Renal tubule 3D printing chip with peripheral capillary and application

Abstract

The invention relates to the technical field of biology, in particular to a renal tubule 3D printing chip with peripheral capillary vessels and application. The invention provides a renal tubule chip for constructing a renal tubule model with peripheral capillary vessels. The invention also discloses a method for constructing a renal tubule model with peripheral capillary vessels by using the renal tubule chip. The renal tubule model with the peripheral capillary vessels constructed by the method has a renal tubule structure and the peripheral capillary vessel network of the renal tubule, the peripheral capillary vessel network of the renal tubule can be highly simulated from two aspects of an anatomical structure and a physiological process, and a more bionic renal tubule chip model is provided; an animal model and a traditional two-dimensional culture mode can be replaced, and a more accurate platform is provided for in-vitro simulated drug kidney toxicity evaluation, renal function mechanism research, kidney disease engineering modeling and kidney disease treatment drug effectiveness test.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a 3D printed chip of renal tubules with peritubular capillaries and its application. Background Art

[0002] Peritubular capillaries are densely distributed around renal tubules, providing oxygen supply, material transport energy supply for nephrons, raw materials for material secretion, transporting reabsorbed substances, being the main nourishing blood vessels of renal tubules and renal interstitium, and also being a key factor in maintaining the normal structure and function of renal tubules. As the main nourishing blood vessels of renal tubules, the damage of peritubular capillaries can reduce peritubular perfusion and the supply of oxygen and nutrients to renal tubular epithelial cells. Constructing an in vitro model of renal tubules with peritubular capillaries is crucial for the research of diseases and effective drug testing. However, the current animal experiment research is not only expensive but also time-consuming, and the conventional two-dimensional cell culture has poor simulation.

[0003] In the past decade, with the rapid development of technologies such as microfluidics, microfabrication, and 3D printing, organ chips based on materials such as polydimethylsiloxane (PDMS) or polymethyl methacrylate (PMMA), with bionic microstructures and capable of precisely controlling the physical and chemical microenvironment, have emerged one after another. However, the vast majority of the reported renal tubule chips do not have a peritubular capillary network, and only a few simulate the peritubular capillary network with a single capillary, which not only leads to incomplete reabsorption ability of renal tubules but also a decrease in the secretion ability of renal tubules. How to effectively and quickly establish a bionic experimental model of peritubular capillaries of renal tubules is very important for the research of kidneys and related diseases. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a 3D printed chip of renal tubules with peritubular capillaries and its application.

[0005] The present invention provides a renal tubule chip with peritubular capillaries, which comprises a first chip (1), a second chip (2), and a third chip (3) stacked from top to bottom;

[0006] A renal tubule inlet (4), a renal tubule outlet (6), a collagen and endothelium inlet (5), a first peritubular auxiliary blood vessel inlet (8), and a second peritubular auxiliary blood vessel inlet (9) are provided at the first end of the first chip (1);

[0007] A collagen and endothelium outlet (7), a first peritubular auxiliary blood vessel outlet (10), and a second peritubular auxiliary blood vessel outlet (11) are provided at the second end of the first chip (1);

[0008] The first end of the second chip (2) is provided with a renal tubule inlet through-hole (12), a renal tubule outlet through-hole (14), a collagen and endothelium inlet through-hole (13), a first peritubular auxiliary blood vessel inlet through-hole (16), and a second peritubular auxiliary blood vessel inlet through-hole (17);

[0009] The second end of the second chip (2) is provided with a collagen and endothelium outlet through-hole (15), a first peritubular auxiliary blood vessel outlet through-hole (18), and a second peritubular auxiliary blood vessel outlet through-hole (19);

[0010] The first end of the third chip is provided with a filtered liquid channel (20), a renal tubule filtered liquid channel (22), a first peritubular auxiliary blood vessel inlet channel (23), a second peritubular auxiliary blood vessel inlet channel (24), and a collagen and endothelium inlet channel (27);

[0011] The second end of the third chip is provided with a first peritubular auxiliary blood vessel outlet channel (25), a second peritubular auxiliary blood vessel outlet channel (26), and a collagen and endothelium outlet channel (28);

[0012] A renal tubule and peritubular capillary network cavity (21) is provided between the first end and the second end of the third chip;

[0013] The glomerular filtered liquid channel (20), the renal tubule filtered liquid channel (22), the first peritubular auxiliary blood vessel inlet channel (23), the second peritubular auxiliary blood vessel inlet channel (24), and the collagen and endothelium inlet channel (27) communicate with one end of the renal tubule and peritubular capillary network cavity (21);

[0014] The first peritubular auxiliary blood vessel outlet channel (25), the second peritubular auxiliary blood vessel outlet channel (26), and the collagen and endothelium outlet (28) communicate with the other end of the renal tubule and peritubular capillary network cavity (21);

[0015] An isolation array (29) arranged at equal intervals in the direction from the first end to the second end is provided in the renal tubule and peritubular capillary network cavity (21);

[0016] The first ends of the first chip, the second chip, and the third chip are far from the second ends.

