CN112776325B - Three-dimensional ultrasonic array support-free cell printing device and printing process thereof - Google Patents

Abstract

A three-dimensional ultrasonic array scaffoldless cell printing device and a printing process thereof, including a cell transport mechanism, a printing platform, a platform driving mechanism, a transport driving mechanism and a three-dimensional ultrasound array micro-potential well generation system, the printing platform is mounted on the platform driving mechanism, and the cell transport mechanism Installed on the conveying drive mechanism, the three-dimensional ultrasonic array micro-potential well generation system is connected to the printing platform, and a plane sound pressure micro-potential well is formed in the printing platform, and a plurality of plane sound pressure micro-potential wells with different heights are formed on the printing platform to come Realize the positioning of cells that need to be printed at different heights, and build a three-dimensional system of cells by constructing a three-dimensional array of acoustic pressure potential wells, which has better controllability and can be used for three-dimensional printing of cells in various cells and environments. Excellent universality.

Description

Three-dimensional ultrasonic array scaffold-free cell printing device and printing process

technical field

The invention belongs to the fields of biological manufacturing and cell printing, and particularly relates to a three-dimensional ultrasonic array scaffold-free cell printing device and a printing process thereof.

Background technique

Cell/Bio 3D printing 3D assembly of cells by gradually depositing living cells, extracellular matrix, biological factors and necessary biological materials, constructing a biologically active 3D multicellular system in vitro, and then culturing and multiplying to achieve tissue regeneration and organ repair It is an effective way to solve the dilemma of traditional tissue engineering, which is of great significance to the development of life science and biomedicine.

In cell printing technology, the first problem to be solved is how to accurately deposit a set number of specific cells to a preset position in three-dimensional space, that is, how to build a three-dimensional system of cells on demand, and facilitate the nutrient delivery for cell culture and reproduction, eventually form an organization. In order to achieve the above goals, existing cell 3D printing usually mixes scaffold materials represented by hydrogels with cell fluids to prepare bioinks for printing, and the hydrogels solidify to become cell-attached biological scaffolds. However, as a carrier for further cell proliferation, bioscaffolds face numerous challenges, such as interference of intercellular communication, uneven cell attachment, effects of scaffold degradation, and poor precision of scaffold materials. However, the current scaffold-free cell printing technology avoids mixing the scaffold material into the cell fluid to make bio-ink for printing, and has significant advantages in intercellular communication. However, the existing scaffold-free cell printing technologies still require the use of agar rods as physical supports during the cell printing process. Therefore, it is of great significance to study the scaffold-free and unsupported cell 3D printing technology.

SUMMARY OF THE INVENTION

In view of the above deficiencies, the technical problem to be solved by the present invention is to provide a three-dimensional ultrasonic array scaffoldless cell printing device and a printing process thereof. A three-dimensional multicellular system with biological activity is constructed in vitro, and then cultured and propagated to achieve the purpose of tissue regeneration and organ repair.

In order to solve the above technical problems, the technical scheme adopted in the present invention is,

A three-dimensional ultrasonic array scaffoldless cell printing device includes a cell transport mechanism, a printing platform, a platform drive mechanism, a transport drive mechanism and a three-dimensional ultrasound array micro-potential well generation system. The printing platform is installed on the platform drive mechanism, and the cell transport mechanism is installed on the transport drive In terms of mechanism, the three-dimensional ultrasonic array micro-potential well generating system is connected with the printing platform, and a plane acoustic pressure micro-potential well array is formed in the printing platform.

Further, the three-dimensional ultrasonic array micro-potential well generation system includes a signal generator, a power amplifier and a piezoelectric transducer array, the piezoelectric transducer array is fixedly installed on the printing platform, and the power amplifier is connected to the piezoelectric transducer array, The signal generator is connected with the power amplifier.

Further, the printing platform includes a chassis and a polygonal resonant cavity, the chassis is connected with the platform driving mechanism, the polygonal resonant cavity is fixedly installed on the chassis, the piezoelectric transducer array is fixedly installed on the inner wall of the polygonal resonant cavity, and is formed in the polygonal resonant cavity. Planar acoustic pressure micro-potential well array.

