A culture method of a tight junction structure-producing adherent cell

Abstract

This invention provides a method for culturing adherent cells that generate tight junction structures, comprising: seeding adherent cells resuspended in culture medium onto a bottom-supporting liquid in a culture container for culture to obtain a membrane-like cell sheet; wherein the density of the bottom-supporting liquid is greater than that of the culture medium and is immiscible with the culture medium. This invention is the first to apply a bottom-supporting liquid to the culture of adherent cells, which can promote the formation of tight junctions between adherent cells and the secretion of a large amount of extracellular matrix to form a high-density membrane-like cell sheet, effectively mimicking the in vivo tissue structure; at the same time, it eliminates the need for phosphate-buffered saline washing and trypsin digestion steps in traditional adherent cell culture methods, preserving the extracellular matrix of adherent cells during growth, better restoring the cell growth microenvironment, making it more suitable for life science and clinical medical research; moreover, this culture method is simple to operate, has low additional costs, and has significant commercial value.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering and biomanufacturing technology under biomedical engineering, and specifically relates to a method for culturing adherent cells that produce tight junction structures. Background Technology

[0002] The establishment of in vitro cell maintenance and growth technologies is a significant milestone in bioscience. In vitro cell culture refers to the process of obtaining cells that would normally grow in vivo, providing them with necessary physicochemical clues and growth factors to maintain their basic life processes outside the body. Cell culture is an important component of life science and clinical medical research. It can conveniently reproduce basic human life processes in vitro, helping us study the formation and function of human tissues or organs outside the body, and providing a convenient and alternative approach for studying pathophysiological processes, disease occurrence, and drug development.

[0003] To facilitate the convenient and high-throughput reproduction of cellular life processes, solid-matrix-based cell culture has become the most widely used and common cell culture method. Solid-matrix cell culture is simple, low-cost, and robust, and has not undergone major changes since its introduction in 1912.

[0004] However, traditional solid-matrix cell culture methods cannot recreate the in vivo cellular microenvironment, leading to changes in cell phenotype and genotype, affecting the reliability and reproducibility of life science and clinical medical research. Some advanced solid-matrix cell culture methods use hydrogels similar to the extracellular matrix as scaffolds for cell culture, providing a suitable three-dimensional microenvironment for cells. However, it is difficult to guarantee high cell densities comparable to human tissues when using three-dimensional solid-matrix cell culture. Furthermore, the experience-dependent and high-cost nature of three-dimensional solid-matrix cell culture methods also limits their widespread application. Using magnetic fields, acoustic fields, or mechanical forces to assemble monodisperse cells into cell microspheres can create high cell densities and tight junctions comparable to human tissues. However, externally mediated solid-matrix cell culture methods have not been widely adopted due to their operational complexity and high learning curve.

[0005] Therefore, it is necessary to provide an easy-to-use cell culture method that can promote the formation of tight junctions between adherent cells and the secretion of large amounts of extracellular matrix to achieve high cell density. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention provides a method for culturing adherent cells that generates tight junction structures. This method is simple to operate, has low additional costs, and can promote the formation of tight junctions between adherent cells and the secretion of a large amount of extracellular matrix to form high-density membrane-like cell sheets.

[0007] The method for culturing adherent cells with tightly connected structures provided by this invention overcomes the limitations of traditional cell culture methods, which cannot simultaneously achieve ease of operation, low additional cost, and effective reconstruction of the in vivo cellular microenvironment, and thus has great commercial value.

[0008] A method for culturing adherent cells that generate tight junction structures includes: seeding adherent cells resuspended in culture medium onto a bottom support liquid in a culture container, and completing the culture on the surface of the bottom support liquid;

[0009] The density of the bottom supporting liquid is greater than that of the culture medium, and it is not miscible with the culture medium.

[0010] The adherent cells described above are cells that have adhered and grown during cell culture. This culture method can be applied to the culture of normal adherent cells as well as to the culture of high-density membranous cell sheets. When this method is used to form membranous cell sheets, the resulting sheets can be used in 3D bioprinting, cell patching, and drug testing.

[0011] Preferably, the bottom support liquid includes, but is not limited to, one or more of the following: fluorinated oil, fluoroalkane compounds, siloxane compounds (such as silicone oil, uncured polydimethylsiloxane), and ester compounds (such as dimethyl carbonate, dimethyl sulfate).

[0012] As a further preferred option, the bottom support liquid is one or a mixture of 3M Novec HFE series fluorinated oil (such as HFE7500), 3M Fluorinert FC series fluorinated oil, TECCEM Fluoronox series fluorinated oil, silicone oil, uncured polydimethylsiloxane, dimethyl carbonate, and dimethyl sulfate.