[0017] Furthermore, in the renal tubule chip of the present invention,

[0018] The openings of the collagen and endothelium inlet (5), the collagen and endothelium inlet through-hole (13), and the collagen and endothelium inlet channel (27) coincide and communicate, and are located on the central axis in the direction from the first end to the second end and close to the first end;

[0019] The openings of the collagen and endothelium outlet (7), the collagen and endothelium outlet through-hole (15), and the collagen and endothelium outlet channel (28) coincide and communicate with each other, and are located on the central axis in the direction from the first end to the second end, and are close to the second end;

[0020] The openings of the renal tubule inlet (4), the renal tubule inlet through-hole (12), and the filtered liquid channel (20) coincide and communicate with each other, are located on the first side perpendicular to the central axis in the direction from the first end to the second end, and are close to the central axis in the direction from the first end to the second end;

[0021] The openings of the renal tubule outlet (6), the renal tubule outlet through-hole (14), and the renal tubule filtered liquid channel (22) coincide and are the same, are located on the second side perpendicular to the central axis in the direction from the first end to the second end, and are close to the central axis in the direction from the first end to the second end;

[0022] The openings of the first peritubular auxiliary blood vessel inlet (8), the first peritubular auxiliary blood vessel inlet through-hole (16), and the first peritubular auxiliary blood vessel inlet channel (23) coincide and communicate with each other, are located on the first side perpendicular to the central axis in the direction from the first end to the second end, and are far from the central axis in the direction from the first end to the second end;

[0023] The openings of the second peritubular auxiliary blood vessel inlet (9), the second peritubular auxiliary blood vessel inlet through-hole (17), and the second peritubular auxiliary blood vessel inlet channel (24) coincide and communicate with each other, are located on the second side perpendicular to the central axis in the direction from the first end to the second end, and are far from the central axis in the direction from the first end to the second end;

[0024] The openings of the first peritubular auxiliary blood vessel outlet (10), the first peritubular auxiliary blood vessel outlet through-hole (18), and the first peritubular auxiliary blood vessel outlet channel (25) coincide and communicate with each other, are located on the first side perpendicular to the central axis in the direction from the first end to the second end, and are far from the central axis in the direction from the first end to the second end;

[0025] The openings of the second peritubular auxiliary blood vessel outlet (11), the second peritubular auxiliary blood vessel outlet through-hole (19), and the second peritubular auxiliary blood vessel outlet channel (26) coincide and communicate with each other, are located on the second side perpendicular to the central axis in the direction from the first end to the second end, and are far from the central axis in the direction from the first end to the second end;

[0026] The first side and the second side are far from each other.

[0027] In the renal tubule chip of the present invention,

[0028] The renal tubule and the peritubular capillary network cavity (21) is a straight notch structure, with a length of 40 - 70 mm, a width of 30 - 60 mm, and a depth of 1 - 6 mm.

[0029] The isolation array (29) is a solid cylinder with an elliptical cross-section. The minor axis length of the elliptical cross-section of the solid cylinder is 0.5 mm to 1.5 mm, the major axis length of the elliptical cross-section of the solid cylinder is 3 to 5 mm; and the height is 1 mm to 6 mm.

[0030] The number of the isolation arrays is multiple; the spacing between each isolation array is 0.3 mm to 1.0 mm.

[0031] The inner diameters of the renal tubule filtrate liquid channel (22), the first peritubular auxiliary blood vessel inlet channel (23), the second peritubular auxiliary blood vessel inlet channel (24), the first peritubular auxiliary blood vessel outlet channel (25), the second peritubular auxiliary blood vessel outlet channel (26), the collagen and endothelium inlet channel (27), and the collagen and endothelium outlet channel (28) are 0.5 mm to 1.5 mm, and the lengths from the opening to the end communicating with the cavity (21) of the renal tubule and the peritubular capillary network are the same, and the length is 40 to 70 mm.

[0032] In the renal tubule chip of the present invention, the first chip, the second layer chip, and the third chip are fastened by threaded fasteners or buckles.

[0033] The present invention provides a method for constructing a renal tubule with peritubular capillaries, which is to construct a renal tubule with peritubular capillaries by using the renal tubule chip of the present invention.

[0034] Furthermore, the construction method includes the following steps:

[0035] Step 1: Prepare two hollow cylindrical hydrogel structures of venous blood vessels, and culture monolayer endothelial cells after coating the outer wall of the venous blood vessels with collagen or laminin; in a specific embodiment of the present invention, the inner diameter of the hollow cylindrical venous blood vessel is less than 1 mm; the wall thickness is 0.1 to 0.2 mm; appropriate tools can be prepared according to requirements to change the wall thickness and inner diameter, and the present invention does not limit this.

[0036] Step 2: Prepare a hollow cylindrical hydrogel structure of a renal tubule. After coating the outer side of the renal tubule with a collagen or laminin layer, culture monolayer renal epithelial cells in the first part starting from one end of the renal tubule, culture monolayer renal principal cells in the third part starting from the other end of the renal tubule, and the second part between the first part and the third part has a mixture of monolayer renal epithelial cells and renal principal cells; the lengths of the first part and the third part are equal, the ratio of the lengths of the first part and the third part of the renal tubule to the length of the second part is (1 to 2):1, and the inner diameters of the first part and the third part of the renal tubule are larger than those of the second part.

[0037] Step 3: The renal tubule enters through the renal tubule inlet (4) of the first chip, makes a U-shaped loop in the renal tubule and peritubular capillary network cavity (21) of the third chip in a manner parallel to the upper surface of the chip, and then exits through the renal tubule outlet (6), ensuring that the first part of the renal tubule is located on the side of the renal tubule inlet (4) of the first chip;

[0038] Step 4: Any one of the venous blood vessels enters through the first peritubular auxiliary blood vessel inlet (8) and exits through the first peritubular auxiliary blood vessel outlet (10); the other one of the venous blood vessels enters through the second peritubular auxiliary blood vessel inlet (9) and exits through the second peritubular auxiliary blood vessel outlet (11);

[0039] Step 5: A mixture containing endothelial cells and collagen is injected through the collagen and endothelium inlet (5), passes through the collagen and endothelium inlet through-hole (13) of the second chip, perfuses the renal tubule and peritubular capillary network cavity (21), and then the excess mixture containing endothelial cells and collagen is discharged through the collagen and endothelium outlet (7);

[0040] Step 6: An EDTA solution is injected through the renal tubule inlet (4), the first peritubular auxiliary blood vessel inlet (8), and the second peritubular auxiliary blood vessel inlet (9) for a decomposition reaction to decompose the calcium alginate hydrogel in the renal tubule and venous blood vessels; the calcium alginate hydrogel is formed by cross-linking sodium alginate and calcium chloride.