Further, piezoelectric transducer arrays are installed on the inner wall of the polygonal resonator, and the piezoelectric transducer arrays are arranged at equal distances in the height direction to form a plurality of sound pressure micro-potential wells of different heights.

Further, the cell delivery mechanism includes a micro-transport nozzle, a pulse-actuating device, a nozzle clip, and a shelf plate, the micro-transport nozzle is fixedly installed on the shelf plate through the nozzle clip, and the pulse-actuating device is fixedly installed on the shelf plate, and is connected with the micro-transporter. The upper part of the nozzle is connected.

Further, a spray head is installed at the lower end of the micro-transport nozzle, and a cell counter is fixedly installed between the micro-transport nozzle and the spray head.

Further, a microscope is fixedly mounted on the frame plate, and the microscope is arranged toward the nozzle.

Further, the platform driving mechanism includes a base plate, a lifting bracket, a lifting screw slider assembly and a lifting motor, the lifting bracket is fixedly installed on the base plate, the lifting screw slider assembly is rotatably installed on the lifting bracket, the lifting motor and the lifting wire are rotatably installed. Rod slider assembly connection.

Further, the conveying driving mechanism includes an X-axis driving member, a Y-axis driving member and a support column, the supporting column is fixedly installed on the base plate, the Y-axis driving member is fixedly installed on the supporting column, and the X-axis driving member is fixedly installed on the Y-axis driving member. On the part, the cell transport mechanism is installed on the X-axis drive part.

The printing process of the above-mentioned three-dimensional ultrasonic array scaffold-free cell printing device includes the following steps:

S1: Sterilize the printing device, keep the temperature constant and appropriate, inject the culture medium into the polygonal resonance cavity, and inject the cell suspension into the micro-nozzle;

S2: According to the pre-designed model, the piezoelectric transducer array works, and the planar array sound pressure micro-potential well is excited on demand in the bottommost layer of the polygonal resonator;

S3: According to the pre-designed model, the control platform drive mechanism moves to make the nozzle reach the specific height required for printing the polygon resonant cavity model, and then controls the movement of the conveying drive mechanism to move the micro-nozzle to the designated sound pressure micro-potential well position;

S4: According to the needs of the model, the cell transport mechanism transports a specific number of cells into the corresponding planar array sound pressure micro-potential well, and the delivered cells are trapped in the sound pressure potential well under the action of the acoustic restoring force in the sound pressure potential well. , to complete the printing of cells in a single potential well, and so on, until the printing of cells in all acoustic potential wells in a layer of planes is completed;

S5: After all the printing of the first layer is completed, the three-dimensional ultrasonic array micro-potential well generating system excites the second-layer array sound pressure micro-potential well, moves the polygonal cultivation platform downward to continue printing, and repeats the steps S2-S4, so as to By analogy, the printing of three-dimensional system of cells can be realized;

S6: After the printing is completed, the three-dimensional cell system is cultured at a suitable temperature, and the cells divide and grow to finally form a cell tissue.

The beneficial effect of the present invention is,

In the present invention, the cells are manipulated by the acoustic radiation force to realize the construction of a three-dimensional system of the cells, and the acoustic radiation force manipulation of the cells has obvious advantages in terms of universality, biocompatibility and manipulation scale. It has no special requirements for cell type, density and shape, and can be propagated in opaque media. It has stronger applicability and can complete three-dimensional manipulation of cells in various environments and environments. The invention adopts the method of constructing a three-dimensional array sound pressure potential well to construct a three-dimensional cell system, which has better controllability, can be used for three-dimensional printing of cells in various cells and various environments, and has excellent universality.

The invention constructs a three-dimensional array of sound pressure micro-potential wells through array transducers, and uses acoustic radiation force to capture cell particles. One micro-potential well is a cell capture point (node), the nodes are arranged in lines, the lines are arranged in planes, and the planes are superimposed. Adults, thereby constructing a three-dimensional cell system, and then culturing to form a cell tissue. This technology does not require biological scaffolds/supports and has significant advantages in terms of cell density, intercellular communication, and cell growth. And there is no scaffold/support degradation process, no degradation by-products, and cells are more likely to survive and grow. In addition, the cell printing process does not generate high temperature, high pressure, strong electric field and other phenomena, and the survival rate after cell printing is further improved, and the survival rate can be guaranteed to be above 90%.

