• 专利附录
  • 专利说明
  • 权利要求
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    • 专利摘要:
    The subject of the invention is a method for printing at least one biological ink, said method using at least one laser-type printing head (102) for depositing at least one droplet of at least one biological ink on a dispensing surface of a receiving substrate (58), characterized in that the printing process uses at least one printing head (104, 104 ', 104' ') with a nozzle for depositing at least one droplet of at least one biological ink on a dispensing surface of the same receiving substrate (58) as the laser-like printing head (102).
    公开号:FR3030361A1
    申请号:FR1462570
    申请日:2014-12-17
    公开日:2016-06-24
    发明作者:Fabien Guillemot
    申请人:Institut National de la Sante et de la Recherche Medicale INSERM；Universite de Bordeaux；
    IPC主号:
    专利说明:

    [0001] The present invention relates to a method for printing biological elements by laser as well as to a device for implementing it. The invention also relates to a method for producing a biological tissue that uses this printing process. According to a first procedure, it is possible to grow cells in vitro on a biodegradable macroporous matrix (scaffold in English) to obtain a biological tissue. This first procedure is not fully satisfactory because the growth of the biological tissue can be limited in the depth of the macroporous matrix due to a defect in cell colonization. In addition, this procedure makes it difficult to deal with tissue complexity (which is characterized by a multiplicity of cell types organized according to specific and generally anisotropic arrangements). To overcome these disadvantages, methods for printing biological elements have been developed to produce a biological tissue in an automated manner. According to the literature, these printing processes are called bio-printing, micro-printing of biological elements or bioprinting in English.
    [0002] According to these methods, the biological tissue is obtained by printing organic ink droplets. To obtain a volume, the droplets are arranged in a layer which are superimposed on each other. According to a first variant, the biological ink is stored in a reservoir and passes through nozzles or capillaries to form droplets that are transferred onto a support. This first so-called nozzle printing variant includes bioextrusion, inkjet printing or microvalve printing. Bioextrusion makes it possible to obtain a large cell density of the order of 100 million cells per milliliter and a resolution of the order of a millimeter. The printing by microvalves makes it possible to obtain a lower cell density of the order of a few million cells per milliliter and a better resolution of the order of 100 μm.
    [0003] The inkjet printing makes it possible to obtain a cell density identical to microvalve printing, less than 10 million cells per milliliter and a better resolution of the order of 10 μm. In the case of bioextrusion, the cells are deposited from a first nozzle and a hydrogel is simultaneously deposited from a second nozzle. Alternatively, the cells and the hydrogel are mixed in a reservoir before extrusion. In the other two cases, the ink is an aqueous medium containing the cells. According to the variants, the bioextrusion makes it possible to deposit the ink continuously in the form of filaments or discontinuously in the form of droplets.
    [0004] According to these nozzle printing modes, the printing resolution being related in particular to the section of the nozzles, only biological inks with given rheological characteristics can be used for high resolutions. Thus, biological inks with high cell density can be difficult to print with high resolution because this printing technique induces at the time of passage through the nozzle significant shear stresses may damage the cells. In addition, with this type of ink, the risk of clogging of the nozzles by the cells is important due in particular to the sedimentation of the cells inside the tanks. To be able to use a wide range of biological inks and achieve a high level of resolution, a method of printing biological elements by laser has been developed. This printing process called laser bio-printing, is also known as "Laser-Assisted Bioprinting" (LAB) in English. The invention relates more specifically to this type of printing process. By way of comparison, laser bio-printing makes it possible to print inks with a high cell density of the order of 100 million cells per milliliter with a resolution of 10 μm.
    [0005] As illustrated in FIG. 1, a device for printing biological elements by laser which is based on the so-called Laser-Induced Forward Transfer (LIFT) technique in English, comprises a pulsed laser source 10 emitting a laser beam 12, a system 14 for focusing and orienting the laser beam 12, a donor support 16 which comprises at least one biological ink 18 and a receiving substrate 20 positioned to receive droplets 22 emitted from the donor support 16. According to this printing technique, the laser beam is pulsed and at each pulse a droplet is generated.