[0041] Step 7: Induce the generation of a capillary network to obtain the renal tubule with peritubular capillaries.

[0042] The hydrogel of the present invention is obtained by cross-linking a biological macromolecule with calcium ions; the biological macromolecule includes but is not limited to sodium alginate, gelatin, hyaluronic acid, etc. In a specific embodiment of the present invention, the biological macromolecule is sodium alginate, which forms a stable three-dimensional structure with good elasticity and mechanical strength through cross-linking with the cross-linking agent calcium ions.

[0043] In the construction method of the present invention, the induction of the generation of a capillary network includes the following steps:

[0044] Culture medium 1 enters the renal tubule and peritubular capillary network cavity (21) through the renal tubule inlet (4) at a speed of 0.2 - 4.0 mL / h and then flows out through the renal tubule outlet (6); the specific speed is 0.2 mL / h;

[0045] Culture medium 2 enters the renal tubule and peritubular capillary network cavity (21) through the first peritubular auxiliary blood vessel inlet (8) at a speed of 0.2 - 4.0 mL / h and then flows out through the first peritubular auxiliary blood vessel outlet (11); the specific speed is 0.2 mL / h;

[0046] The culture medium 3 enters the renal tubule and the peritubular capillary network cavity (21) through the second peritubular auxiliary blood vessel inlet (9) at a rate of 0.1 - 4.0 mL / h and then flows out through the second peritubular auxiliary blood vessel outlet (11). The specific rate is 0.1 mL / h.

[0047] The culture medium 1, culture medium 2, and culture medium 3 are the same and are selected from at least one or a combination of two or more of DMEM, MEM, and DMEM / F - 12.

[0048] In step 1 of the construction method of the present invention,

[0049] Collagen or laminin is coated on the inner cavity wall of the hollow cylindrical venous blood vessel. In a specific embodiment of the present invention, laminin is coated on the inner cavity wall of the hollow cylindrical venous blood vessel; the concentration of the laminin is 0.5 mg / mL; the coating conditions are 37°C, 5% CO2, for 0.5 - 1.5 h, specifically 1 h.

[0050] After coating collagen or laminin on the inner cavity wall of the hollow cylindrical venous blood vessel, monolayer endothelial cells are cultured; the monolayer endothelial cells are renal microvascular endothelial cells; the concentration of the renal microvascular endothelial cells is 1×10 7 cells / mL;

[0051] The inner diameter of the hollow cylindrical venous blood vessel is less than 1 mm; the wall thickness is 0.1 - 0.2 mm.

[0052] In step 2 of the construction method of the present invention,

[0053] Collagen or laminin is coated on the outside of the renal tubule. In a specific embodiment of the present invention, laminin is coated on the outside of the renal tubule; the concentration of the laminin is 0.5 mg / mL; the coating conditions are 37°C, 5% CO2, for 0.5 - 1.5 h, specifically 1 h.

[0054] The inner diameter of the hollow cylindrical renal tubule is less than 1 mm; the wall thickness is 0.1 - 0.2 mm.

[0055] In step 5 of the construction method of the present invention,

[0056] In the mixture containing endothelial cells and collagen, the concentration of endothelial cells is 1×10 5 cells / mL - 3×10 5 cells / mL, specifically 2×10 5 cells / mL; the collagen concentration is 1 mg / mL - 3 mg / mL, specifically 2 mg / mL.

[0057] After perfusion, incubation is required. The conditions for incubation are 37 °C, 5% CO2, for 10 - 20 min; specifically, it is 15 min.

[0058] In step 6 of the construction method described in the present invention,

[0059] The concentration of EDTA in the EDTA solution is 5 - 20 mM; specifically, it is 5 mM;

[0060] The time for the decomposition reaction is 1 - 3 min; specifically, it is 3 min.

[0061] The present invention provides a renal tubule with peritubular capillaries constructed by the described construction method.

[0062] The present invention provides the application of the renal tubule model with peritubular capillaries in in vitro simulation of drug nephrotoxicity assessment, renal function mechanism research, renal disease engineering modeling, and effectiveness testing of nephropathy treatment drugs.

[0063] The present invention provides a renal tubule chip for constructing a renal tubule model with peritubular capillaries; and a method for constructing a renal tubule model with peritubular capillaries using the renal tubule chip; the renal tubule model with peritubular capillaries constructed by the present invention has a renal tubule structure and a peritubular capillary network of the renal tubule, can highly mimic the peritubular capillary network of the renal tubule from two aspects of anatomical structure and physiological process, provides a more biomimetic renal tubule chip model, can replace animal models and traditional two-dimensional culture methods, and provides a more accurate platform for in vitro simulation of drug nephrotoxicity assessment, renal function mechanism research, renal disease engineering modeling, and effectiveness testing of nephropathy treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Shows the exploded view of the renal tubule peritubular capillary chip provided by the embodiment of the present invention;

[0065] Figure 2 Shows the schematic diagram of constructing the peritubular capillary network provided by the embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0066] The present invention provides a 3D printing chip of a renal tubule with peritubular capillaries and its application. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate alterations and combinations to the methods and applications in this article without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0067] In a specific implementation manner of the present invention, the shapes of the first chip, the second chip, and the third chip may be rounded rectangles or rectangles, and are rounded rectangles in specific embodiments of the present invention; the areas of the upper surfaces and the lower surfaces of the first chip, the second chip, and the third chip are equal, and the area is defined by length and width. In a specific embodiment of the present invention, the length is 100 mm and the width is 70 mm;

