Non-alcoholic fatty liver chip for realizing co-culture of different types of cells and use method

Abstract

The invention belongs to the field of organ chips and non-alcoholic fatty liver, and discloses a non-alcoholic fatty liver chip for realizing co-culture of different types of cells and a use method. The non-alcoholic fatty liver chip comprises a lower-layer substrate and a middle-layer chip, the middle-layer chip comprises two different cell culture chambers, namely a rectangular liver chamber and a straight channel blood vessel chamber with two circular ends; the two cell culture chambers interact with each other through the miniature fences on the adjacent sides so as to simulate the physiological process between the liver and the blood vessel in the body. The non-alcoholic fatty liver chip adopts an open top structure design, so that the tedious process that a traditional laminated chip needs to be repeatedly disassembled and assembled is avoided, and the operation convenience of the whole process of a cell culture experiment is remarkably improved; comprising but not limited to improvement of cell inoculation efficiency, more convenient real-time observation in the culture process, more efficient sample extraction and analysis, and simpler equipment cleaning and reuse.

Description

Technical Field

[0001] The present invention belongs to the fields of organ-on-a-chip and non-alcoholic fatty liver disease, and relates to a non-alcoholic fatty liver chip for realizing co-culture of different types of cells and a using method thereof. Background Art

[0002] The pathological features of non-alcoholic fatty liver disease are mainly manifested as abnormal deposition of lipids in the liver, which can gradually develop into different degrees of liver lesions ranging from mild non-alcoholic simple fatty liver to severe non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, and even liver failure. Although certain progress has been made in effective therapies for non-alcoholic steatohepatitis, it is still unable to fully meet the current urgent clinical needs. In addition, due to the lack of a highly predictive in vitro disease model, the drug R & D process faces major challenges.

[0003] Organ-on-a-chip has now become an important platform for dynamically simulating diseases and a powerful means for drug evaluation. There is great promise in the research of using organ-on-a-chip to construct disease models at the preclinical stage, but few studies involve non-alcoholic fatty liver. Therefore, the construction of an in vitro non-alcoholic fatty liver model based on organ-on-a-chip technology undoubtedly provides a new approach for drug evaluation. Summary of the Invention

[0004] To solve the above problems, the purpose of the present invention is to provide a co-culture chip for different types of cells, which supports reproducing the early pathological features of non-alcoholic fatty liver in vitro and studying the physiological changes of endothelial cells during the formation of non-alcoholic fatty liver.

[0005] The technical solution of the present invention:

[0006] A non-alcoholic fatty liver chip for realizing co-culture of different types of cells, including a lower substrate and a middle chip; the length of the middle chip is 28 - 32 mm, the width is 17 - 21 mm, and the height is 5 - 10 mm; the length and width of the lower substrate correspond to those of the middle chip, and the height is 0.5 - 1.5 mm;

[0007] The middle chip includes two different cell culture chambers, namely a rectangular liver chamber and a straight-channel vascular chamber with round ends; the two cell culture chambers interact through a micro fence on the adjacent side to simulate the physiological process between the liver and blood vessels in vivo;

[0008] Furthermore, the width of the micro fence is 100 - 300 μm, and the height is 0.5 - 2 mm;

[0009] Further, the material of the non-alcoholic fatty liver chip for co-culturing different types of cells is one of plastic, resin, glass, quartz, and silicon. Among them, plastic includes rigid plastic and elastic plastic. Rigid plastic includes, but is not limited to, materials such as PMMA, PC, or PS. Elastic plastic includes, but is not limited to, materials such as PDMS, PET, or PVC. Preferably, the rigid plastic is PMMA.

[0010] A method for using a non-alcoholic fatty liver chip for co-culturing different types of cells is as follows:

[0011] S1. Hepatocytes are cultured in a three-dimensional culture manner in the liver chamber of the non-alcoholic fatty liver chip, and endothelial cells are cultured in a two-dimensional culture manner in the vascular chamber, and material exchange is carried out through a micro fence;

[0012] S2. Add serum-free cell culture medium supplemented with a high-fat inducer and HUVEC cell culture medium to the liver chamber and the vascular chamber respectively. The composition of the serum-free cell culture medium is 2 wt.% serum-free substitute and 98 wt.% α-MEM medium, and the composition of the HUVEC cell culture medium is 10 wt.% fetal bovine serum and 90 wt.% RPMI1640 medium;

[0013] S3. Place the chip into a sterile cell culture dish and then place it in a cell culture incubator with a carbon dioxide concentration of 5 vol.% and a temperature of 37 °C for culture. During the culture period, the culture medium in the chamber can be taken for liver function detection, and the cells in each chamber of the chip can be taken for detection of liver function and endothelial cell function biomarkers, and / or determination of lipid accumulation in the liver chamber;