    [0006] The biological ink 18 comprises a matrix, for example an aqueous medium, in which there are elements, for example cells, to be deposited on the receiving substrate 20. The donor support 16 comprises a wavelength-transparent plate 24 the laser beam 12 which is coated with an absorbent layer 26 on which is affixed the biological ink 18 in the form of a film. Absorbent layer 26 converts light energy into kinetic energy. Thus, the laser beam 12 produces a spot heating at the absorbent layer 26 which vaporizes a gas bubble 28 which by expansion causes the ejection of a droplet of biological ink.
    [0007] According to a known arrangement, the laser beam 12 impacts the donor support 16 by being oriented in an approximately vertical direction and in a direction from top to bottom, in the same direction as the gravitational force G. Thus, the biological ink 18 is placed under the blade 24 so as to be oriented downwards towards the receiving substrate 20 which is placed under the donor support 16.
    [0008] Given this arrangement, the biological ink 18 is in the form of a film with a thickness E less than a given threshold to be held on the blade. This threshold varies in particular according to the surface tension, the viscosity and the density of the biological ink. The formation of the droplets 30 from the biological ink film depends on numerous parameters which are particularly related to the laser beam 12 (wavelength, energy, pulse duration, etc.), to the nature of the ink biological 18 (surface tension, viscosity, ...), external conditions (temperature, hygrometry, ...). Even if laser printing makes it possible theoretically to obtain an important printing flow, since it is possible to generate tens of thousands of droplets per second, the production of biological tissues is not fast because these droplets do not only a few dozen picoliters. In addition, the donor media must be replaced frequently because they each contain only a small volume of ink to be printed of the order of a few tens of microliters. According to a second embodiment illustrated in FIG. 2 and described in a publication entitled "Microdroplet deposition through a film-free laser forward technique" published on October 1, 2011 on the site www.elsevier.com, a laser printing device comprises a laser source 32 emitting a laser beam 34, a system 36 for focusing and orienting the laser beam 34, a donor support 38 which contains at least one biological ink 40 and a receiving substrate 42 positioned to receive droplets 44 emitted from the donor support 38. In contrast to the first embodiment, the donor support 38 does not comprise an absorbent layer distinct from the biological ink. According to this embodiment, the donor support 38 comprises a reservoir 46 without an upper wall so that the free surface 48 of the biological ink 40 contained in the reservoir faces the receiving substrate 42. To obtain a free surface 48 that is regular and substantially planar, the biological ink is not in the form of a thin film but a volume having a depth of about 3 mm. In this way, the bottom of the reservoir has no influence on the shape of the free surface 48 of the biological ink and the side walls of the reservoir have an effect limited to the periphery of the free surface 48 because of the Surface tension. Given the depth of the biological ink volume, the free surface 48 is necessarily upward to remain in the reservoir and the receiving substrate 42 is positioned above the biological ink 40. According to this document, to obtain the ejection of a droplet, the laser beam 34 is focused just below the free surface 48 to a depth of the order of 40 to 80 microns. Thus, the droplets emitted from the free surface 48 are projected towards the receiving substrate 42 in a sense of displacement contrary to the direction of the gravitational force G. Even if this second embodiment uses donor supports containing a volume of ink more importantly, it is not necessarily suitable for organic inks. Indeed, as indicated above, these biological inks contain elements to be printed, such as cells, embedded in a matrix, which tend to sedimentation down tank bottom. Since the concentration of printing elements is low close to the free surface, the printed droplets have in fact low concentrations of cells, which is generally detrimental to the printed biological tissue. Moreover, according to this method, it is very difficult to control the number of cells and the concentration of the deposited cells.
    [0009] Also, the present invention aims to overcome the disadvantages of the prior art. For this purpose, the subject of the invention is a method for printing at least one biological ink, said method using at least one laser-type printing head for depositing at least one droplet of at least one biological ink on a surface for depositing a receiving substrate. The printing method is characterized in that it uses at least one nozzle print head to deposit at least one droplet of at least one biological ink on a delivery surface of the same receiving substrate as the print head. laser type. This combination makes it possible to increase the production rate of biological tissues and to obtain more complex biological tissues. For the production of a biological tissue comprising both cells and an extracellular matrix, the materials constituting this extracellular matrix are deposited by the nozzle printing head (s) and in that the cells are deposited by the or the laser print head (s). The invention also relates to a method for producing a biological tissue which is characterized in that it comprises the steps of: - Generating a three-dimensional digital representation of the biological tissue to be produced, said representation comprising several volume zones colored or textured, each color or texture being associated with a biological ink, - Slicing the digital representation into a succession of superimposed layers, each layer comprising colored or textured areas corresponding to the volume zones of the digital representation, - For each layer, determining the position of the droplets to be printed of each biological ink as a function of the colored or textured areas and the expected volume of each of the droplets, - Print the different droplets. Preferably, the layers have a thickness depending on the size of the droplets.
