Biological 3D printing spray head for preparing and collecting high-throughput cell spheres and use method of biological 3D printing spray head

Abstract

The invention relates to a biological 3D printing nozzle for high-throughput cell sphere preparation and collection and a use method thereof, and belongs to the field of three-dimensional cell sphere culture and biological 3D printing, a chip is processed and manufactured by a top layer, a culture solution layer, a microporous layer, a runner layer and a bottom layer of PDMS in a laser cutting mode, the top layer of the chip is provided with at least three sample adding ports, and the bottom layer of the chip is provided with at least three sample adding ports. The micro-pore layer of the chip is communicated with the culture solution layer, the micro-pore layer of the chip comprises a plurality of display micro-pores, continuously bent channels are formed in the positions, corresponding to the bottoms of the micro-pores, of the flow channel layer of the chip, and the tip of an outlet can be in butt joint with a common needle head or a coaxial needle head. According to the biological 3D printing spray head for preparing and collecting the high-flux cell balls and the using method thereof, the cell balls are manufactured in a high-flux mode through a micropore method, liquid replacement is supported, the problem that collection is difficult is solved, the complexity of the cell ball preparation process is reduced, and the biological 3D printing spray head has the advantages of being automatic, low in cost and integrated in ball forming, collecting and printing.

Description

Technical Field

[0001] The present invention relates to the fields of three-dimensional cell sphere culture and biological 3D printing technology, and specifically to a biological 3D printing nozzle for high-throughput cell sphere preparation and collection and a method for using the same. Background Art

[0002] Cellular aggregates, also known as cellular spheroids, are 3D structures formed by co-culturing various types of cells with endogenously secreted extracellular matrix to form tissues, aiming to more realistically summarize the key characteristics of native tissues. Currently, the most widely used in vitro 3D cell model can faithfully reflect the three-dimensional relationship between cells, between cells and extracellular matrix, and between cells and the external microenvironment. It can also well simulate the inherent metabolism of cells (such as nutrients, oxygen, metabolites, etc.) and the proliferation gradient of cells in the natural physiological environment, and can track the diffusion path of drugs or metabolites. These technologies are different in mechanism, and each has its own advantages and limitations.

[0003] Bioprinting has become a key tool for locating these spheres. The currently established methods for constructing artificial biological tissues can be mainly divided into two categories: "bottom-up" and "top-down". The "bottom-up" method is representative of the combination of micro-nano manufacturing, microfluidics, and micro-nano operation technologies to construct biological micro-tissues containing multicellular aggregates. The "top-down" method is a biological manufacturing method represented by biological 3D printing, which directly or indirectly controls the assembly and construction of biological structures of cells, growth factors, and matrix materials. The biological 3D printing strategy also cannot meet the actual size of the tissue and the protection of the anatomical structure.

[0004] Methods for preparing cell spheroids include: hanging drop method, low adhesion substrate method, microfluidics method, bioreactor method, magnetic particle control method, etc. However, the hanging drop method has limited throughput, poor mechanical stability of cell hanging drops, and difficulty in long-term culture. The low adhesion substrate method cannot control the size of the spheroids well. The microwell method often has low throughput, and the cell spheroids are difficult to extract for subsequent analysis. During the medium disturbance in the microfluidics method, the spheroids may separate from the microwells and fuse with each other to form irregular clumps, which may cause repeatability problems between runs. The cell spheroids in the bioreactor method have poor uniformity and are easily damaged by rotation during culture. The addition of magnetic nanoparticles in the magnetic particle control method brings a safety issue. Although the magnetic nanoparticles used are claimed to be non-toxic, it is still unclear whether other substances are non-toxic to cells, and the experimental cost is high. Overall, the current technology is highly manual, the turnaround time is slow, and the process control, throughput and scalability are limited.

