Triple-hole paper-based cell three-dimensional culture chip as well as preparation method and application thereof

Abstract

The invention relates to a triple-hole paper-based three-dimensional cell culture chip as well as a preparation method and application thereof. The chip comprises a paraffin hydrophobic region and a paper-based hydrophilic region. The preparation method mainly comprises the following steps: 1) designing a mask plate; 2) cutting the mask plate by laser; and 3) preparing the triple-hole paper-based chip by hot pressing. The thickness of the chip is 100-200 [mu] m, the porous fiber structure of the paper-based material allows transportation of oxygen and waste, and a three-dimensional microenvironment for in-vivo cell growth can be simulated. Meanwhile, a transparent and liquid-tight supporting layer is fused on the lower layer of the paper-based chip, so that leakage of cells inoculated on the upper layer of the paper-based chip is avoided. The paper-based chip is embedded into a 6-pore plate, and bladder cancer cells (5637), mouse astrocytoma (U87) and human breast cancer cells (MCF-7) are inoculated in a paper-based hydrophilic region respectively. The cell compatibility of the paper-based chip and the growth behaviors of three different types of cells in three-dimensional paper fibers are investigated. It is proved that the chip can be used for researching three-dimensional construction and growth of human and mammalian cells, and the difference between three-dimensional culture and two-dimensional culture is revealed by detecting the proliferation capacity and survival rate of different cells.

Description

Technical Field

[0001] The invention belongs to the field of cell culture and tissue engineering, and relates to a triple-hole paper-based three-dimensional cell culture chip, a preparation method and application thereof. Background Art

[0002] In the physiological and pathological studies of human and mammalian cells, researchers have found that the state of cells is closely related to the cell microenvironment. The cell growth microenvironment is mainly composed of cellular components (such as epithelial cells, fibroblasts, endothelial cells) and non-cellular components (such as extracellular matrix, cytokines, chemokines and growth factors). It is complex, diverse and easy to change. Although traditional two-dimensional (2D) cell culture technology has laid an important foundation for cell tissue engineering research, cells grow in a monolayer in a two-dimensional plane and cannot reconstruct the spatial heterogeneity of tissues and complex cell-to-cell interactions. Three-dimensional (3D) cell culture models are considered to be models that are closer to the in vivo niche. 3D culture can better simulate the structure and function of the cell microenvironment, including close contact between cells, three-dimensional distribution of extracellular matrix, and gradient changes in nutrients and oxygen. It is difficult to reproduce the complex relationship between cells and their microenvironment in a laboratory environment because it requires complex culture conditions to promote the growth and interaction of multiple cell types in a multifaceted environment. Therefore, it is necessary to develop simple and rapid 3D cell culture models in vitro, which can not only provide a platform that reflects the complexity of in vivo tissue structure and function, but also provide more accurate disease models for disease research and drug development.

[0003] To date, common 3D cell culture methods include scaffolding, embedding, hanging drop, 3D printing, and low-adsorption spheroidization. These methods suffer from drawbacks such as high cost, complex operation, and difficulty controlling cell distribution, and are unable to fully reproduce cell-cell and cell-extracellular matrix interactions. Summary of the Invention

[0004] To address the above technical issues, the present invention provides a method for preparing a triple-well paper-based chip and its application. This method, with its simple steps and precise control of cell distribution, provides a 3D cell culture platform, potentially serving as an alternative to difficult-to-obtain organ-on-a-chip models.

[0005] The technical solutions of the present invention are as follows:

[0006] A three-dimensional cell culture chip with three-holes and a paper base comprises a paraffin hydrophobic region and a paper-based hydrophilic region. The paraffin hydrophobic region comprises a paraffin sealing film with a thickness of 100 μm; the paraffin sealing film is readily available and can quickly infiltrate the paper-based material after heat pressing. The hydrophilic region comprises lens paper with a thickness of 38-48 μm. To prevent cells from leaking through the gaps in the paper base, a layer of PE plastic wrap is fused to the bottom of the paper-based material as a support layer. Paraffin sealing film and PE plastic wrap are fused to both sides of the paper-based hydrophilic region, respectively, to construct a paper-based chip with a thickness of 100-200 μm.

