WO2015003404A1 - Cell printing method and cell printing system - Google Patents

Cell printing method and cell printing system Download PDF

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Publication number
WO2015003404A1
WO2015003404A1 PCT/CN2013/079613 CN2013079613W WO2015003404A1 WO 2015003404 A1 WO2015003404 A1 WO 2015003404A1 CN 2013079613 W CN2013079613 W CN 2013079613W WO 2015003404 A1 WO2015003404 A1 WO 2015003404A1
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WO
WIPO (PCT)
Prior art keywords
micro
cell
nozzle
displacement
cells
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PCT/CN2013/079613
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French (fr)
Chinese (zh)
Inventor
林峰
孙伟
赵龙
张磊
张婷
Original Assignee
清华大学
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Application filed by 清华大学 filed Critical 清华大学
Publication of WO2015003404A1 publication Critical patent/WO2015003404A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/04Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062General methods for three-dimensional culture
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2535/00Supports or coatings for cell culture characterised by topography
    • C12N2535/10Patterned coating

Definitions

  • the present invention relates to the field of cell printing and cell extraction, and more particularly to a cell printing method and a cell printing system. Background technique
  • cell printing technology has played an increasingly important role in the fields of tissue engineering, pathological model construction, drug screening and detection, and cell biology research.
  • cell printing technology mainly includes inkjet printing (piezoelectric volume driving and thermal bubble type), laser direct writing, laser induced transfer, electrostatic spraying, focused ultrasonic jetting, micro extrusion, etc.
  • inkjet printing piezoelectric volume driving and thermal bubble type
  • laser direct writing laser induced transfer
  • electrostatic spraying focused ultrasonic jetting
  • micro extrusion etc.
  • the above cell printing technologies exist as follows The problem: First, in the process of cell printing, there will often be instant high temperature, high pressure or instantaneous strong electrostatic field, which is unfavorable to the survival of the cells or to maintain their original biological characteristics.
  • the present invention aims to solve at least one of the technical problems existing in the prior art.
  • Another object of the present invention is to provide a cell printing system that can achieve cell uptake, aggregation, and ejection.
  • a cell printing method comprising the steps of: S1: inserting a micro-spray into a desired cell suspension, the cell suspension being separately contained in a different cell-containing container; S2: utilizing a micro-displacement reciprocating mechanism performs a predetermined cell suction drive on the micro-nozzle to inhale a certain number of cells and combine into a cell print sequence; S3: performing the micro-sprinkler on the micro-spray using the micro-displacement reciprocating mechanism The predetermined cells are collectively driven to align the cells in a micro-nozzle into a close-packed single cell row; S4: moving the micro-nozzle to a desired printing position, using the micro-displacement reciprocating mechanism in the micro-nozzle A predetermined cell ejection drive is performed thereon to eject the cells in the sequence at the printing position. And if it is desired to continue printing the cells corresponding to the cell suspension, the above steps S1-S4 are repeatedly performed using the micro-nozzles until all
  • the cell printing method of the embodiment of the present invention by utilizing the viscous force and the inertial force of the cell suspension to alternately act as a power, that is, to utilize the action of the alternating hysteresis, the cell suspension is absorbed, aggregated, and ejected, thereby In the process of sucking or spraying the cell suspension, no instantaneous high temperature or instantaneous strong electrostatic field is generated, and the damage to the cells is small, and at the same time, since the cell printing method of the present invention uses the advanced ejection sequence to suck and spray cells, Therefore, the same micro-nozzle can be utilized to realize a plurality of cell suction and printing operations, thereby avoiding the complexity of the device due to the use of multiple sets of micro-nozzles, and avoiding the complicated operation process caused by replacing the micro-nozzles.
  • the cells can be micro-driven by the poly-synchronization drive.
  • the single cell array arranged in a close row in the nozzle improves the stability of the stable and controllable single cell printing.
  • the cell printing method according to the present invention has the following additional technical features:
  • the micro-displacement reciprocating mechanism inhales the required cells into the micro by causing the micro-pilot to generate an asymmetric reciprocating motion corresponding to the cell suction driving.
  • the micro-displacement reciprocating mechanism inhales the required cells into the micro by causing the micro-pilot to generate an asymmetric reciprocating motion corresponding to the cell suction driving.
  • the micro-pilot inhales the required cells into the micro by causing the micro-pilot to generate an asymmetric reciprocating motion corresponding to the cell suction driving.
  • the nozzle In the nozzle.
  • the micro-displacement reciprocating mechanism arranges the cells near the micro-pipe outlet by causing the micro-nozzles to generate an asymmetric reciprocating motion corresponding to the cell-collecting drive. Separate rows of single cells.
  • the micro-displacement reciprocating mechanism ejects the cells in the printing by causing the micro-pilot to generate an asymmetric reciprocating motion corresponding to the cell ejection driving. Location.
  • the micro-displacement reciprocating mechanism drives the micro-nozzles to generate asymmetric axial reciprocating motions corresponding to different displacement curves to perform suction driving, coalescing driving, and jet driving of desired cells.
  • the amount of inhalation, the degree of convergence of the desired cells or the degree of convergence of the desired cells is controlled by controlling the voltage, frequency and driving waveform time width of the micro-displacement reciprocating mechanism.
  • the number of shots can reduce the waste of cells.
  • the step S4 is performed in an air medium.
  • the step S4 is performed in a liquid medium or in a gel medium.
  • step S2 the micro-nozzle is moved away from the liquid surface, its acceleration is increased from zero to a first predetermined value, and then the first predetermined value is maintained for a first predetermined time. Finally, returning to the zero value; and the driving signal applied to the micro-displacement reciprocating mechanism causes the absolute value of the slope of the curve of the displacement waveform of the micro-nozzle to gradually increase with time.
  • step S4 the micro-nozzle is moved close to the liquid surface, the acceleration thereof first rises from zero to a second predetermined value, and then the second predetermined value is maintained for a second predetermined time. And finally returning to the zero value; and the driving signal applied to the micro-displacement reciprocating mechanism causes the slope of the curve of the displacement waveform of the micro-nozzle to gradually increase with time.
  • step S3 the acceleration of the micro-nozzle first rises from zero to a third predetermined value, then the third predetermined value is maintained for a third predetermined time, and finally returns to a zero value.
  • a driving signal applied to the micro-displacement reciprocating mechanism such that a displacement waveform of the micro-nozzle assumes a curved shape after a fourth predetermined time and then assumes a linear shape for a fifth predetermined time, and During the fourth predetermined time, the slope of the curve gradually increases with time, and the slope of the straight line is a negative value at the fifth predetermined time.
  • the inner diameter d n of the micro-nozzle. zzle meet ld edl ⁇ d n. Zzle ⁇ 2d edl , where d edl is the single cell diameter in the cell suspension.
  • d edl is the single cell diameter in the cell suspension.
  • a cell printing system comprising: a three-dimensional motion mechanism; a plurality of cell-packing containers, the plurality of cell-containing containers respectively containing different kinds of cell suspensions; a micro-nozzle, the micro Spray a tube is disposed above the three-dimensional movement mechanism, the three-dimensional movement mechanism is moved such that the micro-nozzle is located at a desired printing position or protrudes into the cell-contained container; a micro-displacement reciprocating mechanism, a micro-displacement reciprocating mechanism coupled to the micro-nozzle and applying a desired asymmetric reciprocating motion to the micro-nozzle to perform a suction drive, a coalogenesis drive, or a jet drive to the micro-jet
  • the desired cell suspension is aspirated into the micro-nozzle, the cells are arranged in a micro-nozzle into a close-packed single cell column and the cell suspension is ejected out of the micro-nozzle; and a three-dimensional motion controller, the three-dimensional motion a controller configured to control movement of the
  • the ⁇ -DOM applies a desired drive voltage signal to the micro-nozzle to perform bi-directional actuation of the micro-spray to draw the desired cell suspension into the micro-nozzle and to the cells.
  • the suspension ejects the micro-nozzle, which realizes the printing of the cells in the order of advanced output, so that not only the same nozzle can be used to print different cells, the complexity of the device is reduced, the complexity of the operation is simplified, and the operation can be reduced.
  • the probability of cell infection is beneficial to ensure the activity of cells during printing.
  • the cell suspension is subjected to the action of alternating hysteresis, the cell suspension is sucked and ejected, so that no transient high temperature or instantaneous strong electrostatic field is observed in the process of sucking and ejecting the cell suspension. Produced, less damage to cells.
  • the cell printing system according to the present invention has the following additional technical features:
  • the cell printing system further includes an image capture device that performs real-time observations of the picking, gathering, and printing processes. Thereby, it is possible to detect whether the cell is sucked up - the entire operation of the printing process is normal, and the reliability of the cell printing system is ensured.
  • the cell printing system further includes a micro-nozzle holder through which the micro-nozzle is coupled to the lower end of the micro-displacement reciprocating mechanism.
  • a micro-nozzle holder through which the micro-nozzle is coupled to the lower end of the micro-displacement reciprocating mechanism.
  • Zzle satisfies ld edl ⁇ d n .
  • Zzle ⁇ 2d edl where d edl is the single cell diameter in the cell suspension.
  • the micro-nozzle is moved away from the liquid surface, the acceleration is increased from zero to a first predetermined value, and then the Determining a first predetermined value for a first predetermined time, and finally returning to a zero value; and applying a driving signal on the micro-displacement reciprocating mechanism such that an absolute value of a slope of a displacement waveform of the micro-nozzle is over time Change and gradually increase.
  • the micro-nozzle is moved close to the liquid surface, and the acceleration first rises from zero to a second predetermined value, and then the Determining a second predetermined value for a second predetermined time, and finally returning to a zero value; and applying a driving signal to the micro-displacement reciprocating mechanism such that a slope of a curve of the displacement waveform of the micro-nozzle gradually changes with time Increase.
  • the acceleration of the micro-nozzle in the process of arranging cells in the micro-nozzle into a close-packed single cell row, the acceleration of the micro-nozzle first rises from zero to a third predetermined value, and then maintains Determining a third predetermined value for a third predetermined time, and finally returning to a zero value; and driving signals applied to the micro-displacement reciprocating mechanism such that a displacement waveform of the micro-nozzle assumes a curved shape for a fourth predetermined time After being presented as a straight line in the fifth predetermined time a shape, and during the fourth predetermined time, the slope of the curve gradually increases with time, and the slope of the straight line is a negative value at the fifth predetermined time.
  • the desired printing position is in an air medium, a liquid medium or a gel medium.
  • the micro-displacement reciprocating controller controls the micro-displacement reciprocating mechanism to cause the micro-nozzle to generate an asymmetric reciprocating motion corresponding to the cell suction drive, and to inhale the desired cell suspension In the micro-nozzle.
  • the micro-displacement reciprocating motion controller controls the micro-displacement reciprocating mechanism to cause the micro-nozzle to generate an asymmetric reciprocating motion corresponding to a cell-concentration drive, and the cells are in a micro-nozzle A dense row of single cell columns is formed.
  • the micro-displacement reciprocating controller controls the suction displacement curve to cause the micro-nozzle to generate an asymmetric reciprocating motion corresponding to the cell ejection drive, and print the cell suspension in the The desired print position.
  • the micro-nozzle is located directly above the desired printing position and the distance between the micro-nozzle and the desired printing position is 0 to 5 mm.
  • the micro-displacement reciprocating motion controller controls the inhalation amount, the degree of convergence, or the number of ejections of the desired cell suspension by controlling the voltage, frequency, and driving waveform time width of the micro-displacement reciprocating mechanism. Thereby reducing the waste of cells.
  • FIG. 1 is a flow chart of a cell printing method according to an embodiment of the present invention.
  • FIG. 2 is a schematic diagram of a cell printing system in accordance with an embodiment of the present invention.
  • Figure 3 is a schematic view of the micro-pipe in the cell printing system shown in Figure 2 when it is inserted into the cell-contained container;
  • Figure 4 is the micro-pipe in the cell printing system shown in Figure 2 at the desired printing position
  • Figure 5 is a schematic view showing the waveform of the acceleration of the micro-nozzle during cell printing according to an embodiment of the present invention
  • Figure 6 is a waveform diagram of a voltage applied to a ⁇ -DOM during cell printing in accordance with one embodiment of the present invention
  • Figure 7 is a schematic diagram showing the waveform of the acceleration of the micro-nozzle during cell uptake according to one embodiment of the present invention.
  • Figure 8 is a waveform diagram showing voltages applied to the ⁇ -DOM during cell uptake according to an embodiment of the present invention.
  • Figure 9 is a waveform diagram showing the acceleration of the micro-nozzle during cell aggregation in accordance with one embodiment of the present invention.
  • Figure 10 is a waveform diagram showing the voltage applied to the ⁇ -DOM during cell aggregation in accordance with one embodiment of the present invention.
  • Cell printing system 100 three-dimensional motion mechanism 1, cell-contained container 2, micro-nozzle 3,
  • Micro-displacement reciprocating mechanism 4 micro-displacement reciprocating motion controller 50, industrial computer 51,
  • first and second are used for descriptive purposes only, and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, features defining “first”, “second” may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, “multiple” means two or more unless otherwise stated.
  • the cells printed by the cell printing method of the present invention can be used for producing a tissue engineering product with high precision and preparing a single cell spot.
  • Tissue engineering products can be used to reconstruct complex tissues or organs of the human body in vitro, and can be used for artificial repair of damaged tissues or organs of the human body, while single cell spotting can be used for high-throughput, short-time drug development.
  • drug screening can also be used to construct in vitro tumor pathology models, can also be used to prepare cell-based high-sensitivity biosensors.
  • the cell printing method according to the embodiment of the present invention includes the following steps:
  • the micro-nozzle Insert the micro-nozzle into the desired cell suspension, and the cell suspensions are respectively accommodated in different cell-contained containers, that is, different cell-containing containers can hold different cell suspensions, and the micro-spray can be extended.
  • the micro-nozzle can be a showerhead having a microfluidic channel to enable cells to be arranged in a single column within the microfluidic channel to achieve single cell printing.
  • S2 Performing a predetermined cell suction drive on the micro-spray using a Micro Displacement Oscillating Mechanism ( ⁇ -DOM) to inhale a certain number of cells in the desired cell suspension and combine them into a cell print sequence
  • ⁇ -DOM Micro Displacement Oscillating Mechanism
  • the micro-nozzle is reciprocated, and the cell suspension is sucked into the micro-nozzle based on the principle of the alternating hysteresis.
  • the amount of inhalation of the desired cell suspension is controlled by controlling the voltage, frequency, and drive waveform time width of the ⁇ -DOM to avoid cell waste.
  • the micro-nozzle is reciprocated under the drive of ⁇ -DOM.
  • the micro-spray is first moved in the negative direction, the cells are placed in the container.
  • the viscous force between the cell suspension and the outer wall of the micro-nozzle acts as a power to drive the cell suspension to move in the negative direction, and the inertial force of the cell suspension acts as a resistance to hinder the cell suspension from moving in the negative direction.
  • the inertial force of the cell suspension acts as a power to drive the cell suspension in the cell holding container to continue moving in the negative direction, while the cells hold the cell suspension in the container and the micro-nozzle
  • the viscous force between the outer walls acts as a resistance to hinder the movement of the cell suspension in the negative direction.
  • the acceleration and acceleration time of the movement of the micro-nozzle are controlled, the inertial force of the cell suspension as the resistance when the micro-spray moves in the negative direction is small, and when the micro-spray moves in the positive direction, it is used as the power.
  • the inertial force of the cell suspension is large, and in one exercise cycle, the cell suspension liquid phase generates a negative displacement in the micro-nozzle, and the cell suspension outside the micro-nozzle is sucked into the micro-nozzle.
  • the combination of the step S2 into a cell print sequence means that, according to the printing needs, a cell suspension can be inhaled by using a micro-nozzle, and then the microcell nozzle is used to inhale different cell suspensions, that is, in the cell.
  • different cell suspensions can be sequentially inhaled in the micro-nozzle.
  • a plurality of cell suspensions are sequentially arranged in the micro-nozzle according to the order of the inhalation action, which can make various The cell suspension is combined into a cell print sequence within the microprojection.
  • the micro-nozzle is a non-inertial system, and the micro-nozzle is a non-inertial system, and the micro-nozzle system is outside the cell and extracellular.
  • the matrix is subject to cyclical inertial forces; since this inertial force is a volumetric force, the density of the material is greater than the inertial force of the material with a lower density; because the cell density is greater than the density of the surrounding liquid extracellular matrix Therefore, the inertial force of the cells is greater than the inertial force of the liquid extracellular matrix. Therefore, the cells will collide with the fluid under the action of periodic inertial forces. If the diameter of the micro-nozzle satisfies ld edl ⁇ d n . Zzle ⁇ 2d edl (where ( ⁇ is the diameter of a single cell in a cell suspension), when the aggregation motion reaches a steady state, the cells form a close-packed single cell column in the micro-nozzle.
  • a micro-nozzle with a cell suspension is taken to reciprocate under the cell ejection drive performed by the ⁇ -DOM, when the micro-spray is first directed
  • the viscous force between the cell suspension and the inner wall of the micro-nozzle acts as a power to drive the cell suspension in the micro-spray
  • the negative direction moves, and the inertial force of the cell suspension acts as a resistance to prevent the cell suspension from moving in the negative direction.
  • the inertial force of the cell suspension acts as a power-driven cell suspension to continue to move in the negative direction, and the viscous force between the cell suspension and the inner wall of the micro-nozzle acts as a resistance to hinder cell suspension.
  • the liquid moves in the negative direction. If the time of acceleration and acceleration of the movement of the micro-nozzle is controlled, the inertial force of the cell suspension as a resistance when the micro-jet is moved in the negative direction is small, and the cell which is the power when the micro-jet moves in the positive direction is realized.
  • the inertial force of the suspension is large, then in a movement cycle, the cell suspension liquid phase will produce a negative displacement in the micro-nozzle, so that the cell suspension is ejected from the micro-nozzle to print at the desired printing position. At the office.
  • the micro-nozzle and the ⁇ -DOM should be placed in a sterile chamber for sterilization before the above steps are performed to ensure that the above steps are It is carried out in a sterile environment to avoid bacterial contamination of the cell suspension and to ensure the survival rate and biological properties of the cells.
