A device and method for cell patterning culture based on 3D printing technology

Abstract

The present invention belongs to the field of 3D printing technology and cell culture technology. Specifically, the present invention relates to a device and method for rapid preparation of cell patterned co-culture based on 3D printing. The present application provides a microdevice technology that can be rapidly prepared using 3D printing for cell culture and cell patterned co-culture, and the device can be easily compatible with existing cell biotechnology, such as microscopic imaging, cell immunostaining, etc. It can easily achieve patterning of multiple cells, and can perform microscopic imaging analysis and immunohistochemical analysis on cells to obtain quantitative results. It provides a device and method for scientific researchers to achieve co-culture of multiple cells more quickly and conveniently.

Description

Technical Field

[0001] The present invention belongs to the field of 3D printing technology and cell culture technology. Specifically, the present invention relates to a device and method for rapidly preparing patterned cell co-culture based on 3D printing. Background Art

[0002] The main purpose of cell patterning is to make cells grow in pre-set areas and form specific patterns. Micropatterning technology can control the spatial distribution of cells at the micron scale, so it is of great significance to the research of tissue engineering, biosensor technology, and basic biological problems. Micropatterning technology can directly regulate the size of cell colonies, the distance between colonies, and the interaction between homologous or heterologous cells, and the control of these spatial parameters can directly affect cell metabolism and cell function. For example, the size and space occupied by human umbilical vein endothelial cells will directly affect their differentiation tendency, while the interaction between macrophages, fibroblasts and endothelial cells will promote the angiogenesis tendency of endothelial cells. Therefore, cell micropatterning technology is obviously a powerful biomedical engineering tool for studying cell behavior and function.

[0003] Over the past decade, cell patterning techniques have been widely used to study cell-cell interactions, cell responses to their environment, polarized cell growth, cell differentiation, and other cellular functions. Generally speaking, cell patterning methods can be categorized into two main groups: one involves modifying the surface of culture media using various chemical agents, such as self-assembled monolayers and human fibrin, to either repel or promote cell attachment; the other involves physically anchoring cells to specific areas, typically using microfabricated channels, micropits, partitions, and hooks. Some reports have cleverly combined these two approaches to study cell-cell interactions.

[0004] However, current physical methods for cell patterning are generally complex, such as photolithography and soft lithography. Their preparation processes are complex and require specialized equipment, making them difficult to implement in general laboratories. With the gradual development and advancement of 3D printing technology, it is becoming possible to achieve multi-scale manufacturing. 3D printing, also known as additive manufacturing, is a type of rapid prototyping technology that allows the creation of three-dimensional objects of virtually any shape based on digital model files. 3D printing uses bondable materials such as powdered metal or plastic to construct objects layer by layer, a process known as "layer-by-layer modeling." 3D printing differs from traditional machining techniques, which typically rely on cutting or drilling (subtractive processes). Previously used for model creation in mold making and industrial design, 3D printing is now increasingly being used for the direct manufacture of some products. Parts printed using this technology are already being used in some high-value applications, demonstrating the widespread adoption of 3D printing. 3D printers currently on the market can now produce objects at the micron scale, with precision improving year by year. It is worth noting that the application of 3D printing technology in the field of biomedical engineering is still in the exploratory stage. Many studies focus on the repair of scaffolds and bone joints, while its application in cell biology, tissue engineering and other aspects is rare.

[0005] There are no reports on the invention of using 3D printing to manufacture devices with micron-level precision and apply them to cell biology, especially cell micropatterning. Summary of the Invention

[0006] To address the above issues, this application provides a microdevice technology that can be rapidly prepared using 3D printing for cell culture and patterned cell co-culture. This device is also easily compatible with existing cell biotechnology techniques, such as microscopy and cell immunostaining. It can conveniently pattern a variety of cells and perform microscopic imaging and immunohistochemical analysis on the cells, yielding quantitative results. This provides researchers with a device and method that can more quickly and conveniently achieve co-culture of multiple cells.

