A method of printing cell microspheres
Patent Information
- Application Number
- CN202111270825.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-10-29
- Publication Date
- 2026-10-27
- Estimated Expiration
- 2041-10-29
AI Technical Summary
[0004]鉴于以上所述现有技术的缺点，本发明的目的在于提供一种细胞微球打印方法，用于解决传统类器官制作时遇到的实验操作随机性、重复性不佳、粒径不均一导致培养与分析困难等问题
[0020]如上所述，本发明的细胞微球打印方法利用MEMS生物打印芯片实现微米级范围内的细胞定量接种，实现细胞微球的定量、均一粒径、特定位置的高精度与高通量的接种与培养，在类器官微球应用方面，解决了目前存在的细胞接种后粒径与分布随机性的技术难点，避免了实验操作随机性、重复性不佳、粒径不均一导致培养与分析困难等问题。
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Figure CN116064235B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of microfluidics and biopharmaceuticals, and relates to a method for printing cell microspheres. Background Technology
[0002] Organoids are multicellular, three-dimensional structures cultured in vitro that possess the microscopic anatomical features of their source organs. As a branch of stem cell research, they have gained widespread acceptance and recognition in recent years. Currently, numerous studies on organoids, including various tissues, disease models, and simulated developmental processes, have appeared in the scientific literature. Furthermore, many specialized culture media for culturing organoids, instruments and reagents for analyzing and detecting organoid activity and indicators, and even commercial activities involving organoid models as marketed products have emerged. The value of organoids lies in their potential for culturing disease models in vitro. They are widely recognized as important biological research tools with significant clinical applications. In the fields of tumor biology and new drug development, organoids construct organ samples with three-dimensional structures. Compared to animal models, the human cells used in organoids are more reliable for research reference, and their advantage lies in enabling in vitro experimental studies of human tissues.
[0003] Although organoids represent a promising technological breakthrough for clinical applications, the technology still has certain limitations. These limitations stem from expensive culture reagents, inadequate cell culture microenvironments, and the randomness inherent in experimental techniques. The last limitation is particularly significant because current organoid fabrication methods rely on pipetting, where cells are mixed with Matrigel and then dropped into wells for three-dimensional culture. The cells then grow into multiple microspheres of varying sizes and random locations within the Matrigel, serving as a model for subsequent research. This methodological limitation makes subsequent analysis of cell culture activity and biomarkers difficult. Furthermore, the randomness of cell spheroidization in the Matrigel can lead to cell apoptosis due to differences in particle size or uneven diffusion of drugs within the microspheres. Summary of the Invention
[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a cell microsphere printing method to solve the problems encountered in traditional organoid production, such as randomness of experimental operations, poor reproducibility, and difficulties in culture and analysis caused by uneven particle size.
[0005] To achieve the above and other related objectives, the present invention provides a method for printing cell microspheres, comprising the following steps:
[0006] Cell suspension application step: A MEMS bioprinting chip is provided, and a cell suspension is poured into the MEMS bioprinting chip. The MEMS bioprinting chip is provided with a flow channel and a plurality of nozzles communicating with the flow channel. The cell suspension flows through the flow channel to the nozzles.
[0007] Cell printing steps: The MEMS bioprinting chip is moved along a preset path to sequentially spray cell suspensions from multiple nozzle groups onto a designated location on the surface of the cell culture device to form printed cell clusters. Each nozzle group includes one or more nozzles.
[0008] Cell culture step: Add culture medium to the cell culture device to culture the printed cell clusters into cell microspheres.
[0009] Optionally, in the cell printing step, a nozzle group includes a plurality of nozzles, and the plurality of nozzles in a nozzle group are printed simultaneously or sequentially.
[0010] Optionally, the cell printing step may be repeated at least once to increase the number of cells in the cell cluster printed at the designated location.
[0011] Optionally, the cell printing step is repeated at least once to sequentially form printed cell clusters at multiple designated locations on the surface of the cell culture device.
[0012] Optionally, after the cell culture step, the cell microspheres located at different designated locations have the same particle size, or the difference between the particle size of any designated cell microsphere and the average particle size of all designated cell microspheres in the cell culture device is less than 10% of the average particle size.
[0013] Optionally, the plurality of designated positions are arranged in an array.