  • the cell-filling container containing the cell suspension cannot be placed in the sterile chamber to avoid killing the cells, and can wait for a certain time, such as 5 minutes, after the sterilization is completed.
  • the cell-filled container containing the cell suspension is then placed in a sterile chamber.
  • the ⁇ -DOM can be sterilized by an ultraviolet sterilizing lamp, and the micro-nozzle can be sterilized by high-pressure steam sterilization.
  • the desired printing position may be located in an air medium, a liquid medium or a gel medium.
  • the liquid medium is one of a cell culture medium, a sodium alginate solution, collagen, and a fiber protein.
  • the medium is one of various gels.
  • the micro-nozzle can be suspended at a certain height above the substrate as a carrier for cell printing for cell printing of a specific pattern or trait, or can be inserted into the grid of the tissue engineering scaffold. Cell printing is performed in the cavity to achieve the purpose of planting a specific number of cells at specific locations of the stent.
  • the micro-spray tube is first inserted into a cell-containing container containing the cell suspension, and then the ⁇ -DOM performs a predetermined cell-absorption drive, and the ⁇ -DOM drives the micro-nozzle in the cell.
  • Reciprocating motion corresponding to the cell suction drive is performed in the suspension, so that the cell suspension in the cell-contained container is subjected to the alternating inertia and is sucked into the micro-spray tube under the action of the alternating inertia
  • the above procedure is repeated such that a plurality of cell suspensions are aspirated into the micro-nozzles and a plurality of cell suspensions are combined into a cell print sequence within the micro-nozzles.
  • the micro-nozzles sucking the cell suspension are mixed and driven by the ⁇ -DOM, and the cells are arranged in a micro-pipe to form a close-packed single cell column.
  • the micro-nozzle is then moved to the desired printing position, the ⁇ -DOM performs a predetermined cell ejection drive, and the micro-nozzle is driven by the ⁇ -DOM to perform a reciprocating motion corresponding to the cell ejection drive so that the micro-nozzle is inside
  • the cell suspension is subjected to alternating inertia, and under the action of the alternating inertia, the cell suspension is ejected from the micro-nozzle to be printed at the desired printing position, that is, the cell is absorbed. - The printing process. If you need to continue printing, re-insert the micro-nozzle into the cell suspension and repeat the above steps to complete the multiple-cell capture-print process.
  • the cell suspensions taken during the pipetting-printing process of the cells may be the same or different when the cell-pick-printing process is performed multiple times using the cell printing method described above.
  • the cell suspension is sucked and ejected by using the viscous force and the inertial force of the cell suspension alternately as a power, that is, by utilizing the action of the alternating hysteresis inertia, thereby
  • the process of aspirating or spraying the cell suspension no transient high temperature, high pressure or transient strong electric field is generated, and the damage to the cells is small, and at the same time, since the cell printing method of the present invention uses the advanced ejection sequence to suck and spray cells, Therefore, the same micro-nozzle can be utilized to realize a plurality of cell suction and printing operations, thereby avoiding the increase in equipment cost due to the use of multiple sets of micro-nozzles, and avoiding the complicated operation process caused by the
  • step S2 the ⁇ -DOM draws the desired cell suspension into the micro-nozzle by causing the micro-nozzle to generate an asymmetric reciprocating motion corresponding to the cell suction drive.
  • step S3 the ⁇ -DOM arranges the cells in a close-packed single cell row near the exit of the micro-jet by causing the micro-spray to generate an asymmetric reciprocating motion corresponding to the cell-gathering drive.
  • step S4 the ⁇ -DOM prints the cell suspension at the printing position by causing the micro-nozzle to generate an asymmetrical reciprocating motion corresponding to the cell ejection drive.
  • the ⁇ -DOM driven micro-nozzles generate asymmetric axial reciprocating motions corresponding to different displacement curves to perform jet drive, suction drive, and bunch drive for the desired cell suspension. That is, the axial reciprocating motion of the displacement curve corresponding to the cell suction drive generated by the ⁇ -DOM driven micro-nozzle, and the axial reciprocation of the displacement curve corresponding to the cell aggregation drive generated by the ⁇ -DOM driven micro-nozzle.
  • the axial reciprocating motion of the displacement curve corresponding to the cell ejection drive produced by the motion and ⁇ -DOM driven micro-nozzles is different.
  • step S2 the micro-nozzle is moved away from the liquid surface, its acceleration is increased from zero to a first predetermined value, and then the first predetermined value is maintained for the first predetermined Time, and finally back to zero. Therefore, the speed of the micro-nozzle is continuously changed instead of jumping during the acceleration process to reduce the vibration during the movement of the micro-nozzle. And as shown in FIG. 7, in step S2, the micro-nozzle is moved away from the liquid surface, its acceleration is increased from zero to a first predetermined value, and then the first predetermined value is maintained for the first predetermined Time, and finally back to zero. Therefore, the speed of the micro-nozzle is continuously changed instead of jumping during the acceleration process to reduce the vibration during the movement of the micro-nozzle. And as shown in FIG.
  • step S2 the voltage applied to the ⁇ -DOM is such a voltage waveform, the slope of the curve of the voltage waveform is a negative value, and the absolute value of the slope of the curve gradually increases with time. Since the ⁇ -DOM has a substantially proportional relationship between voltage and displacement, the waveform also represents the displacement waveform of the micro-nozzle in step S2, that is, as shown in FIG. 8, the drive applied to the ⁇ -DOM The signal causes the absolute value of the slope of the curve of the displacement waveform of the micro-nozzle to gradually increase with time.
  • step S2 the acceleration waveform of the micro-nozzle and the voltage waveform applied to the ⁇ -DOM are merely illustrative, and are not specific limitations of the present invention, which should be understood by those skilled in the art.
  • step S2 the acceleration waveform of the micro-nozzle and the voltage waveform applied to the ⁇ -DOM are sucked into the micro-nozzle as long as the cell suspension can be subjected to the alternating inertia and the micro-nozzle is made The vibration during the movement is small.
  • step S4 the micro-nozzle is moved close to the liquid surface, and its acceleration first rises from zero to the first The second predetermined value is then held for a second predetermined value for a second predetermined time, and finally returned to zero value, thereby ensuring that the speed is continuously changed rather than jumped during the acceleration of the micro-nozzle.
  • the voltage applied to the ⁇ -DOM is such a The waveform of the voltage waveform gradually increases with time.
  • the waveform also represents the displacement waveform of the micro-nozzle in step S4, that is, That is, as shown in FIG. 6, the driving signal applied to the ⁇ -DOM causes the slope of the curve of the displacement waveform of the micro-nozzle to gradually increase with time.
  • step S4 the acceleration waveform of the micro-nozzle and the voltage waveform applied to the ⁇ -DOM are merely illustrative, and are not specifically limited to the present invention, and those skilled in the art should understand.
  • the acceleration waveform of the micro-nozzle and the voltage waveform applied to the ⁇ -DOM are such that the cell suspension in the micro-jet is ejected from the micro-nozzle under the action of the alternating hysteresis and makes The vibration of the micro-nozzle during the movement is small.
  • step S3 the acceleration of the micro-nozzle first rises from zero to a third predetermined value, and then The third predetermined value is maintained for a third predetermined time, and finally returned to zero value, thereby ensuring that the speed is continuously changed rather than jumped during the acceleration of the micro-nozzle. And as shown in FIG. 9, in step S3, the acceleration of the micro-nozzle first rises from zero to a third predetermined value, and then The third predetermined value is maintained for a third predetermined time, and finally returned to zero value, thereby ensuring that the speed is continuously changed rather than jumped during the acceleration of the micro-nozzle. And as shown in FIG.
  • the voltage applied to the ⁇ -DOM is a voltage waveform that appears as a curved shape after the fourth predetermined time and then appears as a linear shape in the fifth predetermined time, and is in a fourth predetermined During the time, the slope of the curve gradually increases with time, and the slope of the straight line at the fifth predetermined time is negative and remains constant. Since the ⁇ -DOM has a proportional relationship between voltage and displacement, the waveform also represents the displacement waveform of the micro-nozzle in step S3, that is, as shown in FIG.
  • the driving applied to the micro-displacement reciprocating mechanism causes the displacement waveform of the micro-nozzle to assume a curved shape after the fourth predetermined time and then assumes a linear shape for a fifth predetermined time, and in a fourth predetermined time, the slope of the curve gradually increases with time.
  • the slope of the straight line at the fifth predetermined time is a negative value.
  • step S3 the acceleration waveform of the micro-nozzle and the voltage waveform applied to the ⁇ -DOM are satisfied so that the cells in the cell suspension in the micro-nozzle are concentrated with respect to the fluid under the action of periodic inertial forces. Exercise, and finally make the cells form a dense row of single cell columns in the micro-nozzle.
  • the micro nozzle inner diameter d n. Zzle satisfies ld cdl ⁇ d n . Zzk ⁇ 2d cdl , where ⁇ is the single cell diameter in the cell suspension to ensure that the cells in the cell suspension are arranged in a stable single cell array in the micro-spray tube, preventing the cells from clogging the micro-nozzles or forming local accumulation, and further It is ensured that the cells in the cell suspension are ejected in the shape of a single cell.
  • a cell printing system 100 in accordance with an embodiment of the present invention will now be described with reference to Figs.
  • the cell printing system 100 includes: a three-dimensional motion mechanism 1, a plurality of cell-contained containers 2, a micro-nozzle 3, a ⁇ - ⁇ 4, a three-dimensional motion controller 52, and a micro-displacement reciprocating The motion controller 50, wherein the three-dimensional motion mechanism 1 can be up and down (as shown in FIG. 2), left and right (as shown in FIG. 2), and front and rear (as shown in FIG. 2). Move in the direction. Different cell suspensions are contained in the plurality of cell-containing containers 2, respectively.
  • the micro-pipe 3 is disposed above the three-dimensional moving mechanism 1, and the three-dimensional moving mechanism 1 is moved so that the micro-jet 3 is located at a desired printing position or protrudes into the cell-containing container 2.
  • the desired print position is the upper surface of the slide.
  • the ⁇ - ⁇ 4 is connected to the micro-nozzle 3, and the required asymmetric reciprocating motion is applied to the micro-nozzle 3 to perform suction driving, coalescing driving or jet driving on the micro-jet 3 to bring the desired cell suspension
  • the cells are sucked into the micro-pipe 3, and the cells are arranged in a micro-lance 3 to form a single cell row and the cell suspension is ejected out of the micro-jet 3.
  • the micro-nozzle 3 is connected to the lower end of the ⁇ - ⁇ 4 through the micro-pipe clamp 7, thereby facilitating the mounting of the micro-nozzle 3 and avoiding damage of the micro-nozzle 3.
  • the three-dimensional motion controller 52 is configured to control the movement of the three-dimensional motion mechanism 1.
  • the micro-displacement reciprocating motion controller 50 is configured to control the bi-directional actuation of the micro-nozzle 3 by the ⁇ - ⁇ 4 to perform the suction, collection, and ejection of the cell suspension.
  • the cell printing system 100 further includes an industrial computer 51 connected to the micro-displacement reciprocating motion controller 50 and the three-dimensional motion controller 52, and the micro-displacement reciprocating motion controller 50 and the ⁇ -DOM 4 Connected to provide the driving voltage signal of the ⁇ -DOM 4, the voltage adjustment range of the micro-displacement reciprocating controller 50 is 0-90 ⁇ , and the frequency adjustment range is 1-200 ⁇ .
  • the three-dimensional motion controller 52 receives an instruction from the industrial computer 51 to control the motion of the three-dimensional motion mechanism 1.
  • the cell printing system 100 of the present invention is placed in a sterile chamber, and the three-dimensional motion mechanism 1, the ⁇ - ⁇ 4 and the micro-jet 3 are sterilized before the cell printing system 100 is operated, and ultraviolet sterilization can be used at this time.
  • the lamp sterilizes the three-dimensional motion mechanism 1 and the ⁇ - ⁇ 4, and the sterilization time is about 30 minutes.
  • the micro-lance 3 is sterilized by high-pressure steam and connected to the ⁇ - ⁇ 4.
  • the cell-containing container 2 containing the cell suspension cannot be placed in the aseptic chamber to avoid killing the cells, and can wait for a certain time, such as 5 minutes, after the sterilization is completed.
  • the cell-containing container 2 containing the cell suspension is then placed in a sterile chamber.
  • the three-dimensional motion controller 52 controls the movement of the three-dimensional motion mechanism 1 so that the micro-jet 3 extends into the cell-contained container 2, and then the micro-displacement reciprocating controller 50 controls the ⁇ .
  • the three-dimensional motion controller 52 The three-dimensional motion mechanism 1 is controlled to move so that the micro-jet 3 sucking the cell suspension is located at a desired printing position, and during the movement, the micro-displacement reciprocating controller 50 controls the driving voltage signal required for the ⁇ - ⁇ 4 Applying to the micro-nozzle 3 causes the micro-jet 3 to generate an asymmetric reciprocating motion corresponding to the driving voltage signal, so that the cells are arranged densely in the micro-nozzle 3 Rows of single cells, followed by a micro-displacement reciprocating controller 50 controlling ⁇ - ⁇ 4 to apply a desired drive voltage signal to the micro-nozzle 3 to cause the micro-jet 3 to generate an asymmetric reciprocation corresponding to the drive voltage signal
  • the movement causes the cell suspension in the micro-nozzle 3 to be ejected from the micro-lance 3 in the form of a single cell under the action of the alternating hysteresis, thereby printing the single cells in the desired printing position in sequence, completing Once the cell's aspiration-printing process.
  • the cell suspensions taken during the pipetting-printing process of the cells may be the same or different when the cell-pick-printing process is performed multiple times using the cell printing system described above.
  • the control system 5 controls the inhalation amount or the ejection amount of the desired cell suspension by controlling the voltage, frequency, and driving waveform time width of ⁇ - ⁇ 4 to avoid waste of cells.
  • the process of printing the cell suspension at the printing position can be performed in an air medium or can be performed in a liquid medium or a gel medium to meet different needs.
  • the liquid medium is a cell culture medium, sodium alginate.
  • One of solution, collagen, and fiber protein, and the gel medium is one of various gels.
  • the micro-jet 3 can be printed at a certain height above the substrate as a carrier for cell printing for cell printing of a specific pattern or trait, and can also be inserted into a tissue engineering scaffold. Cell printing is performed in a mesh cavity to achieve the purpose of planting a specific number of cells at specific locations in the scaffold.
  • the micro-jet 3 is located directly above the desired printing position and the distance between the micro-jet 3 and the desired printing position is 0 to 5 mm.
  • ⁇ - ⁇ 4 applies a desired driving voltage signal to the micro-nozzle 3 to perform bidirectional driving on the micro-jet 3 to draw the desired cell suspension into the micro-nozzle 3 and the cell suspension is ejected out of the micro-nozzle 3, that is, the cell printing is realized in the order of advanced output, so that not only the same nozzle can be used for printing different cells, the equipment cost is reduced, and the operation complexity is simplified. At the same time, it can reduce the chance of cell infection, which is beneficial to ensure the activity of cells during printing.
  • the cell suspension is subjected to the action of alternating hysteresis, the cell suspension is sucked and ejected, so that no transient high temperature, high pressure or electric field is generated during the process of sucking and ejecting the cell suspension. , damage to cells is small.
  • the micro-displacement reciprocating motion controller 50 controls ⁇ - ⁇ 4 to cause the micro-lance 3 to generate an asymmetric reciprocating motion corresponding to the cell suction driving, and to inhale the desired cell suspension into the micro-spray 3.
  • the micro-displacement reciprocating motion controller controls ⁇ - ⁇ 4 to cause the micro-lance 3 to generate an asymmetric reciprocating motion corresponding to the cell ejection drive, and to print the cell suspension at the desired printing position.
  • the micro-displacement reciprocating motion controller controls the micro-displacement reciprocating mechanism such that the micro-nozzles generate an asymmetric reciprocating motion corresponding to the cell-gathering drive, and the cells form a close-packed single-cell column in the micro-nozzle.
  • the cell printing system 100 further includes an image capturing device 6 that performs real-time observation of the picking, gathering, and printing processes, thereby detecting whether the entire cell's pick-and-print process is Normal operation ensures the reliability of the cell printing system 100.
  • the camera device 6 can be a CCD camera, thereby having the advantages of high sensitivity, high glare resistance, small distortion, small volume, long life, and anti-vibration.
  • the inner diameter d n of the micro-jet 3 is. Zzle satisfies ld edl ⁇ d n . Zzk ⁇ 2d edl , where d edl is the single cell diameter in the cell suspension, thereby ensuring that the cells in the cell suspension in the micro-bubble 3 are arranged in a stable single cell array, preventing the cells from clogging the micro-spray 3 or forming Local accumulation, thereby ensuring that cells in the cell suspension in the micro-pipe 3 are printed as single cells.
  • the vibration of the micro-nozzle 3 affects both the survival rate of the cells and the printing position accuracy of the cells, thereby reducing the movement of the micro-nozzle 3 Vibration, in some embodiments of the present invention, as shown in Figure 7, during the suction of the cell suspension, the micro-nozzle 3 is moved away from the liquid surface, and the acceleration of the micro-jet 3 is first Zero is added to the first predetermined value, then the first predetermined value is maintained for the first predetermined time, and finally returned to the zero value, thereby ensuring that the speed of the micro-pipe 3 during the acceleration is continuously changed instead of jumping. And as shown in FIG.
  • the voltage applied to the ⁇ - ⁇ 4 during the suction process of the micro-spray tube 3 is such a voltage waveform, the slope of the curve of the voltage waveform is a negative value, and the curve is inclined.
  • the absolute value of the rate gradually increases with time. Since ⁇ - ⁇ 4 has a substantially proportional relationship between voltage and displacement, the waveform also represents the displacement waveform of the micro-nozzle 3, that is, as shown in Fig. 8. It is shown that the driving signal applied to the ⁇ - ⁇ 4 causes the absolute value of the slope of the curve of the displacement waveform of the micro-cavity 3 to gradually increase with time.