    [0010] Advantageously, each layer comprises a set of small elementary polygons that have different colors or textures depending on the zone to which they belong. According to one procedure, to determine the position of each droplet, each zone of the same color or of the same texture is filled by identical ellipses which have a size dependent on the size of the droplets of the biological ink to be printed. in said zone, the center of each ellipse corresponding to the position of the center of a droplet. Preferably, the positioning of the ellipses is performed zone by zone, in descending order of size. The invention also relates to a printing device which comprises at least one receiving substrate with a depositing surface and at least one laser-type printing head and which is characterized in that it comprises at least one printing head. nozzle printing for printing at least one biological ink on the same receiving substrate as the laser printing head (s). According to another characteristic, the printing device comprises an enclosure configured to store at least one pedestal supporting a donor support, said enclosure being equipped with confinement means for retaining inside an atmosphere adapted to biological inks. Preferably, the enclosure has dimensions adapted to store several bases. In this case, the printing device comprises at least one base configured to be stored inside said enclosure, said base comprising a housing for each base.
    [0011] Advantageously, the enclosure comprises on a first face facing the printheads a first opening for removing the bases and on another side a second opening for introducing the bases. According to another characteristic, the printing device comprises a mobile clamp for moving the bases between the enclosure and the laser-type printing head.
    [0012] According to another characteristic, the printing device comprises a mobile frame supporting at least one receiving substrate, a system for guiding and moving the mobile frame with respect to a frame in three directions and a control system for controlling the movements of the mobile frame, said guide and displacement system and said control system having a micrometer accuracy.
    [0013] Other features and advantages will become apparent from the following description of the invention, a description given by way of example only, with reference to the accompanying drawings in which: FIG. 1 is a diagrammatic representation of a printing device by FIG. 2 is a schematic representation of a laser printing device which illustrates another variant of the invention. FIG. 3 is a diagrammatic representation of a device of FIG. 4A to 4D are side views illustrating the formation or not of a droplet according to different regimes, FIGS. 5A to 5D are diagrams which illustrate a droplet at different times of the present invention. its formation, the last FIG. 5D illustrating the moment when a droplet reaches a recipient substrate, FIG. 6 is a section of a donor support illustrating the relationship between the tail 7A and 7B are side views illustrating the formation of a protrusion on the free surface of a biological ink film, prior to the formation of a droplet, at the same instant but produced with different energies for the laser beam, FIG. 8 is a schematic representation of a printing device according to one embodiment of the invention combining at least one head of laser-like printing and at least one inkjet-type printing head, FIG. 9 is a perspective view of a printing device according to an embodiment of the invention combining a printing head 10 is a perspective view of a portion of the printing device of FIG. 9 when printing with one of the printing heads of FIG. inkjet type, Figure 11 is a section of a part of the printing device of FIG. 9 when printing with the laser-type printing head; FIG. 12 is a perspective view of a 3-dimensional representation of a part of a biological tissue 13 is a perspective view of a slice of the representation of FIG. 12, FIG. 14 is a top view of the slice of FIG. 13 illustrating the positioning of the droplets of FIG. organic inks.
    [0014] In FIG. 3, there is shown a printing device 50 for producing at least one biological tissue by assembling, layer by layer, according to a predefined arrangement, different constituents, for example an extracellular matrix and various morphogens.
    [0015] Thus, the printing device 50 makes it possible to deposit layer-by-layer droplets 52 of at least one biological ink 54 on a deposition surface 56 which corresponds to the surface of a receiving substrate 58 for the first layer or to the last layer deposited on said receiving substrate 58 for the following layers.