[0007] The lens cleaning paper has the characteristics of low cytotoxicity, good mechanical properties and physical permeability, and its fiber structure can simulate the natural cell microenvironment, providing good cell attachment points and growth conditions. The lens cleaning paper is Whatman 105 lens cleaning paper.

[0008] The preparation method of the above-mentioned triple-well paper-based three-dimensional cell culture chip comprises the following steps:

[0009] Step 1: Use AutoCAD software to draw the mask template dimensions required for paraffin hot press printing, import it into the laser cutting computer program, place the PET plate on the cutting area and position it, and set the cutting process parameters to 100μm. After laser cutting, a triple hole mask template is formed;

[0010] Step 2: Place the prepared mask on a workbench, place a piece of paraffin sealing film on top, and layer lens cleaning paper and PE plastic wrap on the bottom, stacking them to form a chip prototype. Next, use two PET sheets as lamination supports to press the stacked materials together. Finally, wrap it in aluminum foil and heat it on a high-temperature plywood at 140-200°C for 5-10 minutes to complete the hot pressing process, forming a paper-based chip with both hydrophilic and hydrophobic regions.

[0011] Step 3: Place the prepared paper-based chip into a 6-well cell culture plate and sterilize it in a clean bench under ultraviolet light (wavelength 254 nm) for 1 hour on both sides. Seal the sterilized paper-based material.

[0012] Further characterization, in step 2, the PE plastic wrap is removed and hot pressing is performed. The paper-based material is placed on the test table of a contact angle measuring instrument, and the water output during contact angle measurement is set to 4-6 μL. The contact angles on both sides of the hot-pressed paper-based material are measured to characterize the hydrophilicity and hydrophobicity of the chip surface.

[0013] The aforementioned three-well paper-based 3D cell culture chip was used to seed three different cell types, respectively, onto the hydrophilic area of ​​the paper chip and into a 96-well cell culture plate, to compare the differences between 3D and 2D culture. The three cell types described were cystoma cells (5637), mouse astrocytoma (U87), and human breast cancer cells (MCF-7).

[0014] Further features, the three different types of cell seeding density are all 2.0×10 5 cells / mL, cultured at 37°C in a 5% CO2 environment, in DMEM medium for 24 h and 48 h, respectively, and then MTS assay and live-dead cell staining assay were performed.

[0015] This invention supports three-dimensional co-culture of different cell types and has broad application prospects in pathology research, drug screening, and personalized medicine. Regarding the preparation and material selection of the paper-based chip, the materials used in this invention are non-toxic, odorless, low-cost, and easy to obtain and process. The preparation process is simple and rapid, while also offering significant environmental advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG1 is a structural diagram of a triple-hole paper-based chip;

[0017] Figure 1a It is the size diagram of the mask;

[0018] Figure 1b It is a physical picture of the mask;

[0019] Figure 1c It is a working diagram of hot pressing method;

[0020] Figure 1d This is a physical picture of a triple-well paper-based cell chip.

[0021] Figure 2 is a graph showing the contact angle of a paper-based chip.

[0022] Figure 2a This is the contact angle measurement diagram of the upper side of the hydrophobic area of ​​the paper-based chip;

[0023] Figure 2b This is the contact angle measurement diagram of the lower side of the hydrophobic area of ​​the paper-based chip;

[0024] Figure 2c This is the contact angle measurement diagram of paraffin sealing film;

[0025] Figure 2d It is a statistical graph of contact angle measurement;

[0026] Figure 3 is a morphological diagram of two-dimensional cell culture. Scale bar: 50 μm.

[0027] Figure 3a This is a morphological image of bladder cancer cells (5637) cultured in two dimensions. Scale bar: 50 μm.

[0028] Figure 3b This is a morphological image of a two-dimensional culture of mouse astrocytoma (U87). Scale bar: 50 μm.

[0029] Figure 3c This is a morphological image of human breast cancer cells (MCF-7) cultured in two dimensions. Scale bar: 50 μm.