[0007] The present invention is achieved through the following technical solutions:

[0008] A device for multi-cell patterned culture based on 3D printing technology, characterized in that:

[0009] The device includes multiple groups of concentrically arranged hollow tubes, each group of hollow tubes including an outer hollow tube and at least one inner hollow tube, with the bottom end of the inner hollow tube concentric with the bottom end of the outer hollow tube. The device is manufactured using 3D printing technology, with the bottom ends of the hollow tubes all placed on a cell culture vessel, and the bottom ends of the inner hollow tubes can be removed from the cell culture vessel. The outer diameter of the inner hollow tube is smaller than the inner diameter of the outer hollow tube. Different types of cells are added to the blank areas between the inner hollow tube, the outer hollow tube, and the inner hollow tube. After the cells adhere to the wall, the inner hollow tube is removed, so that multiple cells are patterned adjacent to each other. At the same time, the wall thickness of the inner hollow tube allows for a certain gap between different cell regions. This device can be used to facilitate the observation of migration and interaction between multiple cells.

[0010] Furthermore, the device includes an upper part and a lower part, the lower part includes multiple outer hollow tubes and a lower support part; the upper part includes multiple inner hollow tubes and an upper support part, the inner hollow tubes are connected to the upper support part, the upper support part and the lower support part are detachable and matched with each other, and when the upper support part and the lower support part are matched, each inner hollow tube body is respectively arranged concentrically with the bottom end of an outer hollow tube body; when the upper support part and the lower support part are disassembled, the bottom end of the inner hollow tube body can be pulled out of the cell culture vessel.

[0011] Furthermore, the inner diameter of the upper end of the inner hollow tube is larger than the inner diameter of the bottom end of the inner hollow tube.

[0012] Furthermore, the inner diameter of the hollow tube ranges from micrometer level to centimeter level.

[0013] Furthermore, by splicing and combining the upper and lower support parts, a reversible seal can be achieved.

[0014] Furthermore, the raw materials for 3D printing have good cell compatibility and environmental friendliness.

[0015] A method for multi-cell patterned culture based on 3D printing technology, characterized in that the method uses the device, and the method comprises the following steps: device preparation: using 3D printing technology to prepare a multi-cell patterned culture device, and placing the device on a cell culture vessel;

[0016] Adding cell suspension solution: injecting the cell suspension solution into the inner hollow tube and between the inner hollow tube and the outer hollow tube respectively, and placing them in a cell culture incubator for culture; the cell suspension solutions injected into different hollow tubes may be the same or different;

[0017] Removing the inner hollow tube: After the cells adhere to the wall, vertically remove the inner hollow tube and replace with fresh culture medium to continue culturing and observe cell migration and interaction.

[0018] Furthermore, before use, a layer of a substance that promotes cell adhesion is incubated on the cell culture vessel.

[0019] Furthermore, in the step of adding the cell suspension solution, the cell suspension solution is a suspension of fibroblasts and a suspension of human fibrosarcoma cells, and the cell concentration is 10 5 / ml, the temperature of the cell culture incubator is 37°C, the volume concentration of carbon dioxide is 5%, and the culture time is 4-6 hours.

[0020] Furthermore, the cell culture vessel is a cell culture dish or a culture plate, and the substrate of the cell culture vessel is glass, silicon wafer, metal or polymer material.

[0021] Beneficial technical effects of the present invention:

[0022] The application of 3D printing technology breaks the limitations of traditional device chip preparation for complex technologies such as photolithography. It can be easily implemented in any laboratory, reducing the requirements for clean laboratory space and complex equipment. In addition, it can quickly change the optimized design in real time based on experimental results, eliminating the need to re-engraving the template as in photolithography, thus avoiding the waste of time and money. More importantly, it can quickly design a large number of different models or structures based on one's own experiments. Compared with photolithography, it has lower technical requirements for experimental personnel, improving work efficiency while also achieving the experimental purpose. Using this invention, the relative movement and interaction of multiple cells can be intuitively observed. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a schematic diagram of a 3D printing device;

[0024] Figure 2 It is to infuse different cells at different locations in the 3D printing device;

[0025] Figure 3 is a schematic diagram of the cell pattern formed after removing the superstructure;

[0026] Figure 4 yes Figure 3 A magnified view of one of them;

[0027] Figure 5 It is a time-lapse graph of the effects of different drugs on cell migration;

