A microfluidic chip and a method for constructing a full-thickness corneal model using the same

Abstract

This disclosure provides a microfluidic chip and a method for constructing a full-thickness corneal model using it. The chip includes a detachable lower half and an upper half; the lower half has an upwardly convex curvature dome and a peripheral annular microfluidic channel; the upper half has a central gas-liquid culture well and annularly arranged nerve seeding wells, with the curvature dome located directly below the gas-liquid culture well after assembly. The method includes: embedding corneal stromal cells in a collagen solution and molding them into a dense collagen membrane; seeding endothelial cells on one side and then flipping them over to cover the surface of the curvature dome; implanting nerve tissue into the nerve seeding wells and assembling the chip, pre-culturing to allow nerve fibers to extend to the curvature dome region; seeding epithelial cells on the top surface of the collagen membrane; dynamically perfusing through the annular microfluidic channel and establishing a gas-liquid interface through the gas-liquid culture wells to obtain a full-thickness corneal model with physiological curvature (curvature radius 6-9 mm). This invention can be used for ophthalmic drug screening, ocular irritation toxicity testing, and research on the mechanisms of corneal diseases.

Description

Technical Field

[0001] This invention belongs to the fields of tissue engineering and regenerative medicine, and microfluidic organ-on-a-chip technology. Specifically, it relates to a microfluidic chip and a method for constructing a full-thickness corneal model using the same chip. More specifically, it relates to a microfluidic chip for constructing a full-thickness corneal model with physiological curvature and a method for constructing a full-thickness corneal model using the same microfluidic chip. Background Technology

[0002] The cornea, located at the very front of the eyeball, is the first transparent barrier for light entering the eye. Its surface curvature is closely related to refractive power and is a fundamental structure for maintaining clear vision. Histologically, the human cornea consists of, from the outside in, the corneal epithelium (approximately 50 μm, consisting of 5-7 layers of stratified squamous epithelium), Bowman's membrane (approximately 12 μm), the stroma (approximately 480-500 μm, accounting for about 90% of its thickness, composed of type I collagen and corneal stromal cells), Descemet's membrane (approximately 8-10 μm), and the corneal endothelium (approximately 5 μm, consisting of a single layer of hexagonal cells). The epithelium performs barrier functions; the stroma maintains morphology and transparency; and the endothelium regulates water content through ion pumps. Furthermore, the cornea is one of the most richly innervated tissues in the body. Trigeminal nerve sensory fibers are widely distributed in the stroma and epithelium, participating in reflexes, tear secretion, and epithelial homeostasis, and are closely related to dry eye syndrome and neurotrophic keratitis.

[0003] In drug development and safety evaluation, ophthalmic preparations (especially eye drops) need to be absorbed through or act on the cornea. Therefore, in vitro corneal models are important tools for evaluating drug penetration, barrier function, toxicity, and eye irritation. Traditional methods include animal experiments (such as the Draize rabbit eye test) and two-dimensional cell culture, but both have significant limitations: animal experiments suffer from species differences, ethical restrictions, low throughput, and high costs; two-dimensional cell culture causes cells to lose their three-dimensional structure, mechanical microenvironment, and intercellular interactions, resulting in limited predictability.

[0004] In the field of in vitro corneal modeling, existing technologies mainly include the following categories: Transwell / insertion culture models place porous membranes or collagen gels in chambers, seed corneal epithelial cells on the membrane or gel surface, and, if necessary, seed endothelial cells under the membrane. This can establish an air-liquid interface culture to promote epithelial multilayering. However, this system has a planar structure, lacks physiological curvature, and does not contain neural components. Collagen gel / tissue engineering scaffold models prepare hydrogels by mixing type I collagen with stromal cells, and then seed endothelial and epithelial cells sequentially. However, most of these are still planar or bulky and are not integrated with the microfluidic dynamic environment. Microfluidic corneal chips use polymer materials as substrates and are fabricated through microfabrication to create devices containing microchannels and culture chambers. They integrate collagen matrix to achieve partitioned or co-culture of epithelium, stroma, and endothelium, and simulate dynamic conditions such as aqueous humor circulation through perfusion.

[0005] However, the existing microfluidic corneal chips generally have the following shortcomings: the culture substrate is mostly planar, failing to reproduce the physiological curvature of the cornea; most contain only 1-2 layers of cells or simplified matrix, lacking neural innervation; and dynamic perfusion of the aqueous humor side and exposure of the corneal air-liquid interface have not been integrated simultaneously within the same chip. Although there are reports of using dome-shaped scaffolds to construct ocular surface chips with curvature, these have not constructed full-thickness corneal chips containing the stromal layer, endothelial layer, and nerve ingrowth.

[0006] In summary, existing in vitro corneal models have significant shortcomings in terms of morphological curvature, full-thickness structure, neural innervation, and dynamic microenvironment. There is an urgent need to develop a microfluidic corneal chip and its construction method that can simultaneously achieve physiological curvature shaping, full-thickness cell structure construction, neural innervation integration, and dynamic dual microenvironment synergistic culture. Summary of the Invention

[0007] To address the aforementioned shortcomings of existing in vitro corneal models, this invention aims to provide a microfluidic corneal chip capable of reconstructing the physiological curvature, epithelium, stroma, endothelium, and sensory neural network of the human cornea in vitro. The invention also provides the chip's structure, preparation method, culture method, and its applications in ocular surface drug evaluation and disease research.