[0014] Further, in step S1, the three-dimensional culture method of the hepatocytes is one of hydrogel embedding culture, cell spheroid culture, and liver organoid construction. This method can improve liver function and more effectively simulate the pathological characteristics of in vitro disease models;

[0015] Further, in step S2, the volume of the serum-free cell culture medium added accounts for 90%-95% of the volume of the liver chamber, and the HUVEC cell culture medium added accounts for 90%-95% of the vascular chamber;

[0016] Further, in step S2, the high-fat inducer includes at least one of lipotoxic drugs such as oleic acid, palmitic acid, oleic acid + palmitic acid, lauric acid, linoleic acid, and stearic acid, and is diluted with serum-free cell culture medium or HUVEC cell culture medium to a serum-free cell culture medium and HUVEC cell culture medium with a final concentration of 100-400 μM high-fat inducer;

[0017] Further, in step S3, the liver function detection indexes are AST and ALT, the marker of liver function is albumin, the marker of endothelial cell function is nitric oxide, and the lipid accumulation marker is qualitative analysis by Nile red staining and quantitative analysis of intracellular TG and TC contents.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1. The non-alcoholic fatty liver chip for co-culturing different types of cells of the present invention inoculates liver cells in the liver chamber and endothelial cells in the vascular chamber. The barrier is responsible for the material exchange between the two chambers, mimicking the physiological arrangement of blood vessels and the liver in the body. This chip restores the physiological process between the liver and blood vessels in the body.

[0020] 2. Both hepatocytes and endothelial cells are cultured in independent chambers, which supports applying different types and durations of stimuli to a certain type of cell or cell combination in the chip alone, and supports the separation of specific cells and the acquisition of target samples, thus greatly reducing the difficulty of non-alcoholic fatty liver modeling.

[0021] 3. The chip adopts an open-top structure design, avoiding the cumbersome process of repeated disassembly and assembly required by traditional stacked chips, and significantly improving the operational convenience of the entire process of cell culture experiments, including but not limited to: improving cell seeding efficiency, making real-time observation during the culture process more convenient, making sample extraction and analysis more efficient, and making equipment cleaning and reuse simpler. Description of the Drawings

[0022] Figure 1 Schematic diagram of the structure of the non-alcoholic fatty liver chip for co-culturing different types of cells of the present invention;

[0023] Figure 2 Lipid accumulation of HepG2 cells in the chip during non-alcoholic fatty liver formation in Example 1: Among them, (a)-(d) are Nile red staining of HepG2 cell spheroids in the liver chamber stimulated with sodium palmitate at concentrations of 0, 100, 200, and 400 μM for 24 h, (e) is the intracellular TG content of HepG2 cell spheroids in the liver chamber stimulated with sodium palmitate at concentrations of 0, 100, 200, and 400 μM for 24 h, and (f) is the intracellular TC content of HepG2 cell spheroids in the liver chamber stimulated with sodium palmitate at concentrations of 0, 100, 200, and 400 μM for 24 h;

[0024] Figure 3For the detection of liver function on the chip during the formation of non-alcoholic fatty liver in Example 1: Among them, (a)-(d) are immunofluorescence staining of albumin in HepG2 cell spheroids in the liver chamber stimulated by sodium palmitate at concentrations of 0, 100, 200, and 400 μM, (e) is the content of AST secreted after 24 hours of stimulation of HepG2 cell spheroids in the liver chamber by sodium palmitate at concentrations of 0, 100, 200, and 400 μM, and (f) is the content of ALT secreted after 24 hours of stimulation of HepG2 cell spheroids in the liver chamber by sodium palmitate at concentrations of 0, 100, 200, and 400 μM;

[0025] Figure 4 For the detection of endothelial cell function on the chip during the formation of non-alcoholic fatty liver in Example 1: Among them, (a)-(d) are the detection of nitric oxide after 24 hours of stimulation of HUVEC cells in the vascular chamber by sodium palmitate at concentrations of 0, 100, 200, and 400 μM. Detailed implementation mode

[0026] The following further illustrates the detailed implementation mode of the present invention in combination with the attached drawings and technical solutions.

[0027] Example 1: Construction of a non-alcoholic fatty liver model on the chip

[0028] As Figure 1 shown, a microfluidic chip for co-culturing different types of cells for constructing non-alcoholic fatty liver, the overall size of the microfluidic chip is 30mm * 19mm * 5mm, including a lower substrate and a middle chip. Among them, 1 represents the liver chamber, with a size of 28mm * 8mm; 2 represents the vascular chamber, with a size of 24mm * 4mm; 3 represents the micro fence, with a width of 200μm;

[0029] Specifically, the method for constructing the non-alcoholic fatty liver chip proposed by the present invention includes the following steps:

[0030] S1. The middle chip contains two different cell culture chambers. The cell culture chambers include a liver chamber located on the upper side of the chip and a vascular chamber located on the lower side of the chip. The two chambers arranged up and down can interact through the micro fence on the adjacent side. Hepatocytes are inoculated in the liver chamber, and endothelial cells are inoculated in the vascular chamber; the distance between the two chambers is 2mm, so that the culture solution can be kept in their respective chambers for stimulation without rapid diffusion. There is a 200μm-wide interval between the chambers. At the bottom of the interval, that is, the part connected to the bottom surface of the chamber, there is a micro fence structure to support the material exchange between different chambers through slow penetration.

