A multi-cell controllable biological 3D printer nozzle and method

Abstract

The present invention relates to a multi-cell controllable biological 3D printer nozzle and method, the nozzle includes an extrusion tube, the first end of which is connected to a coaxial needle tube; the second end of which penetrates at least two cell liquid tanks in sequence, and the penetration position is at the bottom of each cell liquid tank, and the top of the corresponding penetration section on the extrusion tube is a notch; at least two cell liquid tanks are used to contain different types of cell balls; a conveying wheel is rotatably arranged in each cell liquid tank, and a plurality of conveying grooves are evenly spaced on the outer circumference of the conveying wheel, and the shape of the conveying grooves matches the notch at the top of the extrusion tube to form a conveying channel; by controlling the rotation rate of the conveying wheel and the extrusion rate of the hydrogel, the density of the cell balls in the extruded hydrogel is adjusted. The controllable temporal and spatial distribution of a single hydrogel cell is achieved, thereby mapping to the control of the cell distribution of the entire surface and multiple layers of the print. The cross-scale method provides a new solution path for the application of artificial biological tissue organs.

Description

Technical Field

[0001] The present invention relates to the technical field of organ manufacturing 3D printing, and more specifically to a multi-cell controllable biological 3D printer nozzle and method. Background Art

[0002] Extrusion-based bio-3D printing technology, during the printing process, the bio-ink is usually composed of a cell-laden hydrogel with shear-thinning properties. When the bio-ink passes through the nozzle, it is affected by shear forces, resulting in a more fluid-like behavior as the viscosity of the material decreases. The shear stress and low viscosity are maintained until the extrusion point, at which time the bio-ink rapidly increases in viscosity and quickly gels, thereby stabilizing the cylindrical fiber shape.

[0003] Cell spheres are tiny spheres produced by encapsulating proteins or cells in hydrogels. The size can range from 10um to 500um and can be produced by microfluidics, centrifugal manufacturing, template manufacturing, electrostatic spraying and other technologies. The purpose of the above technologies is to produce cell spheres with the most uniform size possible to serve organoid technology or biochips. However, the arrangement of cell spheres is difficult to control, and the efficiency of micro-nano manipulation to absorb the arrangement of cell spheres is low. Therefore, there is a lack of a biological 3D printing technology solution that can efficiently control the distribution of cell spheres on demand and control the density of cell spheres. The currently formed artificial biological tissue construction methods can be mainly divided into two categories: "bottom-up" and "top-down". The former is representative of the combination of micro-nano manufacturing, microfluidics, and micro-nano manipulation technologies to construct biological microtissues containing multicellular aggregates. The latter is a biological structure constructed by directly or indirectly controlling the assembly of cells, growth factors and matrix materials using biological manufacturing methods represented by biological 3D printing. However, although biological microtissues have evolved from two dimensions to three and four dimensions, and their components have evolved from single cells to a controllable distribution of multiple cells and matrix materials, they are still limited by the limitations of assembly technology in terms of environmental adaptability, flexibility, controllability, repeatability, and assembly efficiency. Artificial tissue organs obtained by bottom-up assembly of microtissues often cannot break through size limitations. At present, the development of manufacturing precision and active regulation of the cell survival microenvironment is still imperfect. As the size of the tissue increases, the complexity also increases significantly, which makes the problem of effective functional bionics extremely difficult, and is also an important problem that currently plagues the application of artificial biological tissue organs.

[0004] Therefore, how to provide a multi-cell controllable biological 3D printer nozzle and method is a problem that technical personnel in this field urgently need to solve. Summary of the invention

[0005] To this end, one purpose of the present invention is to propose a multi-cell controllable biological 3D printer nozzle to solve the problems that the existing hydrogel can only arrange and distribute single cell balls, the density and spacing of the cell balls are complex to control, and the repeatability of biological 3D printing is low.

[0006] The present invention discloses a multi-cell controllable biological 3D printer nozzle, comprising:

[0007] An extrusion tube, the extrusion tube is cylindrical, a first end of which is connected to a coaxial needle tube and is used to receive the pre-cured hydrogel;

[0008] Cell liquid tanks, the second end of the extrusion tube sequentially penetrates at least two of the cell liquid tanks, and the penetration position is at the bottom of each of the cell liquid tanks, and the top of the corresponding penetration section on the extrusion tube is a notch; at least two of the cell liquid tanks are used to contain different types of cell spheres;

[0009] A conveying wheel, wherein one conveying wheel is rotatably arranged in each of the cell liquid tanks, and a plurality of conveying grooves are evenly spaced on the outer circumference of each of the conveying wheels, and the shape of the conveying grooves matches the notch at the top of the extrusion tube to form a conveying channel;

[0010] The density of the cell spheres in the extruded hydrogel is adjusted by controlling the rotation rate of the conveying wheel and the extrusion rate of the hydrogel.

