further, the chamber size is 10 mm × 10 mm × 2 mm; the pore diameter of the microporous array is 750 μm; the pore size of the semi-permeable porous membrane is 0.4 μm.

[0011] The preparation method of the above-mentioned dual-chamber microfluidic organ chip is characterized by comprising the following steps:

[0012] Prepare sacrificial male molds for the top layer, upper layer and lower layer by 3D printing;

[0013] After mixing the PDMS prepolymer and the curing agent at a weight ratio of 10:1, pour it on the sacrificial male mold for curing, and then separate the PDMS from the sacrificial male mold to obtain the top layer, upper layer and lower layer;

[0014] Perform surface treatment on the top layer, upper layer, middle layer and lower layer by using oxygen plasma;

[0015] Assemble the top layer, upper layer, middle layer and lower layer in sequence to obtain the product.

[0016] The application of the above-mentioned dual-chamber microfluidic organ chip in dual-organ culture.

[0017] Further, the steps of dual-organ culture include:

[0018] 1) Sterilize the chip with cyclohexane;

[0019] 2) Feed two types of organs into the microporous arrays of the upper chamber and the lower chamber respectively through the flow channels;

[0020] 3) Pump the culture medium into the upper chamber and the lower chamber for culture.

[0021] The application of the above-mentioned dual-chamber microfluidic organ chip in constructing a metabolic disease model.

[0022] The application of the above-mentioned dual-chamber microfluidic organ chip in developing drugs or devices for preventing or treating metabolic diseases.

[0023] Further, the metabolic diseases include: diabetes and its complications, metabolic associated fatty liver disease, hyperuricemia or metabolic syndrome.

[0024] The beneficial effects of the present invention:

[0025] 1. The present invention prepares a dual-chamber microfluidic organ chip by a PDMS microfluidic chip manufacturing method assisted by 3D printing templates. The method is simple, has high repeatability, low cost, and is easy to synthesize in batches.

[0026] 2. The present invention adopts a dual-chamber co-culture technique to establish a co-culture system for two organs, promoting the material exchange between the two organs and realizing the long-term survival and function maintenance of liver and pancreatic organoids.

[0027] 3. A dual-chamber microfluidic organ chip prepared by the present invention can be used for the construction of metabolic disease models, mechanism research, and drug screening. By simulating the interaction between the liver and pancreatic organs on the chip, disease research based on a human organ chip is realized. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0029] Figure 1 It is a schematic structural diagram of the dual-chamber microfluidic organ chip of the present invention;

[0030] Figure 2 It is a physical image of the dual-chamber microfluidic organ chip of the present invention;

[0031] Figure 3 It is a simulation diagram of the dual-chamber microfluidic organ chip of the present invention;

[0032] Figure 4 It is an optical microscopic image of the cells cultured in the micropore array of the dual-chamber microfluidic organ chip of the present invention;

[0033] Figure 5 It is the morphology of the liver organoids and islet organoids cultured on the dual-chamber microfluidic organ chip of the present invention;

[0034] Figure 6 It is the function of the liver organoids and islet organoids cultured on the dual-chamber microfluidic organ chip of the present invention;

[0035] Figure 7 It is a MASLD intervention model established based on liver organoids and islet organoids on the dual-chamber microfluidic organ chip of the present invention;

[0036] In the figure: 1 - flow-through channel, 2 - top layer, 3 - upper layer, 4 - middle layer, 5 - lower layer, 6 - upper chamber micropore array, 7 - lower chamber micropore array. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0038] As Figure 1 shown, a double-chamber microfluidic organ-on-a-chip (OoC chip) includes, from top to bottom in sequence: a top layer 2, an upper layer 3, a middle layer 4, and a lower layer 5;

[0039] Chambers are provided in both the upper layer 3 and the lower layer 5, and an upper chamber micropore array 6 and a lower chamber micropore array 7 are respectively provided in the chambers of the upper layer 3 and the lower layer 5 to accommodate biological samples (such as cells, tissues, or organs); and a liquid storage pool is provided in the chamber to store the culture solution, so as to realize the culture of biological samples;

[0040] A plurality of flow channels 1 are opened on the top layer 2 and can be respectively communicated with the upper and lower chambers. The flow channels 1 can realize the entry and exit of biological samples into and out of the upper chamber micropore array 6 and the lower chamber micropore array 7.