[0008] To achieve the aforementioned objectives, in one aspect, the present invention provides a microfluidic chip for constructing a full-thickness corneal model with physiological curvature, comprising:

[0009] The lower half chip has an upwardly protruding curved top and an annular microfluidic channel disposed around the curved top. The annular microfluidic channel is provided with an inlet and an outlet.

[0010] The upper half of the chip has a central gas-liquid culture well and a plurality of nerve inoculation wells arranged in a ring around the central gas-liquid culture well, and the nerve inoculation wells are connected to the central gas-liquid culture well.

[0011] The upper half chip and the lower half chip are detachably assembled and connected, and after assembly, the position of the central gas-liquid culture hole corresponds to the position of the curvature top, with the curvature top located directly below the central gas-liquid culture hole.

[0012] In another aspect, the present invention provides a method for constructing a full-thickness corneal model with physiological curvature using the aforementioned microfluidic chip, comprising the following steps:

[0013] (1) Provide the above-mentioned microfluidic chip;

[0014] (2) Corneal stromal cells are embedded in a collagen solution, and then gelled and molded to form a dense collagen membrane;

[0015] (3) Inoculate corneal endothelial cells on one side of the collagen membrane and culture them until a fused monolayer is formed;

[0016] (4) Flip the collagen membrane inoculated with corneal endothelial cells so that the side inoculated with corneal endothelial cells faces down and covers the top surface of the lower half of the chip, so that it forms a curved surface shape that conforms to the top surface of the curvature.

[0017] (5) The nerve tissue is implanted into the nerve inoculation hole of the upper half chip, and the upper half chip and the lower half chip are aligned and assembled, and pre-cultured so that the nerve fibers emitted by the nerve tissue extend to the curvature top region.

[0018] (6) Inoculate corneal epithelial cells on the top surface of the dense collagen membrane;

[0019] (7) Dynamic perfusion culture is carried out through the annular microfluidic channel of the lower half chip, and gas-liquid interface culture is established through the central gas-liquid culture hole of the upper half chip, so that the top surface of the corneal epithelial cells is exposed to air, thereby obtaining a full-thickness corneal model with physiological curvature on the top surface of the curvature.

[0020] In another aspect, the present invention provides the application of the above-described microfluidic chip or the method for constructing a full-thickness corneal model with physiological curvature using a microfluidic chip in the preparation of corneal models for ophthalmic drug screening, alternative detection of ocular irritation toxicity, or research on the mechanisms of corneal diseases.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] (1) Biomimetic Physiological Curvature. This invention constructs a matrix-endothelial composite layer with a stable physiological curvature through the curvature top design of the lower half of the chip and the collagen molding and shaping process. The curvature radius is 6~9 mm (preferably 7.8 mm), which is consistent with the physiological curvature of the central anterior surface of the human cornea. Cells grow under a near-real biomechanical curvature state, which can more accurately reproduce the tissue morphology and biophysical characteristics of the cornea.

[0023] (2) Complete cell structure throughout the entire corneal layer. This invention achieves three-dimensional co-culture of the entire corneal epithelium, stroma, and endothelium, with each layer having a distinct structure and complete function, solving the problem of incomplete cell structure in existing models. Histological examination results show that the constructed corneal model forms a multilayered epithelial structure, a collagen stroma layer containing stromal cells, and a continuous monolayer of endothelial cells.

[0024] (3) Integration of neural innervation. This invention utilizes a ring-shaped arrangement of nerve inoculation holes around the upper half of the chip to implant nerve tissue and guide nerve fibers to grow along the microchannels into the central curvature tissue, thus achieving the integration of the neural network with the corneal epithelium and stroma. The constructed model exhibits a higher nerve area fraction and branch point density than the natural cornea, while the nerve length density and terminal point density show no statistically significant difference from the natural cornea. This indicates that the corneal model constructed in this invention possesses neural network distribution characteristics highly consistent with those of the natural cornea.

[0025] (4) Synergistic construction of dual microenvironments. This invention simulates the dynamic circulation and material exchange of aqueous humor in the anterior chamber through the lower annular microfluidic channel, while establishing gas-liquid interface culture through the central gas-liquid culture hole in the upper half. This synergistically realizes corneal surface air exposure and dynamic perfusion in the anterior chamber within the same chip, significantly improving the differentiation and maturation of the tissue.

[0026] (5) High structural integration, suitable for standardized applications. The present invention adopts a split PDMS chip design, which is precisely aligned and assembled through positioning posts / positioning holes. The structure is simple and easy to mass-produce, and can serve industrial application scenarios such as ophthalmic drug screening, toxicity substitution testing and corneal disease mechanism research. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in this invention or related technologies, the accompanying drawings used in describing this invention or related technologies will be briefly introduced below. Obviously, the drawings in the following description only relate to some embodiments of the invention. For those skilled in the art, other drawings can be obtained based on these drawings without any inventive effort.

[0028] Figure 1 This is a schematic diagram of the microfluidic chip of the present invention and a physical diagram thereof. Figure 1 In the diagram, A is a schematic diagram of the lower half of the chip of the present invention, wherein A1 is a schematic diagram, and A2~A4 are the front view, left view and top view, showing the curvature top 1, the positioning post 2 and the annular microfluidic groove 3; B is a schematic diagram of the upper half of the chip of the present invention, wherein B1 is a schematic diagram, and B2~B4 are the front view, left view and top view, showing the positioning hole 4, the nerve inoculation hole 5 and the central gas-liquid culture hole 6; C, from left to right, are the physical images of the microfluidic chip (lower half chip and upper half chip) C1, the physical image of the assembled microfluidic chip C2 and the side view C3.