[0013] Preferably, the amount of bottom-supporting liquid added to the culture container is greater than 0.08 mL / cm³. 2 As a further preferred embodiment, the amount of bottom support liquid added is 0.3–0.7 mL / cm³. 2 .

[0014] Preferably, the seeding concentration of the adherent cells is 2 × 10⁻⁶. 4 ~2×10 8 pcs / cm 2 Further preferred is 1×10. 6 pcs / cm 2 ~2×10 6 pcs / cm 2 .

[0015] Preferably, the culture temperature for adherent cells is 35–39°C, and the culture time is 1–28 days. More preferably, the culture temperature is 37°C, and the culture time is 1–14 days.

[0016] Preferably, adherent cells are cultured in a 5% carbon dioxide incubator after seeding.

[0017] Preferably, the adherent cells are cryopreserved cells after thawing or cells after passage and digestion.

[0018] Preferably, the adherent cells are one or more of stem cells, tumor cells, epithelial cells, endothelial cells, glial cells, pericytes, fibroblasts, nerve cells, smooth muscle cells, skeletal muscle cells, cardiomyocytes, hepatocytes, bile duct cells, stellate cells, bone-derived cells, immune-related cells, and other cells derived from various tissues or organs.

[0019] Preferably, the culture medium is changed every 10-15 hours during the culture period, and the volume of the replaced culture medium is 70-90% of the original culture medium volume. More preferably, the culture medium is changed every 12 hours during the culture period.

[0020] As a further preferred option, the new culture medium is preheated before being replaced.

[0021] Preferably, the bottom support liquid is added to the culture container after sterilization.

[0022] As a further preferred option, the bottom support liquid is sterilized using one or more of the following sterilization methods: chemical sterilization, radiation sterilization, dry heat sterilization, moist heat sterilization, and filtration sterilization. Even more preferred is ultraviolet light sterilization.

[0023] The culture container can be a culture plate, a culture dish, or any container suitable for general cell culture. Culture containers can also be made of custom materials, shapes, and structures. Preferably, the culture container is a commercially available multi-well culture plate.

[0024] As a preferred embodiment, a method for culturing adherent cells to generate tight junction structures includes the following steps.

[0025] Step 1: Sterilize the bottom support liquid and add it to the adherent cell culture container in advance;

[0026] Step 2: Resuspend the adherent cells in culture medium and seed them onto a bottom-supporting liquid;

[0027] Step 3: Incubate the inoculated adherent cells at 35-39°C for 1-28 days, changing the culture medium every 10-15 hours during the incubation period.

[0028] The method for culturing adherent cells that generates tight junction structures according to the present invention can be applied to life science and clinical medical research. It can achieve adherent cell culture and promote the formation of tight junctions between adherent cells and secrete a large amount of extracellular matrix to form membrane-like cell sheets.

[0029] The present invention provides a method for culturing adherent cells with tightly connected structures. For the first time, a bottom-supporting liquid is applied to cell culture, enabling more diverse cell culture modes and expanding the substrate types of existing cell culture methods.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] (1) The culture method for producing adherent cells with tight junction structures of the present invention is a supplement to the existing cell culture methods, expands the substrate types of the existing cell culture methods, is simple to operate, has low additional cost, and has great commercial value.

[0032] (2) The culture method of adherent cells that produces a tight junction structure in this invention can promote the formation of tight junctions between adherent cells and secrete a large amount of extracellular matrix to form a high-density membrane cell sheet, which well simulates the in vivo tissue structure, making it more suitable for life science and clinical medical research and has great application prospects.

[0033] (3) The method for culturing adherent cells with tight junctions of the present invention can eliminate the steps of phosphate buffer washing and trypsin digestion in traditional adherent cell culture methods, retain the extracellular matrix of adherent cells during growth, better restore the cell growth microenvironment, and is beneficial to life science and clinical medical research and application.

[0034] (4) The culture method of the present invention for producing adherent cells with tight junction structure can dynamically maintain the entire culture substrate in a horizontal state during the culture process, which helps the cells to be evenly distributed during the culture process. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the process of forming a membrane-like cell sheet in a 12-well culture plate according to an embodiment of the present invention; wherein, 1 is a 12-well culture plate, 2 is the bottom support liquid, 3 is the frozen cells after thawing, 4 is the culture medium, and 5 is the membrane-like cell sheet;

[0036] Figure 2 This is a schematic diagram comparing the method for culturing tightly connected adherent cells according to an embodiment of the present invention with a conventional method for culturing adherent cells; wherein, Figure 2 A is a schematic diagram of the culture method for adhering cells that produce tight junction structures; Figure 2 B is a schematic diagram of the traditional adherent cell culture method;

