Multilevel cell condition culture trapezoidal chip for high-throughput structure screening

Abstract

The utility model relates to a multi-level cell condition culture trapezoidal chip for high-throughput structure screening. The multi-level cell condition culture trapezoidal chip comprises a chip body, the chip body is provided with a plurality of layered condition culture system units, and each layered condition culture system unit is provided with at least two condition culture layers from top to bottom; each conditional culture layer is provided with a plurality of cell culture chambers; each cell culture chamber is provided with a high-throughput structure matrix, the high-throughput structure matrix comprises a plurality of porous materials which are arranged in a matrix manner, and at least one of the curvature, the strut diameter, the pore diameter and the communicating pore diameter of the plurality of porous materials is in gradient change; the upper and lower adjacent cell culture chambers are communicated with each other; a cell culture fluid collecting and analyzing chamber is arranged below the chip body and is communicated with the cell culture chamber. The condition culture hierarchical ladder structure can realize observation of various cell behaviors at different levels, and is applied to analysis and rapid screening of interaction between cells by a tissue repair porous material structure.

Description

Technical Field

[0001] The utility model relates to the technical field of porous materials for tissue repair, in particular to a multi-level cell conditional culture ladder-shaped chip for high-throughput structure screening. Background Art

[0002] The structural design of porous biomaterials has an important influence on biological properties and tissue repair effects. The structural design of tissue repair materials can control nutrient transport, and is also crucial to cell behaviors such as cell adhesion, proliferation and differentiation, which in turn affects the morphological development and functional realization of tissues. In addition, the structure directly affects cell behavior (such as cell paracrine) and will indirectly affect the fate of other cells, thereby affecting the effect of tissue repair (for example, inorganic ions such as calcium and phosphorus released during the degradation of bioceramics have been shown to promote osteoblast differentiation, while inhibiting osteoclast differentiation and bone resorption function, playing a key role in the regeneration and repair of bone tissue). Tissue regeneration is the result of multicellular behavior and interaction. Studying the effect of structure on cell-to-cell interactions is of great significance for the development of porous materials for tissue repair with good performance.

[0003] At present, the study of the effects of different structures on cell interactions is mainly achieved by collecting the culture medium after incubating cells with materials from different experimental groups, and then re-incubating another type of cell with the collected conditioned medium. Such a method is cumbersome, and the time lag in the collection process affects the activity of biological factors in the conditioned medium, and is prone to bacterial contamination. In terms of conditioned cultured cells, some scholars have proposed methods and equipment for filtering and purifying conditioned culture fluids (such as patent CN202222066269.2, a conditioned culture fluid filtration device for dental pulp stem cells and patent CN202110013769.7, a method for separating and purifying microvesicles from cell culture supernatant and biological fluids). The above patents are only methods for filtering conditioned culture media, and co-culture of multiple cells cannot be achieved. In addition, although there are cell culture devices and chips that can achieve cell co-culture (such as CN202311645597.0, a real-time observation system for cell co-culture, and CN202111150248.2, a microfluidic chip device for cell co-culture and a cell co-culture method, etc.), these methods require not only a power device for a flow pump, etc. and a complex preparation process, but also have no structural screening function, and the device materials are biologically inert materials.

[0004] In summary, the existing conditioned culture cell devices have complex preparation processes and usage methods, and the biocompatibility of raw materials is limited, making them unsuitable as conditioned culture research and structural screening tools for the effects of tissue repair material structure on the interaction of different cells. Utility Model Content

[0005] In view of the problems existing in the prior art, the purpose of the utility model is to provide a multi-level cell conditional culture ladder chip for high-throughput structural screening, which can realize multi-level and multi-cell conditional culture and is suitable as a conditional culture research and structural screening tool for the effect of tissue repair material structure on the interaction of different cells.