In the present invention, the technology of cell printing driven by pulse inertial force is adopted. This technology uses the pulse inertial force generated by the pulse actuating device and changes the size of a single pulse to realize the precise delivery of a single cell or a plurality of cells. The precise and controllable delivery ensures the efficient and precise construction of the three-dimensional cell system, which has important potential for in vitro bioengineering applications.

Description of drawings

Figure 1 is a schematic diagram of a three-dimensional ultrasonic array micro-potential well scaffold-free cell 3D printing system.

FIG. 2 is a partial enlarged schematic diagram of a polygonal resonator.

Figures 3 to 5 are schematic diagrams of single-cell and multi-cell printing in a single acoustic potential well.

6 to 9 are schematic diagrams of the principles of the 3D printing process of scaffold-free cells with three-dimensional ultrasonic array micro-potential wells.

Description of drawings: 1. Signal generator; 2. Power amplifier; 3. Observation microscope; 4. Micro-delivery nozzle; 5. Pulse actuating device; 6. Nozzle clip; 7. Shelf plate; 8. Chassis; 9. Ultraviolet sterilization lamp; 12 cell counter; 13 piezoelectric transducer array; 14, polygon resonant cavity; 15, upper computer; 16, spray head.

Detailed ways

The present invention will be further described below with reference to the accompanying drawings.

A three-dimensional ultrasonic array scaffoldless cell printing device includes a cell transport mechanism A, a printing platform B, a platform driving mechanism C, a transport driving mechanism D, and a three-dimensional ultrasonic array micro-potential well generation system E. The printing platform B is installed on the platform driving mechanism C. The cell transport mechanism A is installed on the transport drive mechanism D, and the three-dimensional ultrasonic array micro-potential well generation system E is connected to the printing platform B. A planar acoustic pressure micro-potential well array is formed in the printing platform B, and a plurality of different micro-potential well arrays are formed on the printing platform B. A highly planar acoustic pressure micro-potential well array is used to locate cells that need to be printed at different heights. The three-dimensional system of cells is constructed by constructing a three-dimensional array of acoustic pressure potential wells, which has better controllability and can be used for a variety of cells. , 3D printing of cells in a variety of environments, with excellent universality.

The three-dimensional ultrasonic array micro-potential well generation system includes a signal generator 1, a power amplifier 2 and a piezoelectric transducer array 13, the piezoelectric transducer array 13 is fixedly installed on the printing platform B, and the power amplifier 2 and the piezoelectric transducer The array 13 is connected, the signal generator 1 is connected with the power amplifier 2, the piezoelectric transducer array is fixed on the inner wall of the culture chamber, and is arranged equidistantly in the height direction, and the multiple transducers of each layer are on the same horizontal plane, Thereby, a plurality of plane sound pressure micro-potential well arrays of different heights are formed; the front end of the signal generator 2 is connected with the host computer 15, and the signal generator 2 generates a signal of a specific frequency and a specific waveform under the control of the host computer 15, so The wave signal is amplified into an electrical signal of a specific voltage as required by the power amplifier, and then transmitted to the piezoelectric transducer array of a certain layer of the array piezoelectric transducer array; the piezoelectric transducer array is located in the piezoelectric transducer array. Acoustic waves with specific energy and specific frequency are generated under the drive of electrical signals to form a polygonal plane sound field, and a plane sound pressure micro-potential well array is constructed; the array piezoelectric transducer array forms different piezoelectric transducer arrays by exciting different layers of piezoelectric transducer arrays The plane array sound pressure micro-potential well, the layer-by-layer construction of the plane array sound pressure micro-potential well is superimposed to form a three-dimensional array sound pressure micro-potential well.