[0041] The middle layer 4 is a semi-permeable porous membrane; it allows material exchange and biochemical molecular information exchange between the upper and lower chambers, and realizes the interaction between two organs or tissues;

[0042] In this embodiment, the material of the semi-permeable porous membrane is polycarbonate. In other embodiments, the material of the semi-permeable porous membrane can also be: fluorinated ethylene polymer (FEP), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), or polyvinyl alcohol (PVA).

[0043] In this embodiment, the materials of the top layer 2, the upper layer 3, and the lower layer 5 are made of polydimethylsiloxane (PDMS); while in other embodiments, the materials of the upper layer 3 and the lower layer 5 can also be polymethyl methacrylate (PMMA), hydrogel, collagen, polystyrene, or styrene-ethylene (butene)-styrene copolymer.

[0044] In this embodiment, the chamber sizes of the upper layer 3 and the lower layer 5 are 10 mm × 10 mm × 2 mm; the pore diameters of the micropores in the upper chamber micropore array 6 and the lower chamber micropore array 7 are 750 μm; the pore diameter of the semi-permeable porous membrane is 0.4 μm (Millipore, HTTP02500).

[0045] Embodiment 2

[0046] In this embodiment, a specific preparation method of the double-chamber microfluidic organ-on-a-chip (OoC chip) in Embodiment 1 is disclosed, including the following steps:

[0047] S1. Use 3D Max software to design the 3D model of the OoC chip, and adopt a projection microstereolithography (PμSL) 3D printing system (nanoArch S140) to fabricate the 3D printed sacrificial male molds of the top layer 2, upper layer 3 and lower layer 5 of the OoC chip with a resolution of 2 μm.

[0048] S2. After mixing the PDMS prepolymer and the curing agent (platinum catalyst) at a weight ratio of 10:1, pour it on the 3D printed template (sacrificial male mold), and perform a curing treatment at 80 °C for 4 h to form the negative mold structure of the OoC chip. Separate the PDMS from the template by physical peeling to obtain the top layer 2, upper layer 3 and lower layer 5 of the OoC chip.

[0049] S3. Use a commercial polycarbonate membrane (Millipore, HTTP02500) cut into a semi-permeable porous membrane of 8 mm × 8 mm as the intermediate layer 4;

[0050] S4. Assemble the top layer 2, upper layer 3, intermediate layer 4 and lower layer 5 through oxygen plasma treatment;

[0051] Specifically: Use a plasma surface treatment instrument (VPC100M, Betop) to perform surface treatment on the OoC component. The frequency of the radio frequency plasma generation source of the radio frequency transceiver core circuit is 13.56 MHz, and the vacuum degree is 20 - 60 Pa.

[0052] The physical diagram of the double-chamber microfluidic organ chip prepared in this embodiment is as Figure 2 shown; It can be seen from the figure that the OoC chip has a structure of a top layer 2, an upper layer 3, an intermediate layer 4 and a lower layer 5, wherein the chambers of the upper layer 3 and the lower layer 5 are respectively connected to the flow channels 1 to form a path for the chambers to communicate with the outside world.

[0053] Example 3

[0054] In this embodiment, an application of a double-chamber microfluidic organ chip in organ culture is introduced, as Figure 3 shown, including the following contents:

[0055] 1) Before conducting the culture experiment, sterilize the OoC chip with cyclohexane;

[0056] 2) Culture human induced pluripotent stem cells (hiPSC) in a six-well plate with mTeSR1 medium. The culture plate is pre-coated with Matrigel diluted at 1:50. Use Accutase to separate iPSC cells, and perform subsequent passage after reaching about 80% confluence;

[0057] 3) Differentiate organoids from hiPSCs; first, digest and suspend the hiPSCs, and seed them on a microwell device with a diameter of 200 μm to form embryoid bodies (EBs). Culture the EBs in mTeSR1 medium supplemented with 10 μM Y27632 for 3 days. For definitive endoderm (DE) differentiation, culture the EBs in DMEM / F12 containing 50 ng / mL Activin A, 1% GlutaMAX, 1% P-S, 1% KSR, and 1% B27 for 5 days to prepare for inducing two types of organoids.