[0028] Figure 6 is a schematic cross-sectional view of a 3D printing device;

[0029] In the figure: A. lower part of the device, B. upper part of the device, C. base of the cell culture vessel, a. inner diameter of the bottom end of the inner hollow tube, b. thickness of the wall of the inner hollow tube, c. distance between the outer wall of the inner hollow tube and the inner wall of the outer hollow tube, d. thickness of the wall of the outer hollow tube, e. inner diameter of the upper end of the inner hollow tube, h1. height of the inner hollow tube, h2. height of the outer hollow tube. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0031] On the contrary, the present invention covers any alternatives, modifications, equivalents, and solutions that fall within the spirit and scope of the present invention as defined by the claims. Furthermore, to facilitate a better understanding of the present invention, certain specific details are described in detail below in the detailed description of the present invention. Those skilled in the art will be able to fully understand the present invention without these details.

[0032] Example 1

[0033] like Figure 1 As shown, a multi-cell co-culture device is provided, comprising: a 3D-printed device and a cell culture dish or plate base. The required three-dimensional image is designed using 3D design software, and a corresponding model is manufactured using a 3D printer as the experimental device. The device is primarily a tubular structure with an inner diameter ranging from micrometers to centimeters. The 3D-printed model is placed on the cell culture dish or plate base. The surface of the cell culture dish or plate base is either untreated or coated with a layer of rat tail collagen or other substances that promote cell attachment. The fabrication process does not require complex photolithography techniques, making it simple, convenient, and easy to operate.

[0034] Figure 1 It is a schematic diagram of a 3D printing device, which is mainly composed of two parts, including an upper part and a lower part. The lower part includes nine connected outer hollow tubes with a diameter of 1 cm and a height of 1 mm and a lower support part; the upper part includes multiple inner hollow tubes and an upper support part. The inner hollow tubes are connected to the upper support part. The upper support part and the lower support part are detachable and matched with each other. When the upper support part and the lower support part are matched, each inner hollow tube is respectively arranged concentrically with the bottom end of an outer hollow tube; when the upper support part and the lower support part are disassembled, the bottom end of the inner hollow tube can be pulled out of the cell culture vessel.

[0035] In this embodiment, the lower part includes nine connected outer hollow tubes and a frame located around the nine outer hollow tubes. Grooves are provided on both sides of the frame, and the frame serves as a lower support portion.

[0036] The upper portion consists of an inner hollow tube and a "door"-shaped frame. The "door" frame consists of a horizontal rod and two vertical rods, with the vertical rods perpendicular to the horizontal rods. The upper end of the inner hollow tube is mounted on the horizontal rods, which have holes at corresponding positions that communicate with the hollow holes in the inner hollow tube. The "door"-shaped frame serves as the upper support.

[0037] The groove of the frame body is adapted to the horizontal rod of the "door" shaped frame. Inserting the horizontal rod into the groove can fix and support the inner hollow tube body. At this time, the bottom end of an inner hollow tube body and the bottom end of an outer hollow tube body are concentrically arranged.

[0038] In the present invention, every three outer hollow tubes form a column, and the nine connected outer hollow tubes are divided into three rows, and each lower part must have three identical upper parts, but the present application is not limited to this arrangement.

[0039] The inner diameter of the bottom end of the inner hollow tube is different from the inner diameter of the upper end. The inner diameter of the upper end is larger than the inner diameter of the bottom end, which is convenient for injecting cell fluid.

[0040] In this embodiment, the cross-sectional surface of the hollow tube is circular, but the present invention is not limited to a circular shape. The cross-sectional surface may also be square, prismatic or other irregular shapes.

[0041] Figure 6 It is a schematic diagram of the longitudinal section of the 3D printing device, indicating the dimensions of key locations.

[0042] Here, a is the diameter of the initial circular area occupied by the inner circle of cells; b is the width of the blank circular area between the inner and outer circles of cells, representing the wall thickness of the inner hollow tubular body; c is the width of the initial circular area occupied by the outer circle of cells; d represents the wall thickness of the outer hollow tubular body; e represents the inner width of the groove connecting the inner hollow tubular body, which affects the volume of the inner cell suspension; h1 represents the outer height of the inner hollow tubular body, which affects the volume of the inner and outer cell suspensions; h2 represents the height of the outer hollow tubular body, which affects the volume of the outer cell suspension. The specific dimensions are as follows: a is 800 μm, b is 1000 μm, c is 3600 μm, d is 1000 μm, e is 4000 μm, h1 is 2000 μm, and h2 is 1000 μm.