The printing platform includes a chassis 8 and a polygonal resonant cavity 14. The chassis 8 is connected to the platform drive mechanism C, the polygonal resonant cavity 14 is fixedly installed on the chassis 8, and the piezoelectric transducer array 13 is fixedly installed on the inner wall of the polygonal resonant cavity 14. A planar acoustic pressure micro-potential well array is formed in the polygonal resonant cavity 14, and transducers of different heights are excited to form a plurality of planar array acoustic pressure potential wells, which are superimposed to form a three-dimensional array of acoustic pressure potential wells.

A piezoelectric transducer array 13 is installed on the inner wall of the polygonal resonator cavity 14, and the piezoelectric transducer arrays 13 are equidistantly arranged in the height direction to form a plurality of sound pressure micro-potential wells of different heights, which are superimposed to form a three-dimensional array sound pressure potential trap.

The cell delivery mechanism A includes a micro-delivery nozzle 4, a pulse-actuating device 5, a nozzle clip 6, and a shelf plate 7. The micro-transport nozzle 4 is fixedly installed on the shelf plate 7 through the nozzle clip 6, and the pulse-actuating device 5 is fixedly installed on the shelf plate. 7, and connected to the upper part of the micro-transport nozzle 4, through the pulse inertial force generated by the pulse actuating device, by changing the size of a single pulse, the precise delivery of a single cell or multiple cells can be achieved, and the precise delivery of cells during the printing process can be achieved. Controllable, ensuring the efficient and accurate construction of the cell three-dimensional system.

A spray head 16 is installed at the lower end of the micro-delivery nozzle 4, and a cell counter 12 is fixedly installed between the micro-delivery nozzle 4 and the spray head 16. When the delivered cells reach a specified number, the delivery is stopped.

A microscope 3 is fixedly installed on the shelf plate 7, the microscope 3 is set towards the nozzle 16, the microscope follows the nozzle movement, and is used to observe the cell position state during printing, and the microscope 3 is connected with the host computer 15, and the host computer 15 is based on the detection feedback of the microscope 3. Control the movement of the 3D platform and the delivery of cells.

The platform driving mechanism C includes a base plate 1-1, a lifting bracket 1-2, a lifting screw slider assembly 1-3 and a lifting motor 1-4. The lifting bracket 1-2 is fixedly installed on the base plate 1-1, and the lifting screw The rod slider assembly 1-3 is rotatably installed on the lifting bracket 1-2, the lifting motor 1-4 is connected with the lifting screw slider assembly 1-3, and the printing platform B is fixedly installed on the lifting screw slider assembly 1-3. On the slider, the height of the printing platform B is adjusted through the platform driving mechanism C.

The conveying driving mechanism D includes an X-axis driving member, a Y-axis driving member and a support column 2-1. The supporting column 2-1 is fixedly installed on the base plate 1-1, and the Y-axis driving member is fixedly installed on the supporting column 1-1. The X-axis driver is fixedly mounted on the Y-axis driver, and the cell transport mechanism is mounted on the X-axis driver, and the plane position of the cell transport mechanism A is adjusted through the X-axis driver and the Y-axis driver.

In some preferred manners, the structures of the X-axis drive member and the Y-axis drive member are consistent with the structure of the platform drive mechanism C, including a bracket, a screw slider assembly and a drive motor, and the screw slider assembly is rotatably installed in the bracket , the drive motor is connected with the screw slider assembly.

In some preferred manners, a cover is provided on the outer cover of the device, and an ultraviolet sterilization lamp 9 is fixedly installed on the cover.

The printing process of the above-mentioned three-dimensional ultrasonic array scaffold-free cell printing device includes the following steps:

S1: Sterilize the printing device, keep the temperature constant and appropriate, inject the culture medium into the polygonal resonance cavity, and inject the cell suspension into the micro-nozzle;

S2: According to the pre-designed model, the piezoelectric transducer array works, and the planar array sound pressure micro-potential well is excited on demand in the bottommost layer of the polygonal resonator;

S3: According to the pre-designed model, the control platform drive mechanism moves to make the nozzle reach the specific height required for printing the polygon resonant cavity model, and then controls the movement of the conveying drive mechanism to move the micro-nozzle to the designated sound pressure micro-potential well position;