[0030] Figure 4 The morphology of bladder cancer cells (5637) in three-dimensional culture at different times is shown. Scale bar: 50 μm.

[0031] Figure 5 Figure 3 shows the morphology of mouse astrocytoma (U87) in three-dimensional culture at different times. Scale bar: 50 μm.

[0032] Figure 6 The morphology of human breast cancer cells (MCF-7) cultured in three dimensions at different times is shown. Scale bar: 50 μm.

[0033] Figure 7 This is a graph of different cell proliferation folds at 48 h;

[0034] Figure 8 This is a live-dead staining effect of bladder cancer cells (5637) at 48 hours;

[0035] Figure 9 This is a live-dead staining effect of 48h mouse astrocytoma (U87);

[0036] Figure 10 This is a live-dead staining effect diagram of human breast cancer cells (MCF-7);

[0037] Figure 11 This is a comparison chart of different cell survival rates at 48 hours. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below with reference to the accompanying drawings.

[0039] Example 1: A method for preparing a triple-hole paper-based chip

[0040] Step 1: Use AutoCAD software to create a mask template with dimensions of 30 mm in length, 10 mm in width, and 1 mm in height. This is then imported into a laser cutting computer program. The PET sheet is protected from heat generated by the laser cutting process and deformed. The PET sheet is then positioned in the cutting area, with the cutting parameters set to 100 μm. This creates a triple-hole mask template.

[0041] Step 2: Place the prepared mask on a workbench, place a piece of paraffin sealing film on top, and layer Whatman 105 lens cleaning paper and a layer of PE plastic wrap on the bottom, stacking them to form a chip prototype. Next, use two PET sheets as lamination supports to press the stacked materials together. Finally, wrap it in aluminum foil and heat it on a high-temperature plywood at 140-200°C for 5-10 minutes, forming a paper-based chip with both hydrophilic and hydrophobic regions.

[0042] Step 3: Place the prepared paper-based chip into a 6-well cell culture plate and sterilize it under ultraviolet light (wavelength 254 nm) in a clean bench for 1 h on both the front and back sides.

[0043] Step 4: Remove the PE cling film and perform hot pressing. After cooling to room temperature, place it on the test bench of the contact angle meter, set the water output when measuring the contact angle to 4-6μL, turn on and adjust the shooting light. Measure the contact angles on both sides of the paper-based material after hot pressing to characterize the hydrophilicity and hydrophobicity of the paper-based material surface. The specific operation is: select five different areas on each surface (including top, middle, bottom, left, and right), measure the contact angles separately, and calculate the arithmetic average of these measured values ​​as the final contact angle data for the surface.

[0044] The structure of the triple-hole paper-based chip of this embodiment is shown in Figure 1. The contact angle measured by the paper-based chip of this embodiment is shown in Figure 2.

[0045] Example 2: A triple-well paper-based chip for three-dimensional culture of different cell types

[0046] Use trypsin to digest the three cells from the T75 culture flask; add 4mL DMEM complete medium to the centrifuge tube, and the centrifugation conditions are: 23℃, 1000rpm / min, 5min; separate the supernatant after centrifugation and add 1mL culture medium. Then, take 10μL of cell suspension and mix it with 10μL trypan blue to count the cells. After the cell counting is completed, place the above paper-based chip in a 6-well cell culture plate, making sure that the hydrophilic area of ​​the paper base faces up and the side covered with PE plastic wrap fits tightly with the bottom of the six-well plate. Add 10-20μL, 2×10 5 Similarly, add 10-20 μL, 2×10 cells / mL of the three different cell types, respectively. 5 Cells were suspended at 100 μL / mL in a 96-well plate. 100 μL of culture medium was added to each well. Three experimental groups and three blank control groups were set up for each cell type. Incubate at 37°C, 5% CO₂ for 24 and 48 hours.

[0047] The morphology of the two-dimensional cell culture of this embodiment is shown in Figure 3. Figure 4-6shown.