[0068] The first chip and the second chip should not be too thick or too thin, and the third chip is required to have a certain thickness; in a specific embodiment of the present invention, the thickness of the first chip is 0.5 mm to 4 mm; specifically, it is 1.5 mm; the thickness of the second chip is 0.1 mm to 2 mm; specifically, it is 0.5 mm; the thickness of the third chip is 3 mm to 10 mm; specifically, it is 7 mm;

[0069] The materials of the first chip and the third chip may be polymethyl methacrylate (PMMA) or polydimethylsiloxane (PDMS); the material of the second chip is PDMS. In this embodiment, there are no special restrictions on the types and sources of the PMMA and PDMS. The PMMA and / or PDMS well-known to those skilled in the art can be used and can be obtained by purchasing in the market; for example, in the embodiments of the present invention, the PMMA provided by Suzhou Anheda Plastic Products Co., Ltd. or the PDMS polymer provided by Corning, Midland, Michigan, USA can be used.

[0070] The first chip, the second chip, and the third chip described in the present invention can be fastened by threaded fasteners or by snap fasteners; specifically, in a specific embodiment of the present invention, they are fastened by threaded fasteners; specifically, screw holes are provided on the first chip (1), the second chip (2), and the third chip (3); the number of the screw holes can be 4 to 10, specifically 8; the screw holes are arranged circumferentially around the chip for 1 week;

[0071] An isolation array (29) is arranged in the renal tubule and peritubular capillary network cavity (21). There may be multiple isolation arrays, specifically, there may be 7. The isolation array is a solid column, specifically, it may be a solid column with a circular cross-section or a solid column with an elliptical cross-section; the solid column plays a role of blocking. In the specific implementation process, the isolation array may also be a hollow column structure prepared from an isolation material, and the present invention does not limit this; moreover, in the present invention, the number of the isolation arrays may change according to the size of the renal tubule and peritubular capillary network cavity (21) and the size of a single isolation array, and the present invention does not limit this.

[0072] In the chip of the present invention, since the inlets, inlet through-holes and inlet channels sequentially arranged on the first chip (1), second chip (2) and third chip (3) of the chip are respectively communicated with the corresponding outlets, outlet through-holes and outlet channels through the renal tubule and peritubular capillary network cavity (21);

[0073] Exemplarily, the renal tubule inlet (4) opens downward, sequentially penetrates through the renal tubule inlet through-hole (12) and renal tubule inlet channel (20), and then passes through the renal tubule and peritubular capillary network cavity (21) and is communicated with the renal tubule outlet channel (22), the renal tubule outlet through-hole (14) and the renal tubule outlet (6).

[0074] Exemplarily, the first peritubular auxiliary blood vessel inlet (8) opens downward, sequentially penetrates through the first peritubular auxiliary blood vessel inlet through-hole (16) and first peritubular auxiliary blood vessel inlet channel (23), and then passes through the renal tubule and peritubular capillary network cavity (21) and is communicated with the first peritubular auxiliary blood vessel outlet (25), the first peritubular auxiliary blood vessel outlet through-hole (18) and first peritubular auxiliary blood vessel outlet channel (10).

[0075] In the chip of the present invention, the renal tubule inlet and outlet are respectively located at both ends of the central axis of the chip in the direction from the first section to the second section; the peritubular auxiliary blood vessels are located on both sides of the renal tubule inlet and outlet;

[0076] The renal tubule and peritubular capillary network cavity (21) has a certain space, and the renal tubule inlet, inlet through-hole, inlet channel and renal tubule outlet, outlet through-hole and outlet channel are located at the first end of the chip. After entering from the inlet, the renal tubule forms a U-shaped structure in the renal tubule and peritubular capillary network cavity (21); the inlets, inlet through-holes and inlet channels of the first peritubular auxiliary blood vessel and the second peritubular auxiliary blood vessel are located at the first end of the chip, and the outlets, outlet through-holes and outlet channels are respectively located at the second end of the chip; the first peritubular auxiliary blood vessel and the second peritubular auxiliary blood vessel enter through the inlet, pass through the renal tubule and peritubular capillary network cavity (21) and then pass out through the outlet, and are located on both sides of the U-shaped structure of the renal tubule; the U-shaped structure of the renal tubule and the first peritubular auxiliary blood vessel and the second peritubular auxiliary blood vessel do not cross each other and are independent and non-communicating structures.

[0077] The construction method of the present invention relies on the renal tubule chip. In the construction method, the specific structures inside the first chip (1), the second chip (2) and the third chip (3) corresponding to the renal tubule, the first peritubular auxiliary blood vessel and the second peritubular auxiliary blood vessel are in one-to-one correspondence, and the present invention will not elaborate.

[0078] In the construction method of the present invention, the renal tubules, the first peritubular auxiliary blood vessel, and the second peritubular auxiliary blood vessel are hollow columnar hydrogels; the hollow columnar hydrogels are obtained by 3D printing, specifically coaxial extrusion; after crosslinking the biological macromolecules with calcium ions, they are coaxial extruded to prepare the hollow columnar hydrogels; the biological macromolecules include, but are not limited to, sodium alginate.

[0079] In the specific construction, after culturing endothelial cells on the outer layer of the renal tubules, the first peritubular auxiliary blood vessel, and the second peritubular auxiliary blood vessel, the support structure of the hollow columnar hydrogel is decomposed by EDTA; thus, a biomimetic structure of the renal tubules, the first peritubular auxiliary blood vessel, and the second peritubular auxiliary blood vessel is formed.