S4: According to the needs of the model, the cell transport mechanism transports a specific number of cells into the corresponding planar array sound pressure micro-potential well, and the delivered cells are trapped in the sound pressure potential well under the action of the acoustic restoring force in the sound pressure potential well. , to complete the printing of cells in a single potential well, and so on, until the printing of cells in all acoustic potential wells in a layer of planes is completed;

S5: After all the printing of the first layer is completed, the three-dimensional ultrasonic array micro-potential well generating system excites the second-layer array sound pressure micro-potential well, moves the polygonal cultivation platform downward to continue printing, and repeats the steps S2-S4, so as to By analogy, the printing of three-dimensional system of cells can be realized;

S6: After the printing is completed, the three-dimensional cell system is cultured at a suitable temperature, and the cells divide and grow to finally form a cell tissue.

When printing cells, the cell transport mechanism A outputs single or multiple printed cells, and the printed cells can be single or multiple according to the requirements of the model. When printing cells, the cell fluid concentration is high, and the micro-nozzles contain The concentration of the cell fluid is approximately 10 ⁶ to 10 ⁷ , and the concentration of the cell fluid is adjusted according to the size of the printed cells.

During the printing process, when the nozzle head 16 moves to the designated position of the culture chamber, the nozzle head 16 is not suspended on the acoustic potential well of the array, but in the potential well, and the micro-nozzles directly print cells at the appropriate position in the specific potential well. .

In the present invention, the array ultrasonic transducer is placed on the inner wall of the polygonal resonant cavity, which can be two faces or multiple faces. When placed on multiple faces, the array ultrasonic transducer passes through different faces. The combination of transducers forms different array sound pressure potential wells, and the array ultrasonic transducers can form sound potential wells of different sizes by exciting sound waves of different frequencies, and construct array sound pressure micro-potential wells of different densities. The excitation frequency of the array ultrasonic transducer is MHz level, and the size of the formed potential well is micrometer level.

The above description of the disclosed embodiments enables any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention; thus , the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Although the terms corresponding to the reference numerals in the figures are used more in this paper, the possibility of using other terms is not excluded; these terms are only used to describe and explain the essence of the present invention more conveniently; they are interpreted as any Such additional limitations are contrary to the spirit of the invention.

Claims (9)

