CN106510898A - Multicomponent three-dimensional organism printing device and method based on multi-channel nozzle - Google Patents

Abstract

The invention relates to a multi-component three-dimensional bioprinting device and method based on a multi-channel nozzle, which includes a multi-channel nozzle, the multi-channel nozzle is arranged on a three-axis stepping motor, and the multi-channel nozzle is formed by a plurality of micro-pipes arranged side by side. One end of all micropipes is aligned to form a nozzle, and the other end is connected to each liquid reservoir through a Teflon tube; each fluid drive device is connected to each liquid reservoir to drive the liquid in each liquid reservoir; terminal control The system controls the three-axis stepping motor, various fluid drive equipment and temperature control devices, so that the multi-channel nozzles move and ink out accordingly, and realize three-dimensional printing on the temperature-controlled substrate. The invention can precisely adjust the ratio of different inks by controlling the sampling speed of each channel, and the printed artificial tissue is closer to the real tissue or organ in terms of structure and cell composition, so that it has more advantages in tissue engineering than existing inks. Broader application prospects of the technology.

Description

A multi-component three-dimensional bioprinting device and method based on a multi-channel nozzle

technical field

The present invention relates to a bioprinting device and method, in particular to a multi-component three-dimensional bioprinting device and method based on multi-channel nozzles.

Background technique

Since the concept of tissue engineering was proposed in the 1980s, its related development has greatly promoted the progress of modern biomedicine. Among them, the development of tissue engineering relies heavily on the construction of tissue scaffolds; however, traditional tissue scaffolds are limited by the existing material molding technology and can only obtain some simple structures. With the rapid development of 3D printing technology, many researchers have used 3D printing technology to assist tissue engineering and further applied it to the field of biomedicine, making significant progress. The cells are placed in the tissue scaffold in situ during the printing process, and after being formed, they are cultured in a certain culture environment to obtain the expected active tissue scaffold. Using this 3D printer that can print biomaterials or cells, researchers can quantitatively print cells in tissue scaffolds.

At present, scholars at home and abroad have successively developed a variety of biological 3D printers with different principles, such as liquid extrusion-curing mode, inkjet mode and UV curing molding. Most of the 3D printing based on the liquid extrusion-solidification mode prints a single cell, or uses multiple nozzles to load and alternately print different cells, which greatly reduces the printing speed. The 3D printing based on the inkjet mode can usually mix a variety of cells, but the requirements for the viscosity and surface tension of the solution are high, and the application is severely limited. However, the 3D bioprinting technology based on ultraviolet light curing has certain limitations on the survival rate of cells due to the certain lethality of ultraviolet light on cells.

Contents of the invention

In view of the above problems, the object of the present invention is to provide a multi-component three-dimensional bioprinting device and method based on a multi-channel nozzle, which is simple and flexible, can precisely control the content of each component, print a three-dimensional gel with a specific pattern, and A characteristic that enables quantitative loading of one or more cells at specific locations on a gel.

To achieve the above object, the present invention adopts the following technical solutions: a multi-component three-dimensional bioprinting device based on multi-channel nozzles, characterized in that it includes multi-channel nozzles, three-axis stepping motors, several liquid reservoirs, several fluid Drive equipment, a temperature-controlled substrate composed of a printing substrate and a temperature control device, and a terminal control system; the multi-channel nozzle is arranged on the three-axis stepping motor, and the multi-channel nozzle is formed by a plurality of micro-pipes arranged side by side One end of all the micro-pipelines is aligned to form a nozzle, and the other end is connected to each of the liquid reservoirs through a Teflon tube; each of the fluid drive devices is connected to each of the liquid reservoirs, and each of the liquid The liquid in the reservoir is driven; the terminal control system controls the three-axis stepping motor, each of the fluid driving devices and the temperature control device, so as to realize three-dimensional printing on the temperature control substrate.

Each micropipe adopts a glass capillary or a stainless steel needle tube.