[0058] 4) To differentiate hepatocytes, treat the DE cells with RPMI 1640 supplemented with 20 ng / mL HGF, 100 ng / mL Activin A, 10 ng / mL bFGF, 1% GlutaMAX, 1% P-S, 1% KSR, and 1% B27 for 5 days. Then, treat them with a mixture based on 10 ng / mL OSM and 100 nM Dex in HCM for another 5 days. Finally, to generate mature hepatocytes, culture the HP cells in HCM containing 100 nM Dex.

[0059] 5) To induce pancreatic endoderm (PE), differentiate the cells in DMEM with a mixture of 2 μM dorsomorphin, 5 ng / mL bFGF, 2 μM RA, 10 μM SB431542, 250 nM SANT-1, 1% GlutaMAX, 1% P-S, 1% KSR, and 1% B27 for 6 days. Subsequently, obtain endocrine progenitor cells (EPs) by adding 2 μM dorsomorphin, 10 μM SB431542, 50 μg / mL ascorbic acid, and 10 μM DAPT to DMEM and adding supplements for intervention for 4 days. Finally, to differentiate endocrine cells (ECs), replace the medium with CMRL 1066 and supplement it with 2 μM dorsomorphin, 10 μM SB431542, 50 μg / mL ascorbic acid, 10 mM nicotinamide, 25 mM glucose, 1% P-S, and 1% B27, and culture for 8 days;

[0060] 6) Co-culture the organoids differentiated from hiPSCs on the OoC chip, collect approximately 5×10 6 cells of each type of organoid, and introduce them into the chambers of the chip through the flow channels respectively. Then, deposit the organoids in the microwells on the chip chambers. The co-culture medium is pumped into the OoC chip at a flow rate of 100 μL / h. The co-culture solution consists of 1% GlutaMAX, 1% NEAA, 1% P-S, 1% B27, 1% N2, and 11 mM glucose, and is placed in RPMI 1640.

[0061] In this example, asFigure 4 As shown Figure 4 in it, a i is the cell image in the upper chamber microporous array; Figure 4 in it, a ii is the cell image in the lower chamber microporous array, Figure 4 and b in it is the statistical chart of the area of cell spheroid aggregates formed in the microporous array in the double-chamber; it can be seen that cell spheroid aggregates with uniform size are formed in the upper and lower chamber microporous arrays, indicating that the OoC chip provides a platform for batch manufacturing and high repeatability for organ culture.

[0062] In this embodiment, as Figure 5 shown, on the OoC chip, hiPSCs gradually develop and differentiate to form stem cell-derived liver and pancreatic islet organoids, showing morphological structures at different developmental stages under a light microscope. The results show that the OoC chip can continuously support the induced differentiation of stem cell-derived organoids and create a physiological microenvironment suitable for organoid culture.

[0063] Comparative Example 1

[0064] Comparative Example 1 is to directly culture liver organoids and pancreatic islet organoids on a six-well plate, and then measure the release amounts of albumin and insulin.

[0065] Comparative Example 2

[0066] Comparative Example 2 is to culture liver organoids and pancreatic islet organoids on the OoC chip respectively, and then measure the release amounts of albumin and insulin.

[0067] Measurement of the release amount data of albumin and insulin:

[0068] In Example 3, the functions of culturing liver organoids and pancreatic islet organoids using the chip are as Figure 6 shown, Figure 6 in which a is to measure the hepatocyte function of liver organoids by the albumin release amount; Figure 6 in which b is to measure the β-cell function of pancreatic islet organoids by the insulin release amount; it can be seen that compared with Comparative Example 1, the albumin release amount in Example 3 is significantly increased on the 15th and 20th days; compared with Comparative Example 1 and Comparative Example 2, the insulin release amount in Example 3 is significantly increased on the 15th, 20th, and 25th days. The results show that under the OoC co-culture conditions, the functions of liver organoids and pancreatic islet organoids are improved, and there are statistically significant differences compared with the organoids cultured in a six-well plate and the organoids cultured alone on the OoC.

