3D printing cell culture chip for high-throughput structure screening and preparation method and application thereof
The use of 3D printing technology to prepare cell culture chips solves the problems of complex preparation processes and small structural size in existing technologies, and enables the efficient screening of three-dimensional structures of porous materials for tissue repair, thereby improving detection efficiency and biocompatibility.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SOUTH CHINA UNIV OF TECH
- Filing Date
- 2022-12-14
- Publication Date
- 2026-06-23
AI Technical Summary
Existing cell culture chip fabrication processes are complex, the biocompatibility of raw materials is limited, and the structural size is small, making it impossible to efficiently screen the three-dimensional structure of porous materials for tissue repair.
Cell culture chips, including cell culture blocks, anti-adhesion strips, and cell isolation chambers, are fabricated using 3D printing technology. A gradient-changing structural matrix is designed using ceramic powder, metal powder, or polymeric monomer materials. The chips are formed by digital light processing or laser melting and combined with anti-adhesion materials such as PDMS or agarose to achieve high-throughput structure screening.
It enables efficient and rapid preparation of diverse cell culture chips, suitable for structural screening of porous materials for tissue repair, and can observe the behavior of various cells on different gradient structures, thus improving detection efficiency and biocompatibility.
Smart Images
Figure CN116042390B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of porous materials for tissue repair, specifically relating to a 3D-printed cell culture chip for high-throughput structure screening, its preparation method, and its application. Technical Background
[0002] Specific structures in natural biological tissues play a crucial role in their physiological functions. Similarly, cell behavior and tissue regeneration and repair can be regulated and guided through the optimized design of the substrate structure of tissue-engineered porous materials and other porous implants. The three-dimensional structural design of tissue-engineered porous materials is an important research direction in the field of tissue engineering. Studies have shown that different three-dimensional structures of porous materials significantly affect the behavior, density, and spatial distribution of cells within them, thereby influencing tissue morphology and functional realization. Therefore, the research and determination of the three-dimensional structure of porous materials are crucial in the design and fabrication of tissue-engineered porous materials.
[0003] However, current screening of porous material structures generally involves continuous discrete experiments in a near-spatiotemporal environment, resulting in low detection efficiency and an inability to truly achieve co-culture of cells and porous materials under the same time and culture conditions. To address this, some researchers have proposed cell culture chip technology. These cell culture chips mostly employ replication molding techniques to prepare cell-sized structures (e.g., CN201110310986.9, A 3D Single-Cell Culture Chip Based on PDMS and Its Controllable Preparation Method), used for rapid screening of the effects of different surface micropatterns on cell behavior. Similarly, there are cell culture chips prepared using multiple processes such as photoresist, PDMS casting, and etching (CN200710045997.2, Preparation Method and Application of Cell Culture Chips Based on ITO Glass Substrates), applied to observe cell migration and differentiation behavior. The above cell culture chips suffer from complex fabrication processes and are made from bio-inert materials. Furthermore, although existing soft lithography technology for fabricating cell culture chips (such as CN201611053030.4 Three-dimensional cell culture chip based on soft lithography technology, its fabrication method and application) has a relatively simple fabrication process, the fabricated structures are only 7-15 μm in size, which still limits the observation of cell behavior. In summary, existing structure screening cell chip fabrication processes are complex, the biocompatibility of raw materials is limited, and the structure size is small, making them unsuitable as tools for screening the structure of tissue repair materials. Summary of the Invention
[0004] The present invention aims to overcome the shortcomings of existing structure matrix cell culture chips used for cell behavior observation, such as complex fabrication process, limited biocompatibility of raw materials, and small structural size. The primary objective of the present invention is to provide a 3D printed cell culture chip for high-throughput structure screening.
[0005] Another object of the present invention is to provide a method for preparing the above-mentioned 3D printed cell culture chip for high-throughput structure screening.
[0006] Another object of the present invention is to provide the application of the above-mentioned 3D printed cell culture chip.
[0007] The objective of this invention is achieved through the following technical solutions.
[0008] A 3D-printed cell culture chip for high-throughput structure screening, the cell culture chip being 3D printed, comprising a cell culture block, an anti-adhesion strip, a cell isolation groove, and a substrate; the cell culture block is composed of a structural matrix arrangement of at least one unit structure with gradient changes in curvature, porosity, pore size, and interconnecting pore size and / or parallel groups thereof.
[0009] Preferably, the chip comprises, from top to bottom, a cell culture block, an anti-adhesion strip, a cell isolation groove, and a substrate.
[0010] Preferably, the cell chip is integrally formed by 3D printing from at least one of ceramic powder, metal powder, or polymeric compound monomer.