[0014] Optionally, the cell culture apparatus includes a rigid substrate and a culture medium located on the rigid substrate, wherein the cell microspheres are formed on the surface of the culture medium.
[0015] Optionally, the rigid substrate includes one of a glass slide, a silicon wafer, and a cell culture plate, and the culture medium includes a water-based gel.
[0016] Optionally, the water-based gel includes at least one of rat tail collagen, gelatin, methacrylated gelatin, matrix gelatin, sodium alginate, poloxamer, pranic acid, and polyethylene glycol hydrogel.
[0017] Optionally, in the cell culture step, cell culture is performed after adding a culture medium containing matrix gel to the cell culture device, or cell culture is performed after adding a culture medium to the cell culture device and covering the surface of the culture medium with a layer of matrix gel.
[0018] Optionally, the MEMS bioprinting chip includes a thermally printed chip.
[0019] Optionally, in the cell suspension application step, the number of cells contained in one of the nozzles is 0-5.
[0020] As described above, the cell microsphere printing method of the present invention utilizes MEMS bioprinting chips to achieve quantitative cell seeding within the micrometer range, realizing high-precision and high-throughput seeding and culture of cell microspheres with quantitative, uniform particle size, and specific location. In the application of organoid microspheres, it solves the technical difficulties of randomness in particle size and distribution after cell seeding, and avoids problems such as randomness in experimental operation, poor repeatability, and difficulties in culture and analysis caused by non-uniform particle size. Attached Figure Description
[0021] Figure 1 The flowchart shown is a process for printing cell microspheres according to the present invention.
[0022] Figure 2 This is a planar layout diagram showing a partial location of a thermally printed chip.
[0023] Figure 3 This is another planar layout diagram showing a partial location of a thermally printed chip.
[0024] Figure 4 This is a schematic diagram showing the cross-sectional structure of the cell culture device.
[0025] Figures 5a-5c This is a schematic diagram showing cell printing at a designated location on a cell culture device.
[0026] Figures 6a-6b The diagram shows a printing cycle using a thermal printing chip with 320 nozzles.
[0027] Figure 7a This diagram shows the printed cell clusters obtained at the designated location after the first printing cycle is completed.
[0028] Figure 7b This diagram illustrates a second printing sequence performed at a specified location to increase the particle size of the printed cell clusters.
[0029] Figure 8 The diagram illustrates a process where a thermally printed chip with 320 nozzles is used to perform one printing cycle at a designated location A in a cell culture apparatus to obtain a printed cell cluster, and another printing cycle is performed at another designated location B, spaced apart from location A, to obtain another printed cell cluster.
[0030] Figure 9 The diagram shows multiple printed cell clusters arranged in an array printed on the surface of a cell culture device.
[0031] Figure 10 Displayed as will Figure 9 The diagram shows a lattice of printed cell clusters cultured to obtain a lattice of cell microspheres.
[0032] Figure 11 The image shown is a microscope image of a 3×3 array of printed cell clusters obtained by seeding a hydrogel substrate using the cell microsphere printing method of the present invention.
[0033] Figures 12a to 12d The images shown are microscope images obtained four times by continuously printing a 3×3 cell cluster array on the surface of a hydrogel substrate using the cell microsphere printing method of the present invention.
[0034] Component designation explanation
[0035] 100 Thermal Printing Chips
[0036] 101 flow channel
[0037] 102 nozzles
[0038] 200 cell culture apparatus
[0039] 201 rigid substrate
[0040] 202 Culture Material
[0041] 300 cells
[0042] 400, 400a, 400b Printed cell clusters
[0043] 500 cell microspheres
[0044] 1-1, 1-2, 1-3, 2-1, 2-2, 2-3, 2-4, 2-320, Spray Nozzle
[0045] M, N, A, B specify the position Detailed Implementation
[0046] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.
[0047] Please see Figures 1 to 12dIt should be noted that the illustrations provided in this embodiment are only schematic representations of the basic concept of the present invention. Therefore, the drawings only show the components related to the present invention and are not drawn according to the actual number, shape and size of the components in the actual implementation. In the actual implementation, the form, quantity and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex.