[0005] In order to meet the growing demand for cell spheroid-based applications, future technological developments must improve existing technologies to enable large-scale, consistent and reproducible cell spheroid manufacturing. Such development work should focus on areas such as liquid handling, spheroid transfer, spheroid characterization, process standardization and process scalability. Although spheroids constitute a microenvironment that mimics the cell density in natural tissues, their morphology, anatomy and physiology may not be a good summary of natural tissues. Their mechanical and structural properties are very weak. It is difficult to arrange multiple cell spheroids in a controllable combination. In response to this problem, this application proposes a biological 3D printing nozzle for high-throughput cell spheroid preparation and collection and a method for using the same. Summary of the invention

[0006] 1. Technical issues to be resolved In view of the shortcomings of the prior art, the present invention provides a biological 3D printing nozzle for high-throughput cell sphere preparation and collection and a method of using the same, which has the advantages of large-scale, consistent and repeatable, and solves the problem of greatly reducing labor costs compared to traditional photolithography mold making and reducing the complexity of the cell sphere preparation process.

[0007] (II) Technical solution To achieve the above-mentioned purpose, the present invention provides the following technical solutions: a biological 3D printing nozzle for high-throughput cell ball preparation and collection, the nozzle includes a microfluidic chip made of PDMS material, the chip is made of 5 layers of PDMS including a top layer, a culture liquid layer, a microporous layer, a flow channel layer and a bottom layer by laser cutting, the top layer of the chip is provided with at least 3 sample injection ports, which are connected to the culture liquid layer, the microporous layer of the chip includes a plurality of displayed micropores, the chip flow channel layer corresponds to the position of the bottom of the micropores, forming a continuously curved channel, the tip at the outlet can be connected to an ordinary needle and a coaxial needle, the chip as a whole can be clamped to a general 3-axis biological 3D printer, and the extrusion power is provided by a micro pump or an air pump or other power source.

[0008] Preferably, the top layer of the chip is provided with at least three sample addition ports, which are connected to the culture solution layer, and the microporous layer of the chip includes a plurality of display micropores.

[0009] Preferably, the chip flow channel layer corresponds to the position of the bottom of the micropore, forming a continuously curved channel, the tip of the outlet can be connected to an ordinary needle and a coaxial needle, and the entire chip can be clamped to a universal 3-axis biological 3D printer, and the extrusion power is provided by a micro pump or an air pump or other power source.

[0010] Another problem to be solved by the present invention is a method for using a biological 3D printing nozzle for high-throughput cell spheroid preparation and collection. The cell spheroid preparation process specifically includes the following steps: Step 1) Chip pretreatment: aligning the various layers of the chip for plasma bonding; Step 2) Adding samples: Add the cell suspension from the sample adding port, and the cell suspension flows through the culture medium layer. As the suspension is slowly added, it flows through the entire culture medium layer, and stops when the cell suspension flows to another sample adding port; Step 3) Horizontal standing: After the sample is added, the chip is placed on a horizontal surface and allowed to stand. Some cells fall into the micropores due to gravity. The micropores are through holes, but due to capillary force, the suspension will not flow directly to the next flow channel layer, and will form a U-shaped hanging drop below the micropores. Since the PDMS surface is hydrophobic, cells are not easy to adhere to the micropore surface, which causes the cells to clumping together to form spheres. Step 4) The chip is moved into a cell culture incubator and placed horizontally for 6-24 hours, then the excess cell suspension in the culture medium layer is removed, and new liquid culture medium is added. The culture medium is fused with the cell spheres in the microwells, so that the cell spheres in each microwell can obtain nutrients. The cells are continued to be placed horizontally and cultured until compact cell spheres are formed. The spheroid printing process specifically includes the following steps: Step 5) collecting the cell spheres; Step 6) Extruding the cell spheres for printing; Step 7) The chip is clamped on a three-axis biological 3D printer, and the printer performs path movement. The extruder provides extrusion power, and the chip works as the nozzle of the printer.

[0011] Preferably, in step 5), the next layer of the chip microporous layer is the flow channel layer, and the bioprinting material is added to the flow channel layer inlet, and normally passes into the entire flow channel layer before the hydrogel is solidified, and is left to stand for a period of time. The liquid phase material is mixed with the cell balls and culture fluid in the microporous layer, and the cell balls fall into the flow channel layer under the influence of gravity.