[0011] Furthermore, the gap between the cell liquid tank wall and the conveying wheel is smaller than the diameter of the cell ball.

[0012] Furthermore, the outer circle of the gear teeth formed between adjacent conveying grooves is in an arc transition.

[0013] Furthermore, at least two of the cell liquid tanks are arranged in parallel along the extrusion tube.

[0014] Furthermore, the rotation speed of the conveying wheel is driven by a driving device mechanism.

[0015] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a multi-cell controllable biological 3D printer nozzle, which has the following beneficial effects:

[0016] 1. The gel microspheres or cell spheres are transported from the cell tank to the bottom transport channel through a transport wheel, and embedded in the pre-extruded post-cured hydrogel fibers in the transport channel, so that the cell spheres are finally arranged in an orderly and controllable manner in the extruded single hydrogel. Thereby, the distribution of cells on demand and the cell density are controlled, thereby affecting the cell growth environment, so that the extrusion-type biological 3D printing improves repeatability, has a simple structure, and is easy to control. The present invention carries more than two cell tanks, and realizes multi-cellular biological printing of a single hydrogel. The present invention combines micro-nano manufacturing with microfluidics, biological 3D printing, and micro-nano operation technologies with tissue engineering and regenerative medicine, and integrates technical features with different manufacturing and manipulation capabilities, so as to realize the orderly arrangement of cell spheres on demand, control cell distribution and density, and improve the repeatability of biological printing.

[0017] 2. By controlling the hydrogel extrusion rate and the rotation rate of the conveying wheel, the arrangement order and the distance between multiple cell spheres can be controlled.

[0018] 3. The present invention realizes the controllable temporal and spatial distribution of a single hydrogel cell, thereby mapping to the control of cell distribution on the entire printing surface and multiple layers.

[0019] 4. The present invention realizes the integration of the nozzle, and the front end can be used by connecting the traditional coaxial needle tube.

[0020] Another object of the present invention is to provide a control method for a nozzle of a multi-cell controllable biological 3D printer, comprising the following steps:

[0021] S1. Add different types of prepared spheroid suspensions into different spheroid liquid tanks to ensure that the spheroid suspensions completely submerge the conveying wheel;

[0022] S2, connecting the first end of the extruded tube to the coaxial needle tube for receiving the pre-cured hydrogel;

[0023] S3, the conveying wheel in each cell ball liquid tank rotates to the initial position to ensure that the conveying groove of the conveying wheel and the notch of the extrusion tube form a conveying channel;

[0024] S4, the coaxial needle tube extrude the hydrogel, and according to the preset number and type of cell spheres required, the conveying wheel is controlled to rotate at a corresponding speed, and the hydrogel extrusion is stopped at this time, and the cell spheres are sent to the conveying channel by the conveying wheel, and the hydrogel is continued to be extruded, and the hydrogel with arranged cell spheres is pushed to move along the extrusion tube;

[0025] S5, the hydrogel stops extruding, and the delivery wheel in the next cell ball liquid tank delivers the cell ball to the delivery channel;

[0026] S6. Repeat S4 and S5 to complete the extrusion of multiple cell spheres with preset densities.

[0027] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a control method for a multi-cell controllable biological 3D printer nozzle, which has the advantages of multi-cell printing capability, precise controllability, high versatility, etc., and is suitable for extrusion-type biological 3D printing and organoid manufacturing. Micro-nano manufacturing is combined with microfluidics, biological 3D printing, and micro-nano operation technologies with tissue engineering and regenerative medicine, and technical features with different manufacturing and manipulation capabilities are integrated to achieve orderly arrangement of gel microspheres or cell spheres on demand, control cell distribution and density, and improve bioprinting repeatability. The cross-scale assembly method provides a new solution path for the application of artificial biological tissues and organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0029] Figure 1 The accompanying drawing is a schematic structural diagram of a multi-cell controllable biological 3D printer nozzle provided by the present invention;

[0030] Figure 2 The accompanying drawing is a side cross-sectional view of a nozzle of a multi-cell controllable biological 3D printer provided by the present invention;

[0031] Figure 3 The accompanying drawing shows a schematic diagram of the structure inside the cell tank;

[0032] Figure 4 The accompanying drawings show an embodiment of a conveyor wheel drive;

[0033] Figure 5 The accompanying drawings show schematic diagrams of the extrusion of two cell spheroids;

[0034] Figure 6 The accompanying drawing is a flow chart of a control method for a multi-cell controllable biological 3D printer nozzle provided by the present invention. DETAILED DESCRIPTION