[0043] The substrate of the cell culture dish or plate is glass, silicon wafer, metal or polymer material such as polystyrene, which is easy to obtain and inexpensive.

[0044] The shape and size of the 3D printing device can be flexibly changed, and sizes can range from microns to centimeters.

[0045] The 3D printing device can achieve reversible sealing by splicing and combining the upper and lower support parts.

[0046] The raw materials for 3D printing are biodegradable polylactic acid, ABS engineering plastics or other polymer materials that can be used for 3D printing, which have good cell compatibility and environmental friendliness.

[0047] To achieve the above object, the present invention further provides a method for multi-cell co-culture, using the above device, specifically comprising the following steps:

[0048] Step 1: Prepare the cell culture dish or culture plate base and the 3D printing device, which mainly consists of two parts. The bottom part is nine connected tubular bodies with a diameter of 1 cm and a height of 1 mm. Figure 1 As shown in structure A; concentric with it is a tubular body with a diameter of 800µm, as shown in Figure 1 As shown in structure B, the specific dimensions refer to Figure 6 ;

[0049] Step 2: Prepare rat tail collagen at a concentration of 300µg / ml in water and glacial acetic acid solvent, operate at a low temperature of 4°C, drop the rat tail collagen into the base of the culture dish or culture plate, place it in an incubator for 12 hours, then slowly wash it three times with PBS (1X) solution, let it dry naturally at room temperature, and place the 3D printing device into the base of the cell culture dish or culture plate that has been incubated with a layer of rat tail collagen. The purpose is to make the cells grow on the wall in a more realistic condition in vivo. By splicing and combining the upper and lower support parts, a reversible seal can be formed;

[0050] Step 3: Figure 2 As shown, a cell suspension solution is prepared and injected into the device of the cell culture dish or culture plate base incubated with a layer of rat tail collagen, and then placed in a cell culture incubator to culture cells for 4-6 hours. Figure 2 Different cells are injected into different positions of the 3D printed structure. The dark part at the bottom is the fibroblast suspension, and the light part in the upper groove is the human fibrosarcoma cell suspension. There will be no cross-contamination between the two.

[0051] Step 4: After the cells adhere to the wall, the inner hollow tube is removed vertically and replaced with fresh culture medium to continue culturing. This breaks the limitations of traditional photolithography technology and establishes a multi-cell co-culture method based on 3D printing technology. Figure 3 This is a schematic diagram of the cell pattern formed after removing the upper structure. The light-colored part in the middle is human fibrosarcoma cells, the dark-colored part outside is fibroblasts, and the blank area between the two is the part where the cells migrate together.

[0052] Different cell solutions can be injected into the inner hollow tube and the outer hollow tube according to actual research needs. In this embodiment, human fibrosarcoma cells and fibroblasts are injected, but the present invention is not limited thereto.

[0053] It can also be used to study the effects and functions of different drugs in cellular interactions, such as Figure 5 As shown, the co-culture diagrams under different conditions were obtained, which provided a basis for subsequent biological analysis. Figure 5These are time-lapse images showing the effects of different drugs on cell migration. The light-colored center section represents human fibrosarcoma cells, while the darker sections on the outside represent fibroblasts. The first row shows the control group without drug, the second row shows the experimental group with Cytochalsin D, and the third row shows the experimental group with GM6001. The images show that the drugs inhibit cell migration.

[0054] The cell suspension solution in step 3 is a suspension of fibroblasts and a suspension of human fibrosarcoma cells, and the cell concentration is 10 5 The cell culture incubator temperature is 37° C., the carbon dioxide volume concentration is 5%, and the culture time is 4-6 hours.

[0055] In step 3, use a 1 ml syringe to draw up the cell suspension, such as Figure 2 As shown, the cell suspension of fibroblasts is injected into the blank space between the two tubular bodies, and the suspension of human fibrosarcoma cells is injected into the groove connecting the inner hollow tubular body.