    [0016] In order to simplify the representation, the deposition surface 56 corresponds to the surface of the receiving substrate 58 in FIG. 3. According to one embodiment in FIG. 6, the biological ink 54 comprises a matrix 60, for example a medium aqueous, in which are present elements 62, for example cells or aggregates of cells, to be printed on the dispense surface 56.
    [0017] Depending on the case, a biological ink 54 comprises in the matrix 60 only one kind of printing elements 62 or several kinds of printing elements 62. Alternatively, the biological ink 54 may comprise only one component. By biological ink is meant for the present patent application a biological material or biomaterial. By way of example, the biological ink comprises only an extracellular matrix (for example collagen), an extracellular matrix and elements such as cells or aggregates of cells, an aqueous medium containing elements such as cells or aggregates of cells. The biological ink 54 is not more described because it can have different natures and different rheological characteristics from one ink to another.
    [0018] This printing device comprises a laser source 64 configured to emit a laser beam 66 which is characterized inter alia by its wavelength, its frequency, its energy, its diameter, its pulse duration. Preferably, the laser source 64 is parameterizable in order to adjust at least one characteristic of the laser beam, in particular its energy.
    [0019] In order to form droplets dissociated from each other, the laser source 64 is a pulsed source. To give an order of magnitude, it is possible to eject 10,000 droplets per second. By way of example, the laser source 64 is a laser source with a wavelength of 1064 nm.
    [0020] In addition, from the laser source, the printing device 50 comprises an optical system 68 which allows adjustment of the focusing along an axis Z perpendicular to the deposition surface 56. Advantageously, the optical system 68 comprises a lens which enables focusing the laser beam 66 on an impacted area. Preferably, the optical system 68 includes a mirror for changing the position of the impacted area. Thus, the optical system 68 makes it possible to modify the zone impacted by the laser beam in an impact plane referenced Pi in FIG.
    [0021] The laser source 64 and the optical system 68 are not further described because they are known to those skilled in the art and may be identical to those of the prior art. The printing device 50 also comprises at least one donor support 70 which comprises, according to one embodiment, an absorbent layer 72 at the wavelength of the laser beam 66 on which is affixed a film 74 of at least one biological ink.
    [0022] For the remainder of the description, film is understood to mean that the biological ink occupies a volume with a thickness (dimension in a direction perpendicular to the impact plane Pi) of less than 500 μm. Unlike a reservoir, the fact that the biological ink is packaged in the form of a film makes it possible to avoid sedimentation phenomena.
    [0023] The absorbent layer 72 is made of a material adapted to the wavelength of the laser beam 66 to transform the light energy into a spot heating of the absorbent layer 72. Preferably, the donor support 70 is positioned so that the optical system focuses the laser beam at the level of the absorbent layer 72.
    [0024] According to one embodiment, the absorbent layer 72 is made of gold, titanium, or other depending on the wavelength of the laser beam 66. According to another embodiment, the donor support 70 does not comprise an absorbent layer 72 In this case, the energy of the laser beam 66 is absorbed by the ink. Preferably, the donor support 70 comprises a blade 76 of a material transparent to the wavelength of the laser beam 66 which comprises on one of its faces a coating corresponding to the absorbent layer 72. The presence of the blade 76 confers rigidity the donor support 70 for manipulating and retaining the ink and / or the absorbent layer 72 substantially flat in the plane of impact Pi. The film 74 of biological ink comprises a free surface 78 which is spaced from the absorbent layer 72 of a distance E corresponding to the thickness of the film 74 and which is spaced from the deposition surface 56 by a distance L. The free surface 78 and the deposition surface 56 face each other.
    [0025] As illustrated in FIG. 3, the laser beam 66 is adapted to produce a cavity 80 at the interface between the absorbent layer and the biological ink film 74 which generates a droplet 82 which detaches from the free surface 78 to move towards the dispense surface 56.
    [0026] For the rest of the description, a vertical direction is parallel to the gravitational force G and the up-down direction corresponds to the direction of the gravitational force G. The direction of the laser beam 66 and the direction of the movement of the droplet are parallel to the vertical direction. Pull up: According to one characteristic of the invention, the laser beam 66 and therefore the movement of the droplet 82 are oriented in the opposite direction to the gravitational force G. Thus, the free surface 78 of the film 74 of biological ink is facing up. During its movement of the film 74 of biological ink to the dispense surface 56, the droplet 82 moves upwards, in the low-high direction.