[0006] In order to achieve the above purpose, the utility model adopts the following technical solutions:

[0007] A multi-level cell conditional culture ladder chip for high-throughput structural screening, comprising a chip body;

[0008] A plurality of layered conditional culture system units are arranged horizontally at intervals on the chip body, and each layered conditional culture system unit is provided with at least two conditional culture levels from top to bottom;

[0009] Each conditioned culture level has multiple cell culture chambers;

[0010] Each cell culture chamber is provided with a high-throughput structural matrix, which includes a plurality of porous materials arranged in a matrix, wherein at least one of the curvature, pillar diameter, pore size, and connected pore size of the plurality of porous materials changes in a gradient;

[0011] The upper and lower adjacent cell culture chambers are interconnected;

[0012] A cell culture fluid collection and analysis chamber is arranged below the chip body, and the cell culture fluid collection and analysis chamber is connected to the cell culture chamber.

[0013] Furthermore, a liquid inlet and a first-layer distribution pipe are provided on the top of the chip body. The first-layer distribution pipe extends horizontally, and the liquid inlet is respectively connected to a plurality of layered conditional culture system units through the first-layer distribution pipe.

[0014] Furthermore, each layered conditional culture system unit includes a first conditional culture layer and a second conditional culture layer, the first conditional culture layer is provided with a plurality of first-layer cell culture chambers, the first-layer distribution pipes are respectively connected to the plurality of first-layer cell culture chambers, the second conditional culture layer is provided with a plurality of second-layer cell culture chambers, a plurality of second-layer liquid inlet pipes are provided between the plurality of first-layer cell culture chambers and the plurality of second-layer cell culture chambers, and the upper and lower ends of the plurality of second-layer liquid inlet pipes are respectively connected to the plurality of first-layer cell culture chambers and the plurality of second-layer cell culture chambers in a one-to-one correspondence.

[0015] Furthermore, multiple cell culture fluid collection and analysis chambers and multiple third-layer liquid inlet tubes are provided below the second conditional culture level, and the upper and lower ends of the multiple third-layer liquid inlet tubes are respectively connected to the multiple second-layer cell culture chambers and the multiple cell culture fluid collection and analysis chambers.

[0016] Furthermore, a first tooth-shaped separation opening for easy separation is provided at the bottom of the chip body between the liquid inlet and the first condition culture layer.

[0017] Furthermore, a second tooth-shaped separation opening for easy separation is provided at the bottom of the chip body between the first conditioned culture layer and the second conditioned culture layer.

[0018] Furthermore, a third tooth-shaped separation port for easy separation is provided at the bottom of the chip body between the second conditional culture layer and the cell culture fluid collection and analysis chamber.

[0019] Furthermore, the chip body is integrally formed by 3D printing.

[0020] Furthermore, the 3D printing integrated molding is digital light processing technology molding or laser melting 3D printing molding.

[0021] In general, the utility model has the following advantages:

[0022] (1) The use of 3D printing integrated molding can obtain high-precision, high-quality cell conditional culture chips.

[0023] (2) Existing cell culture chips require an external power system to achieve the flow of culture fluid. The utility model constructs a stepped structure and does not require an additional external power system. After the culture fluid is replenished, the liquid level in the cell culture chamber is made to exceed the groove, so that the conditioned culture fluid can flow to the culture chamber of another type of cell in the next layer under the action of gravity.

[0024] (3) The cell culture chip of the utility model has a stepped structure of conditional culture levels, which can realize the observation of various cell behaviors at different levels and is applied to the analysis and rapid screening of cell-cell interactions in porous material structures for tissue repair.

[0025] (4) The cell culture chip of the present invention is designed with multiple tooth-shaped separation ports, which facilitates the disassembly of the chip and the observation of the adhesion, differentiation, protein expression and other behaviors of different cells at each level. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a three-dimensional structural schematic diagram of the utility model.

[0027] Figure 2 It is a schematic diagram of the planar structure of the utility model.

[0028] Figure 3 It is a schematic diagram of the implementation process of the utility model.