[0080] In the present invention, the first end and the second end are used to clearly describe the structure of the multiple renal tubule chips; for example, taking the first chip (1) as an example; the first end of the first chip can specifically be the end with a renal tubule inlet (4), a renal tubule outlet (6), a first peritubular auxiliary blood vessel inlet (8), a collagen and endothelium inlet (5), and a second peritubular auxiliary blood vessel inlet (9); the second end can specifically be the end with a collagen and endothelium outlet (7), a first peritubular auxiliary blood vessel outlet (10), and a second peritubular auxiliary blood vessel outlet (11); there is also an intermediate part between the first end and the second end.

[0081] The first side of the chip can be described as the two regions on both sides of the upper surface or the lower surface of the chip divided by the central axis line in the direction from the first end to the second end; specifically, taking the first chip as an example; the renal tubule inlet (4), the first peritubular auxiliary blood vessel inlet (8), and the first peritubular auxiliary blood vessel outlet (10) are located on the first side of the first chip (1); the renal tubule outlet (6), one end of the second peritubular auxiliary blood vessel inlet (9), and the second peritubular auxiliary blood vessel outlet (11) are located on the second side of the first chip (1);

[0082] The renal tubule inlet (4), the collagen and endothelium inlet (5), the renal tubule outlet (6), the first peritubular auxiliary blood vessel inlet (8), and the second peritubular auxiliary blood vessel inlet (9) can be located on the same parallel line perpendicular to the central axis line from the first end to the second end, or they can be not on the same horizontal line, and the present invention does not limit this.

[0083] The collagen and endothelium outlet (7), the first peritubular auxiliary blood vessel outlet (10), and the second peritubular auxiliary blood vessel outlet (11) can be located on the same parallel line perpendicular to the central axis line from the first end to the second end, or they can be not on the same horizontal line, and the present invention does not limit this.

[0084] In the present invention, the first end and the second end, and the first side and the second side are two parts that are far away from each other in direction, which are used to clearly describe the renal tubule chip.

[0085] In the present invention, the renal tubule is a hollow cylindrical hydrogel pipe with thick ends and a thin middle formed by inner diameter control; the inner diameters of the first part and the third part are equal and slightly larger than that of the second part, specifically as Figure 2 ; in a specific embodiment of the present invention, the inner diameters of the first part and the third part are approximately 0.5 - 1.5 mm; the inner diameter of the second part is approximately 0.5 - 1.5 mm; the lengths of the first part and the third part are the same, and the ratio of the lengths of the first part and the third part to the second part is (1 - 2):1; in a specific embodiment of the present invention, the inner diameter of the first part and the third part is 0.7 mm, the inner diameter of the second part is approximately 0.6 mm, and the ratio of the lengths of the first part and the third part to the second part is 1.5:1.

[0086] In the renal tubule of the present invention, a single layer of renal epithelial cells is cultured in the first part to simulate the proximal tubule of the renal tubule, ensuring that the proximal tubule is located on one side of the renal tubule inlet (4); a single layer of renal principal cell layer is cultured in the second part to simulate the distal tubule of the renal tubule, which is located on one side of the renal tubule outlet (6);

[0087] In the first chip and the second chip, the inner diameters of each inlet, inlet through - hole, outlet, and outlet through - hole are the same, which is 0.3 mm - 1.0 mm; specifically, it is 0.6 mm;

[0088] During the cross - linking process of sodium alginate and Ca 2+ , the concentration of sodium alginate is 2 mg / mL; the concentration of Ca 2+ is 3 mg / mL.

[0089] All the test materials used in the present invention are ordinary commercially available products and can be purchased in the market. The present invention will be further described below in conjunction with embodiments:

[0090] Example 1 Renal peritubular capillary chip

[0091] The present invention provides a renal tubule chip, which includes a first chip (1), a second chip (2), and a third chip (3) stacked in sequence from top to bottom;

[0092] The first chip (1) has a renal tubule inlet (4), a collagen and endothelium inlet (5), a renal tubule outlet (6), a collagen and endothelium outlet (7), a first peritubular auxiliary blood vessel inlet (8), a second peritubular auxiliary blood vessel inlet (9), a first peritubular auxiliary blood vessel outlet (10), and a second peritubular auxiliary blood vessel outlet (11);

[0093] The second chip (2) is provided with a renal tubule outlet through-hole (12), a collagen and endothelium inlet through-hole (13), a renal tubule outlet through-hole (14), a collagen and endothelium outlet through-hole (15), a first peritubular auxiliary blood vessel inlet through-hole (16), a second peritubular auxiliary blood vessel inlet through-hole (17), a first peritubular auxiliary blood vessel outlet through-hole (18), and a second peritubular auxiliary blood vessel outlet through-hole (19);

[0094] The third chip (3) is provided with a fluid channel, a glomerular filtration fluid channel (20), a renal tubule and peritubular capillary network cavity (21), a renal tubule filtration fluid channel (22), a first peritubular auxiliary blood vessel inlet channel (23), a second peritubular auxiliary blood vessel inlet channel (24), a first peritubular auxiliary blood vessel outlet channel (25), a second peritubular auxiliary blood vessel outlet channel (26), a collagen and endothelium inlet channel (27), a collagen and endothelium outlet channel (28), and an isolation array (29).

[0095] According to the renal tubule peritubular capillary chip described above, the renal tubule and peritubular capillary network cavity (21) is used to accommodate the renal tubule, the first auxiliary blood vessel, and the second auxiliary blood vessel. The two ends of the renal tubule are respectively embedded in the glomerular filtration fluid channel (20) and the renal tubule filtration fluid channel (22), so that the culture medium can only pass through the inner lumen of the renal tubule; the two ends of the first auxiliary blood vessel are respectively embedded in the first peritubular auxiliary blood vessel inlet channel (23) and the first peritubular auxiliary blood vessel outlet channel (25), and the two ends of the second auxiliary blood vessel are respectively embedded in the second peritubular auxiliary blood vessel inlet channel (24) and the second peritubular auxiliary blood vessel outlet channel (26), so that the culture medium can only pass through the inner lumens of the first peritubular auxiliary blood vessel and the second peritubular auxiliary blood vessel.