[0029] Figure 2 This diagram illustrates the process of constructing a full-thickness corneal model using a microfluidic chip, the culture process, and a photograph of the actual full-thickness corneal tissue. A represents the construction process; B represents the dynamic perfusion and gas-liquid interface co-culture after chip assembly; and C represents the top and side views of the actual full-thickness corneal tissue removed from the chip after culture.

[0030] Figure 3 This document presents the tissue structure and characterization results of the full-thickness corneal model constructed in this invention. A shows a full-thickness slide fluorescence staining image after the constructed full-thickness corneal tissue was cut into four flaps; B shows a comparison of the layered staining of the epithelium (ZO-1 / F-actin), nerves (βIII-tubulin), stroma (DAPI-labeled cell nuclei), and endothelium (ZO-1) of the constructed model with that of the natural mouse cornea; C shows the ALDH3A1 (green) section staining and epithelial thickness quantification results.

[0031] Figure 4 This is a comparison between the full-thickness corneal model neural network constructed in this invention and the natural mouse cornea. In this diagram, A shows a panoramic and magnified view of the βIII-tubulin immunofluorescence of the constructed model neural network; B shows a panoramic and magnified view of the βIII-tubulin immunofluorescence of the natural mouse corneal neural network; and C is a bar chart comparing the quantitative comparison of nerve area fraction, nerve length density, branch point density, and terminal point density.

[0032] Figure 5 This image shows a comparison of the transmission electron microscopy (TEM) structure and collagen fiber-like structure density of the corneal stromal cell-containing collagen raw material before and after molding. A represents representative TEM images before and after molding under the same 8000x magnification conditions; B is a quantitative comparison bar chart of the area fraction of the collagen fiber-like structure obtained based on image texture analysis.

[0033] Explanation of reference numerals in the attached figures

[0034] 1-Curvature top; 2-Positioning column; 3-Annular microfluidic channel; 4-Positioning hole; 5-Nerve inoculation hole; 6-Central gas-liquid culture hole Detailed Implementation

[0035] To better illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with non-limiting embodiments. These embodiments (including descriptions mentioned in the embodiments) are intended to illustrate implementation methods of the present invention and are not intended to limit the scope of any claims. According to the present invention, those skilled in the art will understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention, and the same or similar results can still be obtained.

[0036] Unless otherwise stated, the terms used in this specification have the following meanings.

[0037] Organ-on-a-chip: refers to an in vitro model that reconstructs the key structures and functions of an organ on a microfluidic chip.

[0038] Curvature dome: refers to the dome-shaped structure that bulges upward from the center of the lower half of the chip, used to impart the physiological curvature of the collagen membrane.

[0039] Microchannel: refers to an annular flow channel surrounding a curvature dome, used to simulate the circulation of aqueous humor in the foreroom.

[0040] Air-liquid interface: refers to the culture conditions in which the top surface of the cell is exposed to air and the bottom surface is in contact with the culture medium, used to simulate the physiological state of corneal epithelium naturally exposed to air.

[0041] Plastic compression: refers to the process of applying mechanical pressure to collagen hydrogel to expel free water and form a dense film.

[0042] ZO-1 (Zonula Occludens-1, tight junction protein-1): is a tight junction protein.

[0043] βIII-tubulin: a marker of nerve fibers.

[0044] ALDH3A1 (Aldehyde Dehydrogenase 3 Family Member A1): a marker for corneal epithelial cells.

[0045] The present invention provides a microfluidic chip for constructing a full-thickness corneal model with physiological curvature, comprising a lower half chip and an upper half chip.

[0046] Now combined Figure 1 The specific structure of the microfluidic chip of the present invention will be further described. For example... Figure 1 As shown in Figure A, the lower half of the chip has a curved top 1 in the center, an annular microfluidic groove 3 around the periphery, and positioning posts 2 at the four corners; as shown in Figure A. Figure 1 As shown in B, the upper half of the chip has a central gas-liquid culture well 6 in the center, and nerve inoculation wells 5 arranged in a ring around the periphery, with positioning wells 4 at the four corners. Figure 1 C1 shows the lower and upper halves of the chip, C2 shows the assembled overall structure, and C3 shows the side view. The assembled curvature top 1 is located directly below the central gas-liquid culture hole 6.

[0047] Preferably, the lower half of the chip is a square block with a side length of 15-30 mm (preferably 20 mm) and a thickness of 3-8 mm (preferably 5 mm). Its material can be polydimethylsiloxane (PDMS), cyclic olefin copolymer, or polymethyl methacrylate, preferably PDMS. The curvature dome 1 is a dome-shaped protrusion with a curvature radius of 6-9 mm (preferably 7.8 mm), a protrusion height of 0.8-2.5 mm (preferably 1.5 mm), and a bottom diameter of 6-10 mm (preferably 8 mm). The surface of the curvature dome 1 is smooth and continuous, used to support the collagen membrane and impart physiological curvature to it. An annular microfluidic channel 3 is provided around the bottom periphery of the curvature dome 1, with a channel width of 200-800 μm (preferably 500 μm) and a channel depth of 100-500 μm (preferably 300 μm). It is connected to external pipelines via inlets and outlets (not shown) on the chip's sidewall or bottom surface, used to simulate the dynamic circulation and material exchange of aqueous humor in the anterior chamber. The chip has cylindrical fixing posts 2 at its four corners, preferably 2 mm in diameter and 3 mm in height, for alignment and assembly with the upper half of the chip.