[0029] In the figure:

[0030] 1-chip body; 2-first tooth-shaped separation port; 3-second tooth-shaped separation port; 4-third tooth-shaped separation port; 5-liquid inlet; 6-first layer distribution pipe; 7-layered condition culture system unit; 8-first layer cell culture chamber; 9-second layer liquid inlet pipe; 10-second layer cell culture chamber; 11-third layer liquid inlet pipe; 12-cell culture fluid collection and analysis chamber. DETAILED DESCRIPTION

[0031] The utility model will be further described in detail below.

[0032] Example 1

[0033] like Figure 1 , Figure 2 As shown, a multi-level cell conditional culture ladder chip for high-throughput structural screening includes a chip body 1 of a ladder structure;

[0034] The chip body 1 is preferably integrally formed from at least one of ceramic powder, metal powder and polymer compound monomer by 3D printing.

[0035] 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; and the polymer compound monomer is a photocurable polymer compound monomer.

[0036] The 3D printing is digital light processing technology molding or laser melting 3D printing molding.

[0037] The top of the chip body 1 is provided with a liquid inlet 5 for replenishing the cell (or drug-containing) culture fluid and a first layer distribution pipe 6, which extends horizontally and has a microchannel therein. The depth of the liquid inlet and the microchannel is 0.2mm-1mm, and the microchannel extends downward along a trapezoid.

[0038] A plurality of layered conditional culture system units 7 are arranged horizontally at intervals on the chip body 1, each layered conditional culture system unit 7 is provided with at least two conditional culture levels from top to bottom, and each conditional culture level is provided with a plurality of cell culture chambers. The specific number and size can be determined according to the requirements.

[0039] In this embodiment, each layered conditional culture system unit 7 includes two conditional culture levels, namely a first conditional culture level and a second conditional culture level. The first conditional culture level is provided with 8 first-layer cell culture chambers 8, and the liquid inlet 5 is respectively connected to the 8 first-layer cell culture chambers 8 through the first-layer distribution pipe 6. The second conditional culture level is provided with 8 second-layer cell culture chambers 10. 8 second-layer liquid inlet pipes 9 are provided between the 8 first-layer cell culture chambers 8 and the 8 second-layer cell culture chambers 10. The upper and lower ends of the 8 second-layer liquid inlet pipes 9 are respectively connected to the 8 first-layer cell culture chambers 8 and the 8 second-layer cell culture chambers 10, and the upper ends of the second-layer liquid inlet pipes 9 are higher than the bottoms of the first-layer cell culture chambers 8.

[0040] A plurality of cell culture fluid collection and analysis chambers 12 and a plurality of third-layer liquid inlet tubes 11 are provided below the second conditioned culture layer. The upper and lower ends of the plurality of third-layer liquid inlet tubes 11 are respectively connected to the plurality of second-layer cell culture chambers 10 and the plurality of cell culture fluid collection and analysis chambers 12 in a one-to-one correspondence. The upper end of the first-layer liquid inlet tube 11 is higher than the bottom of the second-layer cell culture chamber 10.

[0041] Each cell culture chamber is equipped with a high-throughput structural matrix, which includes multiple porous materials arranged in a matrix. At least one of the curvature, pillar diameter, pore size, and connecting pore size of the multiple porous materials changes in a gradient, thereby facilitating the conditional culture of multiple cells at multiple levels, so as to study the impact of gradient-changing structures on cell-to-cell interactions and achieve rapid structural screening.

[0042] Among them, several parameter definitions of porous materials include:

[0043] Curvature: The degree to which the pillars that make up the porous material deviate from a straight line or the degree of concavity and convexity of the curved surface;

[0044] Pillar diameter: The diameter of the pillars that make up the porous material;

[0045] Pore ​​size: the diameter of the pores in a porous material;

[0046] Connected pore size: In materials with a connectivity rate of <100%, the size of the pores that are connected to each other and to the outside world.