The specific number of the plurality of micro-pipes is 2-7.

The inner diameter range of each micropipe is 1-5mm.

The connection between each micropipe and the Teflon tube is sealed with paraffin.

The fluid reservoir includes a syringe or a reservoir.

The fluid-driven device is a programmable fluid-driven device, including a syringe pump, a peristaltic pump, an air-driven pump or a pressure-driven pump.

The printing substrate includes glass plate, non-woven fabric, glass or plastic petri dish.

A multi-component three-dimensional bioprinting method based on a multi-channel nozzle, characterized in that it comprises the following steps: 1) setting a multi-channel nozzle, the multi-channel nozzle is formed by a plurality of micro-pipelines arranged side by side, and the end of each micro-pipeline discharges ink 2) Connect the other end of each micropipe to the liquid reservoir through a Teflon tube, and the connection between each micropipe and the Teflon tube is sealed with paraffin wax; 3) Inject the liquid into each liquid reservoir The corresponding printing ink, and in the injected printing ink, at least two kinds of reactive printing inks need to exist to realize the transformation of the printing ink from liquid to gel in a short time; On the stepper motor, adjust the three-axis stepper motor so that the multi-channel nozzles are located above the temperature-controlled substrate, and the distance is less than 1mm; 5) Set the temperature of the temperature-controlled device in the terminal control system so that the temperature of the printed substrate is controlled at -10°C In the range of ~40°C, it is convenient for gel forming and storage; 6) According to the printing requirements, the printing speed and liquid injection speed are set in the terminal control system, and the multi-channel Corresponding movement of the nozzle and ink discharge; 7) The liquid in each liquid reservoir flows through each micropipe and then converges at the nozzle, and the different inks ejected from the nozzle are assisted in forming on the temperature-controlled substrate through physical or chemical interactions, layer by layer Stacking enables 3D printing.

Because the present invention adopts the above technical scheme, it has the following advantages: 1. Since the present invention is provided with multi-channel nozzles, various printing inks or cell culture fluids can be input through multiple micro-pipes, which ensures the printing speed. 2. The present invention injects multiple cells or printing inks into the multi-channel nozzle, and through the coordinated control of the three-axis stepping motor and the liquid drive device, it can precisely control the loading point and loading amount of different cells, so that the printed The three-dimensional scaffold is closer to the real tissue or organ, so it has a wider application prospect in tissue engineering than the existing technology. 3. Since the present invention adopts a variety of reactive printing inks, the preparation method is simple, and each printing ink converges at the multi-channel nozzle and undergoes a physical or chemical reaction, realizing the conversion from liquid to gel state in a short time.

Description of drawings

Figure 1 is a schematic diagram of a multi-component three-dimensional bioprinting device based on a multi-channel nozzle of the present invention;

₂ is a schematic diagram of a three-dimensional printing sodium alginate-CaCl hydrogel device in Example 1 of the present invention;

3 is a schematic diagram of a blue sodium alginate- _CaCl2 gel device for three-dimensional printing in Example 2 of the present invention;

4 is a schematic diagram of a sodium alginate-CaCl ₂ gel device loaded with cells by three-dimensional printing in Example 3 of the present invention.

detailed description

The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments.

As shown in Figure 1, it is a multi-component three-dimensional bioprinting device based on a multi-channel nozzle according to the present invention, which includes a multi-channel nozzle 1, a three-axis stepper motor 2, several liquid reservoirs 3, several fluid-driven devices 4, and The temperature control base 5 and the terminal control system 6 constituted by the base and the temperature control device. Wherein, the multi-channel nozzle 1 is arranged on the three-axis stepping motor 2, which is formed by a plurality of micro-pipes 11 arranged side by side, and one end of each micro-pipe 11 is aligned to form a multi-channel nozzle 12, and the other ends pass through a Teflon tube. 13 is connected with each syringe 3. Each fluid driving device 4 is connected with each syringe 3 to drive the liquid stored in each syringe 3 . The terminal control system 6 controls the three-axis stepping motor 2 , each fluid drive device 4 and the temperature control device, so that the multi-channel nozzle 12 moves and inks out accordingly, realizing three-dimensional printing on the temperature-controlled substrate 5 .