[0048] Preferably, the upper half of the chip is a square block made of the same material as the lower half of the chip, and its size matches that of the lower half of the chip. The diameter of the central gas-liquid culture well 6 is 6-10 mm, preferably 8 mm, and the depth is 2-5 mm, preferably 3 mm. It is coaxially aligned with the curvature apex 1 and the center of the collagen membrane of the lower half of the chip to provide space for ALI culture. There are 8-16 (preferably 12) nerve seeding wells 5, with a diameter preferably 1 mm and a depth preferably 2 mm. They are arranged in a ring at equal intervals around the center of the central gas-liquid culture well 6. Each well communicates with the central culture area through an internal connecting microchannel, which is used to implant ganglia and guide nerve fibers to grow into the central curvature membrane. Fixing holes 4 are provided at the four corners of the chip. The diameter of the holes is slightly larger than the diameter of the fixing posts by 0.1-0.3 mm. During assembly, the fixing posts are fitted together to achieve positioning and clamping.

[0049] The lower half chip is detachably assembled and connected to the upper half chip, and after assembly, the central gas-liquid culture hole 6 corresponds to the position of the curvature top 1, with the curvature top 1 located directly below the central gas-liquid culture hole 6.

[0050] The chip fabrication method is as follows: SolidWorks or AutoCAD is used to create a three-dimensional model of the upper and lower halves of the chip and export the STL file; the main mold is prepared by CNC precision machining, high-resolution photopolymerization 3D printing or soft photolithography; PDMS prepolymer and curing agent are mixed at a mass ratio of 10:1, degassed, poured and heat-cured and then demolded.

[0051] This invention also provides a method for constructing a full-thickness corneal model with physiological curvature using the aforementioned microfluidic chip, such as... Figure 2As shown, the key process route for constructing a full-thickness corneal model using this chip includes, in sequence: preparation and molding of collagen hydrogel containing stromal cells, endothelial cell seeding, capping and shaping, neural tissue implantation and chip assembly, epithelial cell seeding, and dynamic perfusion and gas-liquid interface co-culture. The specific steps are as follows:

[0052] Step (1): Provide, for example Figure 1 The microfluidic chip shown

[0053] The lower half chip and the upper half chip are provided as described above.

[0054] Step (2): Preparation and molding of collagen hydrogel containing stromal cells

[0055] Corneal stromal cells are embedded in a collagen solution, and then gelled and molded to form a dense collagen membrane.

[0056] Specifically, the matrix layer composition is a type I collagen hydrogel containing living cells. The final concentration of type I collagen can range from 2 to 6 mg / mL, and the corneal stromal cell density can be 1 × 10⁻⁶ cells / mL. 5 ~5×10 5 Cells / mL. Cells can be obtained from the dissociation of primary rabbit, human, or mouse corneal stroma, passaged 2-5 times, with viability ≥95%.

[0057] Step (3): Construction of the corneal endothelial cell layer

[0058] Corneal endothelial cells were inoculated on one side of the collagen membrane and cultured until a fused monolayer was formed.

[0059] Specifically, primary human corneal endothelial cells, immortalized HCE-2 or B4G12 endothelial cells, etc., are used. Primary cells should be used at passages P1 to P3, and the cell line should not be passaged more than 10 times. The collagen membrane obtained in step (2) is placed on a flat surface of the culture dish, which will be used as the endothelial side thereafter. Using 5 × 10⁻⁶ cells... 4 ~2×10 5 cells / cm 2 (Preferred 1×10) 5 cells / cm 2 Endothelial cells were seeded at a density of approximately 100-200 μL / membrane, and incubated at 37°C with 5% CO2 for 4 hours to promote adhesion. Then, endothelial cell culture medium (such as Opti-MEM-I with 5% FBS and endothelial growth factor) was added, and the medium was changed every 2 days. The cells were cultured for 3-7 days (preferably 5 days) until confluence ≥95% and a hexagonal cobblestone-like morphology was observed under a light microscope. ZnO-1 and Na+ can be used as a substrate. + K + -ATPase immunofluorescence confirms monolayer integrity.

[0060] Step (4): Curvature shaping and lower half chip pre-assembly

[0061] The collagen membrane inoculated with corneal endothelial cells is flipped over so that the side inoculated with the corneal endothelial cells faces downwards, and then placed over the curved top surface of the lower half of the chip, forming a curved surface shape that conforms to the curved top. Combined with... Figure 1 As shown in C2 and C3 of C, after assembly, the central gas-liquid culture hole is coaxially aligned with the curvature top, providing space for subsequent gas-liquid interface culture. Figure 2 A flowchart also illustrates the positional relationship between the composite membrane and the curvature top during the top-forming step.

[0062] Step (5): Upper part assembly and ganglion implantation

[0063] The neural tissue is implanted into the neural inoculation hole of the upper half chip, and the upper half chip is aligned and assembled with the lower half chip.