[0047] A first tooth-shaped separation port 2 for easy separation is provided at the bottom of the chip body 1 between the liquid inlet 5 and the first conditional culture layer. A second tooth-shaped separation port 3 for easy separation is provided at the bottom of the chip body 1 between the first conditional culture layer and the second conditional culture layer. A third tooth-shaped separation port 4 for easy separation is provided at the bottom of the chip body 1 between the second conditional culture layer and the cell culture fluid collection and analysis chamber 12. The cell culture chambers of each level are detachable, which is convenient for observation and detection of different cells.

[0048] The implementation process of this embodiment includes the following steps:

[0049] Step 1: According to Figure 1 and Figure 2 The structural diagram of Figure 3 , Materialise Magics was used to design and generate a high-throughput structural screening cell conditional culture trapezoidal chip body 1 model with gradient pillar diameter diamond structure. The chip body 1 has 8 layered conditional culture system units 7, each layered conditional culture system unit 7 has 2 conditional culture levels and 1 sample collection and analysis level, and the first layer of cell culture chamber 8 contains 8 high-throughput matrices arranged in 4 types of diamond structures (corresponding to D1, D2, D3, D4) with pillar diameters of 150μm, 250μm, 350μm and 450μm, respectively, and pore diameters of 225μm.

[0050] Step 2: Use digital light processing (DLP) 3D printing to directly print tricalcium phosphate β-TCP powder to prepare a tricalcium phosphate β-TCP high-throughput structure screening cell condition culture ladder chip designed in step 1, with a printing accuracy of 100 μm. The chip embryo is repeatedly ultrasonically cleaned in ultrapure water, dried at 50°C overnight, and the chip embryo is subjected to high-temperature treatment at 800-1150°C in a muffle furnace for 3 hours to obtain a high-throughput structure screening cell condition culture ladder chip.

[0051] Step 3: The osteoclast differentiation behavior of mouse mononuclear macrophage leukemia cells (Raw264.7) on the chip and the effect of osteoclast conditioned medium on the behavior of mouse bone marrow mesenchymal stem cells (mBMSCs) were studied to verify the structural screening performance of the high-throughput structural screening cell multi-level conditioned culture ladder chip. The high-throughput structural screening multi-level cell conditioned culture ladder chip obtained in step 2 was sterilized at 121°C for 30 minutes, placed in a well plate or culture dish, and soaked in PBS buffer for 1 hour to wet the chip.

[0052] Step 4: 50 μL of 4×10 5 / mL Raw264.7 cell suspension, 50μL 4×10 5 / mL mBMSCs cell suspension was cultured in a 37°C, 5% CO2 cell culture incubator for 1 hour to allow the cells to adhere to the chip structure matrix and the cell culture chamber.

[0053] Step 5: Add DMEM culture medium containing 10% fetal bovine serum and 50 ng / mL osteoclast differentiation factor (RANKL) and DMEM culture medium containing 10% fetal bovine serum but without RANKL to the first cell culture chamber 8 and the second cell culture chamber 10, respectively, and continue to culture in a 37°C, 5% CO2 cell culture incubator.

[0054] Step six: At a certain flow rate, DMEM culture medium containing 10% fetal bovine serum and 50 ng / mL osteoclast differentiation factor (RANKL) is input into the first cell culture chamber 8 from the liquid inlet 5. When the liquid level in the first cell culture chamber 8 exceeds the height of the second liquid inlet tube 9, the conditioned culture medium containing osteoclasts co-cultured with the diamond structure high-throughput matrix with different pillar diameters is input into the second cell culture chamber 10 through the second liquid inlet tube 9, thereby realizing the conditioned culture of mBMSCs in the second cell culture chamber 10.