[0048] Example 3: A method for detecting proliferation of three cells cultured on a paper-based chip

[0049] Aspirate the culture medium from the paper-based material and the 96-well plate respectively, cut the paper-based material inoculated with cells into three parts and place them in a 24-well plate, add 150 μL / well of MTS detection solution, and then incubate it at 37°C, 5% CO2 environment for 40 minutes; use a pipette to transfer 150 μL of detection solution in each well to the 96-well plate, use a microplate reader to read the OD value at 490 nm, and use Origin software to draw the cell growth curve, as shown in Figure 2. Figure 7 shown.

[0050] Example 4: A method for live-dead cell staining in a paper-based chip

[0051] Prepare AM / PI staining working solution by adding 1 μL of 16 mM PI stock solution to 2 mL of DPBS and vortexing to mix thoroughly. Then, add 1 μL of 4 mM calcein AM stock solution to 2 mL of propidium iodide PI solution and vortex to mix thoroughly. Aspirate the culture medium from the paper substrate and 96-well plate, wash three times with DPBS, and then add AM / PI staining working solution. Incubate at 37°C for 30 minutes. Aspirate the staining working solution to terminate the incubation and photograph using an inverted fluorescence microscope. Figure 8-10 .

[0052] The survival rates of three different types of cells in this example are as follows Figure 11 shown.

Claims

1. A three-dimensional cell culture chip with three-hole paper base, characterized in that: The chip comprises a paraffin hydrophobic region and a paper-based hydrophilic region. The paraffin hydrophobic region is a paraffin sealing film with a thickness of 100 μm. The hydrophilic region is a lens cleaning paper with a thickness of 38-48 μm. A layer of PE plastic wrap is fused to the bottom of the paper-based material as a support layer. Paraffin sealing film and PE plastic wrap are fused to both sides of the paper-based hydrophilic region to construct a paper-based chip with a thickness of 100-200 μm.

2. The three-well paper-based cell three-dimensional culture chip according to claim 1, characterized in that: The lens cleaning paper is Whatman 105 lens cleaning paper.

3. A method for preparing the triple-well paper-based three-dimensional cell culture chip according to claim 1 or 2, characterized in that: Here are the steps: Step 1: Use AutoCAD software to draw the mask template size required for paraffin hot press printing, import it into the laser cutting computer program, place the PET plate on the cutting area for positioning, and set the cutting process parameters to 100μm; after laser cutting, a triple hole mask template is formed; Step 2: Place the prepared mask on a workbench, place a piece of paraffin sealing film on top, and place a layer of lens cleaning paper and a layer of PE plastic wrap on the bottom, stacking them to form a chip prototype. Then, use two PET plates as lamination supports to press the stacked materials together. Finally, wrap it with aluminum foil and heat it on a high-temperature plywood at 140-200°C for 5-10 minutes to complete the hot pressing process, forming a paper-based chip with both hydrophilic and hydrophobic regions. Step 3: Place the prepared paper-based chip into a 6-well cell culture plate and sterilize it in a clean bench under ultraviolet light (wavelength 254 nm) for 1 hour on both sides. Seal the sterilized paper-based material.

4. The preparation method according to claim 3, characterized in that In step 2, the PE plastic wrap is removed and hot pressed; the paper-based material is placed on the contact angle measuring instrument test table, the water output when measuring the contact angle is set to 4-6 μL, and the contact angles on both sides of the hot-pressed paper-based material are measured to characterize the hydrophilicity and hydrophobicity of the chip surface.

5. An application of the triple-well paper-based three-dimensional cell culture chip according to claim 1 or 2, characterized in that: Three different cell types were seeded in the hydrophilic area of ​​a paper-based chip and in a 96-well cell culture plate, respectively, to compare the differences between three-dimensional and two-dimensional cultures; the three different cell types referred to were cystoma cells (5637), mouse astrocytoma (U87), and human breast cancer cells (MCF-7).

6. The use according to claim 5, characterized in that The three different cell types were seeded at a density of 2.0×10 5 cells / mL, cultured at 37°C in a 5% CO2 environment, in DMEM medium for 24 h and 48 h, respectively, and then MTS assay and live-dead cell staining assay were performed.

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