[0011] Preferably, the ceramic powder is at least one of hydroxyapatite, tricalcium phosphate β-TCP, and wollastonite; the metal powder is at least one of titanium alloy, tantalum metal, and stainless steel.
[0012] The polymeric compound monomer is a photocurable polymeric compound monomer;
[0013] The anti-adhesion tape is made of polydimethylsiloxane (PDMS) or agarose film to prevent cross-contamination between cells on different structures, facilitating subsequent observation of cell behavior.
[0014] Preferably, the 3D printing is digital light processing (DLP) or laser melting 3D printing.
[0015] Preferably, the number of structures and the size of the matrix contained in the cell culture block are adjusted according to the requirements, and the length, width and height of the gradient-changing unit structure are 100μm-2000μm.
[0016] Preferably, the isolation groove contains an anti-adhesion strip to prevent cells cultured on different structures from interfering with each other, with a depth of 0.5mm-1.5mm and a width of 0.5mm-1mm;
[0017] The substrate is a cuboid substrate with a thickness of at least 1 mm to 2 mm.
[0018] A method for preparing a 3D-printed cell culture chip for high-throughput structure screening includes the following steps:
[0019] (1) Design a structure with at least one gradient change in curvature, porosity, pore size, and interconnection pore size using 3D modeling software, arrange the above structure and / or its parallel groups into a high-throughput matrix, and design a model of the above matrix with a substrate containing cell isolation grooves.
[0020] (2) Import the model, and the chip raw material powder is 3D printed to prepare the 3D shaped chip. After post-processing and sterilization, sterile agarose or polydimethylsiloxane PDMS is added to the isolation tank. After solidification, an anti-sticking effect is formed, and a 3D printed cell culture chip is obtained.
[0021] Preferably, the post-treatment is at least one of sintering, sandblasting, and ultrasonic cleaning;
[0022] The post-processing involves degreasing or cleaning the 3D-formed chip to remove impurities.
[0023] The sterilization process involves high temperature and high pressure, ultraviolet irradiation, or radiation.
[0024] Preferably, the 3D printing precision in step (2) is at least 100 micrometers.
[0025] The above-mentioned 3D-printed cell culture chip for high-throughput structure screening is used in the in vitro analysis and screening of porous material structures for cell culture and tissue repair.
[0026] Preferably, after the cell culture chip is sterilized, a cell suspension is added, and fluorescent staining is performed at a specific time to observe and statistically analyze the adhesion status, proliferation, differentiation, and other behaviors of cells on different structural surfaces.
[0027] Compared to existing cell culture chips, this high-throughput structural culture chip has the advantages of diverse materials and the ability to be integrated using various 3D printing methods. More importantly, it has the function of screening three-dimensional structures of porous materials for tissue engineering, making it an effective tool for studying the behavior of various cells on different gradient structures.
[0028] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0029] (1) Existing cell culture chip fabrication processes are cumbersome. This invention uses 3D printing to achieve integrated and rapid fabrication of cell culture chips.
[0030] (2) Existing cell culture chips have limited structural types and size (less than 100 micrometers) due to process limitations. The present invention constructs a cell culture chip with a complex three-dimensional structure (greater than 100 micrometers), which is more suitable for the structural screening of porous materials for tissue repair.
[0031] (3) This invention designs a high-throughput structural parameter gradient change matrix to observe various cell behaviors on different structures, and applies it to the in vitro analysis and rapid screening of porous material structures for tissue repair. Attached Figure Description
[0032] Figure 1 A schematic diagram of a high-throughput curvature structure screening chip for 3D printing.
[0033] Figure 2 A fabrication process diagram for high-throughput structure screening cell culture chips using 3D printing.
[0034] Figure 3 The image shows a 3D-printed cell culture chip unit structure model for high-throughput Gaussian curvature structure screening in Example 1, along with an optical microscope image after printing.
[0035] Figure 4 This is a photograph of the cell culture chip used for high-throughput Gaussian curvature structure screening, which was 3D printed in Example 1.
[0036] Figure 5 This is a diagram showing the adhesion state of hBMSCs cells on a cell culture chip with a curvature gradient in Example 1.
[0037] Figure 6 This is an ALP staining image of hBMSCs cells differentiating on a cell culture chip with a curvature gradient, as shown in Example 1.
[0038] Figure 7 The high-throughput structural screening cell culture chip in Example 1 was used to observe the adhesion quantity, spreading area, aspect ratio, and alkaline phosphatase staining normalized composite heatmap of human bone marrow mesenchymal stem cells. Detailed implementation method:
[0039] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.
[0040] Source of bone marrow mesenchymal stem cells (hBMSCs): ATCC Cell Bank
[0041] Fetal bovine serum was purchased from Invitrogen Gibco, USA.