[0096] The renal tubule and peritubular capillary network cavity (21) is of a straight notch structure. One end of the renal tubule and peritubular capillary network cavity (21) with a circular structure is respectively connected to the glomerular filtration fluid channel (20), the renal tubule filtration fluid channel (22), the first peritubular auxiliary blood vessel inlet channel (23), the second peritubular auxiliary blood vessel inlet channel (24), and the collagen and endothelium inlet channel (27), and the channels are not connected to each other; the other end of the renal tubule and peritubular capillary network cavity (21) with a circular structure is respectively connected to the first peritubular auxiliary blood vessel outlet channel (25), the second peritubular auxiliary blood vessel outlet channel (26), and the collagen and endothelium outlet channel (28), and the channels are not connected to each other.

[0097] The isolation array (29) is composed of a plurality of elliptical cylinders that are not connected to each other, and the distance between the plurality of elliptical cylinders is 0.3 mm to 1.0 mm; for the isolation array (29), the width of each cylinder is 0.5 mm to 1.5 mm, and the height is 0.3 mm to 1.0 mm; the axis of the isolation array coincides with the long axis of the renal tubule and the peritubular capillary network cavity (21).

[0098] The widths of the renal tubule filtration liquid channel (22), the first peritubular auxiliary blood vessel inlet channel (23), the second peritubular auxiliary blood vessel inlet channel (24), the first peritubular auxiliary blood vessel outlet channel (25), the second peritubular auxiliary blood vessel outlet channel (26), the collagen and endothelium inlet channel (27), and the collagen and endothelium outlet channel (28) are 0.5 mm to 1.5 mm, and the depths are 0.3 mm to 1.0 mm.

[0099] The renal tubule peritubular capillary chip is formed by irreversible sealing of upper and lower layers. The materials of the upper and lower layers are both polymers of the transparent and breathable biocompatible material polydimethylsiloxane (PDMS).

[0100] The specific preparation steps of the renal tubule peritubular capillary chip are as follows: The chip is prepared using traditional photolithography technology. The formed template is modified with trimethylchlorosilane vapor and baked at 95 °C for 5 min so that the template is hydrophobic and does not adhere to PDMS as much as possible. Then, a PDMS polymer replica mold is used to form the upper and lower layer PDMS chips. The upper and lower layers of the organ chip layer are irreversibly sealed and sterilized by high temperature and high pressure for standby.

[0101] Example 2 Construction of a renal tubule peritubular capillary model

[0102] The present invention provides a technology for constructing a renal tubule peritubular capillary model, and its steps are mainly divided into four parts: the formation of renal tubules, the formation of venous blood vessels, the generation of peritubular capillary networks, and the construction of renal tubule peritubular capillary networks.

[0103] (1) Formation of renal tubules:

[0104] Based on a coaxial co-printing needle, a calcium chloride solution is introduced into the inner needle, and an alginate solution is introduced into the outer needle. By controlling the extrusion speed, a hollow hydrogel tube with thick ends and a thin middle is formed; both ends are blocked with epoxy resin; the outside of the tube is coated with fibronectin (0.5 mg / mL; directly prepared with sterile PBS) and incubated for 1 hour (37 °C, 5% CO2) to support cell adhesion and growth; 100 μL of renal epithelial cell and renal principal cell suspension (1×10 7Cells / mL) were respectively seeded onto the surface of the thick segments of the hollow hydrogel tubes (the thick segments were located at both ends of the renal tubules), and incubated in an incubator for two hours to promote cell adhesion on the surface of the hollow hydrogel tubes; the epoxy resins at both ends were cut off to open the tubes, forming renal tubules, which were implanted into the kidney chip in a U-shape.

[0105] (2) Formation of venous blood vessels

[0106] Based on a coaxial co-printing needle, calcium chloride solution was introduced into the inner needle and sodium alginate solution was introduced into the outer needle. By controlling the extrusion speed, two hollow hydrogel tubes were formed; both ends were blocked with epoxy resin; the outer surface of the tubes was coated with fibronectin (0.5 mg / mL; directly prepared with sterile PBS) for 1 hour (37 °C, 5% CO2) to support cell adhesion and growth; 100 μL of renal microvascular endothelial cell suspension (1×10 7 Cells / mL) were respectively seeded onto the surfaces of the two hollow hydrogel tubes and incubated in an incubator for two hours to promote cell adhesion on the surface of the hollow hydrogel tubes; the epoxy resins at both ends were cut off to open the tubes, forming venous blood vessels, which were implanted on both sides of the renal tubule area of the chip.

[0107] (3) Generation of peritubular capillary networks

[0108] Using a 10 mL syringe, 4 mL of endothelial cell and collagen suspension (2×10 5 Cells / mL; collagen concentration was 2 mg / mL) was injected into the renal tubule area through the endothelial and collagen inlets; the renal tubule chip was placed in a cell incubator (37 °C, 5% CO2) for 15 min to make the cell suspension become a gel state, providing a three-dimensional environment for the growth of endothelial cells.

[0109] (4) Construction of peritubular capillary networks of renal tubules

[0110] 5 mM EDTA was respectively added through the renal tubule inlet (4), the first peritubular auxiliary blood vessel inlet (8), and the second peritubular auxiliary blood vessel inlet (9) on the upper chip (1) and reacted for 3 min to decompose the support structure. The single-layer cells outside the support structure would automatically become the single-layer cells in the collagen channels, forming renal tubules without a support structure; based on microfluidic technology, culture media with different flow rates were introduced from the renal tubules and the two venous blood vessels to form peritubular capillary networks in the renal tubule area; the single-layer endothelial cells in the auxiliary blood vessels would automatically become part of the peritubular capillary networks, thus completing the construction of peritubular capillary networks of renal tubules.