[0064] Specifically, dorsal root ganglion organoids (DRGOs) obtained from induced pluripotent stem cell (iPSC) differentiation are placed in the circular nerve seeding wells of the upper half of the chip, with an appropriate amount of nerve culture medium added to each well. Alternatively, primary dorsal root ganglia (DRGs), trigeminal ganglia (TGs), or other iPSC-derived sensory neurons can be used. The upper half of the chip is flipped over and aligned with the lower half, so that the positioning wells fit over the positioning columns, and the central gas-liquid culture well is coaxially assembled with the curvature dome and collagen membrane.

[0065] Preferably, after aligning and assembling the upper half chip and the lower half chip, a pre-culture is performed for 5 to 10 days (preferably 7 days) to allow the nerve fibers from the nerve tissue to extend along the microchannels and tissue edges to the top of the curvature region before subsequent corneal epithelial cell inoculation.

[0066] Step (6): Corneal epithelial cell inoculation

[0067] Corneal epithelial cells were inoculated on the top surface of the dense collagen membrane.

[0068] Specifically, HCE-T corneal epithelial cells are seeded onto the top surface of the curvature collagen membrane, with a preferred seeding density of 2×10⁶ cells / year. 5 cells / cm 2 After cell attachment, the liquid level in the central culture well is lowered to expose the top surface of the epithelium to air and establish an air-liquid interface.

[0069] Step (7): Dynamic perfusion and gas-liquid interface co-culture

[0070] Dynamic perfusion culture is performed through the annular microfluidic channel of the lower half of the chip, while gas-liquid interface culture is established through the central gas-liquid culture well of the upper half of the chip, so that the top surface of the corneal epithelial cells is exposed to air, thereby obtaining a full-thickness corneal model with physiological curvature on the top surface of the curvature.

[0071] Specifically, the inlet and outlet of the lower microfluidic tank are connected to a peristaltic pump (flow rate range 0.1–100 μL / min) and a preheated storage bottle at 37°C via silicone tubing or Peek tubing with an inner diameter of 0.5–1 mm, forming a circulating or unidirectional perfusion system. The perfusion solution uses anterior chamber simulated culture medium, composed of 1×DMEM / F12, 5% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin, 10 mM HEPES, and 25 mM NaHCO3, with a pH of 7.3–7.4 and an osmotic pressure of 290–310 mOsm / kg.

[0072] Perfusion should begin on the day the ALI is established, with a flow rate of 1–50 μL / min (preferably 10 μL / min) and continuous circulation. The total culture time at the gas-liquid interface is 7–21 days (preferably 14 days), with the perfusion medium replaced or replenished every 2–3 days. Dynamic perfusion simulates the aqueous humor circulation in the anterior chamber, maintaining O2 and glucose supply and metabolic waste removal on the endothelial side, forming a dual microenvironment synergistically with the apical ALI, thereby improving endothelial survival, ZO-1 continuity, and full-thickness tissue maturity.

[0073] After the culture is completed, the chip is removed and the curved full-thickness corneal tissue is collected.

[0074] To further understand the present invention, the microfluidic chip and the method for constructing a full-thickness corneal model with physiological curvature using the same are described in detail below with reference to embodiments. The scope of protection of the present invention is not limited by the following embodiments.

[0075] Example 1

[0076] 1. Fabrication of microfluidic chips

[0077] The upper and lower half of the chip were modeled in 3D using SolidWorks and exported as STL files. The lower half of the chip has a curvature radius of 7.8 mm, a protrusion height of 1.5 mm, and a bottom diameter of 8 mm. The annular microfluidic channel is 500 μm wide and 300 μm deep. Cylindrical positioning posts with a diameter of 2 mm and a height of 3 mm are located at the four corners. The upper half of the chip has a gas-liquid culture well with a diameter of 8 mm and a depth of 3 mm in the center. Twelve nerve seeding wells with a diameter of 1 mm and a depth of 2 mm are arranged in a ring around the periphery, each well communicating with the central culture area via an internal microchannel. Positioning holes that mate with the positioning posts are located at the four corners.

[0078] The main mold was prepared using high-resolution photopolymer 3D printing. PDMS prepolymer and curing agent were mixed at a mass ratio of 10:1, thoroughly stirred, and then degassed under vacuum. The mixture was poured into the mold and heat-cured at 65℃ for 3 hours before demolding to obtain the upper and lower chip halves. After cutting and drilling (inlet and outlet ports), the chips were sterilized by immersion in 75% ethanol, cleaned with sterile deionized water, and irradiated with ultraviolet light for later use.

[0079] 2. Preparation and molding of collagen hydrogels containing stromal cells

[0080] Human corneal stromal cells (CSCs) derived from the stromal lens of the SMILE procedure were passaged to 2-5 generations with viability ≥95%.

[0081] Preparation of CSC-containing type I collagen hydrogel (final volume 1 mL): Take 322 μL of 9.3 mg / mL type I collagen (derived from rat tail, endotoxin ≤10 EU / mg), 100 μL of 10×MEM, 23 μL of 0.1N NaOH, and corneal stromal cell suspension (density 3×10⁻⁶). 6 100 μL (cells / mL), 455 μL sterile deionized water, final pH 7.3, final collagen concentration 3 mg / mL, final cell density 3 × 10⁻⁶ 5 Cells / mL.

[0082] Pre-cool 48-well plates to 4°C, then add 10×MEM, NaOH, cell suspension, and collagen solution sequentially, mixing quickly and gently while maintaining 4°C throughout to prevent premature gelation. Transfer the mixture to a pre-cooled 6 mm diameter circular mold and incubate at 37°C with 5% CO2 for 1 h until complete gelation, forming a disc-shaped hydrogel containing stromal cells. Before and after gelation, place nylon mesh rings on both sides of the collagen gel for subsequent clamping and transfer.