[0042] Culture medium was purchased from: Invitrogen Gibco (USA)
[0043] The ALP staining kit was purchased from Beyotime Biotechnology Co., Ltd.
[0044] Example 1: A high-throughput bioceramic chip with a Gaussian curvature gradient for observing cell adhesion behavior.
[0045] In this embodiment, a high-throughput chip with a Gaussian curvature gradient (material: tricalcium phosphate β-TCP) was designed to culture human bone marrow mesenchymal stem cells (hBMSCs) and to statistically analyze the number of cells adhering to different structures and observe cell adhesion and adhesion status.
[0046] Step 1: Using the 3D software Rhino Grasshopper, design three hyperbolic structures with gradient Gaussian curvature: Hyp3, Hyp2, and Hyp1, with Gaussian curvatures of -20, -12.5, and -5 mm respectively. -2 Three structures were identified: a cylindrical structure (Cyl with a Gaussian curvature of 0 and a diameter of 500 μm), and an ellipsoidal structure (Ell3, Ell2, and Ell1 with Gaussian curvatures of +5, +12.5, and +20 mm, respectively). -2 )(like Figure 3 (1) Four parallel samples of the above structure are arranged into 35 high-throughput matrices, and a rectangular substrate (37mm long, 26mm wide, and 1mm high) with isolation grooves (0.5mm) is designed to load the above matrix model.
[0047] Step Two: Import the model and use Digital Light Processing (DLP) 3D printing technology to directly print tricalcium phosphate β-TCP powder, preparing a high-throughput tricalcium phosphate β-TCP cell culture chip as designed in Step One, achieving a printing precision of 100 micrometers. The chip preform is ultrasonically cleaned in ultrapure water, and then ultrasonically cleaned again. After drying at 50℃ for 12 minutes, the chip preform is subjected to high-temperature treatment in a muffle furnace at 800-1150℃ for 3 hours. The post-processed sample is shown below. Figure 3 (1) Figure 4 .
[0048] Step 3: Place the high-throughput cell culture chip and 2wt% agarose, which were sintered in Step 2, into a 121℃ high-temperature sterilization chamber for 30 minutes.
[0049] Step 4: Add the sterile high-throughput structural cell culture chip from Step 3 into a 12-well plate, then add sterile agarose to cover the chip substrate and fill the isolation chamber. After 5-15 minutes, the agarose solidifies, and then soak in a medium containing 10% fetal bovine serum for 1 hour.
[0050] Step 5: Add 1 ml of 1.2 × 10⁻⁶ m³ ... to the high-throughput structural cell culture chip soaked in Step 4. 5 / ml cell suspension.
[0051] Step Six: After 12 hours, change the medium for the cells cultured on the chip in Step Five.
[0052] Step 7: After culturing the cells cultured on the chip in Step 6 at 37°C and 5% CO2 for 1 day, fix the cell culture chips and stain the cell nuclei with fluorescence. Observe the cell adhesion behavior on different structural matrices using laser confocal microscopy or an inverted fluorescence microscope (e.g., ...). Figure 5 ) and ALP staining osteogenic differentiation analysis (e.g. Figure 6 ImageJ statistical analysis was used to process the cell behavior observation data from high-throughput cell microarrays using normalization formulas, and heatmaps were generated to enable structural behavior analysis and screening (e.g., ...). Figure 7 ).
[0053] Example 2: A high-throughput titanium alloy cell culture chip with a porosity gradient and its preparation method
[0054] Step 1: In this embodiment, the 3D software Rhino Grasshopper is used to design a model of 16 structural arrays with cylindrical holes, diamond structures, spherical holes, and overlapping cylindrical units, with a unit size of 1mm and four porosity gradients of 80%, 70%, 60%, and 50%, and a cuboid substrate (22mm long, 22mm wide, and 1.5mm high) containing isolation grooves (0.7mm).
[0055] Step 2: Import the model and use laser melting 3D printing to directly print titanium alloy powder to prepare a high-throughput structured cell culture chip as designed in Step 1, with a printing accuracy of 100 micrometers. After that, the formed chip is subjected to high-temperature annealing, followed by sanding and ultrasonic cleaning at 20Hz for 1 hour.
[0056] Step 3: Place the high-throughput cell culture chip, which has been ultrasonically cleaned in Step 2, into a 121°C high-temperature sterilization chamber.
[0057] Step 4: Add the sterile high-throughput structural cell culture chip from Step 3 into a 12-well plate, add polydimethylsiloxane (PDMS) to cover the chip substrate, incubate overnight under UV light until the PDMS solidifies, add 70% alcohol for UV sterilization for 6 hours, then soak in culture medium containing 10% fetal bovine serum for 4 hours. This chip is now ready for cell culture.