[0111] The sterile peristaltic tube is connected to a culture medium bottle containing 60 mL of cell culture medium and placed in an incubator. The culture medium is driven by a peristaltic pump at a rate of 0.2 mL / h, enters the peritubular vascular region through the renal tubule inlet (4) on the upper chip (1), and finally returns to the culture medium bottle through the renal tubule outlet (6) on the upper chip (1).

[0112] Connected to a container containing 20 mL of cell culture medium, placed in an incubator, the first culture medium is driven by an injection pump at a rate of 0.2 mL / h, enters the first peritubular vascular region through the first peritubular auxiliary vascular inlet (8) on the upper chip (1), and finally returns to the collection bottle through the first peritubular auxiliary vascular outlet on the upper chip (1); the second culture medium is driven by an injection pump at a rate of 0.1 mL / h, enters the first peritubular vascular through the second peritubular auxiliary vascular inlet (9) on the upper chip (1), enters the second peritubular vascular region through the first peritubular auxiliary vascular inlet (8) on the upper chip (1), and finally returns to the collection bottle through the second peritubular auxiliary vascular outlet on the upper chip (1).

[0113] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A renal tubule chip with peritubular capillaries, characterized in that, It includes a first chip (1), a second chip (2), and a third chip (3) stacked from top to bottom; At the first end of the first chip (1), there are a renal tubule inlet (4), a renal tubule outlet (6), a collagen and endothelium inlet (5), a first peritubular auxiliary blood vessel inlet (8), and a second peritubular auxiliary blood vessel inlet (9); At the second end of the first chip (1), there are a collagen and endothelium outlet (7), a first peritubular auxiliary blood vessel outlet (10), and a second peritubular auxiliary blood vessel outlet (11); At the first end of the second chip (2), there are a renal tubule inlet through-hole (12), a renal tubule outlet through-hole (14), a collagen and endothelium inlet through-hole (13), a first peritubular auxiliary blood vessel inlet through-hole (16), and a second peritubular auxiliary blood vessel inlet through-hole (17); At the second end of the second chip (2), there are a collagen and endothelium outlet through-hole (15), a first peritubular auxiliary blood vessel outlet through-hole (18), and a second peritubular auxiliary blood vessel outlet through-hole (19); At the first end of the third chip, there are a filtered liquid channel (20), a renal tubule filtered liquid channel (22), a first peritubular auxiliary blood vessel inlet channel (23), a second peritubular auxiliary blood vessel inlet channel (24), and a collagen and endothelium inlet channel (27); At the second end of the third chip, there are a first peritubular auxiliary blood vessel outlet channel (25), a second peritubular auxiliary blood vessel outlet channel (26), and a collagen and endothelium outlet channel (28); Between the first end and the second end of the third chip, there is a renal tubule and peritubular capillary network cavity (21); The glomerular filtered liquid channel (20), the renal tubule filtered liquid channel (22), the first peritubular auxiliary blood vessel inlet channel (23), the second peritubular auxiliary blood vessel inlet channel (24), and the collagen and endothelium inlet channel (27) communicate with one end of the renal tubule and peritubular capillary network cavity (21); The first peritubular auxiliary blood vessel outlet channel (25), the second peritubular auxiliary blood vessel outlet channel (26), and the collagen and endothelium outlet (28) communicate with the other end of the renal tubule and peritubular capillary network cavity (21); In the renal tubule and peritubular capillary network cavity (21), there is an isolation array (29) arranged at equal distances from the first end to the second end direction; The first ends of the first chip, the second chip, and the third chip are far from the second ends.

2. The renal tubule chip according to claim 1, wherein, The openings of the collagen and endothelium inlet (5), the collagen and endothelium inlet through-hole (13), and the collagen and endothelium inlet channel (27) coincide and communicate, and are located on the central axis in the direction from the first end to the second end, and are close to the first end; The openings of the collagen and endothelium outlet (7), the collagen and endothelium outlet through-hole (15), and the collagen and endothelium outlet channel (28) coincide and communicate, and are located on the central axis in the direction from the first end to the second end, and are close to the second end; The openings of the renal tubule inlet (4), the renal tubule inlet through hole (12), and the filtered liquid channel (20) coincide and communicate with each other, are located on the first side perpendicular to the central axis in the direction from the first end to the second end, and are close to the central axis in the direction from the first end to the second end; The openings of the renal tubule outlet (6), the renal tubule outlet through hole (14), and the renal tubule filtered liquid channel (22) coincide and are the same, are located on the second side perpendicular to the central axis in the direction from the first end to the second end, and are close to the central axis in the direction from the first end to the second end; The openings of the first peritubular auxiliary blood vessel inlet (8), the first peritubular auxiliary blood vessel inlet through hole (16), and the first peritubular auxiliary blood vessel inlet channel (23) coincide and communicate with each other, are located on the first side perpendicular to the central axis in the direction from the first end to the second end, and are far from the central axis in the direction from the first end to the second end; The openings of the second peritubular auxiliary blood vessel inlet (9), the second peritubular auxiliary blood vessel inlet through hole (17), and the second peritubular auxiliary blood vessel inlet channel (24) coincide and communicate with each other, are located on the second side perpendicular to the central axis in the direction from the first end to the second end, and are far from the central axis in the direction from the first end to the second end; The openings of the first peritubular auxiliary blood vessel outlet (10), the first peritubular auxiliary blood vessel outlet through hole (18), and the first peritubular auxiliary blood vessel outlet channel (25) coincide and communicate with each other, are located on the first side perpendicular to the central axis in the direction from the first end to the second end, and are far from the central axis in the direction from the first end to the second end; The openings of the second peritubular auxiliary blood vessel outlet (11), the second peritubular auxiliary blood vessel outlet through hole (19), and the second peritubular auxiliary blood vessel outlet channel (26) coincide and communicate with each other, are located on the second side perpendicular to the central axis in the direction from the first end to the second end, and are far from the central axis in the direction from the first end to the second end; The first side and the second side are far from each other.