In the above embodiments, the micro-pipe 11 is made of glass capillary or stainless steel needle tube, and the specific number of the plurality of micro-pipes 11 is 2-7.

In the above-mentioned embodiments, the inner diameter of the micropipe 11 ranges from 1 to 5 mm.

In the above-mentioned embodiments, the connection between each micropipe and the Teflon tube is sealed with paraffin wax 14 to prevent leakage.

In the above embodiments, the liquid storage 3 includes a syringe or a liquid storage pool.

In the above-mentioned embodiments, each fluid-driven device 4 is a programmable fluid-driven device, including a syringe pump, a peristaltic pump, a pneumatic-driven pump or a pressure-driven pump.

In each of the above-mentioned embodiments, the printing substrates include glass plates, non-woven fabrics, glass or plastic petri dishes; and the above four kinds of printing substrates that are surface-treated by physical or chemical methods, such as plasma treatment to obtain a hydrophilic surface; chemical modification means Use chemical means to carry out hydrophilic and hydrophobic treatment on the printing substrate or change the surface adhesion performance.

Based on the above multi-component three-dimensional bioprinting device based on multi-channel nozzles, the present invention also provides a multi-component three-dimensional bioprinting method based on multi-channel nozzles, comprising the following steps:

1) A multi-channel nozzle 1 is provided. The multi-channel nozzle is formed by a plurality of micro-pipes 11 arranged side by side, and the ink outlets at the ends of the micro-pipes 11 are aligned to form nozzles 12 .

2) The other ends of the micro-pipes 11 are respectively connected to the liquid reservoir 3 through the Teflon tube 13, and the joints between the micro-pipes 11 and the Teflon tube 13 are sealed with paraffin wax 14 to prevent liquid leakage. (In the present invention, only 3 micropipes are used as an example to introduce, but not limited thereto)

3) Inject corresponding printing inks into each liquid reservoir 3, and at least two reactive printing inks must exist in the injected printing inks, so as to realize the transformation of the printing inks from liquid to gel in a short time.

Printing inks include clay inks, hydroxyapatite graphite water, biogel inks, ion inks, and cellular inks. Among them, clay ink, hydroxyapatite graphite water, biogel ink, and ion ink can react with each other to form a gel, and in addition to biogel ink and ion ink, other types of ink can be mixed with each other in any proportion to obtain a new ink.

Wherein, the clay ink refers to the aqueous dispersion liquid of nano-clay particles, and its mass fraction is in the range of 0.1% to 10%. The hydroxyapatite graphite water refers to the aqueous phase dispersion liquid of hydroxyapatite nanoparticles, and its mass fraction is in the range of 0.1% to 10%. Biogel inks refer to aqueous solutions of a range of commonly used biocompatible materials, including agarose, sodium alginate, hyaluronic acid, chitosan, collagen, fibrin, gelatin, dextran, polyacrylamide, cellulose, For polyethylene glycol, polypeptide and the derivatives of the above 12 kinds of substances, the mass fraction of the solute in the aqueous solution is in the range of 0.1% to 40%. The ion ink includes an aqueous solution of calcium chloride (CaCl ₂ ) and magnesium chloride (MgCl ₂ ), the mass fraction of which is in the range of 0.5% to 20%. Cell ink includes cells and suspensions, and its cell types include mesenchymal stem cells, induced pluripotent stem cells, fibroblasts, renal endothelial cells, mesangial cells, vascular endothelial cells, vascular epithelial cells, etc., and the suspension contains corresponding cells Culture medium, cell culture medium includes various substances related to cell culture, division and differentiation.