[0069] Example 4

[0070] In this embodiment, a MASLD model is constructed using a double-chamber microfluidic organ chip and intervened with drugs. The specific content includes:

[0071] Cultivate liver organoids in the upper chamber of the OoC chip and pancreatic islet organoids in the lower chamber. Expose the liver organoids to a mixture of free fatty acids (FFAs) and fructose to establish a MASLD model. Treat the liver organoids with a medium containing oleic acid and palmitic acid fatty acids (1 mM, 1:1 ratio) and fructose (10 mM) for 48 hours. Subsequently, intervene in the MASLD model with 10 μM Dorzagliatin. On the OoC chip, perform a glucose tolerance test (GTT). Pump a medium containing 11 mM glucose into the OoC chip and detect the glucose and insulin levels in the medium pumped out of the chip to evaluate the glucose sensitivity and insulin resistance of the co-cultured organoids on the OoC.

[0072] The detection results are as Figure 7 shown. Figure 7 Figure a in it shows that the glucose consumption in the MASLD group is lower than that in the normal group, indicating a decrease in glucose sensitivity and an increase in insulin resistance. After intervening in the MASLD model with Dorzagliatin, the results show that its glucose consumption is comparable to that in the normal control group. Figure 7 Figure b in it shows that Dorzagliatin enhances insulin secretion in the MASLD model, and the difference is statistically significant compared with the normal control group. The above results suggest that Dorzagliatin can improve glucose sensitivity and insulin resistance in the MASLD model and has the potential to treat metabolic diseases. Therefore, from the perspective of protein secretion function, the OoC system quantitatively evaluates the efficacy of drugs and provides a good model for disease mechanism and drug research.

[0073] In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials, or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0074] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art of this industry should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. A dual-chamber microfluidic organ-on-a-chip, characterized in that From top to bottom, it successively includes: a top layer, an upper layer, a middle layer, and a lower layer; chambers are provided in both the upper layer and the lower layer, and a microporous array for accommodating biological samples is provided in the chambers; the chip further includes a plurality of flow channels and can be respectively communicated with the chambers of the upper and lower layers to enable biological samples to enter and exit the microporous array. The middle layer is a semi-permeable porous membrane.

2. The dual-chamber microfluidic organ-on-a-chip according to claim 1, wherein The material of the semi-permeable porous membrane is polycarbonate, fluorinated ethylene polymer, polytetrafluoroethylene, polyvinyl chloride, or polyvinyl alcohol.

3. The dual-chamber microfluidic organ-on-a-chip according to claim 1, characterized in that, The materials of the upper layer and the lower layer are polydimethylsiloxane, polymethyl methacrylate, hydrogel, collagen, polystyrene, or styrene-ethylene(butylene)-styrene copolymer.

4. A dual-chamber microfluidic organ-on-a-chip according to claim 1, wherein, The size of the chamber is 10mm×10mm×2mm; the diameter of the micropores in the microporous array is 750μm; the pore diameter of the semi-permeable porous membrane is 0.4μm.

5. A method for preparing a dual-chamber microfluidic organ-on-a-chip according to any one of claims 1-4, characterized in that, It includes the following steps: Prepare sacrificial male molds for the top layer, upper layer, and lower layer by 3D printing. After mixing the PDMS prepolymer and the curing agent at a weight ratio of 10:1, pour it on the sacrificial male mold, cure it, and then separate the PDMS from the sacrificial male mold to obtain the top layer, upper layer, and lower layer. Perform surface treatment on the top layer, upper layer, middle layer, and lower layer using oxygen plasma. Assemble the top layer, upper layer, middle layer, and lower layer in sequence to obtain it.

6. Application of a double-chamber microfluidic organ chip according to any one of claims 1-4 in dual-organ culture.

7. The application according to claim 6, wherein The steps of dual-organ culture include: 1) Sterilize the chip with cyclohexane. 2) Feed two types of organs into the microporous arrays of the upper chamber and the lower chamber respectively through the flow channels. 3) Pump the culture medium into the upper chamber and the lower chamber for culture.

8. Application of a double-chamber microfluidic organ chip according to any one of claims 1-4 in constructing a metabolic disease model.

9. Application of a double-chamber microfluidic organ chip according to any one of claims 1-4 in developing drugs or devices for preventing or treating metabolic diseases.

10. The application according to claim 9, characterized in that The metabolic diseases: diabetes and its complications, metabolic associated fatty liver disease, hyperuricemia, or metabolic syndrome.

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