[0048] Example 1
[0049] This embodiment provides a method for printing cell microspheres. Please refer to [link to relevant documentation]. Figure 1 The flowchart of this method is shown below, including the following steps:
[0050] Cell suspension application step: A MEMS bioprinting chip is provided, and a cell suspension is poured into the MEMS bioprinting chip. The MEMS bioprinting chip is provided with a flow channel and a plurality of nozzles communicating with the flow channel. The cell suspension flows through the flow channel to the nozzles.
[0051] Cell printing steps: The MEMS bioprinting chip is moved along a preset path to sequentially spray cell suspensions from multiple nozzle groups onto a designated location on the surface of the cell culture device to form printed cell clusters. Each nozzle group includes one or more nozzles.
[0052] Cell culture step: Add culture medium to the cell culture device to culture the printed cell clusters into cell microspheres.
[0053] Specifically, the MEMS bioprinting chip preferably uses a thermal bubble printing chip, whose nozzle is controlled by the underlying circuit. The instantaneous high temperature of the heating film in the nozzle causes the liquid above the heating film to vaporize, generating bubbles that drive the liquid flow and are ejected from the nozzle, thereby quickly and gently extracting cells. Subsequently, the liquid is replenished under the action of capillary force, providing power for the continuous flow of liquid.
[0054] Specifically, compared to extrusion-based printed chips, thermal inkjet printed chips offer higher precision. This is because extrusion-based chips print hydrogels mixed with cells, achieving cell spheroidization and culture within tiny volumes by extruding minute volumes of hydrogel (containing cells). However, this method typically produces hydrogels with multiple cell microspheres of varying sizes within nanoliter-sized volumes, making it difficult to achieve the precision of thermal inkjet printed chips. Furthermore, compared to piezoelectric printed chips, thermal inkjet printed chips can achieve higher throughput. Although both piezoelectric and thermal inkjet printed chips utilize inkjet printing technology and can achieve comparable printing precision, the piezoelectric printhead structure results in lower throughput, making it more difficult to achieve the hundreds of nozzles on a single chip like thermal inkjet printed chips.
[0055] For example, please refer to Figure 2The image shows a planar layout of a portion of the thermally printed chip 100, wherein nozzles 102 are distributed on both sides of the flow channel 101.
[0056] For example, please refer to Figure 3 This is shown as another planar layout view of a partial location of the thermally printed chip 100, relative to... Figure 2 Multiple nozzles 102 on one side of the central channel 101 are arranged in a straight line. Figure 3 In the middle, multiple nozzles 102 are arranged in an alternating pattern on one side of the flow channel 101.
[0057] It should be noted that the flow channels and nozzles in the thermal printing chip 100 can also adopt other layouts, not limited to those shown in Figure 2. Figure 3 The above is the limit.
[0058] As an example, one of the aforementioned MEMS bioprinted chips has 160 to 1280 micrometer-sized nozzles, such as... Figure 3 As shown, the chip is filled with a cell suspension to be printed. The cell suspension flows through the channel 101 into the nozzle 102. This suspension can be a common isotonic cell buffer such as PBS, HEPES, or NaCl. Due to the size of the printing nozzle, each nozzle usually contains 0, 1, or several cells, for example, 0-5 cells. Figure 3 The data presented a scenario where there were 0, 1, 2, or 3 cells in a nozzle, with a total of 300 cells.
[0059] For example, please refer to Figure 4 The diagram shows a cross-sectional view of the cell culture apparatus 200, which includes a rigid substrate 201 and a culture medium 202 located on the rigid substrate 201, and the cell microspheres are subsequently formed on the surface of the culture medium 202.
[0060] As an example, the rigid substrate 201 includes, but is not limited to, one of glass slides, silicon wafers, and cell culture plates. The culture medium 202 has a special surface that can adhere to printed cells and perform three-dimensional cell culture. Its material includes, but is not limited to, water-based gels, such as mixtures of commonly used three-dimensional culture materials such as rat tail collagen, gelatin, methacrylated gelatin, matrix gelatin, sodium alginate, poloxamer, pranic acid, and polyethylene glycol hydrogel.
[0061] As an example, a method for forming a cell culture apparatus 200 includes: coating a three-dimensional culture material mixture onto the surface of the rigid substrate 201, and then curing it to obtain the culture medium 202.