4) Fix the multi-channel nozzle 1 on the three-axis stepping motor 2, adjust the three-axis stepping motor 2 so that the nozzles 12 are located above the temperature-controlled substrate 5, and the distance is less than 1 mm.

5) Set the temperature of the temperature control device in the terminal control system 6, so that the temperature of the printing substrate is controlled within the range of -10°C to 40°C, which is convenient for gel formation and storage.

6) According to the printing requirements, the printing speed and liquid injection speed are set in the terminal control system 6, and the multi-channel nozzle 1 moves and inks out through the coordinated control of the three-axis stepping motor 2 and each fluid drive device 4 .

The terminal control system 6 can set up the three-axis stepping motor 2 and each liquid drive device 4 to precisely control the ejection of printing ink or cell suspension in each liquid reservoir 3, thereby realizing the loading points of different cells and The loading capacity makes the printed three-dimensional scaffold closer to the real tissue or organ.

7) The liquid in each liquid reservoir 3 flows through each micropipe 11 and then converges at the nozzle 12, and the different inks ejected from the nozzle 12 are assisted in forming on the temperature-controlled substrate 5 through physical or chemical interactions, and are stacked layer by layer. 3D printing.

In order to further illustrate the present invention, the multi-component three-dimensional bioprinting method based on multi-channel nozzles of the present invention will be specifically described below in conjunction with examples, but they should not be construed as limiting the protection scope of the present invention.

Example 1

As shown in FIG. 2 , it is a schematic diagram of the device of the three-channel nozzle provided by Embodiment 1 of the present invention. In this embodiment, three glass capillaries are arranged side by side to form a multi-channel nozzle 1 , and the inner diameters of the glass capillaries are all 500 μm. The top of each glass capillary is connected with a liquid reservoir 3 (not shown in FIG. 2 ) with a Teflon tube 13 and sealed with paraffin wax 14 . Ink No. 1 selected in this embodiment is a 5% sodium alginate solution; ink No. 2 and No. 3 are both calcium chloride (CaCl ₂ ) aqueous solutions with a mass fraction of 5%.

After connecting the liquid channels respectively, the multi-channel nozzle 1 is fixed on the three-axis stepping motor 2, and the z-axis height is adjusted so that the distance between the multi-channel nozzle 1 and the printing substrate is less than 1mm. Start the printing program, set the printing speed to 20mm/s, and the liquid injection speed to 0.2mL/min, then a piece of hydrogel with a grid spacing of 3mm can be printed, and the system of sodium alginate and calcium chloride can be used to print The resulting hydrogel has a resolution of less than 500 μm.

Example 2

As shown in Figure 3, change the No. 3 ink in Example 1 to ordinary blue ink, print with other parameters unchanged, and a piece of blue gel can be printed, similarly, multiple gels can be mixed at different positions Different inks to print colored gels.

Example 3

As shown in Figure 4, the No. 3 ink in Example 1 was replaced with rat aortic endothelial cell suspension, and printing was performed with the same parameters, and a gel evenly loaded with cells could be obtained.

Embodiment 4～10

Change the three kinds of inks in Example 1 into different components, wherein No. 3 ink can be selected arbitrarily, No. 1 and No. 2 inks can be replaced with the inks in the following table 1, and print with the same parameters as in Example 1. Gels can be printed and molded smoothly.

Table 1:

Example ink 1 ink 2 4 0.5～10% sodium alginate 5~40% gelatin 5 0.5～10% sodium alginate 1～5% Chitosan 6 0.5～10% sodium alginate 0.5～20% _MgCl2 7 0.5～10% sodium hyaluronate 0.5～20% _CaCl2 8 0.5～10% sodium hyaluronate 0.5～20% _MgCl2 9 0.5～10% sodium hyaluronate 5~40% gelatin 10 0.5～10% sodium hyaluronate 1～5% Chitosan

The above-mentioned embodiments are only used to illustrate the present invention, wherein the structure, connection mode and manufacturing process of each component can be changed to some extent, and any equivalent transformation and improvement carried out on the basis of the technical solution of the present invention should not excluded from the protection scope of the present invention.