[0012] Preferably, in step 6), the sample addition port is combined with the biological 3D printing extrusion structure to continue extruding the hydrogel material, further pushing the flow channel layer forward, and the hydrogel and the cell spheres are extruded along the path in the flow channel layer (4) and finally reach the outlet position. The outlet can assist the hydrogel to further solidify and extrude long hydrogel fibers, which contain uniformly arranged cell sphere aggregates.

[0013] Preferably, in step 7), The chip microporous layer contains 330 micropores, the number of which can be added as needed. The use of laser cutting can greatly increase the manufacturing format and is not limited by the size of traditional photolithography molds. The shape of the microporous layer can include circular, elliptical, polygonal or other desired two-dimensional graphics. The chip flow channel layer can customize the required path, and the channel width is not less than the micropore diameter to prevent the cell sphere from falling into the flow channel; The chip material includes silicon, glass, polymethyl methacrylate PMMA, polydimethylsiloxane PDMS, polycarbonate PC, polyethylene terephthalate PET, cyclic olefin copolymer COC, polystyrene PS or epoxy resin and other materials suitable for laser cutting; The thickness of the chip is 1-5 mm, depending on the maximum cutting thickness of laser cutting; the assembled chip is sterilized at 120° high temperature and high pressure or ultraviolet, and the cell suspension added in step 2) contains 10^5-10^7 cells per 1 ml.

[0014] (III) Beneficial effects Compared with the prior art, the present invention provides a biological 3D printing nozzle for high-throughput cell spheroid preparation and collection and a method for using the same, which has the following beneficial effects: 1. The biological 3D printing nozzle and its use method for high-throughput cell ball preparation and collection do not require complex and expensive equipment (such as a pipetting manipulator device) by operating the microfluidic chip. The micropore method is used to manufacture cell balls with high throughput and support liquid replacement, and solve the problem of difficult collection. The laser cutting processing method is used to greatly reduce labor costs compared to traditional photolithography mold re-molding, and reduce the complexity of the cell ball preparation process. After the cell ball is prepared, it can be directly collected and printed to avoid contamination during the removal process.

[0015] 2. The biological 3D printing nozzle and its use method for high-throughput cell spheroid preparation and collection are expected to promote the widespread application of three-dimensional cell spheroid models in tissue engineering through the advantages of automation, low cost, and integrated spheroid collection and printing. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the decomposition of each layer of the chip of the present invention; Figure 2 A top view of the chip of the present invention after the various layers are combined; Figure 3 This is a schematic diagram of using the chip of the present invention as a nozzle of a biological 3D printer; Figure 4 A flow chart is used for the present invention.

[0017] exist Figures 1 to 4 Middle: 1. Top layer; 2. Culture medium layer; 3. Microporous layer; 4. Flow channel layer; 5. Bottom layer; 6. Flow channel layer sample port; 7. Culture medium layer sample port; 8. Coaxial needle; 9. Ordinary needle; 10. Extruder; 11. Three-axis biological 3D printer. DETAILED DESCRIPTION