1. A three-dimensional ultrasonic array scaffoldless cell printing device, characterized in that it includes a cell transport mechanism (A), a printing platform (B), a platform drive mechanism (C), a transport drive mechanism (D) and a three-dimensional ultrasound array micro-potential well generator System (E), the printing platform (B) is installed on the platform driving mechanism (C), the cell transporting mechanism (A) is installed on the transporting driving mechanism (D), the three-dimensional ultrasonic array micro-potential well generation system (E) and the printing platform (B) connection to form a planar acoustic pressure micro-potential well array in the printing platform (B); The three-dimensional ultrasonic array micro-potential well generation system (E) includes a signal generator (1), a power amplifier (2) and a piezoelectric transducer array (13), and the piezoelectric transducer array (13) is fixedly installed on a printing platform ( B), the power amplifier (2) is connected with the piezoelectric transducer array (13), and the signal generator (1) is connected with the power amplifier (2); the front end of the signal generator is connected with a host computer, and the signal generator Under the control of the host computer, a signal of a specific frequency and a specific waveform is generated, and the wave signal is amplified into an electrical signal of a specific voltage as required by the power amplifier, and then transmitted to the array of a certain layer of the piezoelectric transducer array; the piezoelectric transducer The transducer array generates sound waves of specific energy and frequency under the drive of the electrical signal, forming a polygonal plane sound field, and constructs a plane sound pressure micro-potential well array; the piezoelectric transducer array forms different planes by exciting arrays of different layers. An array of sound pressure micro-potential wells, the plane array of sound pressure micro-potential wells is constructed and superposed layer by layer to form a three-dimensional array of sound pressure micro-potential wells. 2. The three-dimensional ultrasonic array scaffoldless cell printing device according to claim 1, wherein the printing platform (B) comprises a chassis (8) and a polygonal resonant cavity (14), the chassis (8) and the platform drive mechanism (C) ) connection, the polygonal resonant cavity (14) is fixedly installed on the chassis (8), the piezoelectric transducer array (13) is fixedly installed on the inner wall of the polygonal resonant cavity (14), and a plane is formed in the polygonal resonant cavity (14) Sound pressure micro-potential well array. 3 . The three-dimensional ultrasonic array scaffoldless cell printing device according to claim 2 , wherein the piezoelectric transducer arrays ( 13 ) are equidistantly arranged in the height direction to form a plurality of sound pressure micro-potential wells of different heights. 4 . array. 4. The three-dimensional ultrasonic array scaffoldless cell printing device according to claim 1, characterized in that, the cell delivery mechanism (A) comprises a micro-delivery nozzle (4), a pulse actuating device (5), a nozzle clip (6), The shelf plate (7), the micro-transport nozzles (4) are fixedly mounted on the shelf plate (7) through the nozzle clips (6), and the pulse actuating device (5) is fixedly mounted on the shelf plate (7), and is connected with the micro-transport nozzles (4) Upper connection. 5 . The three-dimensional ultrasonic array scaffoldless cell printing device according to claim 4 , wherein a nozzle ( 16 ) is installed at the lower end of the micro-transport nozzle ( 4 ), and the nozzle ( 4 ) and the nozzle ( 16 ) are located between the micro-transport nozzle ( 4 ) and the nozzle ( 16 ). 6 . A cell counter (12) is fixedly installed. 6 . The three-dimensional ultrasonic array scaffoldless cell printing device according to claim 5 , wherein a microscope ( 3 ) is fixedly installed on the shelf plate ( 7 ), and the microscope ( 3 ) is arranged toward the nozzle ( 16 ). 7 . 7. The three-dimensional ultrasonic array scaffoldless cell printing device according to claim 1, wherein the platform driving mechanism (C) comprises a base plate, a lifting bracket, a lifting screw slider assembly and a lifting motor, and the lifting bracket is fixedly installed on the On the base plate, the lifting screw slider assembly is rotatably mounted on the lifting bracket, and the lifting motor is connected with the lifting screw slider assembly. 8 . The three-dimensional ultrasonic array scaffoldless cell printing device according to claim 1 , wherein the conveying driving mechanism (D) comprises an X-axis driving member, a Y-axis driving member and a support column, and the support column is fixedly installed on the base plate. 9 . , the Y-axis driving member is fixedly installed on the support column, the X-axis driving member is fixedly installed on the Y-axis driving member, and the cell conveying mechanism (A) is installed on the X-axis driving member. 9. A printing process based on the three-dimensional ultrasonic array scaffold-free cell printing device according to any one of claims 1-8, characterized in that, comprising the following steps: S1: Sterilize the printing device, keep the temperature constant and appropriate, inject the culture medium into the polygonal resonance cavity, and inject the cell suspension into the micro-nozzle; S2: According to the pre-designed model, the piezoelectric transducer array works, and the planar array sound pressure micro-potential well is excited on demand in the bottommost layer of the polygonal resonator; S3: According to the pre-designed model, control the movement of the platform driving mechanism to make the nozzle reach the specific height required for the printing of the polygon resonant cavity model, and then control the movement of the conveying driving mechanism to move the micro-nozzle to the specified sound pressure micro-potential well position; S4: According to the needs of the model, the cell transport mechanism transports a specific number of cells into the corresponding planar array sound pressure micro-potential well, and the delivered cells are trapped in the sound pressure potential well under the action of the acoustic restoring force in the sound pressure potential well. , to complete the printing of cells in a single potential well, and so on, until the printing of cells in all acoustic potential wells in a layer of planes is completed; S5: After all the printing of the first layer is completed, the three-dimensional ultrasonic array micro-potential well generation system excites the second-layer array of acoustic pressure micro-potential well arrays, moves the polygonal cultivation platform downward to continue printing, and repeats steps S2-S4 to And so on to realize the printing of cell three-dimensional system; S6: After the printing is completed, the three-dimensional cell system is cultured at a suitable temperature, and the cells divide and grow to finally form a cell tissue.

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