  • acceleration waveform of the micro-pipe 3 and the voltage waveform applied to the ⁇ - ⁇ 4 during the suction of the cell suspension by the micro-nozzle 3 are merely illustrative, rather than the present invention. Specific limitations, those skilled in the art should understand that in the process of sucking the cell suspension by the micro-nozzle 3, the acceleration waveform of the micro-nozzle 3 and the voltage waveform applied to the ⁇ - ⁇ 4 are satisfied as long as the cell suspension is satisfied. The liquid can be sucked into the micro-pipe 3 by the action of the alternating hysteresis and the vibration of the micro-jet 3 during the movement is small.
  • the micro-nozzle 3 is in the process of single cell printing, and the micro-spray 3 is close to the liquid surface. Movement, the acceleration of the micro-nozzle 3 first rises from zero to a second predetermined value, then maintains a second predetermined value for a second predetermined time, and finally returns to zero value, thereby ensuring the speed during the acceleration of the micro-jet 3 It is a constant change rather than a jump change. And as shown in Fig.
  • the voltage applied to ⁇ - ⁇ 4 is such a voltage waveform, the slope of the curve of the voltage waveform gradually increases with time, and since ⁇ - ⁇ 4 has a substantially proportional relationship between voltage and position. Therefore, the waveform also represents the displacement waveform of the micro-nozzle 3, that is, as shown in FIG. 6, the driving signal applied to the ⁇ - ⁇ 4 causes the slope of the curve of the displacement waveform of the micro-cavity 3 to change with time. And gradually increase.
  • the above-mentioned acceleration waveform of the micro-nozzle 3 and the voltage waveform applied to the ⁇ - ⁇ 4 during the single-cell printing process of the micro-nozzle 3 are merely illustrative, and are not specific limitations of the present invention. It should be understood by those skilled in the art that in the single cell printing process of the micro-nozzle 3, the acceleration waveform of the micro-nozzle 3 and the voltage waveform applied to the ⁇ - ⁇ 4 are satisfied as long as they are satisfied in the micro-spray tube 3.
  • the cell suspension is ejected from the micro-spray 3 under the action of alternating hysteresis and makes the micro-lance 3 less vibrating during the movement.
  • the voltage applied to the ⁇ - ⁇ 4 is a voltage waveform which appears as a curved shape in the fourth predetermined time and then appears as a linear shape in the fifth predetermined time, and is in a fourth predetermined During the time, the slope of the curve gradually increases with time, and the slope of the straight line at the fifth predetermined time is negative and remains constant. Since ⁇ - ⁇ 4 has a proportional relationship between voltage and displacement, the waveform also represents the displacement waveform of the micro-nozzle, that is, as shown in Fig.
  • the driving signal applied to the micro-displacement reciprocating mechanism 4 makes micro
  • the displacement waveform of the nozzle 3 assumes a curved shape after the fourth predetermined time and then assumes a linear shape for a fifth predetermined time, and in a fourth predetermined time, the slope of the curve gradually increases with time,
  • the slope of the line of the fifth predetermined time is a negative value.
  • acceleration waveform of the micro-cavity 3 and the voltage waveform applied to the ⁇ - ⁇ 4 in the process of arranging the cells in the micro-lance 3 into a close-packed single cell row are merely exemplary.
  • the values of the first predetermined value, the first predetermined time, the second predetermined value, the second predetermined time, the third predetermined time, the third predetermined value, the fourth predetermined time, and the fifth predetermined time It can be specifically set according to the characteristics of cells of different cell suspensions to meet different needs.

Abstract

Disclosed are a cell printing method and a cell printing system. The cell printing method comprises: S1: inserting a micro-nozzle into a desired cell suspension; S2: performing a predetermined cell-drawing drive on the micro-nozzle by using a micro-displacement reciprocating motion mechanism to draw in a certain number of cells and combine them into a cell printing sequence; S3: performing a predetermined cell-gathering drive on the micro-nozzle by using the micro-displacement reciprocating motion mechanism to arrange the cells into a closely packed single-cell column in the micro-nozzle; and S4: moving the micro-nozzle to a desired printing position and performing a predetermined cell-spraying drive on the micro-nozzle by using the micro-displacement reciprocating motion mechanism to spray the cells onto the printing position in sequence.

Description

细胞打印方法及细胞打印系统  Cell printing method and cell printing system
技术领域 Technical field
本发明涉及细胞打印和细胞提取领域, 尤其是涉及一种细胞打印方法及细胞打印系 统。 背景技术  The present invention relates to the field of cell printing and cell extraction, and more particularly to a cell printing method and a cell printing system. Background technique
近年来, 细胞打印技术在组织工程学、 病理模型构建、 药物筛选与检测、 细胞生物 学研究等领域发挥着越来越重要的作用。 目前, 细胞打印技术主要包括喷墨打印 (压电 容积驱动式和热气泡式) 、 激光直写、 激光诱导转移、 静电喷射、 聚焦超声波喷射、 微 挤出等, 上述的这些细胞打印技术存在如下的问题: 一、在细胞打印过程中往往会产生 瞬间高温、高压或瞬间强静电场,对细胞的成活或保持其原本的生物学特性等不利。二、 在喷射多种类型细胞时, 往往需要采用多组喷射机构或更换多个喷头, 从而增加了设备 复杂程度或加大了操作的复杂范围。三、多数工艺每次打印的最小细胞数量仍在数个至 数十个范围, 在稳定、 高效的单细胞打印方面还没有得到充分发展。 发明内容  In recent years, cell printing technology has played an increasingly important role in the fields of tissue engineering, pathological model construction, drug screening and detection, and cell biology research. At present, cell printing technology mainly includes inkjet printing (piezoelectric volume driving and thermal bubble type), laser direct writing, laser induced transfer, electrostatic spraying, focused ultrasonic jetting, micro extrusion, etc. The above cell printing technologies exist as follows The problem: First, in the process of cell printing, there will often be instant high temperature, high pressure or instantaneous strong electrostatic field, which is unfavorable to the survival of the cells or to maintain their original biological characteristics. Second, when spraying multiple types of cells, it is often necessary to use multiple sets of injection mechanisms or to replace multiple nozzles, thereby increasing the complexity of the equipment or increasing the complexity of the operation. Third, the minimum number of cells printed per process in many processes is still in the range of several to tens of, and has not been fully developed in stable and efficient single-cell printing. Summary of the invention
本发明旨在至少解决现有技术中存在的技术问题之一。  The present invention aims to solve at least one of the technical problems existing in the prior art.
为此, 本发明的一个目的在于提出一种可实现细胞吸取、 聚齐和喷射的细胞打印方 法。  To this end, it is an object of the present invention to provide a cell printing method which enables cell uptake, aggregation and ejection.
本发明的另一个目的在于提出可实现细胞吸取、 聚齐和喷射的细胞打印系统。  Another object of the present invention is to provide a cell printing system that can achieve cell uptake, aggregation, and ejection.
根据本发明第一方面实施例的细胞打印方法, 包括如下步骤: S 1 : 将微喷管插入所 需细胞悬浮液中, 所述细胞悬浮液分别容纳在不同的细胞盛装容器中; S2: 利用微位移 往复运动机构在所述微喷管上执行预定的细胞吸取驱动,以吸入一定数量的细胞并组合 成细胞打印序列; S3 : 利用所述微位移往复运动机构在所述微喷管上执行预定的细胞聚 齐驱动, 将细胞在微喷管内排列成密排单细胞列; S4: 将所述微喷管移动至所需的打印 位置, 利用所述微位移往复运动机构在所述微喷管上执行预定的细胞喷射驱动, 以将所 述细胞按序列喷射在所述打印位置处。以及如果需要继续打印与所述细胞悬浮液对应的 细胞, 则利用所述微喷管重复执行上述步骤 S 1-S4, 直至完成所有的细胞二维图形或三 维结构的细胞打印。  A cell printing method according to an embodiment of the first aspect of the present invention, comprising the steps of: S1: inserting a micro-spray into a desired cell suspension, the cell suspension being separately contained in a different cell-containing container; S2: utilizing a micro-displacement reciprocating mechanism performs a predetermined cell suction drive on the micro-nozzle to inhale a certain number of cells and combine into a cell print sequence; S3: performing the micro-sprinkler on the micro-spray using the micro-displacement reciprocating mechanism The predetermined cells are collectively driven to align the cells in a micro-nozzle into a close-packed single cell row; S4: moving the micro-nozzle to a desired printing position, using the micro-displacement reciprocating mechanism in the micro-nozzle A predetermined cell ejection drive is performed thereon to eject the cells in the sequence at the printing position. And if it is desired to continue printing the cells corresponding to the cell suspension, the above steps S1-S4 are repeatedly performed using the micro-nozzles until all cell two-dimensional or three-dimensional structure cell printing is completed.
根据本发明实施例的细胞打印方法, 通过利用细胞悬浮液的粘滞力和惯性力交替地 作为动力即利用交变滞惯力的作用来实现细胞悬浮液的吸取、聚齐和喷射, 从而无论是 在细胞悬浮液的吸取或喷射的过程中, 均无瞬间高温或瞬间强静电场等产生, 对细胞的 损伤较小, 同时由于本发明的细胞打印方法采用先进后出的顺序吸取和喷射细胞, 从而 可利用同一个微喷管以实现多种细胞的吸取和打印操作,避免了因采用多组微喷管而使 设备复杂程度增加, 避免了因更换微喷管而造成的操作过程复杂化, 同时避免了使用其 他工具进行上述操作, 可减少细胞的染菌几率, 并且能实现"即吸即打印"的连贯细胞操 作, 有利于保证打印过程中的细胞的活性; 同时, 通过聚齐驱动, 可以使细胞在微喷管 内排列成密排的单细胞列, 从而为稳定、 可控的单细胞打印提高了有力保证。 According to the cell printing method of the embodiment of the present invention, by utilizing the viscous force and the inertial force of the cell suspension to alternately act as a power, that is, to utilize the action of the alternating hysteresis, the cell suspension is absorbed, aggregated, and ejected, thereby In the process of sucking or spraying the cell suspension, no instantaneous high temperature or instantaneous strong electrostatic field is generated, and the damage to the cells is small, and at the same time, since the cell printing method of the present invention uses the advanced ejection sequence to suck and spray cells, Therefore, the same micro-nozzle can be utilized to realize a plurality of cell suction and printing operations, thereby avoiding the complexity of the device due to the use of multiple sets of micro-nozzles, and avoiding the complicated operation process caused by replacing the micro-nozzles. At the same time avoid using it His tools perform the above operations, which can reduce the chance of cell infection, and can realize the "snap-and-print" coherent cell operation, which is beneficial to ensure the activity of cells during printing. At the same time, the cells can be micro-driven by the poly-synchronization drive. The single cell array arranged in a close row in the nozzle improves the stability of the stable and controllable single cell printing.
另外, 根据本发明的细胞打印方法还具有如下附加技术特征:  In addition, the cell printing method according to the present invention has the following additional technical features:
具体地, 在所述步骤 S2 中, 所述微位移往复运动机构通过使所述微喷管产生与所 述细胞吸取驱动相对应的非对称的往复运动, 而将所需的细胞吸入所述微喷管中。  Specifically, in the step S2, the micro-displacement reciprocating mechanism inhales the required cells into the micro by causing the micro-pilot to generate an asymmetric reciprocating motion corresponding to the cell suction driving. In the nozzle.
具体地, 在所述步骤 S3 中, 所述微位移往复运动机构通过使所述微喷管产生与所 述细胞聚齐驱动相对应的非对称的往复运动,而将细胞在微喷管出口附近排列成密排单 细胞列。  Specifically, in the step S3, the micro-displacement reciprocating mechanism arranges the cells near the micro-pipe outlet by causing the micro-nozzles to generate an asymmetric reciprocating motion corresponding to the cell-collecting drive. Separate rows of single cells.
具体地, 在所述步骤 S4 中, 所述微位移往复运动机构通过使所述微喷管产生与所 述细胞喷射驱动相对应的非对称的往复运动, 而将所述细胞喷射在所述打印位置处。  Specifically, in the step S4, the micro-displacement reciprocating mechanism ejects the cells in the printing by causing the micro-pilot to generate an asymmetric reciprocating motion corresponding to the cell ejection driving. Location.
具体地, 所述微位移往复运动机构驱动所述微喷管产生与不同的位移曲线相对应的 非对称的轴向往复运动, 以执行对所需的细胞的吸取驱动、 聚齐驱动和喷射驱动。  Specifically, the micro-displacement reciprocating mechanism drives the micro-nozzles to generate asymmetric axial reciprocating motions corresponding to different displacement curves to perform suction driving, coalescing driving, and jet driving of desired cells.
在本发明的一些实施例中, 在所述步骤 S2、 S3和 S4中, 通过控制所述微位移往复 运动机构的电压、频率和驱动波形时间宽度来控制所需细胞的吸入量、聚齐程度或者喷 射数量, 从而可减少细胞的浪费。  In some embodiments of the present invention, in the steps S2, S3 and S4, the amount of inhalation, the degree of convergence of the desired cells or the degree of convergence of the desired cells is controlled by controlling the voltage, frequency and driving waveform time width of the micro-displacement reciprocating mechanism. The number of shots can reduce the waste of cells.
在本发明的一些示例中, 所述步骤 S4在空气介质中执行。  In some examples of the invention, the step S4 is performed in an air medium.
在本发明的另一些示例中, 所述步骤 S4在液体介质中或凝胶介质中执行。  In other examples of the invention, the step S4 is performed in a liquid medium or in a gel medium.
在本发明的一些实施例中, 在步骤 S2 中, 所述微喷管做离开液面的运动, 其加速 度由零增加到第一预定值, 接着保持所述第一预定值第一预定时间, 最后再回到零值; 以及施加在所述微位移往复运动机构上的驱动信号使得所述微喷管的位移波形的曲线 斜率的绝对值随着时间的变化而逐渐增大。  In some embodiments of the present invention, in step S2, the micro-nozzle is moved away from the liquid surface, its acceleration is increased from zero to a first predetermined value, and then the first predetermined value is maintained for a first predetermined time. Finally, returning to the zero value; and the driving signal applied to the micro-displacement reciprocating mechanism causes the absolute value of the slope of the curve of the displacement waveform of the micro-nozzle to gradually increase with time.
在本发明的一些实施例中, 在步骤 S4 中, 所述微喷管做靠近液面的运动, 其加速 度先由零上升到第二预定值,接着保持所述第二预定值第二预定时间,最后再回到零值; 以及施加在所述微位移往复运动机构上的驱动信号使得所述微喷管的位移波形的曲线 斜率随着时间的变化而逐渐增大。  In some embodiments of the present invention, in step S4, the micro-nozzle is moved close to the liquid surface, the acceleration thereof first rises from zero to a second predetermined value, and then the second predetermined value is maintained for a second predetermined time. And finally returning to the zero value; and the driving signal applied to the micro-displacement reciprocating mechanism causes the slope of the curve of the displacement waveform of the micro-nozzle to gradually increase with time.
在本发明的一些实施例中, 在步骤 S3 中, 所述微喷管的加速度先由零上升到第三 预定值, 接着保持所述第三预定值第三预定时间, 最后再回到零值; 以及施加在所述微 位移往复运动机构上的驱动信号使得所述微喷管的位移波形在第四预定时间内呈现为 曲线形状后在第五预定时间内呈现为直线形状, 且在所述第四预定时间内, 所述曲线的 斜率随着时间的变化而逐渐增大, 在所述第五预定时间所述直线的斜率为负值。  In some embodiments of the present invention, in step S3, the acceleration of the micro-nozzle first rises from zero to a third predetermined value, then the third predetermined value is maintained for a third predetermined time, and finally returns to a zero value. And a driving signal applied to the micro-displacement reciprocating mechanism such that a displacement waveform of the micro-nozzle assumes a curved shape after a fourth predetermined time and then assumes a linear shape for a fifth predetermined time, and During the fourth predetermined time, the slope of the curve gradually increases with time, and the slope of the straight line is a negative value at the fifth predetermined time.
优选地, 所述微喷管的内径 dnzzle满足 ldedl<dnzzle<2dedl, 其中 dedl为所述细胞悬浮 液中的单细胞直径。从而可避免细胞堵塞微喷管或形成局部堆积, 保证细胞悬浮液中的 细胞以单细胞的形式打印。 Preferably, the inner diameter d n of the micro-nozzle. zzle meet ld edl <d n. Zzle <2d edl , where d edl is the single cell diameter in the cell suspension. Thereby, the cells can be prevented from clogging the micro-nozzles or forming local accumulation, and the cells in the cell suspension can be printed in the form of single cells.
根据本发明第二方面实施例的细胞打印系统, 包括: 三维运动机构; 多个细胞盛装 容器, 所述多个细胞盛装容器中分别容纳有不同种类的细胞悬浮液; 微喷管, 所述微喷 管设在所述三维运动机构的上方,所述三维运动机构移动以使得所述微喷管位于所需的 打印位置处或伸入到所述细胞盛装容器内; 微位移往复运动机构, 所述微位移往复运动 机构与所述微喷管相连,且将所需的非对称往复运动施加至所述微喷管上以对所述微喷 管执行吸取驱动、 聚齐驱动或者喷射驱动, 以将所需的细胞悬浮液吸入所述微喷管内、 将细胞在微喷管内排列成密排单细胞列和将所述细胞悬浮液喷射出所述微喷管;以及三 维运动控制器, 所述三维运动控制器配置成控制所述三维运动机构的移动; 微位移往复 运动控制器,所述微位移往复运动控制器配置成控制所述微位移往复运动机构对所述微 喷管的双向驱动, 以执行所述细胞悬浮液的吸取、 聚齐和喷射。 A cell printing system according to an embodiment of the second aspect of the present invention, comprising: a three-dimensional motion mechanism; a plurality of cell-packing containers, the plurality of cell-containing containers respectively containing different kinds of cell suspensions; a micro-nozzle, the micro Spray a tube is disposed above the three-dimensional movement mechanism, the three-dimensional movement mechanism is moved such that the micro-nozzle is located at a desired printing position or protrudes into the cell-contained container; a micro-displacement reciprocating mechanism, a micro-displacement reciprocating mechanism coupled to the micro-nozzle and applying a desired asymmetric reciprocating motion to the micro-nozzle to perform a suction drive, a coalogenesis drive, or a jet drive to the micro-jet The desired cell suspension is aspirated into the micro-nozzle, the cells are arranged in a micro-nozzle into a close-packed single cell column and the cell suspension is ejected out of the micro-nozzle; and a three-dimensional motion controller, the three-dimensional motion a controller configured to control movement of the three-dimensional motion mechanism; a micro-displacement reciprocating motion controller configured to control bidirectional driving of the micro-nozzle by the micro-displacement reciprocating mechanism to perform The cell suspension is aspirated, pooled, and sprayed.