[0055] Step 7: The secretion of coupling factors, such as platelet-derived growth factor BB (PDGF-BB), bone morphogenetic protein 6 (BMP6), sphingosine-1-phosphate (S1P), etc., is detected by enzyme-linked immunosorbent assay (ELISA) using the conditioned cell culture fluid collected in the cell culture fluid collection and analysis chamber 12 of the third layer. In addition, the cell culture chambers of each layer are separated along the separation port reserved at the bottom of the chip, so as to facilitate the observation of cells in each layer of the cell culture chamber separately. TRAP staining is performed on the cells in the first layer of the cell culture chamber 8 to observe the effect of tricalcium phosphate β-TCP with different pillar diameters in the chip structure matrix on the differentiation of Raw264.7 osteoclasts; ALP staining is used to observe the osteogenic differentiation of mBMSCs on the chip structure matrix in the second layer of the cell culture chamber 10, so as to study the effect of osteoclast conditioned medium on the osteogenic differentiation of mBMSCs. Finally, the structure screening function is realized according to the cell behavior detection and analysis data.

[0056] Example 2

[0057] Step 1: Use Materialise Magics to design and generate a high-throughput structural screening cell conditional culture trapezoidal chip body 1 model with gradient pore size diamond structure. The chip body 1 has 14 layered conditional culture system units 7, each layered conditional culture system unit 7 has 2 conditional culture levels and 1 sample collection and analysis level, and the first layer of cell culture chamber 8 contains 14 high-throughput matrices arranged in 7 diamond structures (corresponding to P1, P2, P3, P4, P5, P6, P7) with pore sizes of 220μm, 340μm, 460μm, 580μm, 700μm, 820μm, 940μm, and porosity of 78.5%.

[0058] Step 2: Use laser melting 3D printing to directly print titanium alloy powder to prepare the high-throughput structural cell culture chip designed in step 1, with a printing accuracy of 100 microns. After that, the molded chip is subjected to high-temperature annealing, frosting and 20Hz 1 hour ultrasonic cleaning to obtain a high-throughput structural screening cell conditional culture trapezoidal chip.

[0059] Step 3: Using mouse bone marrow mesenchymal stem cells (mBMSCs) and human umbilical vein endothelial cells (HUVECs) as research objects, the effects of different pore sizes on the levels of paracrine factors in mBMSCs angiogenesis and the further effects of these paracrine factors on HUVEC angiogenesis were studied to verify the structural screening performance of the high-throughput structural screening multi-level cell conditioned culture ladder chip. The high-throughput structural screening multi-level cell conditioned culture ladder chip obtained in step 2 was sterilized at 121°C for 30 minutes, placed in a well plate or culture dish, and soaked in PBS buffer for 1 hour to wet the chip.

[0060] Step 4: 50 μL of 5×10 4 / mL Raw264.7 cell suspension, 50μL 5×10 4 / mL HUVEC cell suspension was cultured in a 37°C, 5% CO2 cell culture incubator for 1 hour to allow the cells to adhere to the chip structure matrix and the cell culture chamber.

[0061] Step 5: Add DMEM culture medium containing 10% fetal bovine blood to the first layer cell culture chamber 8 and the second layer cell culture chamber 10 respectively, and continue to culture in a cell culture incubator at 37° C. and 5% CO 2 .

[0062] Step six: At a certain flow rate, DMEM culture medium containing 10% fetal bovine serum is input into the first-layer cell culture chamber 8 from the liquid inlet 5. When the liquid level in the first-layer cell culture chamber 8 exceeds the height of the second-layer liquid inlet tube 9, the conditioned medium containing mBMSCs co-cultured with the high-throughput matrix with diamond structure having different pore sizes is input into the second-layer cell culture chamber 10 through the second-layer liquid inlet tube 9 to realize the conditioned culture of HUVEC in the second-layer cell culture chamber 10.