[0083] The collagen gel containing CSC was placed on filter paper and subjected to a pressure of 100 kPa for 30 min to expel free water and form a dense collagen film with a thickness of 100 μm.

[0084] 3. Construction of the corneal endothelial cell layer

[0085] Place the dense collagen membrane obtained in step 2 on a flat surface of the culture dish (this surface will subsequently serve as the endothelial side). Use B4G12 corneal endothelial cell line (passaged no more than 10 times) at a ratio of 1×10⁻⁶. 5 cells / cm 2The collagen membrane was inoculated at a density of approximately 100-200 μL / membrane. The membrane was then incubated at 37°C with 5% CO2 for 4 hours to promote adhesion. Opti-MEM-I medium containing 5% fetal bovine serum (FBS) and endothelial growth factor was added, and the medium was changed every 2 days. The membrane was cultured for 5 days until confluence was ≥95%, exhibiting a hexagonal paving stone-like morphology under a light microscope.

[0086] 4. Curvature shaping and lower half chip pre-assembly

[0087] Using sterile forceps, flip the collagen membrane inoculated with corneal endothelial cells so that the endothelial cell layer faces down and the collagen matrix surface faces up; remove the nylon mesh rings located on both sides of the collagen membrane. Prefill the lower half of the chip's annular microfluidic groove with culture medium and remove air bubbles, then gently cover the curved top surface with the composite membrane, allowing the membrane to form a dome-shaped three-dimensional structure under elastic and geometric constraints. Pre-equilibrate at 37℃ and 5% CO2 for 30 min to ensure the membrane fully adheres and the endothelial side is in contact with the perfusion fluid.

[0088] 5. Upper body assembly and ganglion implantation

[0089] Dorsal root ganglion organoids (DRGOs) obtained from induced pluripotent stem cell (iPSC) differentiation were placed in 10 circular nerve seeding wells of the upper half of the chip, with an appropriate amount of nerve culture medium added to each well. The upper half of the chip was flipped over and aligned with the lower half of the chip, so that the positioning wells fit the positioning columns, and the central gas-liquid culture well was coaxially assembled with the curvature top and collagen membrane.

[0090] After assembly, microfluidic perfusion culture was initiated for 7 days, allowing the nerve bundles emitted by DRGO to extend along the microchannels and tissue edges and enter the central corneal construct.

[0091] 6. Corneal epithelial cell seeding and air-liquid interface culture

[0092] After 7 days of DRGO pre-culture, HCE-T corneal epithelial cells were seeded onto the top surface of the curvature collagen membrane at a seeding density of 2×10⁶ cells / day. 5 cells / cm 2 After cell attachment, the culture medium in the central gas-liquid culture well was aspirated or naturally evaporated, exposing the top surface of the epithelium to humidified air containing 5% CO2 to establish a gas-liquid interface. The basal side was wetted by the collagen membrane and the microfluidic fluid, maintaining a relative humidity of ≥85%. Simultaneously, dynamic perfusion was initiated in the lower annular microfluidic tank. The perfusion medium was anterior chamber simulated culture medium (1×DMEM / F12, 5% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin, 10 mM HEPES, 25 mM NaHCO3, pH 7.3–7.4, osmotic pressure 290–310 mOsm / kg), with a flow rate of 10 μL / min, continuously circulated. ALI and perfusion were co-cultured for 14 days, with the perfusion medium replaced or replenished every 2–3 days.

[0093] After the culture is completed, the chip is removed and the curved full-thickness corneal tissue is collected.

[0094] 7. Histological characteristics

[0095] Immunofluorescence staining was performed on the constructed full-thickness corneal model. For example... Figure 3 As shown in the full-thickness slice of A, the periphery of the tissue reveals DRGO corresponding to the location of the circular nerve seeding hole and nerve bundles extending centrally (βIII-tubulin-labeled nerve fibers, red). Figure 3 Immunofluorescence of βIII-tubulin, ZO-1, F-actin, DAPI, and ALDH3A1 in B showed that the construct had an epithelial layer (ZO-1 / F-actin labeled cell boundaries, gray / green) and its accompanying nerves (βIII-tubulin, red), a stromal layer containing CSCs (DAPI labeled cell nuclei, blue), and a continuous endothelial cell layer (ZO-1, gray), with the layered relationship corresponding to that of the natural mouse cornea.

[0096] like Figure 3 The slice measurements of C showed that the epithelial thickness of the constructed model was 23.24±3.63 μm (n=5), ALDH3A1 expression was detected (green), and multilayered epithelial-like structures were observed, indicating that the model formed a corneal-associated cell phenotype and layered tissue structure.

[0097] like Figure 4 Quantitative analysis of the neural network in AC showed that the neural area fraction of the constructed model and the mouse cornea were 0.2146±0.0167 and 0.1736±0.0110, respectively (n=4, p=0.00635); the neural length densities were 17.92±3.09 and 14.86±2.35 mm / mm, respectively. 2 (n=4, p=0.1662); the branch point densities were 811.38±138.15 and 566.86±102.77 points / mm, respectively. 2 (n=4, p=0.0296); the terminal point densities were 1504.67±187.22 and 1454.65±137.88 points / mm, respectively. 2 (n=4, p=0.6821). Figure 4 The quantitative comparison of the bar charts for C shows that the neural area fraction and branch point density of the constructed model are significantly higher than those of the natural cornea (p<0.05), while the neural length density and terminal point density are not statistically different from those of the natural cornea (p>0.05), indicating that the corneal model constructed in this invention has neural network distribution characteristics comparable to those of the natural cornea.