根据本发明实施例的细胞打印系统, μ-DOM 将所需的驱动电压信号施加至微喷管 上以对微喷管执行双向驱动,以将所需的细胞悬浮液吸入微喷管内和将细胞悬浮液喷射 出微喷管, 即以先进后出的顺序实现了细胞的打印, 从而不仅可用同一个喷头实现不同 细胞的打印, 降低了设备复杂程度和简化了操作的复杂程度, 同时可减小细胞的染菌几 率, 有利于保证打印过程中细胞的活性。又由于通过使细胞悬浮液受到交变滞惯力的作 用而实现细胞悬浮液的吸取和喷射, 从而无论是细胞悬浮液的吸取和喷射的过程中, 均 无瞬间的高温或瞬间强静电场等产生, 对细胞的损伤较小。  In accordance with a cell printing system in accordance with an embodiment of the present invention, the μ-DOM applies a desired drive voltage signal to the micro-nozzle to perform bi-directional actuation of the micro-spray to draw the desired cell suspension into the micro-nozzle and to the cells. The suspension ejects the micro-nozzle, which realizes the printing of the cells in the order of advanced output, so that not only the same nozzle can be used to print different cells, the complexity of the device is reduced, the complexity of the operation is simplified, and the operation can be reduced. The probability of cell infection is beneficial to ensure the activity of cells during printing. Moreover, since the cell suspension is subjected to the action of alternating hysteresis, the cell suspension is sucked and ejected, so that no transient high temperature or instantaneous strong electrostatic field is observed in the process of sucking and ejecting the cell suspension. Produced, less damage to cells.
另外, 根据本发明的细胞打印系统还具有如下附加技术特征:  In addition, the cell printing system according to the present invention has the following additional technical features:
在本发明的一些实施例中, 细胞打印系统还包括摄像装置, 所述摄像装置对所述吸 取、 聚齐和打印过程进行实时观测。 从而可检测细胞的吸取 -打印的整个操作过程是否 正常运行, 保证细胞打印系统的可靠性。  In some embodiments of the invention, the cell printing system further includes an image capture device that performs real-time observations of the picking, gathering, and printing processes. Thereby, it is possible to detect whether the cell is sucked up - the entire operation of the printing process is normal, and the reliability of the cell printing system is ensured.
进一步地, 细胞打印系统还包括微喷管夹具, 所述微喷管通过所述微喷管夹具与所 述微位移往复运动机构的下端相连。从而可便于微喷管的安装,避免对微喷管造成损坏。  Further, the cell printing system further includes a micro-nozzle holder through which the micro-nozzle is coupled to the lower end of the micro-displacement reciprocating mechanism. Thereby, the installation of the micro-nozzle can be facilitated, and damage to the micro-nozzle can be avoided.
优选地, 所述微喷管的内径 dnzzle满足 ldedl<dnzzle<2dedl, 其中 dedl为所述细胞悬浮 液中的单细胞直径。从而可避免细胞堵塞微喷管或形成局部堆积, 保证细胞悬浮液中的 细胞以单细胞的形式打印。 Preferably, the inner diameter d n of the micro-nozzle. Zzle satisfies ld edl <d n . Zzle <2d edl , where d edl is the single cell diameter in the cell suspension. Thereby, the cells can be prevented from clogging the micro-nozzles or forming local accumulation, and the cells in the cell suspension can be printed in the form of single cells.
在本发明的一些实施例中, 在所述微喷管进行细胞悬浮液的吸取过程中, 所述微喷 管做离开液面的运动, 其加速度由零增加到第一预定值, 接着保持所述第一预定值第一 预定时间, 最后再回到零值; 以及施加在所述微位移往复运动机构上的驱动信号使得所 述微喷管的位移波形的曲线斜率的绝对值随着时间的变化而逐渐增大。  In some embodiments of the present invention, during the suction of the cell suspension by the micro-nozzle, the micro-nozzle is moved away from the liquid surface, the acceleration is increased from zero to a first predetermined value, and then the Determining a first predetermined value for a first predetermined time, and finally returning to a zero value; and applying a driving signal on the micro-displacement reciprocating mechanism such that an absolute value of a slope of a displacement waveform of the micro-nozzle is over time Change and gradually increase.
在本发明的一些实施例中, 在所述微喷管进行单细胞的打印过程中, 所述微喷管做 靠近液面的运动, 其加速度先由零上升到第二预定值, 接着保持所述第二预定值第二预 定时间, 最后再回到零值; 以及施加在所述微位移往复运动机构上的驱动信号使得所述 微喷管的位移波形的曲线斜率随着时间的变化而逐渐增大。  In some embodiments of the present invention, during the single cell printing process of the micro-nozzle, the micro-nozzle is moved close to the liquid surface, and the acceleration first rises from zero to a second predetermined value, and then the Determining a second predetermined value for a second predetermined time, and finally returning to a zero value; and applying a driving signal to the micro-displacement reciprocating mechanism such that a slope of a curve of the displacement waveform of the micro-nozzle gradually changes with time Increase.
在本发明的一些实施例中, 在将细胞在所述微喷管内进行排列成密排单细胞列的过 程中, 所述微喷管的加速度先由零上升到第三预定值, 接着保持所述第三预定值第三预 定时间, 最后再回到零值; 以及施加在所述微位移往复运动机构上的驱动信号使得所述 微喷管的位移波形在第四预定时间内呈现为曲线形状后在第五预定时间内呈现为直线 形状, 且在所述第四预定时间内, 所述曲线的斜率随着时间的变化而逐渐增大, 在所述 第五预定时间所述直线的斜率为负值。 In some embodiments of the present invention, in the process of arranging cells in the micro-nozzle into a close-packed single cell row, the acceleration of the micro-nozzle first rises from zero to a third predetermined value, and then maintains Determining a third predetermined value for a third predetermined time, and finally returning to a zero value; and driving signals applied to the micro-displacement reciprocating mechanism such that a displacement waveform of the micro-nozzle assumes a curved shape for a fourth predetermined time After being presented as a straight line in the fifth predetermined time a shape, and during the fourth predetermined time, the slope of the curve gradually increases with time, and the slope of the straight line is a negative value at the fifth predetermined time.
可选地, 所述所需的打印位置处位于空气介质中、 液体介质中或者凝胶介质中。 具体地, 所述微位移往复运动控制器控制所述微位移往复运动机构, 以使所述微喷 管产生与细胞吸取驱动相对应的非对称的往复运动,而将所需的细胞悬浮液吸入所述微 喷管中。  Optionally, the desired printing position is in an air medium, a liquid medium or a gel medium. Specifically, the micro-displacement reciprocating controller controls the micro-displacement reciprocating mechanism to cause the micro-nozzle to generate an asymmetric reciprocating motion corresponding to the cell suction drive, and to inhale the desired cell suspension In the micro-nozzle.
具体地, 所述微位移往复运动控制器控制所述微位移往复运动机构, 以使所述微喷 管产生与细胞聚齐驱动相对应的非对称的往复运动,而将所述细胞在微喷管中形成密排 单细胞列。  Specifically, the micro-displacement reciprocating motion controller controls the micro-displacement reciprocating mechanism to cause the micro-nozzle to generate an asymmetric reciprocating motion corresponding to a cell-concentration drive, and the cells are in a micro-nozzle A dense row of single cell columns is formed.
具体地, 所述微位移往复运动控制器控制所述吸取位移曲线的, 以使所述微喷管产 生与细胞喷射驱动相对应的非对称的往复运动,而将所述细胞悬浮液打印在所需的打印 位置处。  Specifically, the micro-displacement reciprocating controller controls the suction displacement curve to cause the micro-nozzle to generate an asymmetric reciprocating motion corresponding to the cell ejection drive, and print the cell suspension in the The desired print position.
在发明的一些实施例中, 所述微喷管位于所需的打印位置处的正上方且所述微喷管 与所述所需的打印位置处之间的距离为 0〜5mm。  In some embodiments of the invention, the micro-nozzle is located directly above the desired printing position and the distance between the micro-nozzle and the desired printing position is 0 to 5 mm.
具体地, 所述微位移往复运动控制器通过控制所述微位移往复运动机构的电压、频 率和驱动波形时间宽度来控制所需细胞悬浮液的吸入量、聚齐程度或者喷射数量。从而 可减少细胞的浪费。  Specifically, the micro-displacement reciprocating motion controller controls the inhalation amount, the degree of convergence, or the number of ejections of the desired cell suspension by controlling the voltage, frequency, and driving waveform time width of the micro-displacement reciprocating mechanism. Thereby reducing the waste of cells.
本发明的附加方面和优点将在下面的描述中部分给出, 部分将从下面的描述中变得 明显, 或通过本发明的实践了解到。 附图说明  The additional aspects and advantages of the invention will be set forth in part in the description which follows. DRAWINGS
本发明的上述和 /或附加的方面和优点从结合下面附图对实施例的描述中将变得明 显和容易理解, 其中:  The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from
图 1为根据本发明实施例的细胞打印方法的流程图;  1 is a flow chart of a cell printing method according to an embodiment of the present invention;
图 2为根据本发明实施例的细胞打印系统的示意图;  2 is a schematic diagram of a cell printing system in accordance with an embodiment of the present invention;
图 3为图 2所示的细胞打印系统中的微喷管伸入到细胞盛装容器内时的示意图; 图 4为图 2所示的细胞打印系统中的微喷管位于所需打印位置处的示意图; 图 5 为根据本发明一个实施例的处于细胞打印过程中微喷管的加速度的波形示意 图;  Figure 3 is a schematic view of the micro-pipe in the cell printing system shown in Figure 2 when it is inserted into the cell-contained container; Figure 4 is the micro-pipe in the cell printing system shown in Figure 2 at the desired printing position Figure 5 is a schematic view showing the waveform of the acceleration of the micro-nozzle during cell printing according to an embodiment of the present invention;
图 6为根据本发明一个实施例的细胞打印过程中施加在 μ-DOM上的电压的波形示 意图;  Figure 6 is a waveform diagram of a voltage applied to a μ-DOM during cell printing in accordance with one embodiment of the present invention;
图 7 为根据本发明一个实施例的处于细胞吸取过程中微喷管的加速度的波形示意 图;  Figure 7 is a schematic diagram showing the waveform of the acceleration of the micro-nozzle during cell uptake according to one embodiment of the present invention;
图 8为根据本发明一个实施例的细胞吸取过程中施加在 μ-DOM上的电压的波形示 意图;  Figure 8 is a waveform diagram showing voltages applied to the μ-DOM during cell uptake according to an embodiment of the present invention;
图 9 为根据本发明一个实施例的处于细胞聚齐过程中微喷管的加速度的波形示意 图; Figure 9 is a waveform diagram showing the acceleration of the micro-nozzle during cell aggregation in accordance with one embodiment of the present invention. Figure
图 10为根据本发明一个实施例的细胞聚齐过程中施加在 μ-DOM上的电压的波形示 意图。 附图标记:  Figure 10 is a waveform diagram showing the voltage applied to the μ-DOM during cell aggregation in accordance with one embodiment of the present invention. Reference mark:
细胞打印系统 100、 三维运动机构 1、 细胞盛装容器 2、 微喷管 3、  Cell printing system 100, three-dimensional motion mechanism 1, cell-contained container 2, micro-nozzle 3,
微位移往复运动机构 4、 微位移往复运动控制器 50、 工控机 51、  Micro-displacement reciprocating mechanism 4, micro-displacement reciprocating motion controller 50, industrial computer 51,
三维运动控制器 52、 摄像装置 6、 微喷管夹具 7 具体实施方式  Three-dimensional motion controller 52, imaging device 6, micro-jet clamp 7
下面详细描述本发明的实施例, 所述实施例的示例在附图中示出, 其中自始至终相 同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附 图描述的实施例是示例性的, 仅用于解释本发明, 而不能理解为对本发明的限制。  The embodiments of the present invention are described in detail below, and the examples of the embodiments are illustrated in the drawings, wherein the same or similar reference numerals are used to refer to the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the drawings are intended to be illustrative only and not to limit the invention.
在本发明的描述中, 需要理解的是, 术语"中心"、 "上"、 "下"、 "前"、 "后"、 "左" 、 "右" 、 "竖直" 、 "水平" 、 "顶" 、 "底" "内" 、 "外"等指示的方位 或位置关系为基于附图所示的方位或位置关系, 仅是为了便于描述本发明和简化描述, 而不是指示或暗示所指的装置或元件必须具有特定的方位、 以特定的方位构造和操作, 因此不能理解为对本发明的限制。  In the description of the present invention, it is to be understood that the terms "center", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", The orientation or positional relationship of the "top", "bottom", "inside", "outside" and the like is based on the orientation or positional relationship shown in the drawings, and is merely for the convenience of describing the present invention and simplifying the description, rather than indicating or implying The device or component referred to has a particular orientation, is constructed and operated in a particular orientation and is therefore not to be construed as limiting the invention.
需要说明的是, 术语 "第一" 、 "第二 "仅用于描述目的, 而不能理解为指示或暗 示相对重要性或者隐含指明所指示的技术特征的数量。 由此, 限定有 "第一"、 "第二" 的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地,在本发明的描述中, 除非另有说明, "多个" 的含义是两个或两个以上。  It should be noted that the terms "first" and "second" are used for descriptive purposes only, and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, features defining "first", "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, "multiple" means two or more unless otherwise stated.
下面参考图 1、 图 5-图 10描述根据本发明实施例的一种细胞打印方法。其中, 用本 发明的细胞打印方法打印出的细胞可用于高精度地制造组织工程产品和制备单细胞点 样。组织工程产品可以用于在体外重建出人体的复杂组织或器官, 可以用于人体坏损组 织或器官的人工修复, 而单细胞点样的制备则可用于高通量、耗时短的药物开发和药物 筛选,也可以用于构建体外肿瘤病理模型,还可用于制备细胞基的高灵敏度生物传感器。  A cell printing method according to an embodiment of the present invention will be described below with reference to FIGS. 1 and 5 to 10. Among them, the cells printed by the cell printing method of the present invention can be used for producing a tissue engineering product with high precision and preparing a single cell spot. Tissue engineering products can be used to reconstruct complex tissues or organs of the human body in vitro, and can be used for artificial repair of damaged tissues or organs of the human body, while single cell spotting can be used for high-throughput, short-time drug development. And drug screening, can also be used to construct in vitro tumor pathology models, can also be used to prepare cell-based high-sensitivity biosensors.
根据本发明实施例的细胞打印方法, 如图 1所示, 包括如下步骤:  The cell printing method according to the embodiment of the present invention, as shown in FIG. 1, includes the following steps:
S1 : 将微喷管插入所需细胞悬浮液中, 细胞悬浮液分别容纳在不同的细胞盛装容器 中, 也就是说不同的细胞盛装容器内可盛放不同的细胞悬浮液, 微喷管可伸入到盛有所 需的细胞悬浮液的细胞盛放容器内。 优选地, 该微喷管可为具有微流体通道的喷头, 以 使细胞在微流体通道内实现单列排列, 从而实现单细胞打印。  S1: Insert the micro-nozzle into the desired cell suspension, and the cell suspensions are respectively accommodated in different cell-contained containers, that is, different cell-containing containers can hold different cell suspensions, and the micro-spray can be extended. Into the container containing the desired cell suspension. Preferably, the micro-nozzle can be a showerhead having a microfluidic channel to enable cells to be arranged in a single column within the microfluidic channel to achieve single cell printing.
S2: 利用微位移往复运动机构( Micro Displacement Oscillating Mechanism, μ-DOM) 在微喷管上执行预定的细胞吸取驱动,以吸入所需的细胞悬浮液中的一定数量的细胞并 组合成细胞打印序列,具体地, 当利用 μ-DOM在微喷管上执行预定的细胞吸取驱动时, 微喷管做往复运动, 细胞悬浮液会基于交变滞惯力原理而被吸入到微喷管内。 具体地, 通过控制 μ-DOM的电压、 频率和驱动波形时间宽度来控制所需细胞悬浮液的吸入量, 以避免细胞的浪费。 S2: Performing a predetermined cell suction drive on the micro-spray using a Micro Displacement Oscillating Mechanism (μ-DOM) to inhale a certain number of cells in the desired cell suspension and combine them into a cell print sequence Specifically, when a predetermined cell suction drive is performed on the micro-pipe using the μ-DOM, the micro-nozzle is reciprocated, and the cell suspension is sucked into the micro-nozzle based on the principle of the alternating hysteresis. specifically, The amount of inhalation of the desired cell suspension is controlled by controlling the voltage, frequency, and drive waveform time width of the μ-DOM to avoid cell waste.
值得说明的是,利用交变滞惯力进行细胞的吸取过程的原理如下:微喷管在 μ-DOM 的驱动下做往复运动, 当微喷管首先向负方向运动时, 细胞盛放容器内的细胞悬浮液与 微喷管的外壁之间的粘滞力作为动力驱动细胞悬浮液向负方向运动,而细胞悬浮液的惯 性力作为阻力阻碍细胞悬浮液向负方向运动。 当微喷管接着向正方向运动时, 细胞悬浮 液的惯性力作为动力驱动细胞盛放容器内的细胞悬浮液继续向负方向运动,而细胞盛放 容器内的细胞悬浮液与微喷管的外壁之间的粘滞力作为阻力阻碍细胞悬浮液向负方向 运动。此时如果通过控制微喷管运动的加速度和加速时间, 实现当微喷管向负方向运动 时作为阻力的细胞悬浮液的惯性力较小,而当微喷管向正方向运动时作为动力的细胞悬 浮液的惯性力较大, 那么在一个运动周期内, 细胞悬浮液相对于微喷管产生一段沿负方 向的位移, 微喷管外的细胞悬浮液就被吸入到微喷管内。  It is worth noting that the principle of cell aspiration using the alternating hysteresis is as follows: the micro-nozzle is reciprocated under the drive of μ-DOM. When the micro-spray is first moved in the negative direction, the cells are placed in the container. The viscous force between the cell suspension and the outer wall of the micro-nozzle acts as a power to drive the cell suspension to move in the negative direction, and the inertial force of the cell suspension acts as a resistance to hinder the cell suspension from moving in the negative direction. When the micro-spray is then moved in the positive direction, the inertial force of the cell suspension acts as a power to drive the cell suspension in the cell holding container to continue moving in the negative direction, while the cells hold the cell suspension in the container and the micro-nozzle The viscous force between the outer walls acts as a resistance to hinder the movement of the cell suspension in the negative direction. At this time, if the acceleration and acceleration time of the movement of the micro-nozzle are controlled, the inertial force of the cell suspension as the resistance when the micro-spray moves in the negative direction is small, and when the micro-spray moves in the positive direction, it is used as the power. The inertial force of the cell suspension is large, and in one exercise cycle, the cell suspension liquid phase generates a negative displacement in the micro-nozzle, and the cell suspension outside the micro-nozzle is sucked into the micro-nozzle.