3. The renal tubule chip according to claim 1, wherein The renal tubule and the peritubular capillary network cavity (21) is a straight notch structure, with a length of 40 - 70 mm, a width of 30 - 60 mm, and a depth of 1 - 6 mm; The isolation array (29) is a solid cylinder with an elliptical cross-section. The length of the minor axis of the elliptical cross-section of the solid cylinder is 0.5 mm - 1.5 mm, the length of the major axis of the elliptical cross-section of the solid cylinder is 3 - 5 mm; and the height is 1 mm - 6 mm; The number of the isolation arrays is multiple; the spacing between each isolation array is 0.3 mm - 1.0 mm.

4. The renal tubule chip according to claim 1, characterized in that, The inner diameters of the renal tubule filtered liquid channel (22), the first peritubular auxiliary blood vessel inlet channel (23), the second peritubular auxiliary blood vessel inlet channel (24), the first peritubular auxiliary blood vessel outlet channel (25), the second peritubular auxiliary blood vessel outlet channel (26), the collagen and endothelium inlet channel (27), and the collagen and endothelium outlet channel (28) are 0.5 mm - 1.5 mm, and the lengths from the openings to the ends connecting to the renal tubule and the peritubular capillary network cavity (21) are the same, and the length is 40 - 70 mm.

5. The renal tubule chip according to any one of claims 1 to 5, characterized in that, The first chip, the second layer chip and the third chip are fastened by threaded fasteners or snap fasteners.

6. A method for constructing a renal tubule model with peritubular capillaries, characterized in that, To construct a renal tubule with peritubular capillaries using the renal tubule chip according to any one of claims 1 to 5.

7. The construction method according to claim 6, wherein Including the following steps: Step 1: Prepare two venous blood vessels with a hollow cylindrical hydrogel structure; after coating the outer wall of the venous blood vessels with collagen or laminin, culture a monolayer of endothelial cells. Step 2: Prepare a renal tubule with a hollow cylindrical hydrogel structure. After coating the outer side of the renal tubule with a collagen or laminin layer, culture a monolayer of renal epithelial cells in the first part starting from one end of the renal tubule and a monolayer of principal renal cells in the third part starting from the other end of the renal tubule. The second part located between the first part and the third part has a mixture of monolayer renal epithelial cells and principal renal cells; the lengths of the first part and the third part are equal, and the ratio of the lengths of the first part and the third part of the renal tubule to the length of the second part is (1 to 2):1, and the inner tube diameters of the first part and the third part are larger than those of the second part. Step 3: The renal tubule enters through the renal tubule inlet (4) of the first chip, makes a U-shaped loop in the renal tubule and peritubular capillary network cavity (21) of the third chip in a manner parallel to the upper surface of the chip, and then exits from the renal tubule outlet (6), ensuring that the first part of the renal tubule is located on the side of the renal tubule inlet (4) of the first chip. Step 4: Let any one of the venous blood vessels enter through the first peritubular auxiliary blood vessel inlet (8) and exit from the first peritubular auxiliary blood vessel outlet (10); let the other venous blood vessel enter through the second peritubular auxiliary blood vessel inlet (9) and exit from the second peritubular auxiliary blood vessel outlet (11). Step 5: A mixture containing endothelial cells and collagen is injected through the collagen and endothelial inlet (5), passes through the collagen and endothelial inlet through-hole (13) of the second chip, perfuses the renal tubule and peritubular capillary network cavity (21), and then the excess mixture containing endothelial cells and collagen is discharged through the collagen and endothelial outlet (7). Step 6: Inject an EDTA solution through the renal tubule inlet (4), the first peritubular auxiliary blood vessel inlet (8), and the second peritubular auxiliary blood vessel inlet (9) to carry out a decomposition reaction to decompose the calcium alginate hydrogel in the renal tubule and the venous blood vessels. Step 7: Induce the formation of a capillary network to obtain the renal tubule with peritubular capillaries.

8. The preparation method according to claim 7, wherein, The inducing the formation of a capillary network includes the following steps: Culture medium 1 enters the renal tubule and peritubular capillary network cavity (21) through the renal tubule inlet (4) at a rate of 0.2 to 4.0 mL / h and then flows out through the renal tubule outlet (6). Culture medium 2 enters the renal tubule and peritubular capillary network cavity (21) through the first peritubular auxiliary blood vessel inlet (8) at a rate of 0.2 to 4.0 mL / h and then flows out through the first peritubular auxiliary blood vessel outlet (11). The culture medium 3 enters the renal tubule and peritubular capillary network cavity (21) through the second peritubular auxiliary blood vessel inlet (9) at a rate of 0.1 to 4.0 mL / h and then flows out through the second peritubular auxiliary blood vessel outlet (11); The culture medium 1, culture medium 2, and culture medium 3 are the same and are selected from at least one or a combination of two or more of DMEM, MEM, and DMEM / F-12.

9. A renal tubule model with peritubular capillaries prepared by the preparation method according to any one of claims 6 to 8.

10. Use of the renal tubule model with peritubular capillaries according to claim 9 in in vitro simulation of drug nephrotoxicity assessment, renal function mechanism research, renal disease engineering modeling, and effectiveness testing of drugs for treating kidney diseases.

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