[0098] 8. Verification of Collagen Compression Effect

[0099] Transmission electron microscopy was used to observe the CSC-containing raw materials before and after molding. Figure 5 A). Under the same 8000x magnification conditions, the area fraction of collagen fiber-like structures obtained based on image texture segmentation is as follows: Figure 5 As shown in Figure B, the percentage of collagen matrix before compression was 19.39 ± 7.79% (n = 10 figures), and after compression it was 27.95 ± 5.26% (n = 5 figures), representing a relative increase of approximately 44.2% and an absolute increase of 8.56 percentage points (exploratory exact replacement test p = 0.0496). The results indicate that compression treatment can significantly improve the density of the collagen matrix.

[0100] Example 2

[0101] The chip was fabricated and a full-thickness cornea was constructed using the same process as in Example 1, except that the chip parameters and process conditions were changed as follows:

[0102] The curvature radius at the top is 6 mm, the protrusion height is 0.8 mm, and the bottom diameter is 6 mm. There are 8 nerve inoculation holes. The annular microfluidic channel has a width of 200 μm and a depth of 100 μm.

[0103] The concentration of type I collagen was 2 mg / mL, and the corneal stromal cell density was 1×10⁻⁶. 5 Cells / mL. The compression conditions were: pressure 50 kPa, time 2 h. The corneal endothelial cell seeding density was 5 × 10⁻⁶ cells / mL. 4 cells / cm 2 The cultivation period is 7 days.

[0104] The neural pre-culture time is 5 days.

[0105] The dynamic perfusion flow rate was 1 μL / min, and the total incubation time at the gas-liquid interface was 7 days.

[0106] The constructed full-thickness corneal model also possesses physiological curvature, full-thickness cell structure, and nerve innervation.

[0107] Example 3

[0108] The chip was fabricated and a full-thickness cornea was constructed using the same process as in Example 1, except that the chip parameters and process conditions were changed as follows:

[0109] The curvature radius at the top is 9 mm, the protrusion height is 2.5 mm, and the bottom diameter is 10 mm. There are 16 nerve inoculation holes. The annular microfluidic channel has a width of 800 μm and a depth of 500 μm.

[0110] The concentration of type I collagen was 6 mg / mL, and the corneal stromal cell density was 5 × 10⁻⁶. 5Cells / mL. The compression conditions were: pressure 200 kPa, time 10 min.

[0111] The corneal endothelial cell seeding density is 2×10 5 cells / cm 2 The cultivation period is 3 days.

[0112] The neural pre-culture time is 10 days.

[0113] The dynamic perfusion flow rate was 50 μL / min, and the total incubation time at the gas-liquid interface was 21 days.

[0114] The constructed full-thickness corneal model also possesses physiological curvature, full-thickness cell structure, and nerve innervation.

[0115] Example 4

[0116] The chip was fabricated and a full-thickness cornea was constructed using the same process as in Example 1, except for the source of the neural tissue. Primary dorsal root ganglia (DRGs) were used instead of iPSC-derived DRGs for implantation into the neural inoculation wells. The DRGs were obtained from newborn mice, separated, minced, digested with collagenase, and then inoculated into the neural inoculation wells.

[0117] The constructed full-thickness corneal model also exhibits the characteristic of neural networks growing into the central corneal construct.

[0118] Example 5

[0119] The chip was prepared and a full-thickness cornea was constructed using the same process as in Example 1, except for the chip material. Cyclic olefin copolymer (COC) was used instead of PDMS as the chip material, and the upper and lower halves of the chip were prepared using an injection molding process.

[0120] The constructed full-thickness corneal model also possesses physiological curvature, full-thickness cell structure, and nerve innervation.

[0121] Comparative Example 1 (without curvature apex)

[0122] The chip was prepared and a full-thickness cornea was constructed using the same process as in Example 1, except that the lower half of the chip did not have a curvature top and a planar culture substrate was used.

[0123] A collagen membrane inoculated with corneal endothelial cells was laid flat on a planar culture medium substrate. The results showed that the constructed corneal model was a planar structure, and the cells grew on the planar substrate, failing to form a physiological curvature morphology.

[0124] Comparative Example 2 (without nerve inoculation)

[0125] The chip was prepared and a full-thickness cornea was constructed using the same process as in Example 1, except that the upper half of the chip did not have a nerve inoculation hole and no nerve tissue was implanted.

[0126] The results showed that the constructed corneal model did not contain neural components and could not reproduce the neural distribution characteristics of the natural cornea.

[0127] Comparative Example 3 (without plastic compression treatment)

[0128] The chip was prepared and the full-thickness cornea was constructed using the same process as in Example 1. The difference was that in step 2, the collagen gel was not subjected to compression treatment, but was directly covered with uncompressed collagen hydrogel on the top surface of the curvature.

[0129] The results showed that the unpressed collagen hydrogel was thick and lacked mechanical strength, making it difficult to adhere tightly to the top surface of the curvature. Some areas showed wrinkles or detachment, affecting the curvature shaping effect and the consistency of cell culture.