其中, 步骤 S2 中的组合成细胞打印序列, 指的是根据打印需要, 可在利用微喷管 吸入一种细胞悬浮液后接着利用微喷管吸入不同的细胞悬浮液, 也就是说, 在细胞吸取 过程中, 微喷管内可先后吸入不同的细胞悬浮液, 此时, 由于微喷管的结构限制, 多种 细胞悬浮液根据吸入动作的先后顺序在微喷管内顺序排列,即可使得多种细胞悬浮液在 微喷管内组合呈细胞打印序列。  Wherein, the combination of the step S2 into a cell print sequence means that, according to the printing needs, a cell suspension can be inhaled by using a micro-nozzle, and then the microcell nozzle is used to inhale different cell suspensions, that is, in the cell. During the suction process, different cell suspensions can be sequentially inhaled in the micro-nozzle. At this time, due to the structural limitation of the micro-nozzle, a plurality of cell suspensions are sequentially arranged in the micro-nozzle according to the order of the inhalation action, which can make various The cell suspension is combined into a cell print sequence within the microprojection.
S3: 利用 μ-DOM在所述微喷管上执行预定的细胞聚齐驱动, 将细胞在微喷管内排 列成密排单细胞列。  S3: Performing a predetermined cell aggregation drive on the micro-nozzle by using μ-DOM, and arranging the cells in a micro-nozzle into a close-packed single cell column.
值得说明的是, 在每一个驱动信号周期内, 由于微喷管在 μ-DOM驱动下做非对称 的往复运动, 所以微喷管是一个非惯性系, 微喷管体系内的细胞和细胞外的基质则受到 周期性的惯性力; 由于这种惯性力是一种体积力, 密度大的物质受到的惯性力大于密度 小的物质受到的惯性力; 由于细胞密度大于周围液态细胞外基质的密度, 所以细胞受到 的惯性力大于液态细胞外基质受到的惯性力。 因此, 细胞就会在周期性惯性力作用下相 对于流体发生聚齐运动。 如果微喷管的直径满足 ldedl<dnzzle<2dedl (其中 (^^为细胞悬 浮液中的单细胞直径) , 当聚齐运动达到稳定状态时, 细胞就会在微喷管内形成密排单 细胞列。 It is worth noting that during each drive signal period, the micro-nozzle is a non-inertial system, and the micro-nozzle is a non-inertial system, and the micro-nozzle system is outside the cell and extracellular. The matrix is subject to cyclical inertial forces; since this inertial force is a volumetric force, the density of the material is greater than the inertial force of the material with a lower density; because the cell density is greater than the density of the surrounding liquid extracellular matrix Therefore, the inertial force of the cells is greater than the inertial force of the liquid extracellular matrix. Therefore, the cells will collide with the fluid under the action of periodic inertial forces. If the diameter of the micro-nozzle satisfies ld edl <d n . Zzle <2d edl (where (^^ is the diameter of a single cell in a cell suspension), when the aggregation motion reaches a steady state, the cells form a close-packed single cell column in the micro-nozzle.
S4: 将微喷管移动至所需的打印位置, 利用 μ-DOM在微喷管上执行预定的细胞喷 射驱动, 微喷管在该细胞喷射驱动下做往复运动, 此时微喷管内的细胞会在交变滞惯力 驱动下稳定喷射出, 从而将细胞按序列喷射在打印位置处。如果需要继续打印与细胞悬 浮液对应的细胞, 则利用微喷管重复执行上述步骤 S1-S4, 直至完成所有的细胞打印。 具体地, 可通过控制 μ-DOM的电压、 频率和驱动波形时间宽度来控制所需细胞悬浮液 的吸入量、 聚齐程度或者喷射数量, 以避免细胞的浪费。  S4: moving the micro-nozzle to the desired printing position, performing a predetermined cell ejection drive on the micro-nozzle by using the μ-DOM, and the micro-nozzle is reciprocated under the driving of the cell, and the cells in the micro-spray tube at this time The ejection is stably driven by the alternating hysteresis, so that the cells are sequentially ejected at the printing position. If it is necessary to continue printing the cells corresponding to the cell suspension, the above steps S1-S4 are repeatedly performed using a micro-nozzle until all cell printing is completed. Specifically, the amount of inhalation, the degree of aggregation, or the number of ejections of the desired cell suspension can be controlled by controlling the voltage, frequency, and driving waveform time width of the μ-DOM to avoid cell waste.
值得说明的是, 利用交变滞惯力进行的细胞的喷射过程的原理如下: 吸取有细胞悬 浮液的微喷管在 μ-DOM执行的细胞喷射驱动下做往复运动, 当微喷管首先向负方向运 动时,细胞悬浮液与微喷管的内壁之间的粘滞力作为动力驱动微喷管内的细胞悬浮液向 负方向运动, 而细胞悬浮液的惯性力作为阻力阻碍细胞悬浮液向负方向运动。 当微喷管 接着向正方向运动时, 细胞悬浮液的惯性力作为动力驱动细胞悬浮液继续向负方向运 动, 而细胞悬浮液与微喷管的内壁之间的粘滞力作为阻力阻碍细胞悬浮液向负方向运 动。如果通过控制微喷管运动的加速度和加速度的时间, 实现当微喷管向负方向运动时 作为阻力的细胞悬浮液的惯性力较小,而当微喷管向正方向运动时作为动力的细胞悬浮 液的惯性力较大, 那么在一个运动周期内, 细胞悬浮液相对于微喷管就要产生一段沿负 方向的位移, 从而细胞悬浮液从微喷管内喷射出以打印在所需打印位置处。 It is worth noting that the principle of the cell ejection process using the alternating hysteresis is as follows: A micro-nozzle with a cell suspension is taken to reciprocate under the cell ejection drive performed by the μ-DOM, when the micro-spray is first directed When moving in the negative direction, the viscous force between the cell suspension and the inner wall of the micro-nozzle acts as a power to drive the cell suspension in the micro-spray The negative direction moves, and the inertial force of the cell suspension acts as a resistance to prevent the cell suspension from moving in the negative direction. When the micro-spray is then moved in the positive direction, the inertial force of the cell suspension acts as a power-driven cell suspension to continue to move in the negative direction, and the viscous force between the cell suspension and the inner wall of the micro-nozzle acts as a resistance to hinder cell suspension. The liquid moves in the negative direction. If the time of acceleration and acceleration of the movement of the micro-nozzle is controlled, the inertial force of the cell suspension as a resistance when the micro-jet is moved in the negative direction is small, and the cell which is the power when the micro-jet moves in the positive direction is realized. The inertial force of the suspension is large, then in a movement cycle, the cell suspension liquid phase will produce a negative displacement in the micro-nozzle, so that the cell suspension is ejected from the micro-nozzle to print at the desired printing position. At the office.
其中, 值得理解的是, 根据本发明实施例的细胞打印方法在进行上述的步骤前, 应 将微喷管和 μ-DOM放入到无菌腔室内进行灭菌处理, 以保证上述步骤是在无菌的环境 下进行, 避免细菌对细胞悬浮液的污染, 保证细胞的成活率和生物学性状。 其中, 值得 注意的是, 在进行杀菌过程中, 不能将盛放有细胞悬浮液的细胞盛放容器放置在无菌腔 室内, 以免将细胞杀死, 可在杀菌完成后等待一定时间如 5分钟后再将盛放有细胞悬浮 液的细胞盛装容器放入到无菌腔室内。 具体地, 可采用紫外线灭菌灯对 μ-DOM进行灭 菌, 且可采用高压蒸汽灭菌方式对微喷管进行灭菌处理。  It should be understood that, in the cell printing method according to the embodiment of the present invention, the micro-nozzle and the μ-DOM should be placed in a sterile chamber for sterilization before the above steps are performed to ensure that the above steps are It is carried out in a sterile environment to avoid bacterial contamination of the cell suspension and to ensure the survival rate and biological properties of the cells. Among them, it is worth noting that during the sterilization process, the cell-filling container containing the cell suspension cannot be placed in the sterile chamber to avoid killing the cells, and can wait for a certain time, such as 5 minutes, after the sterilization is completed. The cell-filled container containing the cell suspension is then placed in a sterile chamber. Specifically, the μ-DOM can be sterilized by an ultraviolet sterilizing lamp, and the micro-nozzle can be sterilized by high-pressure steam sterilization.
同时该所需的打印位置处可位于空气介质中、 液体介质中或凝胶介质中, 具体地, 该液体介质为细胞培养基、 海藻酸钠溶液、 胶原、 纤维蛋白质中的一种, 凝胶介质为各 种凝胶中的一种。 同时, 在将细胞悬浮液打印在所需打印位置时, 微喷管可以悬于作为 细胞打印的载体的基板上方的一定高度进行特定图案或性状的细胞打印,也可以插入组 织工程支架的网格空洞中进行细胞打印,以实现在支架特定位置种植特定数量细胞的目 的。  At the same time, the desired printing position may be located in an air medium, a liquid medium or a gel medium. Specifically, the liquid medium is one of a cell culture medium, a sodium alginate solution, collagen, and a fiber protein. The medium is one of various gels. At the same time, when the cell suspension is printed at the desired printing position, the micro-nozzle can be suspended at a certain height above the substrate as a carrier for cell printing for cell printing of a specific pattern or trait, or can be inserted into the grid of the tissue engineering scaffold. Cell printing is performed in the cavity to achieve the purpose of planting a specific number of cells at specific locations of the stent.
具体而言, 当需要进行细胞打印时, 首先将微喷管插入到盛放有细胞悬浮液的细胞 盛装容器内, 接着 μ-DOM执行预定的细胞吸取驱动, μ-DOM驱动微喷管在细胞悬浮 液内进行与该细胞吸取驱动对应的往复运动,以使得细胞盛装容器内的细胞悬浮液受到 交变滞惯力的作用,且在该交变滞惯力的作用下被吸入到微喷管内,如此重复上述过程, 从而将多种细胞悬浮液吸入到微喷管内,且使得多种细胞悬浮液在微喷管内组合成细胞 打印序列。  Specifically, when cell printing is required, the micro-spray tube is first inserted into a cell-containing container containing the cell suspension, and then the μ-DOM performs a predetermined cell-absorption drive, and the μ-DOM drives the micro-nozzle in the cell. Reciprocating motion corresponding to the cell suction drive is performed in the suspension, so that the cell suspension in the cell-contained container is subjected to the alternating inertia and is sucked into the micro-spray tube under the action of the alternating inertia The above procedure is repeated such that a plurality of cell suspensions are aspirated into the micro-nozzles and a plurality of cell suspensions are combined into a cell print sequence within the micro-nozzles.
然后将吸取有细胞悬浮液的微喷管在 μ-DOM的聚齐驱动下做聚齐运动, 使细胞在 微喷管中排列成密排单细胞列。  Then, the micro-nozzles sucking the cell suspension are mixed and driven by the μ-DOM, and the cells are arranged in a micro-pipe to form a close-packed single cell column.
然后将微喷管移动到所需的打印位置处, μ-DOM 执行预定的细胞喷射驱动, 微喷 管在 μ-DOM的驱动下进行与该细胞喷射驱动对应的往复运动, 以使得微喷管内的细胞 悬浮液会受到交变滞惯力的作用,且在该交变滞惯力的作用下细胞悬浮液从微喷管内喷 射出以打印在所需的打印位置处, 即完成一次细胞的吸取 -打印过程。 如果需要继续进 行打印, 可将微喷管重新插入到细胞悬浮液内重复执行上述的步骤, 以完成多次细胞的 吸取 -打印过程。  The micro-nozzle is then moved to the desired printing position, the μ-DOM performs a predetermined cell ejection drive, and the micro-nozzle is driven by the μ-DOM to perform a reciprocating motion corresponding to the cell ejection drive so that the micro-nozzle is inside The cell suspension is subjected to alternating inertia, and under the action of the alternating inertia, the cell suspension is ejected from the micro-nozzle to be printed at the desired printing position, that is, the cell is absorbed. - The printing process. If you need to continue printing, re-insert the micro-nozzle into the cell suspension and repeat the above steps to complete the multiple-cell capture-print process.
其中,值得理解的是,在利用上述的细胞打印方法进行多次细胞的吸取-打印过程时, 每次细胞的吸取-打印过程中的吸取的细胞悬浮液可相同也可不同。 根据本发明实施例的的细胞打印方法, 通过利用细胞悬浮液的粘滞力和惯性力交替 地作为动力即利用交变滞惯力的作用来实现细胞悬浮液的吸取和喷射,从而无论是在细 胞悬浮液的吸取或喷射的过程中, 均无瞬间高温、 高压或瞬间强电场等产生, 对细胞的 损伤较小, 同时由于本发明的细胞打印方法采用先进后出的顺序吸取和喷射细胞, 从而 可利用同一个微喷管以实现多种细胞的吸取和打印操作,避免了因采用多组微喷管而使 设备成本增加, 避免了因更换微喷管而造成的操作过程复杂化, 同时避免了使用其他工 具进行上述操作, 可减少细胞的染菌几率, 并且能实现"即吸即打印"的连贯细胞操作, 有利于保证打印过程中的细胞的活性。 Among them, it is understood that the cell suspensions taken during the pipetting-printing process of the cells may be the same or different when the cell-pick-printing process is performed multiple times using the cell printing method described above. According to the cell printing method of the embodiment of the present invention, the cell suspension is sucked and ejected by using the viscous force and the inertial force of the cell suspension alternately as a power, that is, by utilizing the action of the alternating hysteresis inertia, thereby During the process of aspirating or spraying the cell suspension, no transient high temperature, high pressure or transient strong electric field is generated, and the damage to the cells is small, and at the same time, since the cell printing method of the present invention uses the advanced ejection sequence to suck and spray cells, Therefore, the same micro-nozzle can be utilized to realize a plurality of cell suction and printing operations, thereby avoiding the increase in equipment cost due to the use of multiple sets of micro-nozzles, and avoiding the complicated operation process caused by the replacement of the micro-nozzles, and at the same time It avoids the use of other tools for the above operations, can reduce the chance of cell infection, and can achieve "snap-and-print" coherent cell operation, which is beneficial to ensure the activity of cells during printing.
具体地, 在步骤 S2中, μ-DOM通过使微喷管产生与细胞吸取驱动相对应的非对称 的往复运动, 而将所需的细胞悬浮液吸入微喷管中。 在步骤 S3中, μ-DOM通过使微喷 管产生与细胞聚齐驱动相对应的非对称的往复运动,而将细胞在微喷管出口附近排列成 密排单细胞列。在步骤 S4中, μ-DOM通过使微喷管产生与细胞喷射驱动相对应的非对 称的往复运动, 而将细胞悬浮液打印在打印位置处。  Specifically, in step S2, the μ-DOM draws the desired cell suspension into the micro-nozzle by causing the micro-nozzle to generate an asymmetric reciprocating motion corresponding to the cell suction drive. In step S3, the μ-DOM arranges the cells in a close-packed single cell row near the exit of the micro-jet by causing the micro-spray to generate an asymmetric reciprocating motion corresponding to the cell-gathering drive. In step S4, the μ-DOM prints the cell suspension at the printing position by causing the micro-nozzle to generate an asymmetrical reciprocating motion corresponding to the cell ejection drive.
具体地, μ-DOM 驱动微喷管产生与不同的位移曲线相对应的非对称的轴向往复运 动, 以执行对所需的细胞悬浮液的喷射驱动、 吸取驱动和聚齐驱动。 也就是说, μ-DOM 驱动微喷管产生的与细胞吸取驱动相对应的位移曲线的轴向往复运动、 μ-DOM驱动微 喷管产生的与细胞聚齐驱动相对应的位移曲线的轴向往复运动和 μ-DOM驱动微喷管产 生的与细胞喷射驱动相对应的位移曲线的轴向往复运动是不同的。  Specifically, the μ-DOM driven micro-nozzles generate asymmetric axial reciprocating motions corresponding to different displacement curves to perform jet drive, suction drive, and bunch drive for the desired cell suspension. That is, the axial reciprocating motion of the displacement curve corresponding to the cell suction drive generated by the μ-DOM driven micro-nozzle, and the axial reciprocation of the displacement curve corresponding to the cell aggregation drive generated by the μ-DOM driven micro-nozzle The axial reciprocating motion of the displacement curve corresponding to the cell ejection drive produced by the motion and μ-DOM driven micro-nozzles is different.