[0035] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0036] The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0037] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0038] Since only a single cell ball is arranged and distributed on a single hydrogel in the prior art, the density control of the cell ball is complicated. In view of this, the embodiment of the present invention discloses a multi-cell controllable biological 3D printer nozzle, see the attached Figure 1-3 ,include:

[0039] An extrusion tube 1, wherein the extrusion tube 1 is cylindrical, and a first end thereof is connected to a coaxial needle tube 4 for receiving the pre-cured hydrogel;

[0040] Cell liquid tank 2, the second end of the extrusion tube 1 sequentially penetrates at least two of the cell liquid tanks 2, and the penetration position is at the bottom of each of the cell liquid tanks 2, and the top of the corresponding penetration section on the extrusion tube 1 is a notch; at least two of the cell liquid tanks 2 are used to contain different types of cell spheres;

[0041] A conveying wheel 3, wherein one conveying wheel 3 is rotatably disposed in each of the cell liquid tanks 2, and a plurality of conveying grooves 31 are evenly spaced on the outer circumference of each of the conveying wheels 3, and the shape of the conveying grooves 31 matches the notch at the top of the extrusion tube 1 to form a conveying channel;

[0042] Thus, by controlling the rotation rate of the conveying wheel 3 and the extrusion rate of the hydrogel, the density distribution of the cell spheres in the extruded hydrogel can be adjusted. The coaxial needle tube is a traditional needle tube on the printer, and its center line is on the same installation straight line as the center line of the extrusion tube. The extrusion rate of the hydrogel can be controlled by the existing gel extrusion device, and the rotation rate of the conveying wheel 3 can be achieved by controlling the rotation of the conveying wheel through a set of mechanisms. At the same time, the present invention has multiple cell liquid tanks 2, which can accommodate different types of cell spheres as needed, realize the same hydrogel for multiple cell spheres, and achieve efficient control of the distribution of cell spheres on demand and control of the density of cell spheres.

[0043] In the above embodiment, the gap between the wall of the cell liquid tank 2 and the conveying wheel 3 is smaller than the diameter of the cell spheroid, thereby ensuring the effective transportation of the cell spheroid.

[0044] Advantageously, the outer circle of the gear teeth formed between adjacent conveying grooves 31 has an arc transition to prevent damage to the cell spheres.

[0045] See attached Figure 1 and 5 In a specific embodiment of the present invention, an embodiment of two cell liquid tanks 2 is provided, and the two cell liquid tanks 2 are arranged in parallel along the extrusion tube 1.

[0046] In other embodiments, the cell liquid tanks 2 may be arranged along the circumferential direction of the extrusion tube 1 according to the number and arrangement space of the cell liquid tanks 2 .

[0047] In a specific embodiment of the present invention, the rotation speed of the conveying wheel 3 is driven by a driving device mechanism. Figure 4 , a schematic diagram of the structure of the driving device mechanism is given. A transmission wheel 5 coaxial with the conveying wheel 3 can be arranged outside the cell tank 2, and the transmission wheel 5 is directly driven by the motor 6. If the installation space is considered, the driving force of the motor 6 can also be used to drive the transmission wheel 5 to rotate through a belt or chain 7 and other structures, thereby synchronously driving the conveying wheel 3. The present invention does not limit the specific structure of the driving device mechanism, as long as the mechanism can realize the control of the synchronous rotation of the conveying wheel 3.

[0048] The present invention also provides a control method for a nozzle of a multi-cell controllable biological 3D printer, comprising the following steps:

[0049] S1. Add different types of prepared spheroid suspensions into different spheroid liquid tanks to ensure that the spheroid suspensions completely immerse the conveying wheel;

[0050] S2, connecting the first end of the extruded tube to the coaxial needle tube for receiving the pre-cured hydrogel;

[0051] S3, the conveying wheel in each cell ball liquid tank rotates to the initial position to ensure that the conveying groove of the conveying wheel and the notch of the extrusion tube form a conveying channel;

[0052] S4, the coaxial needle tube extrude the hydrogel, and according to the preset number and type of cell spheres required, the conveying wheel is controlled to rotate at a corresponding speed, and the hydrogel extrusion is stopped at this time, and the cell spheres are sent to the conveying channel by the conveying wheel, and the hydrogel is continued to be extruded, and the hydrogel with arranged cell spheres is pushed to move along the extrusion tube;

[0053] S5, the hydrogel stops extruding, and the conveying wheel in the next cell ball liquid tank conveys the cell ball to the conveying channel;

[0054] S6. Repeat S4 and S5 to complete the extrusion of multiple cell spheres with preset densities.