[0062] As an example, in the cell printing step, one of the nozzle groups contains one nozzle, meaning that the thermal printing chip 100 is moved once for each nozzle printed. See also... Figures 5a-5c The diagram shows a cell printing process at a designated location M in the cell culture apparatus 200, wherein... Figure 5a The diagram shows the process of moving the thermally printed chip 100 filled with cell suspension above the cell culture device 200, aligning the nozzle 1-1 with a designated position M on the cell culture device 200, and spraying the cell suspension in the nozzle 1-1 onto the designated position M. Figure 5b The diagram shows the movement of the thermal printing chip 100, aligning the nozzles 1-2 with the designated position M, and spraying the cell suspension in the nozzles 1-2 to the designated position M. Figure 5c The diagram illustrates the process of moving the thermal printing chip 100 again, aligning the nozzles 1-3 with the designated position M, and spraying the cell suspension within the nozzles 1-3 onto the designated position M. This process is repeated until a printed cell cluster of a preset particle size or preset cell number is obtained at the designated position M.
[0063] As an example, if all the nozzles of the thermally printed chip 100 have been printed, but the printed cell cluster at the designated position M has not yet reached the preset particle size or preset cell number, the cell printing step can be repeated (the MEMS bioprinting chip can be moved again along the preset path) until the printed cell cluster at the designated position M reaches the preset particle size or preset cell number.
[0064] As an example, printing from the first nozzle to the last nozzle of the thermal printhead 100 constitutes one printing cycle. See also... Figures 6a to 6b The diagram shows a printing cycle performed using a thermal printing chip with 320 nozzles. Figure 6a The diagram illustrates the process of spraying cell suspension from the first nozzle 2-1 to the designated location N. The thermal printing chip 100 is then moved step-by-step to sequentially print from the second nozzle 2-2, the third nozzle 2-3, the fourth nozzle 2-4, and so on, until the cell suspension from the last nozzle 2-320 is sprayed to the designated location N (e.g., ...). Figure 6b As shown), this completes one printing sequence. Figure 6b Arrows are used to indicate the direction of movement of the thermally printed chip.
[0065] For example, please refer to Figure 7a and Figure 7b ,in, Figure 7aThe diagram shows the printed cell cluster 400 obtained at the designated position N after the first printing sequence is completed. Figure 7b The diagram illustrates a second printing sequence at the designated location N to increase the particle size of the printed cell cluster 400. It can be seen that the particle size of the printed cell cluster at the designated location can be increased by repeating the cell printing step at least once, for example, by continuously seeding hundreds to thousands of cells in the same small area, thereby increasing the particle size of the cell microspheres obtained after culture.
[0066] As an example, the cell printing step can be repeated at least once to sequentially form printed cell clusters at multiple designated locations on the surface of the cell culture device. After culturing, cell microspheres can be obtained at these multiple designated locations on the surface of the cell culture device. Please refer to [link to relevant documentation]. Figure 8 The diagram shows a printing cycle performed at a designated location A in a cell culture apparatus 200 using a thermal bubble printing chip 100 with 320 nozzles to obtain a printed cell cluster 400a, and another printing cycle performed at another designated location B, which is spaced apart from the designated location A (e.g., spaced 100 micrometers to 1000 micrometers), to obtain another printed cell cluster 400b.
[0067] As an example, the multiple designated positions are arranged in an array. See also Figure 9 The diagram shows a plurality of printed cell clusters 400 arranged in an array printed on the surface of the cell culture apparatus 200. Please refer to [link / reference]. Figure 10 The diagram shows a schematic of culturing the printed cell cluster array shown in Figure 9 to obtain a cell microsphere array of 500 dots.
[0068] For example, please refer to Figure 11 The image shown is a microscope image of a 3×3 array of printed cell clusters obtained by seeding a hydrogel substrate using the cell microsphere printing method of the present invention. It can be seen that each printed cell cluster has a uniform particle size and is arranged regularly.
[0069] For example, please refer to Figures 12a to 12d The images shown are microscope images obtained by printing a 3×3 cell cluster array on the surface of a hydrogel substrate four times using the cell microsphere printing method of the present invention. It can be seen that the printing method of the present invention has high repeatability.
[0070] As an example, in the cell culture step, cell culture is performed after adding a culture medium containing matrix gel to the cell culture apparatus 200, or cell culture is performed after adding a culture medium to the cell culture apparatus and covering the surface of the culture medium with a layer of matrix gel.