由于在微喷管运动的过程中往往会存在振动问题, 而微喷管的振动既会影响细胞的 成活率又会影响细胞的打印位置精度, 所以为了减小微喷管运动过程中的振动, 在本发 明的一些实施例中, 如图 7所示, 在步骤 S2中, 微喷管做离开液面的运动, 其加速度 由零增加到第一预定值, 接着保持第一预定值第一预定时间, 最后再回到零值。 从而保 证微喷管在加速的过程中, 速度是连续变化而不是跳跃变化的, 以减小微喷管运动过程 中的振动。 以及如图 8所示, 在步骤 S2中, 施加在 μ-DOM上的电压为这样的电压波 形, 电压波形的曲线斜率为负值, 且曲线斜率的绝对值随着时间的变化而逐渐增大, 由 于 μ-DOM具有电压和位移基本呈正比例的关系,所以该波形也代表了微喷管在步骤 S2 中的位移波形, 也就是说, 如图 8所示, 施加在 μ-DOM上的驱动信号使得微喷管的位 移波形的曲线斜率的绝对值随着时间的变化而逐渐增大。其中值得说明的是, 上述的在 步骤 S2中微喷管的加速度波形和施加在 μ-DOM上的电压波形仅为示例性说明, 而不 是对本发明的具体限制, 本领域的技术人员应该理解的是, 在步骤 S2中, 微喷管的加 速度波形和施加在 μ-DOM上的电压波形只要满足使得细胞悬浮液可受到交变滞惯力的 作用而被吸取到微喷管内且使得微喷管在运动过程中的振动较小即可。  Since the vibration problem often occurs during the movement of the micro-nozzle, the vibration of the micro-nozzle affects both the survival rate of the cell and the printing position accuracy of the cell, so in order to reduce the vibration during the movement of the micro-nozzle, In some embodiments of the present invention, as shown in FIG. 7, in step S2, the micro-nozzle is moved away from the liquid surface, its acceleration is increased from zero to a first predetermined value, and then the first predetermined value is maintained for the first predetermined Time, and finally back to zero. Therefore, the speed of the micro-nozzle is continuously changed instead of jumping during the acceleration process to reduce the vibration during the movement of the micro-nozzle. And as shown in FIG. 8, in step S2, the voltage applied to the μ-DOM is such a voltage waveform, the slope of the curve of the voltage waveform is a negative value, and the absolute value of the slope of the curve gradually increases with time. Since the μ-DOM has a substantially proportional relationship between voltage and displacement, the waveform also represents the displacement waveform of the micro-nozzle in step S2, that is, as shown in FIG. 8, the drive applied to the μ-DOM The signal causes the absolute value of the slope of the curve of the displacement waveform of the micro-nozzle to gradually increase with time. It should be noted that the acceleration waveform of the micro-nozzle and the voltage waveform applied to the μ-DOM in the above step S2 are merely illustrative, and are not specific limitations of the present invention, which should be understood by those skilled in the art. Yes, in step S2, the acceleration waveform of the micro-nozzle and the voltage waveform applied to the μ-DOM are sucked into the micro-nozzle as long as the cell suspension can be subjected to the alternating inertia and the micro-nozzle is made The vibration during the movement is small.
同时为了减少微喷管运动过程中的振动, 在本发明的一些实施例中, 如图 5所示, 在步骤 S4中, 微喷管做靠近液面的运动, 其加速度先由零上升到第二预定值, 接着保 持第二预定值第二预定时间, 最后再回到零值, 从而保证在微喷管加速的过程中, 速度 是连续变化而不是跳跃变化的。 以及如图 6所示, 施加在 μ-DOM上的电压为这样的电 压波形, 电压波形的曲线斜率随着时间的变化而逐渐增大, 由于 μ-DOM具有电压和位 移呈正比例的关系, 所以该波形也代表了微喷管在步骤 S4中的位移波形, 也就是说, 如图 6所示, 施加在 μ-DOM上的驱动信号使得微喷管的位移波形的曲线斜率随着时间 的变化而逐渐增大。 At the same time, in order to reduce the vibration during the movement of the micro-nozzle, in some embodiments of the present invention, as shown in FIG. 5, in step S4, the micro-nozzle is moved close to the liquid surface, and its acceleration first rises from zero to the first The second predetermined value is then held for a second predetermined value for a second predetermined time, and finally returned to zero value, thereby ensuring that the speed is continuously changed rather than jumped during the acceleration of the micro-nozzle. And as shown in Figure 6, the voltage applied to the μ-DOM is such a The waveform of the voltage waveform gradually increases with time. Since the μ-DOM has a proportional relationship between voltage and displacement, the waveform also represents the displacement waveform of the micro-nozzle in step S4, that is, That is, as shown in FIG. 6, the driving signal applied to the μ-DOM causes the slope of the curve of the displacement waveform of the micro-nozzle to gradually increase with time.
其中值得说明的是,上述的在步骤 S4中微喷管的加速波形和施加在 μ-DOM上的电 压波形仅为示例性说明,而不是对本发明的具体限制,本领域的技术人员应该理解的是, 在步骤 S4中, 微喷管的加速度波形和施加在 μ-DOM上的电压波形只要满足使得微喷 管内的细胞悬浮液在交变滞惯力的作用下从微喷管内喷射出且使得微喷管在运动过程 中的振动较小即可。  It should be noted that the above-mentioned acceleration waveform of the micro-nozzle and the voltage waveform applied to the μ-DOM in the above step S4 are merely illustrative, and are not specifically limited to the present invention, and those skilled in the art should understand. Yes, in step S4, the acceleration waveform of the micro-nozzle and the voltage waveform applied to the μ-DOM are such that the cell suspension in the micro-jet is ejected from the micro-nozzle under the action of the alternating hysteresis and makes The vibration of the micro-nozzle during the movement is small.
进一步地, 为了减少微喷管运动过程中的振动, 在本发明的一些实施例中, 如图 9 所示, 在步骤 S3中, 微喷管的加速度先由零上升到第三预定值, 接着保持第三预定值 第三预定时间, 最后再回到零值, 从而保证在微喷管加速的过程中, 速度是连续变化而 不是跳跃变化的。 以及如图 10所示, 施加在 μ-DOM上的电压为这样的电压波形, 电 压波形在第四预定时间内呈现为曲线形状后在第五预定时间内呈现为直线形状,且在第 四预定时间内, 曲线的斜率随着时间的变化而逐渐增大, 在第五预定时间直线的斜率为 负值且保持为定值。 由于 μ-DOM具有电压和位移呈正比例的关系, 所以该波形也代表 了微喷管在步骤 S3中的位移波形, 也就是说, 如图 10所示, 施加在微位移往复运动机 构上的驱动信号使得微喷管的位移波形在第四预定时间内呈现为曲线形状后在第五预 定时间内呈现为直线形状, 且在第四预定时间内, 曲线的斜率随着时间的变化而逐渐增 大, 在第五预定时间直线的斜率为负值。  Further, in order to reduce the vibration during the movement of the micro-nozzle, in some embodiments of the present invention, as shown in FIG. 9, in step S3, the acceleration of the micro-nozzle first rises from zero to a third predetermined value, and then The third predetermined value is maintained for a third predetermined time, and finally returned to zero value, thereby ensuring that the speed is continuously changed rather than jumped during the acceleration of the micro-nozzle. And as shown in FIG. 10, the voltage applied to the μ-DOM is a voltage waveform that appears as a curved shape after the fourth predetermined time and then appears as a linear shape in the fifth predetermined time, and is in a fourth predetermined During the time, the slope of the curve gradually increases with time, and the slope of the straight line at the fifth predetermined time is negative and remains constant. Since the μ-DOM has a proportional relationship between voltage and displacement, the waveform also represents the displacement waveform of the micro-nozzle in step S3, that is, as shown in FIG. 10, the driving applied to the micro-displacement reciprocating mechanism The signal causes the displacement waveform of the micro-nozzle to assume a curved shape after the fourth predetermined time and then assumes a linear shape for a fifth predetermined time, and in a fourth predetermined time, the slope of the curve gradually increases with time. The slope of the straight line at the fifth predetermined time is a negative value.
其中值得说明的是,上述的在步骤 S3中微喷管的加速波形和施加在 μ-DOM上的电 压波形仅为示例性说明,而不是对本发明的具体限制,本领域的技术人员应该理解的是, 在步骤 S3 中, 微喷管的加速度波形和施加在 μ-DOM上的电压波形只要满足使得微喷 管内的细胞悬浮液中的细胞会在周期性惯性力的作用下相对于流体发生聚齐运动,最后 使得细胞在微喷管内形成密排单细胞列即可。  It should be noted that the above-mentioned acceleration waveform of the micro-nozzle and the voltage waveform applied to the μ-DOM in the above step S3 are merely illustrative, and are not specific limitations of the present invention, which should be understood by those skilled in the art. Yes, in step S3, the acceleration waveform of the micro-nozzle and the voltage waveform applied to the μ-DOM are satisfied so that the cells in the cell suspension in the micro-nozzle are concentrated with respect to the fluid under the action of periodic inertial forces. Exercise, and finally make the cells form a dense row of single cell columns in the micro-nozzle.
在本发明的一些实施例中, 微喷管的内径 dnzzle满足 ldcdl<dnzzk<2dcdl, 其中 ^^为 细胞悬浮液中的单细胞直径,以保证细胞悬浮液内的细胞在微喷管内呈稳定的单细胞列 排列, 防止细胞堵塞微喷管或形成局部堆积, 进而保证细胞悬浮液内的细胞以单细胞的 形状喷射出。 In some embodiments of the present invention, the micro nozzle inner diameter d n. Zzle satisfies ld cdl <d n . Zzk <2d cdl , where ^^ is the single cell diameter in the cell suspension to ensure that the cells in the cell suspension are arranged in a stable single cell array in the micro-spray tube, preventing the cells from clogging the micro-nozzles or forming local accumulation, and further It is ensured that the cells in the cell suspension are ejected in the shape of a single cell.
下面参考图 2-图 10描述根据本发明实施例的一种细胞打印系统 100。  A cell printing system 100 in accordance with an embodiment of the present invention will now be described with reference to Figs.
根据本发明实施例的细胞打印系统 100, 如图 2所示, 包括: 三维运动机构 1、 多 个细胞盛装容器 2、 微喷管 3、 μ-ϋΟΜ 4、 三维运动控制器 52和微位移往复运动控制器 50, 其中, 三维运动机构 1可在上下 (如图 2所示的 Ζ向) 、 左右 (如图 2所示的 Υ 向) 和前后 (如图 2所示的 X向) 六个方向上移动。 多个细胞盛装容器 2中分别容纳 有不同种类的细胞悬浮液。微喷管 3设在三维运动机构 1的上方, 三维运动机构 1移动 以使得微喷管 3位于所需的打印位置处或伸入到细胞盛装容器 2内。在本发明的一些示 例中, 所需的打印位置处为载玻片的上表面。 μ-ϋΟΜ4与微喷管 3相连, 且将所需的非 对称往复运动施加至微喷管 3上以对微喷管 3执行吸取驱动、 聚齐驱动或者喷射驱动, 以将所需的细胞悬浮液吸入微喷管 3内、将细胞在微喷管 3内排列成密排单细胞列和将 细胞悬浮液喷射出微喷管 3。优选地, 微喷管 3通过微喷管夹具 7与 μ-ϋΟΜ4的下端相 连, 从而便于微喷管 3的安装, 避免微喷管 3的损坏。 三维运动控制器 52配置成控制 三维运动机构 1的移动。 微位移往复运动控制器 50配置成控制 μ-ϋΟΜ4对微喷管 3的 双向驱动, 以执行细胞悬浮液的吸取、 聚齐和喷射。 The cell printing system 100 according to an embodiment of the present invention, as shown in FIG. 2, includes: a three-dimensional motion mechanism 1, a plurality of cell-contained containers 2, a micro-nozzle 3, a μ-ϋΟΜ 4, a three-dimensional motion controller 52, and a micro-displacement reciprocating The motion controller 50, wherein the three-dimensional motion mechanism 1 can be up and down (as shown in FIG. 2), left and right (as shown in FIG. 2), and front and rear (as shown in FIG. 2). Move in the direction. Different cell suspensions are contained in the plurality of cell-containing containers 2, respectively. The micro-pipe 3 is disposed above the three-dimensional moving mechanism 1, and the three-dimensional moving mechanism 1 is moved so that the micro-jet 3 is located at a desired printing position or protrudes into the cell-containing container 2. Some indications in the invention In the example, the desired print position is the upper surface of the slide. The μ-ϋΟΜ4 is connected to the micro-nozzle 3, and the required asymmetric reciprocating motion is applied to the micro-nozzle 3 to perform suction driving, coalescing driving or jet driving on the micro-jet 3 to bring the desired cell suspension The cells are sucked into the micro-pipe 3, and the cells are arranged in a micro-lance 3 to form a single cell row and the cell suspension is ejected out of the micro-jet 3. Preferably, the micro-nozzle 3 is connected to the lower end of the μ-ϋΟΜ4 through the micro-pipe clamp 7, thereby facilitating the mounting of the micro-nozzle 3 and avoiding damage of the micro-nozzle 3. The three-dimensional motion controller 52 is configured to control the movement of the three-dimensional motion mechanism 1. The micro-displacement reciprocating motion controller 50 is configured to control the bi-directional actuation of the micro-nozzle 3 by the μ-ϋΟΜ4 to perform the suction, collection, and ejection of the cell suspension.
在本发明的一些实施例中, 细胞打印系统 100还包括工控机 51, 工控机 51与微位 移往复运动控制器 50和三维运动控制器 52相连,微位移往复运动控制器 50与 μ-DOM 4相连用于提供 μ-DOM 4的驱动电压信号, 微位移往复运动控制器 50的电压调节范围 为 0-90ν, 频率调节范围为 1-200Ηζ。 三维运动控制器 52接收工控机 51发出的指令以 控制三维运动机构 1的运动。  In some embodiments of the present invention, the cell printing system 100 further includes an industrial computer 51 connected to the micro-displacement reciprocating motion controller 50 and the three-dimensional motion controller 52, and the micro-displacement reciprocating motion controller 50 and the μ-DOM 4 Connected to provide the driving voltage signal of the μ-DOM 4, the voltage adjustment range of the micro-displacement reciprocating controller 50 is 0-90 ν, and the frequency adjustment range is 1-200 Ηζ. The three-dimensional motion controller 52 receives an instruction from the industrial computer 51 to control the motion of the three-dimensional motion mechanism 1.
具体地, 本发明的细胞打印系统 100放置在无菌腔室内, 在细胞打印系统 100工作 之前, 先对三维运动机构 1、 μ-ϋΟΜ4和微喷管 3进行杀菌, 此时可使用紫外线灭菌灯 对三维运动机构 1和 μ-ϋΟΜ4进行灭菌, 灭菌时间约为 30分钟, 微喷管 3采用高压蒸 汽灭菌后与 μ-ϋΟΜ4 相连。 其中, 值得注意的是, 在进行杀菌过程中, 不能将盛放有 细胞悬浮液的细胞盛装容器 2放置在无菌腔室内, 以免将细胞杀死, 可在杀菌完成后等 待一定时间如 5分钟后再将盛放有细胞悬浮液的细胞盛装容器 2放入到无菌腔室内。  Specifically, the cell printing system 100 of the present invention is placed in a sterile chamber, and the three-dimensional motion mechanism 1, the μ-ϋΟΜ4 and the micro-jet 3 are sterilized before the cell printing system 100 is operated, and ultraviolet sterilization can be used at this time. The lamp sterilizes the three-dimensional motion mechanism 1 and the μ-ϋΟΜ4, and the sterilization time is about 30 minutes. The micro-lance 3 is sterilized by high-pressure steam and connected to the μ-ϋΟΜ4. Among them, it is worth noting that during the sterilization process, the cell-containing container 2 containing the cell suspension cannot be placed in the aseptic chamber to avoid killing the cells, and can wait for a certain time, such as 5 minutes, after the sterilization is completed. The cell-containing container 2 containing the cell suspension is then placed in a sterile chamber.
细胞打印系统 100工作时, 如图 3所示, 首先三维运动控制器 52控制三维运动机 构 1移动以使得微喷管 3伸入到细胞盛装容器 2内, 接着微位移往复运动控制器 50控 制 μ-ϋΟΜ4将所需的驱动电压信号施加到微喷管 3上以使得微喷管 3产生与该驱动电 压信号对应的非对称往复运动,使得细胞盛装容器 2内的细胞悬浮液在交变滞惯力的作 用下被吸入到微喷管 3 内并重复执行上述步骤, 以将多种细胞悬浮液吸入到微喷管 3 内以组合成细胞打印序列, 然后如 4所示, 三维运动控制器 52控制三维运动机构 1移 动以使得吸取有细胞悬浮液的微喷管 3位于所需的打印位置处, 在移动的过程中, 微位 移往复运动控制器 50控制 μ-ϋΟΜ4将所需的驱动电压信号施加到微喷管 3上使得微喷 管 3产生与该驱动电压信号对应的非对称的往复运动,使得细胞在微喷管 3内排列成密 排的单细胞列, 接着微位移往复运动控制器 50控制 μ-ϋΟΜ4将所需的驱动电压信号施 加到微喷管 3上以使得微喷管 3产生与该驱动电压信号对应的非对称的往复运动,使得 微喷管 3内的细胞悬浮液在交变滞惯力的作用下以单细胞的形式从微喷管 3内喷射出, 从而将单细胞按序列打印在所需的打印位置, 完成一次的细胞的吸取 -打印过程。 此时 若需要继续打印细胞, 则重复执行上述步骤以进行多次细胞的吸取 -打印过程。  When the cell printing system 100 is in operation, as shown in FIG. 3, first, the three-dimensional motion controller 52 controls the movement of the three-dimensional motion mechanism 1 so that the micro-jet 3 extends into the cell-contained container 2, and then the micro-displacement reciprocating controller 50 controls the μ. - ϋΟΜ 4 applies a desired driving voltage signal to the micro-nozzle 3 to cause the micro-jet 3 to generate an asymmetric reciprocating motion corresponding to the driving voltage signal, so that the cell suspension in the cell-contained container 2 is subjected to alternating hysteresis The force is absorbed into the micro-spray 3 and the above steps are repeated to inhale a plurality of cell suspensions into the micro-jet 3 to be combined into a cell print sequence, and then, as shown in FIG. 4, the three-dimensional motion controller 52 The three-dimensional motion mechanism 1 is controlled to move so that the micro-jet 3 sucking the cell suspension is located at a desired printing position, and during the movement, the micro-displacement reciprocating controller 50 controls the driving voltage signal required for the μ-ϋΟΜ4 Applying to the micro-nozzle 3 causes the micro-jet 3 to generate an asymmetric reciprocating motion corresponding to the driving voltage signal, so that the cells are arranged densely in the micro-nozzle 3 Rows of single cells, followed by a micro-displacement reciprocating controller 50 controlling μ-ϋΟΜ4 to apply a desired drive voltage signal to the micro-nozzle 3 to cause the micro-jet 3 to generate an asymmetric reciprocation corresponding to the drive voltage signal The movement causes the cell suspension in the micro-nozzle 3 to be ejected from the micro-lance 3 in the form of a single cell under the action of the alternating hysteresis, thereby printing the single cells in the desired printing position in sequence, completing Once the cell's aspiration-printing process. At this time, if it is necessary to continue printing the cells, the above steps are repeated to perform the cell-absorption-printing process.