[0037] Figure 3 This is a photograph of cells forming a membrane-like cell sheet in a 12-well culture plate, as shown in an embodiment of the present invention; wherein, Figure 3 A is a side-view of the formation of a membrane-like cell sheet by cells in a 12-well culture plate according to an embodiment of the present invention. The membrane-like cell sheet produced by adherent cells that generate a tight junction structure is inside the dashed circle. Figure 3 B is a top view of the formation of a membrane-like cell sheet in a 12-well culture plate according to an embodiment of the present invention;

[0038] Figure 4 This is a partial electron micrograph of a membrane-like cell sheet cultured for 3 days in an embodiment of the present invention; wherein, Figure 4 A is a partial electron microscope image of a sheet of membranous cells magnified 400 times; Figure 4 B is a partial electron microscope image of a sheet of membranous cells magnified 2000 times; Figure 4 C is a partial electron microscope image of a sheet of membranous cells magnified 5000 times;

[0039] Figure 4 D is a partial electron microscope image of a sheet of membranous cells magnified 8000 times;

[0040] Figure 5 In the middle: A is a comparison of cell viability under different traditional adherent cell culture conditions under bottom-supported liquid conditions; B is the detection result of cell live / dead percentage in bottom-supported liquid in the embodiments of the present invention;

[0041] Figure 6 The images show actual cell sheets formed in cell culture containers of different materials using the culture method of this invention.

[0042] Figure 7 This is a photograph of a membrane-like cell sheet formed in a custom-shaped cell culture container using the culture method of this invention; wherein, Figure 7 A is a top view of forming a membrane-like cell sheet in a custom-shaped cell culture container using the culture method of an embodiment of the present invention; Figure 7 B is a side view of a cell culture container of a custom shape formed using the culture method of this invention. The area within the dashed square represents the bottom supporting liquid.

[0043] Figure 8 A photograph of a membrane-like cell sheet formed in a custom-structured cell culture container using the culture method of an embodiment of the present invention.

[0044] Figure 9 Micrographs showing the growth of different cells under the culture conditions described in this invention embodiment; wherein, Figure 9 A is a micrograph showing the growth of human foreskin fibroblasts (HFF) derived from the ectoderm; Figure 9B is a micrograph showing the growth of human umbilical vein endothelial cells (HUVECs) derived from the mesoderm; Figure 9 C is a micrograph showing the growth of endoderm-derived liver cancer cells (HepG2); Figure 9 D is a micrograph showing the growth of bone marrow mesenchymal stem cells (BMSCs) derived from stem cells. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0046] like Figure 1 As shown, a method for culturing adherent cells that generate tight junction structures includes the following steps:

[0047] 1. Place the bottom-supporting liquid HFE7500 into a transparent centrifuge tube and sterilize with ultraviolet light for 2 hours.

[0048] 2. Resuspend the revived frozen cells in culture medium and centrifuge at 1000 rpm for 3 min.

[0049] 3. After discarding the supernatant, resuspend the cells in 1 mL of culture medium, mix well, and then take a sample for cell counting.

[0050] 4. Take a brand new 12-well culture plate and fill each well with 0.5 mL / cm³ of water. 2 Add bottom support liquid HFE7500, with each hole having an area of ​​3.8 cm². 2 Therefore, each well requires 1.9 mL of bottom support liquid HFE7500.

[0051] 5. The resuspended cell suspension was prepared at a ratio of 1×10⁻⁶. 6 pcs / cm 2 The inoculum was evenly applied to the bottom-supporting liquid HFE7500, with each well having an area of ​​3.8 cm². 2 Therefore, each well needs to be inoculated with 3.8 × 10⁻⁶ seeds. 6 Each cell.

[0052] 6. Place the 12-well culture plate in a carbon dioxide incubator at 37°C and 5% carbon dioxide concentration, and let the cells naturally settle to the bottom support liquid HFE7500 surface for growth.

[0053] 7. During the culture period, the preheated cell culture medium should be replaced every 12 hours, and the volume of the replaced medium should be 80% of the original volume.

[0054] Product characteristics:

[0055] After the above cultivation, samples can be collected in the incubator as follows: Figure 3 The membrane-like cell sheet shown. Figure 4 This is a sheet of membranous cells collected after 3 days of culture, photographed under an electron microscope. Figure 4 As can be seen, the membranous cell sheets are formed by the tight connections between cells, creating a dense tissue structure.