Claims (9)

1. a kind of multicomponent three dimensional biological printing equipment based on multi-channel nozzle, it is characterised in that：It include multi-channel nozzle, Three-axis stepping motor, some liquid memories, some fluid drives equipment, the temperature control base being made up of printed substrates and attemperating unit Bottom and terminal control system；

The multi-channel nozzle is arranged on the three-axis stepping motor, and the multi-channel nozzle is set side by side by multiple microchannels Put and form, one end alignment of all microchannels constitutes nozzle, and the other end is stored up with each liquid by teflon pipe respectively Storage connects；Each fluid drives equipment is connected with each liquid memory, to the liquid in each liquid memory It is driven；The terminal control system is filled to the three-axis stepping motor, each fluid drives equipment and the temperature control Put and be controlled, realize the 3 D-printing in the temperature control substrate.

2. a kind of multicomponent three dimensional biological printing equipment based on multi-channel nozzle as claimed in claim 1, it is characterised in that： Each microchannel adopts capillary glass tube or rustless steel needle tubing.

3. a kind of multicomponent three dimensional biological printing equipment based on multi-channel nozzle as claimed in claim 1, it is characterised in that： The particular number of the plurality of microchannel is 2～7.

4. a kind of multicomponent three dimensional biological printing equipment based on multi-channel nozzle as claimed in claim 1, it is characterised in that： The inside diameter ranges of each microchannel are 1～5mm.

5. a kind of multicomponent three dimensional biological printing equipment based on multi-channel nozzle as claimed in claim 1, it is characterised in that： Each microchannel is sealed using paraffin with the teflon pipe junction.

6. a kind of multicomponent three dimensional biological printing equipment based on multi-channel nozzle as claimed in claim 1, it is characterised in that： The liquid memory includes syringe or liquid storage tank.

7. a kind of multicomponent three dimensional biological printing equipment based on multi-channel nozzle as claimed in claim 1, it is characterised in that： The fluid drives equipment for can programme controlled fluid drives equipment, including syringe pump, peristaltic pump, air pressure transfer tube or pressure Transfer tube.

8. a kind of multicomponent three dimensional biological printing equipment based on multi-channel nozzle as claimed in claim 1, it is characterised in that： The printed substrates include glass plate, non-woven fabrics, glass or plastic culture dish.

9. a kind of multicomponent three dimensional biological Method of printing based on multi-channel nozzle, is characterised by which comprises the following steps：

1) multi-channel nozzle is set, and the multi-channel nozzle is set up in parallel by multiple microchannels and is formed, and each microchannel end is out of ink Mouth alignment constitutes nozzle；

2) respectively each microchannel other end is connected with liquid memory by teflon pipe, and each microchannel is connected with teflon pipe The place of connecing is sealed using paraffin；

3) inject in corresponding marking ink, and the marking ink for injecting in each liquid memory, need at least there are two kinds of tools There is the marking ink of reactivity, to realize transformation of the marking ink by liquid to gel in the short time；

4) multi-channel nozzle is fixedly installed on three-axis stepping motor, adjusts three-axis stepping motor and multi-channel nozzle is located at The top of temperature control substrate, and it is smaller than 1mm；

5) temperature of attemperating unit is set in terminal control system so that printed substrates temperature control is in -10 DEG C～40 DEG C models In enclosing, it is easy to gel forming and preservation；

6) according to requirement is printed, print speed and liquid infusion speed are set, by three axle steppings electricity in terminal control system The Collaborative Control of machine and each fluid drives equipment so that multi-channel nozzle corresponding sports with it is out of ink；

7) converge at nozzle after the liquid in each liquid memory flows through each microchannel, the different inks sprayed by nozzle pass through Physically or chemically interact the assistant formation in temperature control substrate, and successively stacking realizes 3 D-printing.