[0031] S2. In the chambers of the non-alcoholic fatty liver chip described above, HepG2 cells are cultured using a microporous culture microarray, which is a commonly used three-dimensional cell culture method. This method can improve the functional expression of cells, making HepG2 cells closer to the in vivo liver and enhancing the mimicry. After the cells form spheroids, they are transferred to the liver chamber; in the vascular chamber, endothelial cells are cultured two-dimensionally;

[0032] S3. Cell culture media with sodium palmitate concentrations of 0, 100, 200, and 400 μM are added to different chambers respectively to simulate the free fatty acid stimulation response and the cell-cell interaction under the damaged state that cells undergo during the in vivo non-alcoholic fatty liver process in the chip;

[0033] S4. The chip is placed in a cell culture incubator with a carbon dioxide concentration of 5 vol.% and a temperature of 37 °C. After the non-alcoholic fatty liver chip is induced for 24 h, the culture media in the liver chamber are collected and the liver injury is evaluated using AST and ALT assay kits. The HepG2 cell spheroids in the liver chamber are collected and the liver function is evaluated using immunofluorescence staining. TG, TC assay kits and Nile red staining are used to examine the lipid accumulation; the nitric oxide production of HUVEC cells in the vascular chamber of the chip after 24 h of stimulation is measured to evaluate the endothelial cell function.

[0034] In summary, the non-alcoholic fatty liver chip based on co-culture of different types of cells in the embodiments of the present invention can highly mimic the early characteristics of the disease.

[0035] The above-described embodiments are merely preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims

1. A non-alcoholic fatty liver chip for realizing co-culture of different types of cells, characterized in that, The non-alcoholic fatty liver chip includes a lower substrate and a middle chip; the middle chip contains two different cell culture chambers, namely a rectangular liver chamber and a straight-channel vascular chamber with round ends; the two cell culture chambers interact through the micro-fences on the adjacent sides to simulate the physiological process between the liver and blood vessels in vivo.

2. The non-alcoholic fatty liver chip according to claim 1, wherein The middle chip has a length of 28 - 32 mm, a width of 17 - 21 mm, and a height of 5 - 10 mm; the lower substrate has corresponding length and width to the middle chip and a height of 0.5 - 1.5 mm.

3. The non-alcoholic fatty liver chip according to claim 1, characterized in that The width of the micro-fence is 100 - 300 μm and the height is 0.5 - 2 mm.

4. The non-alcoholic fatty liver chip according to claim 1, wherein The non-alcoholic fatty liver chip is made of one of plastic, resin, glass, quartz, and silicon.

5. A method for using the non-alcoholic fatty liver chip for co-culturing different types of cells as described in claims 1-4, characterized in that, The steps are as follows: S1. The liver cells are cultured in a three-dimensional culture manner in the liver chamber of the non-alcoholic fatty liver chip, and the endothelial cells are cultured in a two-dimensional culture manner in the vascular chamber. The substances are exchanged between different chambers through the micro-fences. S2. Add serum-free cell culture medium supplemented with high-fat inducer and HUVEC cell culture medium to the liver chamber and the vascular chamber respectively. The serum-free cell culture medium consists of 2 wt.% serum-free substitute and 98 wt.% α-MEM medium, and the HUVEC cell culture medium consists of 10 wt.% fetal bovine serum and 90 wt.% RPMI 1640 medium. S3. Place the non-alcoholic fatty liver chip into a sterile cell culture dish and then put it into a cell culture incubator with a carbon dioxide concentration of 5 vol.% and a temperature of 37 °C for culture.

6. The usage method according to claim 5, characterized in that, In step S1, the three-dimensional culture manner of the liver cells is one of hydrogel embedding culture, cell spheroid culture, and liver organoid construction.

7. The usage method according to claim 5, characterized in that, In step S2, the volume of the added serum-free cell culture medium accounts for 90% - 95% of the volume of the liver chamber, and the added HUVEC cell culture medium accounts for 90% - 95% of the volume of the vascular chamber.

8. The usage method according to claim 5, characterized in that, In step S2, the high-fat inducer includes at least one of oleic acid, palmitic acid, oleic acid + palmitic acid, lauric acid, linoleic acid, and stearic acid, and is diluted with serum-free cell culture medium or HUVEC cell culture medium to a serum-free cell culture medium and HUVEC cell culture medium with a final concentration of 100 - 400 μM high-fat inducer.

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