    [0027] This configuration provides the following advantages: It limits the appearance of sedimentation phenomena, the biological ink being in the form of a film, It makes it possible to obtain a thickness E for the film 74 of substantially constant biological ink, the influence of the gravitational force G on the shape of the free surface 78 of the film 74 being limited by the fact that the free surface 78 is oriented upwards, It makes it possible to use a wide range of biological ink when an absorbent layer 72 independent of the film 74 of biological ink is used to transform the light energy into a punctual heating.
    [0028] Kinetic energy virtually zero at the time of deposition of a droplet on the receiving substrate: The formation of a droplet 82 from a biological ink film will depend on many parameters, mainly the characteristics of the biological ink, characteristics of the laser beam and the conditions of realization.
    [0029] FIGS. 4A to 4D show the evolution over time of the deformation of the free surface of the biological ink film, resulting or not in the formation of a droplet, for different values of the energy of the beam laser 66, the latter having an energy of 21 μl for FIG. 4A, of FIG. 35 for FIG. 4B, of 40 μJ for FIG. 4C and 43 μ.1 for FIG. 4D. For the same biological ink and under the same conditions of realization, it is noted that there are several regimes depending on the energy of the laser beam. As illustrated in FIG. 4A, if the energy of the laser beam is lower than a lower threshold, the droplet does not come off the film 74 of biological ink. The maximum height of the deformation 84 generated at the free surface 78 of the film 74 of the ink being less than the distance L between the film 74 and the deposition surface 56 no element is printed. According to the example chosen, the lower threshold is between 21 u..1 and 35 u. As illustrated in FIG. 4D, if the energy of the laser beam is greater than an upper threshold, the gas bubble 80 produced at the Inside the film bursts at the level of the free surface causing the uncontrolled projection of microdroplets. According to the example chosen, the upper threshold is between 40 μl and 43 μd. Between the lower and upper thresholds, as illustrated in FIGS. 4B and 4C, there is in the presence of a regime allowing the formation of a jet. . If the distance L between the film 74 and the deposition surface 56 is sufficient, this regime allows the formation of a droplet.
    [0030] Preferably, the distance L is of the order of 1 to 2 mm to allow the formation of a droplet and not a continuous stream that stretches from the film to the dispensing surface. This configuration limits the risks of contamination of the biological tissue produced by the biological ink. According to another characteristic of the invention, for the same biological ink and under the same conditions of realization, the distance L separating the film 74 from biological ink and the deposition surface 56 and / or the energy of the laser beam 66 are adjusted so that the kinetic energy of the droplet is almost zero when the droplet 82 touches the dispense surface 56, as shown in Figure 5D. This configuration limits the risk of damage to the print elements that are cells.
    [0031] By almost zero, it is meant that the kinetic energy is zero or very slightly positive to allow the droplet to be fixed on the deposition surface 56.
    [0032] This circumstance is made possible by the fact that the droplet 82 moves in the opposite direction to the gravitational force G. Preferably, the distance L between the organic ink film 74 and the dispense surface 56 is fixed. Consequently, the energy of the laser beam 66 is adjusted so that the kinetic energy of the droplet is almost zero when the droplet 82 touches the dispense surface 56. Calibration technique: As indicated above, the formation of the droplet is not solely related to the energy of the laser beam. It is also related to the nature of the biological ink, in particular its viscosity, its surface tension and the conditions of production. FIGS. 5A to 5D, 7A to 7D illustrate a calibration method for determining the energy of the laser beam to obtain an optimum regime for the formation and deposition of the droplets, in particular a regime which leads to a deposition at zero velocity at a given distance L.
    [0033] FIGS. 5A to 5D show some of the steps of forming a droplet 82 between the moment of the impact of the laser beam illustrated in FIG. 5A and the deposition of the droplet 82 on the deposition surface 56. According to one characteristic of the invention, the calibration method for adjusting the energy of the laser comprises the steps of measuring a peak angle θ of a deformation 86 of the free surface 78 of the film 74 of the biological ink. at a fixed instant Ti after the impact of the laser beam 66 and to adjust the energy of the laser beam 66 as a function of the measured value of the tip angle O. As illustrated in FIGS. 5B, 7A and 7B, the deformation 86 has a symmetrical shape with respect to a median axis Am parallel to the vertical direction. This deformation 86 comprises a vertex S centered with respect to the median axis Am. This vertex S corresponds to the zone of the deformation 86 farthest from the remainder of the free surface 78 of the film 74. In a plane containing the median axis Am, the vertex S is extended by a first sidewall 88 on one side of the median axis Am and by a second sidewall 88 'on the other side of the central axis Am, the two sidewalls 88, 88' being symmetrical with respect to the median axis Am.