[0058] Note:
[0059] 1. Curvature is a fundamental geometric parameter in mathematical geometry that describes local information about a shape. In two dimensions, curvature is the reciprocal of the radius of the tangent circle at any point on a curve, used to describe the degree to which a line deviates from a straight line at any point. Points on a surface will have different curvature values in different directions. The maximum curvature κ1 and the minimum curvature κ2 are called principal curvatures and are used to calculate the mean curvature H and the Gaussian curvature K. The calculation formulas are as follows:
[0060] K = κ1 × κ2(1)
[0061] H=(κ1+k2) / 2(2)
[0062] 2. Normalization: The levels of each group in different tests are normalized using a normalization formula (test values are processed to 0-1) and applied to structural screening and comparative analysis.
[0063] Zi=(Xi-Min(X)) / (Max(X)-Min(X))
[0064] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A 3D-printed cell culture chip for high-throughput structure screening, characterized in that, The chip consists of, from top to bottom, a cell culture block, an anti-adhesion strip, a cell isolation groove, and a substrate; The cell culture block is composed of a structural matrix arrangement of unit structures with gradient curvature and parallel groups thereof; The number of structures and the size of the matrix contained in the cell culture block can be adjusted according to the requirements. The length, width and height of the gradient-changing unit structure are 100μm-2000μm. The chip is integrally formed by 3D printing from at least one of ceramic powder, metal powder, or polymer compound monomer; the anti-adhesion tape is made of polydimethylsiloxane or agarose film. The gradient curvature structures are: three hyperbolic structures, three cylindrical structures, and three ellipsoidal structures. Four parallel samples of these structures are arranged into 35 high-throughput matrices. The Gaussian curvatures of the three hyperbolic structures are -20, -12.5, and -5 mm. -2 The cylindrical structure has a Gaussian curvature of 0 and a diameter of 500 μm; the three ellipsoidal structures have Gaussian curvatures of +5, +12.5, and +20 mm, respectively. -2 ; The method for fabricating the 3D-printed cell culture chip for high-throughput structure screening includes the following steps: (1) Design a structure with gradient curvature using 3D modeling software, arrange the above structure and its parallel group into a high-throughput matrix, and design a model of the above matrix with a substrate containing cell isolation grooves. (2) Import the model, and the chip raw material powder is 3D printed to prepare the 3D shaped chip. After post-processing and sterilization, sterile agarose or polydimethylsiloxane is added to the isolation tank. After solidification, an anti-adhesion band is formed to obtain the 3D printed cell culture chip.
2. The 3D-printed cell culture chip for high-throughput structure screening according to claim 1, characterized in that, The ceramic powder is at least one of hydroxyapatite, tricalcium phosphate, and wollastonite; the metal powder is at least one of titanium alloy, tantalum metal, and stainless steel. The polymer monomer is a photocurable polymer monomer.
3. The 3D-printed cell culture chip for high-throughput structure screening according to claim 1, characterized in that, The 3D printing is formed using digital light processing technology or laser melting 3D printing.
4. The 3D-printed cell culture chip for high-throughput structure screening according to claim 1, characterized in that, The cell isolation groove contains an anti-adhesion tape with a depth of 0.5 mm-1.5 mm and a width of 0.5 mm-1 mm; the substrate is a cuboid substrate with a thickness of at least 1 mm-2 mm.
5. A method for preparing a 3D-printed cell culture chip for high-throughput structure screening as described in any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Design a structure with gradient curvature using 3D modeling software, arrange the above structure and its parallel group into a high-throughput matrix, and design a model of the above matrix with a substrate containing cell isolation grooves. (2) Import the model, and the chip raw material powder is 3D printed to prepare the 3D shaped chip. After post-processing and sterilization, sterile agarose or polydimethylsiloxane is added to the isolation tank. After solidification, an anti-adhesion band is formed to obtain the 3D printed cell culture chip.
6. The method for preparing a 3D-printed cell culture chip for high-throughput structure screening according to claim 5, characterized in that, The post-treatment is at least one of sintering, sandblasting, and ultrasonic cleaning. The sterilization process involves high temperature and high pressure, ultraviolet irradiation, or radiation.
7. The method for preparing a 3D-printed cell culture chip for high-throughput structure screening according to claim 5, characterized in that, The precision of the 3D printing in step (2) is at least 100 micrometers.
8. The application of the 3D-printed cell culture chip for high-throughput structure screening as described in any one of claims 1 to 4 in the in vitro analysis and screening of porous material structures for cell culture and tissue repair.