[0063] Step 7: The conditioned cell culture fluid collected in the cell culture fluid collection and analysis chamber 12 of the third layer is used to perform enzyme-linked immunosorbent assay (ELISA) on the secretion of angiogenic factors, such as vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1). In addition, the cell culture chambers of each layer are separated along the separation port reserved at the bottom of the chip, so that the cells in each layer of the cell culture chamber can be observed separately. Calcein / PI staining is used for the mBMSCs in the first layer of the culture chamber, and the cell activity and adhesion behavior of the mBMSCs on the chip structure matrix are observed under an inverted fluorescence microscope; RT-qPCR experiments are used for HUVEC in the second layer of the culture chamber to detect the expression levels of angiogenesis-related genes, such as platelet-derived growth factor (PDGF) and transforming growth factor β (TGF-β), and finally the structure screening function is realized based on the detection and analysis data.

[0064] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A multi-level cell conditional culture ladder chip for high-throughput structural screening, characterized in that: including a chip body; A plurality of layered conditional culture system units are arranged horizontally at intervals on the chip body, and each layered conditional culture system unit is provided with at least two conditional culture levels from top to bottom; Each conditioned culture level has multiple cell culture chambers; Each cell culture chamber is provided with a high-throughput structural matrix, which includes a plurality of porous materials arranged in a matrix, wherein at least one of the curvature, pillar diameter, pore size, and connected pore size of the plurality of porous materials changes in a gradient; The upper and lower adjacent cell culture chambers are interconnected; A cell culture fluid collection and analysis chamber is arranged below the chip body, and the cell culture fluid collection and analysis chamber is connected to the cell culture chamber.

2. The multi-level cell conditional culture ladder chip for high-throughput structural screening according to claim 1, characterized in that: A liquid inlet and a first-layer distribution pipe are arranged on the top of the chip body. The first-layer distribution pipe extends horizontally, and the liquid inlet is respectively connected to a plurality of layered conditional culture system units through the first-layer distribution pipe.

3. The multi-level cell conditional culture ladder chip for high-throughput structural screening according to claim 2, characterized in that: Each layered conditioned culture system unit includes a first conditioned culture layer and a second conditioned culture layer, the first conditioned culture layer is provided with a plurality of first-layer cell culture chambers, the first-layer distribution pipes are respectively connected to the plurality of first-layer cell culture chambers, the second conditioned culture layer is provided with a plurality of second-layer cell culture chambers, a plurality of second-layer liquid inlet pipes are arranged between the plurality of first-layer cell culture chambers and the plurality of second-layer cell culture chambers, and the upper and lower ends of the plurality of second-layer liquid inlet pipes are respectively connected to the plurality of first-layer cell culture chambers and the plurality of second-layer cell culture chambers in a one-to-one correspondence.

4. The multi-level cell conditional culture ladder chip for high-throughput structural screening according to claim 3, characterized in that: A plurality of cell culture fluid collection and analysis chambers and a plurality of third-layer liquid inlet tubes are arranged below the second conditional culture layer. The upper and lower ends of the plurality of third-layer liquid inlet tubes are respectively connected to the plurality of second-layer cell culture chambers and the plurality of cell culture fluid collection and analysis chambers in a one-to-one correspondence.

5. The multi-level cell conditional culture ladder chip for high-throughput structural screening according to claim 3, characterized in that: A first tooth-shaped separation opening for easy separation is arranged between the liquid inlet and the first condition culture layer at the bottom of the chip body.

6. The multi-level cell conditional culture ladder chip for high-throughput structural screening according to claim 3, characterized in that: A second tooth-shaped separation opening for easy separation is provided between the first condition culture layer and the second condition culture layer at the bottom of the chip body.

7. The multi-level cell conditional culture ladder chip for high-throughput structural screening according to claim 3, characterized in that: A third tooth-shaped separation port for easy separation is arranged at the bottom of the chip body between the second conditional culture layer and the cell culture fluid collection and analysis chamber.

8. The multi-level cell conditional culture ladder chip for high-throughput structural screening according to claim 1, characterized in that: The chip body is integrally formed by 3D printing.

9. The multi-level cell conditional culture ladder chip for high-throughput structural screening according to claim 8, characterized in that: The 3D printing integrated molding is digital light processing technology molding or laser melting 3D printing molding.

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