其中,值得理解的是,在利用上述的细胞打印系统进行多次细胞的吸取-打印过程时, 每次细胞的吸取-打印过程中的吸取的细胞悬浮液可相同也可不同。 具体地, 控制系统 5通过控制 μ-ϋΟΜ4的电压、频率和驱动波形时间宽度来控制所需细胞悬浮液的吸入量 或者喷射量, 以避免细胞的浪费。 将细胞悬浮液打印在打印位置处的动作过程可在空气介质中执行, 也可在液体介质 或凝胶介质中执行以满足不同的需求, 具体地, 该液体介质为细胞培养基、海藻酸钠溶 液、 胶原、 纤维蛋白质中的一种, 凝胶介质为各种凝胶中的一种。 同时, 在将细胞悬浮 液打印在所需打印位置处时,微喷管 3可以悬于作为细胞打印的载体的基板上方的一定 高度进行特定图案或性状的细胞打印,可也可以插入组织工程支架的网格空洞中进行细 胞打印, 以实现在支架特定位置种植特定数量细胞的目的。 在本发明的一些实施例中, 微喷管 3位于所需的打印位置处的正上方且微喷管 3与所需的打印位置处之间的距离为 0〜5mm。 Among them, it is understood that the cell suspensions taken during the pipetting-printing process of the cells may be the same or different when the cell-pick-printing process is performed multiple times using the cell printing system described above. Specifically, the control system 5 controls the inhalation amount or the ejection amount of the desired cell suspension by controlling the voltage, frequency, and driving waveform time width of μ-ϋΟΜ4 to avoid waste of cells. The process of printing the cell suspension at the printing position can be performed in an air medium or can be performed in a liquid medium or a gel medium to meet different needs. Specifically, the liquid medium is a cell culture medium, sodium alginate. One of solution, collagen, and fiber protein, and the gel medium is one of various gels. Meanwhile, when the cell suspension is printed at a desired printing position, the micro-jet 3 can be printed at a certain height above the substrate as a carrier for cell printing for cell printing of a specific pattern or trait, and can also be inserted into a tissue engineering scaffold. Cell printing is performed in a mesh cavity to achieve the purpose of planting a specific number of cells at specific locations in the scaffold. In some embodiments of the invention, the micro-jet 3 is located directly above the desired printing position and the distance between the micro-jet 3 and the desired printing position is 0 to 5 mm.
根据本发明实施例的细胞打印系统 100, μ-ϋΟΜ4将所需的驱动电压信号施加至微 喷管 3上以对微喷管 3执行双向驱动,以将所需的细胞悬浮液吸入微喷管 3内和将细胞 悬浮液喷射出微喷管 3, 即以先进后出的顺序实现了细胞的打印, 从而不仅可用同一个 喷头实现不同细胞的打印, 降低了设备成本和简化了操作的复杂程度, 同时可减小细胞 的染菌几率, 有利于保证打印过程中细胞的活性。又由于通过使细胞悬浮液受到交变滞 惯力的作用而实现细胞悬浮液的吸取和喷射,从而无论是细胞悬浮液的吸取和喷射的过 程中, 均无瞬间的高温、 高压或电场等产生, 对细胞的损伤较小。  According to the cell printing system 100 of the embodiment of the present invention, μ-ϋΟΜ4 applies a desired driving voltage signal to the micro-nozzle 3 to perform bidirectional driving on the micro-jet 3 to draw the desired cell suspension into the micro-nozzle 3 and the cell suspension is ejected out of the micro-nozzle 3, that is, the cell printing is realized in the order of advanced output, so that not only the same nozzle can be used for printing different cells, the equipment cost is reduced, and the operation complexity is simplified. At the same time, it can reduce the chance of cell infection, which is beneficial to ensure the activity of cells during printing. Moreover, since the cell suspension is subjected to the action of alternating hysteresis, the cell suspension is sucked and ejected, so that no transient high temperature, high pressure or electric field is generated during the process of sucking and ejecting the cell suspension. , damage to cells is small.
具体地, 微位移往复运动控制器 50控制 μ-ϋΟΜ4, 以使微喷管 3产生与细胞吸取 驱动相对应的非对称的往复运动, 而将所需的细胞悬浮液吸入微喷管 3中。微位移往复 运动控制器控制 μ-ϋΟΜ4, 以使微喷管 3产生与细胞喷射驱动相对应的非对称的往复运 动, 而将细胞悬浮液打印在所需的打印位置处。微位移往复运动控制器控制微位移往复 运动机构, 以使得微喷管产生与细胞聚齐驱动相对应的非对称的往复运动, 而将细胞在 微喷管中形成密排的单细胞列。  Specifically, the micro-displacement reciprocating motion controller 50 controls μ-ϋΟΜ4 to cause the micro-lance 3 to generate an asymmetric reciprocating motion corresponding to the cell suction driving, and to inhale the desired cell suspension into the micro-spray 3. The micro-displacement reciprocating motion controller controls μ-ϋΟΜ4 to cause the micro-lance 3 to generate an asymmetric reciprocating motion corresponding to the cell ejection drive, and to print the cell suspension at the desired printing position. The micro-displacement reciprocating motion controller controls the micro-displacement reciprocating mechanism such that the micro-nozzles generate an asymmetric reciprocating motion corresponding to the cell-gathering drive, and the cells form a close-packed single-cell column in the micro-nozzle.
在本发明的一些实施例中, 如图 1所示, 细胞打印系统 100还包括摄像装置 6, 摄 像装置 6对吸取、 聚齐和打印过程进行实时观测, 从而可检测整个细胞的吸取-打印过 程是否正常运行,保证了细胞打印系统 100的可靠性。优选地, 该摄像装置 6可为 CCD 摄像机, 从而具有灵敏度高、 抗强光、 畸变小、 体积小、 寿命长、 抗震动的优点。  In some embodiments of the present invention, as shown in FIG. 1, the cell printing system 100 further includes an image capturing device 6 that performs real-time observation of the picking, gathering, and printing processes, thereby detecting whether the entire cell's pick-and-print process is Normal operation ensures the reliability of the cell printing system 100. Preferably, the camera device 6 can be a CCD camera, thereby having the advantages of high sensitivity, high glare resistance, small distortion, small volume, long life, and anti-vibration.
优选地, 微喷管 3的内径 dnzzle满足 ldedl<dnzzk<2dedl, 其中 dedl为细胞悬浮液中的 单细胞直径, 从而可保证微喷管 3内的细胞悬浮液中的细胞呈稳定的单细胞列排列, 防 止细胞堵塞微喷管 3或形成局部堆积,进而保证微喷管 3内的细胞悬浮液中的细胞以单 细胞的形式打印。 Preferably, the inner diameter d n of the micro-jet 3 is. Zzle satisfies ld edl <d n . Zzk <2d edl , where d edl is the single cell diameter in the cell suspension, thereby ensuring that the cells in the cell suspension in the micro-bubble 3 are arranged in a stable single cell array, preventing the cells from clogging the micro-spray 3 or forming Local accumulation, thereby ensuring that cells in the cell suspension in the micro-pipe 3 are printed as single cells.
由于微喷管 3在运动的过程中往往存在振动的问题, 而微喷管 3的振动既会影响细 胞的成活率又会影响细胞的打印位置精度, 从而为了减少微喷管 3运动中产生的振动, 在本发明的一些实施例中, 如图 7所示, 微喷管 3在进行细胞悬浮液的吸取过程中, 微 喷管 3做离开液面的运动, 微喷管 3的加速度先由零增加到第一预定值, 接着保持第一 预定值第一预定时间, 最后再回到零值, 从而保证微喷管 3在加速的过程中, 速度是连 续变化而不是跳跃变化的。以及如图 8所示,微喷管 3在进行细胞悬浮液的吸取过程中, 施加在 μ-ϋΟΜ4 上的电压为这样的电压波形, 电压波形的曲线斜率为负值, 且曲线斜 率的绝对值随着时间的变化而逐渐增大, 由于 μ-ϋΟΜ4 具有电压和位移基本呈正比例 的关系, 所以该波形也代表了微喷管 3 的位移波形, 也就是说, 如图 8所示, 施加在 μ-ϋΟΜ4上的驱动信号使得微喷管 3的位移波形的曲线斜率的绝对值随着时间的变化而 逐渐增大。 Since the micro-pipe 3 often has vibration problems during the movement, the vibration of the micro-nozzle 3 affects both the survival rate of the cells and the printing position accuracy of the cells, thereby reducing the movement of the micro-nozzle 3 Vibration, in some embodiments of the present invention, as shown in Figure 7, during the suction of the cell suspension, the micro-nozzle 3 is moved away from the liquid surface, and the acceleration of the micro-jet 3 is first Zero is added to the first predetermined value, then the first predetermined value is maintained for the first predetermined time, and finally returned to the zero value, thereby ensuring that the speed of the micro-pipe 3 during the acceleration is continuously changed instead of jumping. And as shown in FIG. 8, the voltage applied to the μ-ϋΟΜ4 during the suction process of the micro-spray tube 3 is such a voltage waveform, the slope of the curve of the voltage waveform is a negative value, and the curve is inclined. The absolute value of the rate gradually increases with time. Since μ-ϋΟΜ4 has a substantially proportional relationship between voltage and displacement, the waveform also represents the displacement waveform of the micro-nozzle 3, that is, as shown in Fig. 8. It is shown that the driving signal applied to the μ-ϋΟΜ4 causes the absolute value of the slope of the curve of the displacement waveform of the micro-cavity 3 to gradually increase with time.
其中值得说明的是, 上述的在微喷管 3进行对细胞悬浮液的吸取过程中微喷管 3的 加速度波形和施加在 μ-ϋΟΜ4 上的电压波形仅为示例性说明, 而不是对本发明的具体 限制, 本领域的技术人员应该理解的是, 在微喷管 3进行对细胞悬浮液的吸取过程中, 微喷管 3 的加速度波形和施加在 μ-ϋΟΜ4上的电压波形只要满足使得细胞悬浮液可受 到交变滞惯力的作用而被吸取到微喷管 3内且使得微喷管 3在运动过程中的振动较小即 可。  It should be noted that the above-mentioned acceleration waveform of the micro-pipe 3 and the voltage waveform applied to the μ-ϋΟΜ4 during the suction of the cell suspension by the micro-nozzle 3 are merely illustrative, rather than the present invention. Specific limitations, those skilled in the art should understand that in the process of sucking the cell suspension by the micro-nozzle 3, the acceleration waveform of the micro-nozzle 3 and the voltage waveform applied to the μ-ϋΟΜ4 are satisfied as long as the cell suspension is satisfied. The liquid can be sucked into the micro-pipe 3 by the action of the alternating hysteresis and the vibration of the micro-jet 3 during the movement is small.
同时为了减少微喷管 3运动过程产生的振动, 在本发明的一些实施例中, 如图 5所 示, 微喷管 3在进行单细胞的打印过程中, 微喷管 3做靠近液面的运动, 微喷管 3的加 速度先由零上升到第二预定值, 接着保持第二预定值第二预定时间, 最后再回到零值, 从而可保证在微喷管 3加速的过程中, 速度是持续变化而不是跳跃变化的。 以及如图 6 所示, 施加在 μ-ϋΟΜ4 上的电压为这样的电压波形, 电压波形的曲线斜率随着时间的 变化而逐渐增大, 且由于 μ-ϋΟΜ4 具有电压和位置基本呈正比例的关系, 所以该波形 也代表了微喷管 3的位移波形, 也就是说, 如图 6所示, 施加在 μ-ϋΟΜ4上的驱动信 号使得微喷管 3的位移波形的曲线斜率随着时间的变化而逐渐增大。  At the same time, in order to reduce the vibration generated during the movement of the micro-nozzle 3, in some embodiments of the present invention, as shown in FIG. 5, the micro-nozzle 3 is in the process of single cell printing, and the micro-spray 3 is close to the liquid surface. Movement, the acceleration of the micro-nozzle 3 first rises from zero to a second predetermined value, then maintains a second predetermined value for a second predetermined time, and finally returns to zero value, thereby ensuring the speed during the acceleration of the micro-jet 3 It is a constant change rather than a jump change. And as shown in Fig. 6, the voltage applied to μ-ϋΟΜ4 is such a voltage waveform, the slope of the curve of the voltage waveform gradually increases with time, and since μ-ϋΟΜ4 has a substantially proportional relationship between voltage and position. Therefore, the waveform also represents the displacement waveform of the micro-nozzle 3, that is, as shown in FIG. 6, the driving signal applied to the μ-ϋΟΜ4 causes the slope of the curve of the displacement waveform of the micro-cavity 3 to change with time. And gradually increase.
其中值得说明的是, 上述的在微喷管 3进行单细胞的打印过程中微喷管 3的加速波 形和施加在 μ-ϋΟΜ4 上的电压波形仅为示例性说明, 而不是对本发明的具体限制, 本 领域的技术人员应该理解的是, 在微喷管 3进行单细胞的打印过程中, 微喷管 3的加速 度波形和施加在 μ-ϋΟΜ4上的电压波形只要满足使得微喷管 3 内的细胞悬浮液在交变 滞惯力的作用下从微喷管 3内喷射出且使得微喷管 3在运动过程中的振动较小就行。  It should be noted that the above-mentioned acceleration waveform of the micro-nozzle 3 and the voltage waveform applied to the μ-ϋΟΜ4 during the single-cell printing process of the micro-nozzle 3 are merely illustrative, and are not specific limitations of the present invention. It should be understood by those skilled in the art that in the single cell printing process of the micro-nozzle 3, the acceleration waveform of the micro-nozzle 3 and the voltage waveform applied to the μ-ϋΟΜ4 are satisfied as long as they are satisfied in the micro-spray tube 3. The cell suspension is ejected from the micro-spray 3 under the action of alternating hysteresis and makes the micro-lance 3 less vibrating during the movement.
进一步地, 为了减少微喷管 3运动过程中的振动, 在本发明的一些实施例中, 如图 9所示, 在将细胞在微喷管 3内进行排列成密排单细胞列的过程中, 微喷管 3的加速度 先由零上升到第三预定值, 接着保持第三预定值第三预定时间, 最后再回到零值, 从而 保证在微喷管 3加速的过程中, 速度是连续变化而不是跳跃变化的。 以及如图 10所示, 施加在 μ-ϋΟΜ4 上的电压为这样的电压波形, 电压波形在第四预定时间内呈现为曲线 形状后在第五预定时间内呈现为直线形状, 且在第四预定时间内, 曲线的斜率随着时间 的变化而逐渐增大, 在第五预定时间直线的斜率为负值且保持为定值。 由于 μ-ϋΟΜ4 具有电压和位移呈正比例的关系, 所以该波形也代表了微喷管的位移波形, 也就是说, 如图 10所示, 施加在微位移往复运动机构 4上的驱动信号使得微喷管 3的位移波形在 第四预定时间内呈现为曲线形状后在第五预定时间内呈现为直线形状,且在第四预定时 间内, 曲线的斜率随着时间的变化而逐渐增大, 在第五预定时间直线的斜率为负值。  Further, in order to reduce the vibration during the movement of the micro-nozzle 3, in some embodiments of the present invention, as shown in FIG. 9, in the process of arranging the cells in the micro-spray 3 into a close-packed single cell column The acceleration of the micro-nozzle 3 first rises from zero to a third predetermined value, then maintains a third predetermined value for a third predetermined time, and finally returns to a zero value, thereby ensuring that the speed is continuous during the acceleration of the micro-nozzle 3 Change instead of jumping. And as shown in FIG. 10, the voltage applied to the μ-ϋΟΜ4 is a voltage waveform which appears as a curved shape in the fourth predetermined time and then appears as a linear shape in the fifth predetermined time, and is in a fourth predetermined During the time, the slope of the curve gradually increases with time, and the slope of the straight line at the fifth predetermined time is negative and remains constant. Since μ-ϋΟΜ4 has a proportional relationship between voltage and displacement, the waveform also represents the displacement waveform of the micro-nozzle, that is, as shown in Fig. 10, the driving signal applied to the micro-displacement reciprocating mechanism 4 makes micro The displacement waveform of the nozzle 3 assumes a curved shape after the fourth predetermined time and then assumes a linear shape for a fifth predetermined time, and in a fourth predetermined time, the slope of the curve gradually increases with time, The slope of the line of the fifth predetermined time is a negative value.
其中值得说明的是, 上述的在将细胞在微喷管 3内进行排列成密排的单细胞列的过 程中微喷管 3 的加速波形和施加在 μ-ϋΟΜ4上的电压波形仅为示例性说明, 而不是对 本发明的具体限制, 本领域的技术人员应该理解的是, 在将细胞在微喷管 3内进行排列 成密排的单细胞列的过程中中, 微喷管 3 的加速度波形和施加在 μ-ϋΟΜ4上的电压波 形只要满足使得微喷管 3 内的细胞悬浮液中的细胞会在周期性惯性力的作用下相对于 流体发生聚齐运动, 最后使得细胞在微喷管 3内形成密排的单细胞列即可。 It should be noted that the above-mentioned acceleration waveform of the micro-cavity 3 and the voltage waveform applied to the μ-ϋΟΜ4 in the process of arranging the cells in the micro-lance 3 into a close-packed single cell row are merely exemplary. Description, not right Specific limitations of the present invention, it will be understood by those skilled in the art that in the process of arranging cells in a single row of cells arranged in a micro-spray 3, the acceleration waveform of the micro-lance 3 is applied to the μ The voltage waveform on the -4 is satisfied so that the cells in the cell suspension in the micro-spray 3 will collide with the fluid under the action of periodic inertial forces, and finally the cells are formed in the micro-spray 3 in a close-packed manner. Single cell column can be.
其中, 在本发明的描述中, 第一预定值、 第一预定时间、 第二预定值、 第二预定时 间、 第三预定时间、 第三预定值、第四预定时间和第五预定时间的数值可根据不同的细 胞悬浮液的细胞的特性等具体设定, 以满足不同的需求。  Wherein, in the description of the present invention, the values of the first predetermined value, the first predetermined time, the second predetermined value, the second predetermined time, the third predetermined time, the third predetermined value, the fourth predetermined time, and the fifth predetermined time It can be specifically set according to the characteristics of cells of different cell suspensions to meet different needs.