    [0034] Each side 88, 88 'comprises a point of inflection.
    [0035] The first sidewall 88 comprises at its inflection point a first tangent Tg1 and the second sidewall 88 'comprises at its point of inflection a second tangent Tg2, the two tangents Tg1 and Tg2 being intersecting at a point of the median axis Am. The tip angle θ corresponds to the angle formed by the tangents Tg1 and Tg2 and oriented towards the film 74 (ie downwards). To obtain the formation of a droplet, the tip angle θ must be less than or equal to a first threshold 01. Thus, as illustrated in FIG. 7A, if the tip angle θ is greater than the first threshold θ1, energy of the laser beam is not sufficient to generate a droplet. On the contrary, as illustrated in FIG. 7B, if the tip angle θ is smaller than the first threshold θ1, the energy of the laser beam is sufficient to generate a droplet. To obtain a quasi-zero kinetic energy at the moment when the droplet formed reaches the deposition surface 56 placed at a distance L from the free surface 78 of the film 74, the tip angle θ must be greater than or equal to a second threshold O 2.
    [0036] Preferably, the value of the tip angle θ is determined by taking a picture at the instant T1 of the deformation 86. According to one embodiment, the shooting is carried out by means of a camera whose axis aiming line is perpendicular to the vertical direction. The instant T1 is a function of the thickness of the film and varies very little from one ink to another. Advantageously, the instant T1 is of the order of 4 to 5 us since the impact of the laser beam for a film thickness E of the order of 40 to 50 μm. This moment T1 corresponds to FIG. 5B. The first threshold 01 is approximately equal to 1050. Thus, if at the instant T1 the point angle θ is less than or equal to 1050, the energy of the laser beam is sufficient to generate a droplet 82.
    [0037] The second threshold 02 is a function of the distance L between the dispense surface 56 and the free surface 78 of the film 74 of the ink. The second threshold 02 is inversely proportional to the distance L. The second threshold 02 is high and equal to approximately 80 ° for a low distance L of the order of 1 mm. The choice of a relatively small distance L will be preferred to reduce the stresses in the jet and at the moment of contact of the droplets with the dispensing surface. The second threshold 02 is low and equal to approximately 500 for a significant distance L of the order of 10 mm. The choice of a relatively large distance L will be preferred if it is desired to print at long distance, for example if the donor support 70 has larger dimensions than those of the well at the bottom of which the dispensing surface 56 is positioned. the energy of the laser beam makes it possible to optimize the speed of the jet by reducing it in order to limit the risk of damage to the elements contained in the biological ink, especially at the time of deposition on the dispense surface 56. Thickness of the ink film Preferably, the biological ink comprises a high concentration of printing elements 62 to obtain a biological tissue with a high concentration of cells.
    [0038] In this case, as illustrated in FIG. 3, the droplet 82 comprises a high volume fraction of elements to be printed 62. For high concentration biological inks, the thickness E of the film 74 is of the order of 40 to 6011m. Advantageously, to improve the accuracy of the deposition of the elements to be printed, the film 74 of biological ink has a thickness E between 1.5D and 2D, where D is the diameter of the elements to be printed 62 which have an approximately spherical shape or the diameter a sphere in which a print element 62 is inscribed. According to one embodiment, the film 74 of biological ink has a thickness E greater than or equal to 20 μm for the smallest elements to be printed which have a diameter of the order of 10 to 15 μm. The thickness E of the film may be of the order of 400 μm when the elements to be printed 62 are aggregates of cells. Generally, the thickness E of the film is less than 100 μm when the elements to be printed 62 are unit cells. A printing technique combining a laser-type print head and a nozzle-type print head: According to another characteristic of the invention, the printing method uses at least one laser-type print head for at least a first biological ink and at least one nozzle print head for at least one second biological ink. This combination makes it possible to increase the production rate.