[0018] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Embodiment 1: A biological 3D printing nozzle for high-throughput cell ball preparation and collection, the nozzle includes a microfluidic chip made of PDMS material, and the chip is made by laser cutting from a total of 5 layers of PDMS, including a top layer 1, a culture liquid layer 2, a microporous layer 2, a flow channel layer 4 and a bottom layer 5. The top layer 1 of the chip is provided with at least 3 sample injection ports, which are connected to the culture liquid layer 2, and the microporous layer 2 of the chip includes a plurality of displayed micropores. The flow channel layer 4 of the chip corresponds to the position of the bottom of the micropore, forming a continuously curved channel, and the tip at the outlet can be connected to an ordinary needle 9 and a coaxial needle 8. The entire chip can be clamped to a general 3-axis biological 3D printer, and the extrusion power is provided by a micro pump or an air pump or other power source. The specific steps are as follows: Step 1) Chip pretreatment: aligning the various layers of the chip for plasma bonding; Step 2) Adding samples: Add the cell suspension from the sample adding port, and the cell suspension flows through the culture liquid layer 2. As the suspension is slowly added, it flows through the entire culture liquid layer 2, and stops when the cell suspension flows to another sample adding port; Step 3) Horizontal standing: After the sample is added, the chip is placed on a horizontal surface and allowed to stand. Some cells fall into the micropores due to gravity. The micropores are through holes, but due to capillary force, the suspension will not flow directly to the next flow channel layer 4, but will form a U-shaped hanging drop below the micropores. Since the PDMS surface is hydrophobic, cells are not easy to adhere to the micropore surface, which causes the cells to cling to each other to form spheres. Step 4) The chip is moved into a cell culture incubator and placed horizontally for 6-24 hours, then the excess cell suspension in the culture medium layer 2 is removed, and new liquid culture medium is added. The culture medium is fused with the cell spheres in the microwells, so that the cell spheres in each microwell can obtain nutrients. The cells are continued to be placed horizontally and cultured until compact cell spheres are formed. The spheroid printing process specifically includes the following steps: Step 5) Collect the cell spheres. The next layer of the microporous layer 2 of the chip is the flow channel layer 4. Add bioprinting materials (such as hydrogel materials such as Gelma and sodium alginate) to the inlet of the flow channel layer 4. Before the hydrogel is solidified, normally pass it into the entire flow channel layer 4 and let it stand for a period of time. The liquid phase material is mixed with the cell spheres and culture fluid in the microporous layer 2. Under the influence of gravity, the cell spheres fall to the flow channel layer 4. Step 6) Extruding the cell spheres for printing. The sample injection port is combined with the biological 3D printing extrusion structure to continue extruding the hydrogel material, further pushing the flow channel layer 4 forward, and the hydrogel and the cell spheres are extruded along the path in the flow channel layer 4, and finally reach the outlet position. The outlet can assist the hydrogel to be further cured (such as UV curing or coaxial needle 8 adding a crosslinking agent for curing), and extrude long hydrogel fibers, which contain uniformly arranged cell sphere aggregates; Step 7) The chip is clamped on the three-axis biological 3D printer 11, and the printer performs path movement. The extruder 10 provides extrusion power. The chip works as the nozzle of the printer.

[0020] Embodiment 2 This embodiment provides a biological 3D printing nozzle for high-throughput cell spheroid preparation and collection.

[0021] The nozzle is a microfluidic chip made of PDMS material. The chip microporous layer 2 contains 330 micropores. The number of micropores can be added as needed. The use of laser cutting can greatly increase the manufacturing format and is not limited by the size of traditional photolithography molds. The shape of the microporous layer 2 can include circular, elliptical, polygonal or other desired two-dimensional graphics. The chip flow channel layer 4 can customize the required path, and the channel width is not less than the micropore diameter to prevent the cell ball from falling into the flow channel.

[0022] The beneficial effects of the present invention are: 1. The biological 3D printing nozzle and its use method for high-throughput cell ball preparation and collection do not require complex and expensive equipment (such as a pipetting manipulator device) by operating the microfluidic chip. The micropore method is used to manufacture cell balls with high throughput and support liquid replacement, and solve the problem of difficult collection. The laser cutting processing method is used to greatly reduce labor costs compared to traditional photolithography mold re-molding, and reduce the complexity of the cell ball preparation process. After the cell ball is prepared, it can be directly collected and printed to avoid contamination during the removal process.

[0023] 2. The biological 3D printing nozzle and its use method for high-throughput cell spheroid preparation and collection are expected to promote the widespread application of three-dimensional cell spheroid models in tissue engineering through the advantages of automation, low cost, and integrated spheroid collection and printing.

[0024] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A biological 3D printing nozzle for high-throughput cell spheroid preparation and collection, characterized in that: The nozzle includes a microfluidic chip made of PDMS material. The chip is made of five layers of PDMS, namely a top layer (1), a culture liquid layer (2), a microporous layer (2), a flow channel layer (4) and a bottom layer (5), by laser cutting.

2. A biological 3D printing nozzle for high-throughput cell spheroid preparation and collection according to claim 1, characterized in that: The top layer (1) of the chip is provided with at least three sample addition ports, which are connected to the culture liquid layer (2); the microporous layer (2) of the chip comprises a plurality of display micropores.