在本说明书的描述中, 参考术语"一个实施例"、 "一些实施例"、 "示意性实施例"、 "示 例"、 "具体示例"、 或 "一些示例"等的描述意指结合该实施例或示例描述的具体特征、 结 构、 材料或者特点包含于本发明的至少一个实施例或示例中。 在本说明书中, 对上述术语 的示意性表述不一定指的是相同的实施例或示例。 而且, 描述的具体特征、 结构、 材料或 者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。  In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "illustrative embodiment", "example", "specific example", or "some examples", etc. Particular features, structures, materials or features described in the examples or examples are included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.
尽管已经示出和描述了本发明的实施例,本领域的普通技术人员可以理解:在不脱离本 发明的原理和宗旨的情况下可以对这些实施例进行多种变化、 修改、 替换和变型, 本发明 的范围由权利要求及其等同物限定。  While the embodiments of the present invention have been shown and described, the embodiments of the invention may The scope of the invention is defined by the claims and their equivalents.

Claims

权利要求书 claims
1、 一种细胞打印方法, 其特征在于, 包括如下步骤: 1. A cell printing method, characterized in that it includes the following steps:
S 1 : 将微喷管插入所需细胞悬浮液中, 所述细胞悬浮液分别容纳在不同的细胞盛装 容器中; S 1: Insert the microspray tube into the required cell suspension, which is contained in different cell holding containers;
S2: 利用微位移往复运动机构在所述微喷管上执行预定的细胞吸取驱动, 以吸入一 定数量的细胞并组合成细胞打印序列; S2: Use a micro-displacement reciprocating mechanism to perform a predetermined cell suction drive on the micro-nozzle to suck in a certain number of cells and combine them into a cell printing sequence;
S3: 利用所述微位移往复运动机构在所述微喷管上执行预定的细胞聚齐驱动, 将细 胞在微喷管内排列成密排单细胞列; S3: Use the micro-displacement reciprocating mechanism to perform a predetermined cell gathering drive on the micro-nozzle, and arrange the cells into densely packed single-cell rows in the micro-nozzle;
S4: 将所述微喷管移动至所需的打印位置, 利用所述微位移往复运动机构在所述微 喷管上执行预定的细胞喷射驱动, 以将所述细胞按序列喷射在所述打印位置处; 以及 如果需要继续打印与所述细胞悬浮液对应的细胞, 则利用所述微喷管重复执行上述 步骤 S 1-S4, 直至完成所有的细胞二维图形或三维结构的细胞打印。 S4: Move the micro-nozzle to the required printing position, and use the micro-displacement reciprocating mechanism to perform a predetermined cell ejection drive on the micro-nozzle to eject the cells in sequence on the printing surface. position; and if it is necessary to continue printing cells corresponding to the cell suspension, use the micro-nozzle to repeat the above steps S1-S4 until all cell printing of two-dimensional cell patterns or three-dimensional structures is completed.
2、 根据权利要求 1所述的细胞打印方法, 其特征在于, 在所述步骤 S2中, 所述微 位移往复运动机构通过使所述微喷管产生与所述细胞吸取驱动相对应的非对称的往复 运动, 而将所需的细胞吸入所述微喷管中。 2. The cell printing method according to claim 1, characterized in that, in the step S2, the micro-displacement reciprocating mechanism causes the micro-nozzle to generate an asymmetrical motion corresponding to the cell suction drive. reciprocating motion to inhale the desired cells into the micronozzle.
3、 根据权利要求 1所述的细胞打印方法, 其特征在于, 在所述步骤 S3中, 所述微 位移往复运动机构通过使所述微喷管产生与所述细胞聚齐驱动相对应的非对称的往复 运动, 而将细胞在微喷管出口附近排列成密排单细胞列。 3. The cell printing method according to claim 1, characterized in that, in the step S3, the micro-displacement reciprocating mechanism causes the micro-nozzle to generate an asymmetrical movement corresponding to the cell gathering drive. The reciprocating motion arranges the cells into densely packed single-cell rows near the outlet of the micronozzle.
4、 根据权利要求 1所述的细胞打印方法, 其特征在于, 在所述步骤 S4中, 所述微 位移往复运动机构通过使所述微喷管产生与所述细胞喷射驱动相对应的非对称的往复 运动, 而将所述细胞喷射在所述打印位置处。 4. The cell printing method according to claim 1, characterized in that, in the step S4, the micro-displacement reciprocating mechanism causes the micro-nozzle to generate an asymmetrical motion corresponding to the cell ejection drive. reciprocating motion to eject the cells at the printing position.
5、 根据权利要求 1 所述的细胞打印方法, 其特征在于, 所述微位移往复运动机构 驱动所述微喷管产生与不同的位移曲线相对应的非对称的轴向往复运动,以执行对所需 的细胞的吸取驱动、 聚齐驱动和喷射驱动。 5. The cell printing method according to claim 1, wherein the micro-displacement reciprocating mechanism drives the micro-nozzle to generate asymmetric axial reciprocating motion corresponding to different displacement curves to perform the cell printing. Required cell uptake drive, accumulation drive and ejection drive.
6、 根据权利要求 1所述的细胞打印方法, 其特征在于, 在所述步骤 S2、 S3和 S4 中, 通过控制所述微位移往复运动机构的电压、频率和驱动波形时间宽度来控制所需细 胞的吸入量、 聚齐程度或者喷射数量。 6. The cell printing method according to claim 1, characterized in that, in the steps S2, S3 and S4, the required voltage, frequency and driving waveform time width of the micro-displacement reciprocating mechanism are controlled. Cell intake, degree of aggregation, or number of ejections.
7、 根据权利要求 1所述的细胞打印方法, 其特征在于, 所述步骤 S4在空气介质中 执行。 7. The cell printing method according to claim 1, characterized in that step S4 is performed in air medium.
8、 根据权利要求 1所述的细胞打印方法, 其特征在于, 所述步骤 S4在液体介质或 凝胶介质中执行。 8. The cell printing method according to claim 1, characterized in that step S4 is performed in a liquid medium or a gel medium.
9、 根据权利要求 1所述的细胞打印方法, 其特征在于, 在步骤 S2中, 所述微喷管 做离开液面的运动, 其加速度由零增加到第一预定值, 接着保持所述第一预定值第一预 定时间, 最后再回到零值; 以及 施加在所述微位移往复运动机构上的驱动信号使得所述微喷管的位移波形的曲线 斜率的绝对值随着时间的变化而逐渐增大。 9. The cell printing method according to claim 1, characterized in that, in step S2, the micro-nozzle moves away from the liquid surface, and its acceleration increases from zero to a first predetermined value, and then maintains the first predetermined value. a predetermined value for a predetermined time, and finally back to zero; and The driving signal applied to the micro-displacement reciprocating mechanism causes the absolute value of the curve slope of the displacement waveform of the micro-nozzle to gradually increase as time changes.
10、 根据权利要求 1所述的细胞打印方法, 其特征在于, 在步骤 S4中, 所述微喷 管做靠近液面的运动, 其加速度先由零上升到第二预定值, 接着保持所述第二预定值第 二预定时间, 最后再回到零值; 以及 10. The cell printing method according to claim 1, characterized in that, in step S4, the micro-nozzle moves close to the liquid surface, and its acceleration first increases from zero to a second predetermined value, and then maintains the a second predetermined value for a second predetermined time, and finally returns to zero; and
施加在所述微位移往复运动机构上的驱动信号使得所述微喷管的位移波形的曲线 斜率随着时间的变化而逐渐增大。 The driving signal applied to the micro-displacement reciprocating mechanism causes the curve slope of the displacement waveform of the micro-nozzle to gradually increase as time changes.
11、 根据权利要求 1所述的细胞打印方法, 其特征在于, 在步骤 S3中, 所述微喷 管的加速度先由零上升到第三预定值, 接着保持所述第三预定值第三预定时间, 最后再 回到零值; 以及 11. The cell printing method according to claim 1, characterized in that, in step S3, the acceleration of the micro-nozzle first increases from zero to a third predetermined value, and then maintains the third predetermined value. time, and finally returns to zero; and
施加在所述微位移往复运动机构上的驱动信号使得所述微喷管的位移波形在第四 预定时间内呈现为曲线形状后在第五预定时间内呈现为直线形状,且在所述第四预定时 间内, 所述曲线的斜率随着时间的变化而逐渐增大, 在所述第五预定时间所述直线的斜 率为负值。 The driving signal applied to the micro-displacement reciprocating mechanism causes the displacement waveform of the micro-nozzle to assume a curved shape within a fourth predetermined time and then assume a linear shape within a fifth predetermined time, and in the fourth predetermined time Within the predetermined time, the slope of the curve gradually increases with time, and the slope of the straight line is negative at the fifth predetermined time.
12、根据权利要求 1所述的细胞打印方法,其特征在于,所述微喷管的内径 dnzzle满 足 ldedl<dnzzle<2dedl, 其中 d ^为所述细胞悬浮液中的单细胞直径。 12. The cell printing method according to claim 1, wherein the inner diameter of the micro-nozzle is dn . zzle satisfies ld edl <d n . zzle <2d edl , where d^ is the diameter of a single cell in the cell suspension.
13、 一种细胞打印系统, 其特征在于, 包括: 13. A cell printing system, characterized by including:
三维运动机构; Three-dimensional motion mechanism;
多个细胞盛装容器, 所述多个细胞盛装容器中分别容纳有不同种类的细胞悬浮液; 微喷管, 所述微喷管设在所述三维运动机构的上方, 所述三维运动机构移动以使得 所述微喷管位于所需的打印位置处或伸入到所述细胞盛装容器内; A plurality of cell holding containers, the plurality of cell holding containers respectively contain different types of cell suspensions; a micro nozzle, the micro nozzle is located above the three-dimensional movement mechanism, and the three-dimensional movement mechanism moves to Make the micro-nozzle located at the desired printing position or extend into the cell holding container;
微位移往复运动机构, 所述微位移往复运动机构与所述微喷管相连, 且将所需的非 对称往复运动施加至所述微喷管上以对所述微喷管执行吸取驱动、聚齐驱动或者喷射驱 动, 以将所需的细胞悬浮液吸入所述微喷管内、将细胞在微喷管内排列成密排单细胞列 和将所述细胞悬浮液喷射出所述微喷管; 以及 Micro-displacement reciprocating motion mechanism, the micro-displacement reciprocating motion mechanism is connected to the micro-nozzle, and applies the required asymmetric reciprocating motion to the micro-nozzle to perform suction driving and gathering of the micro-nozzle. Driving or ejection driving to draw the desired cell suspension into the micronozzle, arrange the cells into close-packed single-cell rows in the micronozzle, and eject the cell suspension out of the micronozzle; and
三维运动控制器, 所述三维运动控制器配置成控制所述三维运动机构的移动; 微位移往复运动控制器, 所述微位移往复运动控制器配置成控制所述微位移往复运 动机构对所述微喷管的双向驱动, 以执行所述细胞悬浮液的吸取、 聚齐和喷射。 a three-dimensional motion controller, the three-dimensional motion controller is configured to control the movement of the three-dimensional motion mechanism; a micro-displacement reciprocating motion controller, the micro-displacement reciprocating motion controller is configured to control the micro-displacement reciprocating motion mechanism to the The micro-spray is bidirectionally driven to perform aspiration, concentration and ejection of the cell suspension.
14、 根据权利要求 13 所述的细胞打印系统, 其特征在于, 还包括摄像装置, 所述 摄像装置对所述吸取、 聚齐和打印过程进行实时观测。 14. The cell printing system according to claim 13, further comprising a camera device for real-time observation of the absorbing, gathering and printing processes.
15、 跟进权利要求 13 所述的细胞打印系统, 其特征在于, 还包括微喷管夹具, 所 述微喷管通过所述微喷管夹具与所述微位移往复运动机构的下端相连。 15. The cell printing system of claim 13, further comprising a micro-nozzle clamp, and the micro-nozzle is connected to the lower end of the micro-displacement reciprocating mechanism through the micro-nozzle clamp.
16、 根据权利要求 13所述的细胞打印系统, 其特征在于, 所述微喷管的内径 dnzzle 满足 ldedl<dnzzle<2dedl, 其中 d ^为所述细胞悬浮液中的单细胞直径。 16. The cell printing system according to claim 13, wherein the inner diameter of the micro-nozzle is dn . zzle satisfies ld edl <d n . zzle <2d edl , where d^ is the diameter of a single cell in the cell suspension.
17、 根据权利要求 13 所述的细胞打印系统, 其特征在于, 在所述微喷管进行细胞 悬浮液的吸取过程中,所述微喷管做离开液面的运动,其加速度由零增加到第一预定值, 接着保持所述第一预定值第一预定时间, 最后再回到零值; 以及 17. The cell printing system according to claim 13, characterized in that, during the process of absorbing the cell suspension by the micro-nozzle, the micro-nozzle moves away from the liquid surface, and its acceleration increases from zero to first predetermined value, Then maintain the first predetermined value for a first predetermined time, and finally return to the zero value; and
施加在所述微位移往复运动机构上的驱动信号使得所述微喷管的位移波形的曲线 斜率的绝对值随着时间的变化而逐渐增大。 The driving signal applied to the micro-displacement reciprocating mechanism causes the absolute value of the curve slope of the displacement waveform of the micro-nozzle to gradually increase as time changes.
18、 根据权利要求 13 所述的细胞打印系统, 其特征在于, 在所述微喷管进行单细 胞的打印过程中, 所述微喷管做靠近液面的运动, 其加速度先由零上升到第二预定值, 接着保持所述第二预定值第二预定时间, 最后再回到零值; 以及 18. The cell printing system according to claim 13, characterized in that, during the printing process of single cells by the micro-nozzle, the micro-nozzle moves close to the liquid surface, and its acceleration first increases from zero to a second predetermined value, then maintain the second predetermined value for a second predetermined time, and finally return to the zero value; and
施加在所述微位移往复运动机构上的驱动信号使得所述微喷管的位移波形的曲线 斜率随着时间的变化而逐渐增大。 The driving signal applied to the micro-displacement reciprocating mechanism causes the curve slope of the displacement waveform of the micro-nozzle to gradually increase as time changes.
19、 根据权利要求 13 所述的细胞打印系统, 其特征在于, 在将细胞在所述微喷管 内进行排列成密排单细胞列的过程中, 所述微喷管的加速度先由零上升到第三预定值, 接着保持所述第三预定值第三预定时间, 最后再回到零值; 以及 19. The cell printing system according to claim 13, wherein during the process of arranging the cells into a densely packed row of single cells in the micro-nozzle, the acceleration of the micro-nozzle first increases from zero to a third predetermined value, then maintain the third predetermined value for a third predetermined time, and finally return to the zero value; and
施加在所述微位移往复运动机构上的驱动信号使得所述微喷管的位移波形在第四 预定时间内呈现为曲线形状后在第五预定时间内呈现为直线形状,且在所述第四预定时 间内, 所述曲线的斜率随着时间的变化而逐渐增大, 在所述第五预定时间所述直线的斜 率为负值。 The driving signal applied to the micro-displacement reciprocating mechanism causes the displacement waveform of the micro-nozzle to assume a curved shape within a fourth predetermined time and then assume a linear shape within a fifth predetermined time, and in the fourth predetermined time Within the predetermined time, the slope of the curve gradually increases with time, and the slope of the straight line is negative at the fifth predetermined time.
20、 根据权利要求 13 所述的细胞打印系统, 其特征在于, 所述所需的打印位置处 位于空气介质中、 液体介质中或者凝胶介质中。 20. The cell printing system according to claim 13, characterized in that the required printing position is located in air medium, liquid medium or gel medium.
21、 根据权利要求 13 所述的细胞打印系统, 其特征在于, 所述微位移往复运动控 制器控制所述微位移往复运动机构,以使所述微喷管产生与细胞吸取驱动相对应的非对 称的往复运动, 而将所需的细胞悬浮液吸入所述微喷管中。 21. The cell printing system according to claim 13, characterized in that the micro-displacement reciprocating motion controller controls the micro-displacement reciprocating motion mechanism so that the micro-nozzle generates an abnormal motion corresponding to the cell suction drive. Symmetrical reciprocating motion, and the required cell suspension is sucked into the micro-nozzle.
22、 根据权利要求 13 所述的细胞打印系统, 其特征在于, 所述微位移往复运动控 制器控制所述微位移往复运动机构,以使得所述微喷管产生与细胞聚齐驱动相对应的非 对称的往复运动, 而将所述细胞在微喷管中形成密排单细胞列。 22. The cell printing system according to claim 13, wherein the micro-displacement reciprocating motion controller controls the micro-displacement reciprocating motion mechanism so that the micro-nozzle generates non-linear motion corresponding to the cell gathering drive. Symmetrical reciprocating motion causes the cells to form densely packed rows of single cells in the microspray tube.
23、 根据权利要求 13 所述的细胞打印系统, 其特征在于, 所述微位移往复运动控 制器控制所述微位移往复运动机构,以使所述微喷管产生与细胞喷射驱动相对应的非对 称的往复运动, 而将所述细胞悬浮液打印在所需的打印位置处。 23. The cell printing system according to claim 13, wherein the micro-displacement reciprocating motion controller controls the micro-displacement reciprocating motion mechanism so that the micro-nozzle generates a non-linear motion corresponding to the cell ejection drive. Symmetrical reciprocating motion to print the cell suspension at the desired printing position.
24、 根据权利要求 13 所述的细胞打印系统, 其特征在于, 所述微喷管位于所需的 打印位置处的正上方且所述微喷管与所述所需的打印位置处之间的距离为 0〜5mm。 24. The cell printing system according to claim 13, wherein the micro-nozzle is located directly above the desired printing position and the distance between the micro-nozzle and the desired printing position is The distance is 0~5mm.
25、 根据权利要求 13所述的细胞打印系统, 其特征在于, 所述微位移往复运动控 制器通过控制所述微位移往复运动机构的电压、频率和驱动波形时间宽度来控制所需细 胞悬浮液的吸入量、 聚齐程度或者喷射量。 25. The cell printing system according to claim 13, wherein the micro-displacement reciprocating motion controller controls the required cell suspension by controlling the voltage, frequency and driving waveform time width of the micro-displacement reciprocating motion mechanism. The intake volume, the degree of concentration or the injection volume.
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