[0130] Comparative Example 4 (no neural pre-culture, simultaneous inoculation)

[0131] The chip was prepared and a full-thickness cornea was constructed using the same process as in Example 1. The difference was that the 7-day pre-culture of nerves was not performed in step 5. Instead, nerve tissue was implanted at the same time as epithelial cells were inoculated in step 6, and the epithelium and nerves were cultured simultaneously.

[0132] The results showed that under synchronous culture conditions, the extension distance and density of nerve fibers into the central corneal construct were significantly lower than those in the pre-culture group, indicating that nerve pre-culture is significantly preferred for guiding nerves to fully ingrow into the corneal tissue.

[0133] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A microfluidic chip for constructing a full-thickness corneal model with physiological curvature, characterized in that, include: The lower half chip has an upwardly protruding curved top and an annular microfluidic channel disposed around the curved top. The annular microfluidic channel is provided with an inlet and an outlet. The upper half of the chip has a central gas-liquid culture well and a plurality of nerve inoculation wells arranged in a ring around the central gas-liquid culture well, and the nerve inoculation wells are connected to the central gas-liquid culture well. The upper half chip and the lower half chip are detachably assembled and connected, and after assembly, the position of the central gas-liquid culture hole corresponds to the position of the curvature top, with the curvature top located directly below the central gas-liquid culture hole.

2. The microfluidic chip according to claim 1, characterized in that, The radius of curvature of the curvature apex is 6-9 mm; the height of the curvature apex is 0.8-2.5 mm; and the bottom diameter of the curvature apex is 6-10 mm.

3. The microfluidic chip according to claim 1, characterized in that, The number of the plurality of nerve inoculation wells is 8 to 16, which are arranged in a ring at equal intervals around the center of the central gas-liquid culture well.

4. The microfluidic chip according to claim 1, characterized in that, The width of the annular microfluidic channel is 200~800μm; the depth of the annular microfluidic channel is 100~500μm.

5. The microfluidic chip according to claim 1, characterized in that, The lower half chip and / or the upper half chip are made of polydimethylsiloxane, cyclic olefin copolymer or polymethyl methacrylate.

6. The microfluidic chip according to claim 1, characterized in that, The lower half chip is provided with a positioning post, and the upper half chip is provided with a positioning hole that cooperates with the positioning post. The upper half chip and the lower half chip are assembled and positioned by the fitting of the positioning post and the positioning hole.

7. A method for constructing a full-thickness corneal model with physiological curvature using a microfluidic chip according to any one of claims 1 to 6, characterized in that, Includes the following steps: (1) A microfluidic chip as described in any one of claims 1 to 6 is provided; (2) Corneal stromal cells are embedded in a collagen solution, and then gelled and molded to form a dense collagen membrane; (3) Inoculate corneal endothelial cells on one side of the collagen membrane and culture them until a fused monolayer is formed; (4) Flip the collagen membrane inoculated with corneal endothelial cells so that the side inoculated with corneal endothelial cells faces down and covers the top surface of the lower half of the chip, so that it forms a curved surface shape that conforms to the top surface of the curvature. (5) The nerve tissue is implanted into the nerve inoculation hole of the upper half chip, the upper half chip and the lower half chip are aligned and assembled, and pre-cultured so that the nerve fibers emitted by the nerve tissue extend to the curvature top region. (6) Inoculate corneal epithelial cells on the top surface of the dense collagen membrane; (7) Dynamic perfusion culture is carried out through the annular microfluidic channel of the lower half chip, and gas-liquid interface culture is established through the central gas-liquid culture hole of the upper half chip, so that the top surface of the corneal epithelial cells is exposed to air, thereby obtaining a full-thickness corneal model with physiological curvature on the top surface of the curvature.

8. The method according to claim 7, characterized in that, In step (2), the concentration of type I collagen in the collagen solution is 2-6 mg / mL; the density of corneal stromal cells is 1×10⁻⁶ mg / mL. 5 ~5×10 5 Cells / mL; the conditions for the plastic compression treatment are: pressure 50~200 kPa, time 10 min~2 h; the thickness of the dense collagen membrane is 80~150 μm.

9. The method according to claim 7, characterized in that, In step (3), the seeding density of the corneal endothelial cells is 5 × 10⁻⁶. 4 ~2×10 5 cells / cm 2 The culture time is 3 to 7 days, until the confluence is ≥95% and the morphology is hexagonal paving stone-like under a light microscope.

10. The method according to claim 7, characterized in that, In step (5), after aligning and assembling the upper half chip and the lower half chip, pre-culture for 5 to 10 days is performed to allow the nerve fibers from the nerve tissue to extend to the top of the curvature region before corneal epithelial cell inoculation in step (6).

11. The method according to claim 7, characterized in that, In step (7), the flow rate of the dynamic perfusion culture is 1~50 μL / min; the total time of the gas-liquid interface culture is 7~21 days.

12. The method according to claim 7, characterized in that, The corneal stromal cells are derived from the stromal lens of the SMILE surgery; the neural tissue is a dorsal root ganglion organoid (DRGO) obtained by induced pluripotent stem cell differentiation, a primary dorsal root ganglion, or a trigeminal ganglion.

13. The use of the microfluidic chip according to any one of claims 1 to 6 or the method according to any one of claims 7 to 12 in the preparation of corneal models for ophthalmic drug screening, alternative detection of ocular irritation toxicity, or study of corneal disease mechanisms.

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