3. A biological 3D printing nozzle for high-throughput cell spheroid preparation and collection according to claim 1, characterized in that: The chip flow channel layer (4) corresponds to the position of the bottom of the micropore, forming a continuously curved channel, the tip of the outlet can be connected to a common needle (9) and a coaxial needle (8), and the entire chip can be clamped to a universal 3-axis biological 3D printer, and the extrusion power is provided by a micro pump.

4. A method for using a biological 3D printing nozzle for high-throughput cell spheroid preparation and collection, characterized in that: The specific steps include: Step 1) Chip pretreatment: aligning the various layers of the chip for plasma bonding; Step 2) adding samples: adding the cell suspension from the sample adding port, and the cell suspension flows through the culture liquid layer (2). As the suspension is slowly added, it flows through the entire culture liquid layer (2), and stops when the cell suspension flows to another sample adding port; Step 3) Horizontal standing: After the sample is added, the chip is placed on a horizontal surface and allowed to stand. Some cells fall into the micropores due to gravity. The micropores are through holes, but due to capillary force, the suspension will not flow directly to the next flow channel layer (4), but will form a U-shaped hanging drop below the micropores. Since the PDMS surface is hydrophobic, it is difficult for cells to adhere to the micropore surface, which causes the cells to cling to each other to form spheres. Step 4) The chip is moved into a cell culture incubator and placed horizontally for 6-24 hours, then the excess cell suspension in the culture medium layer (2) is removed and new liquid culture medium is added. The culture medium is fused with the cell spheres in the microwells, so that the cell spheres in each microwell can obtain nutrients. The cells are continued to be placed horizontally and cultured until compact cell spheres are formed. The spheroid printing process specifically includes the following steps: Step 5) collecting the cell spheres; Step 6) Extruding the cell spheres for printing; Step 7) The chip is clamped on a three-axis biological 3D printer (11), and the printer performs path movement. The extruder (10) provides extrusion power, and the chip works as a nozzle of the printer.

5. The method for using a biological 3D printing nozzle for high-throughput cell spheroid preparation and collection according to claim 4, characterized in that: In step 5), the next layer of the chip microporous layer (2) is the flow channel layer (4), and the bioprinting material is added to the sample inlet of the flow channel layer (4), and is normally passed into the entire flow channel layer (4) before the hydrogel is solidified, and is left to stand for a period of time. The liquid phase material is mixed with the cell balls and culture fluid in the microporous layer (2), and the cell balls fall into the flow channel layer (4) under the influence of gravity.

6. The method for using a biological 3D printing nozzle for high-throughput cell spheroid preparation and collection according to claim 4, characterized in that: In step 6), the sample injection port is combined with the biological 3D printing extrusion structure to continue to extrude the hydrogel material, further pushing the flow channel layer (4) forward, and the hydrogel and the cell spheres are extruded along the path in the flow channel layer (4) and finally reach the outlet position. The outlet can assist the hydrogel to further solidify and extrude long hydrogel fibers containing uniformly arranged cell sphere aggregates.

7. The method for using a biological 3D printing nozzle for high-throughput cell spheroid preparation and collection according to claim 4, characterized in that: In step 7), The chip microporous layer (2) comprises 330 microporous holes, the number of which can be increased as needed. The manufacturing area can be greatly increased by laser cutting, and is not limited by the size of traditional photolithography molds. The shape of the microporous layer (2) can include a circle, an ellipse, a polygon or any other desired two-dimensional shape. The chip flow channel layer (4) can customize the required path, and the channel width is not less than the micropore diameter to prevent the cell spheres from falling into the flow channel; The chip material includes silicon, glass, polymethyl methacrylate PMMA, polydimethylsiloxane PDMS, polycarbonate PC, polyethylene terephthalate PET, cyclic olefin copolymer COC, polystyrene PS or epoxy resin and other materials suitable for laser cutting; The thickness of the chip is 1-5 mm, depending on the maximum cutting thickness of laser cutting; the assembled chip is sterilized at 120° high temperature and high pressure or ultraviolet, and the cell suspension added in step 2) contains 10^5-10^7 cells per 1 ml.

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