[0055] See attached Figure 5 and 6 , two types of cell spheres are used as examples for explanation, where cell sphere 1-reference numeral 8, cell sphere 2-reference numeral 9, and reference numeral 10 is the output hydrogel containing multiple cells. First, prepare the required cell sphere 1 suspension and cell sphere 2 suspension according to printing needs, and then pour the prepared cell sphere suspensions into cell sphere liquid tank No. 1 and cell sphere liquid tank No. 2 respectively, both covering the conveying wheels; at this time, conveying wheels No. 1 and No. 2 return to their positions and remain stationary; determine whether cell sphere 1 needs to be printed according to the printing instructions, if the conveying wheel No. 1 in the cell sphere liquid tank No. 1 rotates, and conveys cell sphere 1 to the conveying channel to embed it in the hydrogel, and then continues to extrude it from the coaxial needle tube through the hydrogel to push out cell sphere 1; next, determine whether the number of cell spheres 1 to be printed is required according to the printing instructions , if not satisfied, continue to repeat the above steps; if satisfied, is it necessary to print cell ball 2, if so, the No. 2 conveying wheel in the No. 2 cell ball liquid tank rotates, and the cell ball 2 is conveyed to the conveying channel, and is embedded with the hydrogel that has been pushed out, and then the hydrogel is continued to be pushed to push out the hydrogel carrying the cell ball 2. If the number meets the requirement, the program ends, if not satisfied, repeat the above steps, and so on. According to actual needs, different types of cell balls are loaded on a single hydrogel, and the temporal and spatial distribution of cells in a single hydrogel is controllable, thereby mapping to the printing of the entire surface and multi-layer cell distribution control. The cross-scale assembly method of the present invention provides a new solution path for the application of artificial biological tissue organs. The method can be applied to 3D bioprinting of artificial organs or tissues.

[0056] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0057] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A multi-cell controllable biological 3D printer nozzle, characterized in that: include: An extrusion tube (1), the extrusion tube (1) being cylindrical, with a first end connected to a coaxial needle tube (4) for receiving the pre-cured hydrogel; Cell liquid tanks (2), the second end of the extrusion tube (1) sequentially penetrates at least two of the cell liquid tanks (2), and the penetration position is located at the bottom of each of the cell liquid tanks (2), and the top of the corresponding penetration section on the extrusion tube (1) is a notch; at least two of the cell liquid tanks (2) are used to contain different types of cell spheres; A conveying wheel (3), wherein one conveying wheel (3) is rotatably disposed in each of the cell liquid tanks (2), and a plurality of conveying grooves (31) are evenly spaced on the outer circumference of each of the conveying wheels (3), and the shape of the conveying grooves (31) matches the notch at the top of the extrusion tube (1) to form a conveying channel; The density of the cell spheres in the extruded hydrogel is adjusted by controlling the rotation rate of the conveying wheel (3) and the extrusion rate of the hydrogel.

2. The multi-cell controllable biological 3D printer nozzle according to claim 1, characterized in that: The gap between the wall of the cell liquid tank (2) and the conveying wheel (3) is smaller than the diameter of the cell ball.

3. The multi-cell controllable biological 3D printer nozzle according to claim 1, characterized in that: The outer circle of the gear teeth formed between adjacent conveying grooves (31) is in an arc transition.

4. The multi-cell controllable biological 3D printer nozzle according to claim 1, characterized in that: At least two of the cell liquid tanks (2) are arranged in parallel along the extrusion tube (1).

5. A multi-cell controllable biological 3D printer nozzle according to any one of claims 1 to 4, characterized in that: The rotation speed of the conveying wheel (3) is driven by a driving device mechanism.

6. A control method for a nozzle of a multi-cell controllable biological 3D printer, characterized in that: The following steps are involved: S1. Add different types of prepared spheroid suspensions into different spheroid liquid tanks to ensure that the spheroid suspensions completely submerge the conveying wheel; S2, connecting the first end of the extruded tube to the coaxial needle tube for receiving the pre-cured hydrogel; S3, the conveying wheel in each cell ball liquid tank rotates to the initial position to ensure that the conveying groove of the conveying wheel and the notch of the extrusion tube form a conveying channel; S4, the coaxial needle tube extrude the hydrogel, and according to the preset number and type of cell spheres required, the conveying wheel is controlled to rotate at a corresponding speed, and the hydrogel extrusion is stopped at this time, and the cell spheres are sent to the conveying channel by the conveying wheel, and the hydrogel is continued to be extruded, and the hydrogel with arranged cell spheres is pushed to move along the extrusion tube; S5, the hydrogel stops extruding, and the delivery wheel in the next cell ball liquid tank delivers the cell ball to the delivery channel; S6. Repeat S4 and S5 to complete the extrusion of multiple cell spheres with preset densities.

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