[0071] Specifically, after the cell culture step, the cell microspheres located at different designated positions have the same particle size, or the difference between the particle size of a cell microsphere at any designated position and the average particle size of cell microspheres at all designated positions in the cell culture device is less than 10% of the average particle size. That is, the present invention can achieve a cell microsphere array with uniform particle size, uniform cell number, and regular arrangement, which can be applied to high-throughput three-dimensional culture of organoids.
[0072] Example 2
[0073] This embodiment uses essentially the same technical solution as Embodiment 1, the difference being that in the cell printing step of Embodiment 1, each nozzle group contains one nozzle, meaning the thermal printing chip moves once after printing one nozzle. In this embodiment, however, each nozzle group contains multiple nozzles, and the thermal printing chip moves only after all nozzles in the nozzle group have been printed. For example, if a nozzle group contains two nozzles, the thermal printing chip moves once after printing two nozzles; if a nozzle group contains four nozzles, the thermal printing chip moves once after printing four nozzles. The number of nozzles in a nozzle group can be determined according to the size of the cell microspheres to be inoculated.
[0074] As an example, multiple nozzles within a nozzle group can be printed simultaneously or sequentially, which can be achieved through circuit control.
[0075] In summary, the cell microsphere printing method of this invention utilizes MEMS bioprinting chips to achieve quantitative cell seeding within the micrometer range, enabling high-precision and high-throughput seeding and culture of cell microspheres with uniform particle size and specific locations. In the application of organoid microspheres, it solves the technical difficulties of random particle size and distribution after cell seeding, avoiding problems such as randomness in experimental operations, poor reproducibility, and difficulties in culture and analysis caused by non-uniform particle size. Therefore, this invention effectively overcomes the various shortcomings of existing technologies and has high industrial application value.
[0076] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.
Claims
1. A method for printing cell microspheres, characterized in that, Includes the following steps: Cell suspension application step: A MEMS bioprinting chip is provided, the MEMS bioprinting chip includes a thermal bubble printing chip, a cell suspension is poured into the MEMS bioprinting chip, the MEMS bioprinting chip is provided with a flow channel and a plurality of nozzles communicating with the flow channel, the cell suspension flows through the flow channel to the nozzles, and the number of cells contained in one nozzle is 0-5. Cell printing steps: The MEMS bioprinting chip is moved along a preset path to sequentially spray cell suspensions from multiple nozzle groups onto a designated location on the surface of a cell culture device to form printed cell clusters. Each nozzle group includes one or more nozzles. After each nozzle group is completed, the MEMS bioprinting chip is moved so that multiple nozzle groups are sequentially aligned with the same designated location for spraying. The cell printing steps are repeated at least once to increase the number of cells in the printed cell cluster at the designated location, and the cell printing steps are repeated at least once to sequentially form printed cell clusters at multiple designated locations on the surface of the cell culture device, wherein the multiple designated locations are arranged in an array. Cell culture step: Add culture medium to the cell culture device to culture the printed cell cluster into cell microspheres; after the cell culture step, the cell microspheres located at different designated locations have the same particle size, or the difference between the particle size of the cell microsphere at any designated location and the average particle size of the cell microspheres at all designated locations in the cell culture device is less than 10% of the average particle size.
2. The cell microsphere printing method according to claim 1, characterized in that: In the cell printing step, a group of nozzles includes a plurality of nozzles, and the plurality of nozzles in a group of nozzles are printed simultaneously or sequentially.
3. The cell microsphere printing method according to claim 1, characterized in that: The cell culture apparatus includes a rigid substrate and a culture medium located on the rigid substrate, wherein cell microspheres are formed on the surface of the culture medium.
4. The cell microsphere printing method according to claim 3, characterized in that: The rigid substrate includes one of a glass slide, a silicon wafer, and a cell culture plate, and the culture medium includes a water-based gel.
5. The cell microsphere printing method according to claim 4, characterized in that: The water-based gel includes at least one of rat tail collagen, gelatin, methacrylated gelatin, matrix gelatin, sodium alginate, poloxamer, pranic acid, and polyethylene glycol hydrogel.
6. The cell microsphere printing method according to claim 1, characterized in that: In the cell culture step, cell culture is performed after adding a culture medium containing matrix gel to the cell culture device, or cell culture is performed after adding a culture medium to the cell culture device and covering the surface of the culture medium with a layer of matrix gel.