[0056] Step 4: Figure 4 As shown, after the cells adhered, the inner hollow tubular body was removed, and it was observed that human fibrosarcoma cells only grew within a range of about 800µm in diameter in the center of the pattern. The outer circle was a blank circular area of about 1mm, and the outermost layer was fibroblasts. The non-adherent cells were washed with PBS three times, and fresh culture medium was replaced. The culture medium was then placed in the cell culture incubator to observe the migration and interaction of the two types of cells. In this way, by calculating the changes in the area occupied by human fibrosarcoma cells, we can obtain the migration changes of cancer cells under the action of fibroblasts.

[0057] Due to the use of 3D printing technology, the inner diameter of the hollow tube can be as large as centimeters or as small as micrometers, and is not limited to the numbers in this embodiment.

[0058] Example 2

[0059] This embodiment is basically the same as Example 1, except that the device prepared by 3D printing technology includes two inner hollow tubes.

Claims

1. A device for multi-cell patterned culture based on 3D printing technology, characterized in that: The required three-dimensional drawings are designed using three-dimensional design software, and the corresponding models are manufactured using a 3D printer as an experimental device. The device includes multiple groups of concentrically arranged hollow tubes, each group of hollow tubes includes an outer hollow tube and an inner hollow tube, and the bottom end of the inner hollow tube is concentric with the bottom end of the outer hollow tube. The device is prepared using 3D printing technology, and the bottom ends of the hollow tubes are all set on the cell culture vessel, and the bottom end of the inner hollow tube can be removed from the cell culture vessel. The outer diameter of the inner hollow tube is smaller than the inner diameter of the outer hollow tube. The device includes an upper part and a lower part, the lower part includes a plurality of connected outer hollow tubes and a lower support part; the upper part includes a plurality of inner hollow tubes and an upper support part, the inner hollow tubes are connected to the upper support part, the upper support part and the lower support part are detachable and matched with each other, and when the upper support part and the lower support part cooperate, each inner hollow tube is respectively arranged concentrically with the bottom end of an outer hollow tube; when the upper support part and the lower support part are disassembled, the bottom end of the inner hollow tube can be removed from the cell culture device. The upper support portion includes a horizontal rod and two vertical rods, and the vertical rods are perpendicular to the horizontal rods.

2. The device according to claim 1, characterized in that The inner diameter of the upper end of the inner hollow tube body is larger than the inner diameter of the bottom end of the inner hollow tube body.

3. The device according to claim 1, characterized in that The inner diameter of the hollow tube is from 1*10 -6 ~1*10 -2 rice.

4. The device according to claim 1, characterized in that By splicing and combining the upper and lower support parts, a reversible seal can be achieved.

5. The device according to claim 1, characterized in that The raw materials for 3D printing have good cell compatibility and environmental friendliness.

6. A method for multi-cell patterned culture based on 3D printing technology, characterized in that: The method adopts the device of claim 1, and the method comprises the following steps: Device preparation: Use 3D printing technology to prepare a multi-cell patterned culture device and place the device on a cell culture vessel; Adding cell suspension solution: injecting the cell suspension solution into the inner hollow tube and between the inner hollow tube and the outer hollow tube respectively, and placing them in a cell culture incubator for culture; the cell suspension solutions injected into different hollow tubes may be the same or different; Removing the inner hollow tube: After the cells adhere to the wall, vertically remove the inner hollow tube and replace with fresh culture medium to continue culturing and observe cell migration and interaction.

7. The method according to claim 6, wherein: Before use, incubate the cell culture vessels with a layer of substance that promotes cell attachment.

8. The method according to claim 6, wherein: In the step of adding the cell suspension solution, the cell suspension solution The suspension of fibroblasts and human fibrosarcoma cells were prepared with a cell concentration of 10 5 The cell culture incubator temperature was 37°C, the carbon dioxide concentration was 5%, and the culture time was 4-6 hours.

9. The method according to claim 6, wherein: The cell culture apparatus is a cell culture dish or a culture plate, and the base of the cell culture apparatus is glass, silicon wafer, metal or polymer material.

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