[0056] Product functionality testing:

[0057] The difference in metabolic activity between membranous cell sheets collected after 1, 3, and 5 days of culture under the conditions described in this example (bottom-supported liquid) and conventional adherent cell cultures under the same conditions was determined using the CCK8 assay. The results are as follows: Figure 5 As shown in Figure A. Figure 5 The results in A show that the membrane-like cell sheets cultured in this embodiment have no significant difference in metabolic activity compared to cells cultured using traditional adherent cell culture.

[0058] The percentage of viable cells in membranous cell sheets collected after 1, 3, 5, 7, 14, and 21 days of culture under the conditions described in this example (bottom-supported liquid) was determined using a live / dead staining assay. The results are as follows: Figure 5 As shown in B. Figure 5 The results in B show that the membrane cell sheets cultured in this embodiment can maintain a viable cell percentage of over 90% after 1 to 21 days of culture.

[0059] Figure 6 The images show actual specimens of membranous cell sheets cultured in culture containers made of different materials (aluminum, copper, iron, wood, and polydimethylsiloxane) using the culture method of this embodiment. Figure 6 As can be seen, cells can be tightly connected to form dense membrane-like cell sheets in culture containers of different materials. This indicates that the adherent cell culture method for generating tightly connected structures provided in this embodiment can be applied to culture containers of various materials and can cultivate membrane-like cell sheets with high cell density. This solves the problem that traditional adherent cell culture can only be carried out in specially treated PS (polystyrene) materials.

[0060] Figure 7 This is a photograph of adherent cells cultured in a custom-shaped culture container using the culture method of this embodiment. Figure 7 It can be seen that cells can successfully form high-density membrane-like cell sheets in this custom-shaped culture container.

[0061] Figure 8 This image shows the state of adherent cells cultured in a custom-structured culture container using the culture method of this embodiment. Figure 8It can be seen that cells can successfully form membrane-like cell sheets in the culture container with this custom structure.

[0062] Figure 7 and 8 The culture results demonstrate that the culture method for producing adherent cells with tight junctions in this embodiment can be applied to cell culture containers with custom shapes and structures to meet the application requirements of membrane cell sheets with different shapes and structures.

[0063] Using the culture method of this embodiment, four typical adherent cell types (human foreskin fibroblasts derived from ectoderm, human umbilical vein endothelial cells derived from mesodermis, liver cancer cells derived from endoderm, and bone marrow mesenchymal stem cells derived from stem cells) were selected for cell culture. The culture results are as follows: Figure 9 As shown. By Figure 9 It can be seen that the four types of cells can form high-density membranous cell sheets after being cultured using the culture method of this embodiment, indicating that the culture method of this embodiment is applicable to the culture of a variety of different adherent cells and has strong applicability.

[0064] In summary, this embodiment uses fluorinated oil (HFE7500) as the bottom support liquid for the culture of adherent cells. It is simple to operate, has low additional cost, and can promote the formation of tight junctions between adherent cells and secrete a large amount of extracellular matrix to form a high-density membrane-like cell sheet, which well simulates the in vivo tissue structure. This makes it more suitable for life science and clinical medical research and has great application prospects and commercial value.

[0065] The above-described embodiments are merely preferred embodiments of the present invention. It should be noted that the above embodiments are exemplary and should not be construed as limiting the present invention. For those skilled in the art, several changes, modifications, substitutions, and variations can be made without departing from the principle of the present invention, and these improvements and refinements should also be considered within the scope of protection of the present invention.

Claims

1. A method for culturing adherent cells to generate tight junction structures, characterized in that, include: Adherent cells resuspended in culture medium were seeded onto the bottom support liquid in the culture container and cultured on the surface of the bottom support liquid. The density of the bottom supporting liquid is greater than that of the culture medium, and it is not miscible with the culture medium; The bottom support fluid is HFE7500; The amount of bottom support liquid added is 0.3~0.7 mL / cm³. 2 ; The seeding concentration of the adherent cells was 2 × 10⁻⁶. 4 ~2×10 8 pcs / cm 2 .

2. The method for culturing adherent cells to generate tight junction structures according to claim 1, characterized in that, Bottom support liquid was pre-added to the culture container.

3. The method for culturing adherent cells to generate tight junction structures according to claim 1, characterized in that, The culture temperature for adherent cells is 35~39℃, and the culture time is 1~28 days.

4. The method for culturing adherent cells to generate tight junction structures according to claim 1, characterized in that, Adherent cells are either cryopreserved cells after thawing or cells after passage and digestion.

5. The method for culturing adherent cells to generate tight junction structures according to claim 1, characterized in that, The culture medium should be changed every 10-15 hours during the culture period, and the volume of the replaced medium should be 70-90% of the original volume.

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