    [0039] By nozzle print head is meant a print head which includes an orifice through which the second biological ink passes. Thus, a nozzle-type print head may be an inkjet-type print head, a microvalve print head, a bioextrusion-type print head.
    [0040] Preferably, each laser-type print head is identical to that described in FIG. 3. However, the invention is not limited to this laser-type printing head. Thus, it is conceivable to use the laser-type printing heads described in FIGS. 1 and 2 or others. The nozzle-type print heads are no longer described because they are preferably identical to those of the prior art. In the case of a biological tissue comprising disjunct cells separated by extracellular materials, the extracellular materials are preferably deposited by the nozzle printing head (s) and the cells are preferably deposited by the head (s). (s) Laser type printing.
    [0041] Since extracellular materials are less sensitive to shear effects, it is possible to deposit them with a nozzle print head. The biological ink cartridges for nozzle-type printheads having a volume very much greater than the volume of ink (of the order of 400) supported by a donor support 70 for a laser-type printing head, it is possible to deposit the materials of the extracellular matrix with a high flow rate. Even if a nozzle-type print head is capable of depositing the inks with a high flow rate, each donor support intended for a laser-type printing head supporting a very small volume of ink, it is necessary to change them frequently. which tends to increase the time of removal compared to a nozzle print head.
    [0042] Printing device comprising a donor support storage chamber: In FIGS. 8 to 11, a printing device is shown according to one embodiment of the invention. This printing device comprises a frame 100 supporting a laser-type printing head 102 and a plurality of inkjet-type printing heads 104, 104 ', 104 ", which frame 100 comprises an X, Y, Z mark. , the Z axis being oriented in the vertical direction, the X, Y plane corresponding to a horizontal plane.
    [0043] The printheads 102, 104, 104 ', 104 "are fixed relative to the frame 100 and positioned so that the droplets are emitted vertically, upwardly. 104 "are offset in a first direction parallel to the Y axis. According to one embodiment, the inkjet-type printheads 104, 104 ', 104" are placed one against the other. Laser printing 102 is discarded from the jet type printheads 104, 104 ', 104 ". The printing device also comprises a mobile frame 106, a system for guiding and moving the mobile frame 106 with respect to the frame 100 in three directions parallel to the X, Y, Z axes and a control system making it possible to control the movements of the movable frame 106. The guidance and displacement system and the control system are chosen so as to achieve a micrometric precision concerning the displacements of the mobile frame 106 with respect to the frame. As illustrated in Figure 10, the movable frame 106 comprises a frame 108 for releasably securing at least one receiving substrate 58. When secured to the movable frame, the displacements of the receiving substrate 58 are controlled with a micrometer accuracy. The laser-like printing head 102 comprises a cylindrical hollow body 110, fixed with respect to the frame, which contains a portion of an optical system and which is surmounted by a tubular portion 112 which comprises an upper end 114 placed which opens in a horizontal plane. These elements are configured so that a laser beam guided by the optical system scans the section of the upper end 114. Each donor support 70 has the shape of a disk positioned on a base 116. According to one embodiment illustrated in Figure 11, each base 116 has the shape of a tube which comprises at its upper edge a recess 118 which has a diameter identical to that of a donor support 70 and a height sufficient to maintain it. Thus, this recess 118 makes it possible to position a donor support 70 with respect to the pedestal that receives it. The upper end 114 and the base 116 have shapes that cooperate with each other so that the base 116 is immobilized at a given position with respect to the upper end 114 and therefore relative to the X, Y, Z mark of the frame. According to one embodiment, the base 116 comprises an outer collar 120 which bears against the upper end 114 and makes it possible to position the base along the axis Z. Below the collar 120, the base 116 comprises a frustoconical surface 122 which cooperates with a frustoconical portion provided inside the tubular portion 112. These forms make it possible to center the base 116 relative to the tubular portion 112 and to position it in an XY plane. Preferably, it is possible to use magnetic materials to improve the positioning of the base 116 relative to the tubular portion 112. Advantageously, the printing device comprises an enclosure 124 configured to store at least one base 116. This enclosure 124 comprises at least one opening 125 for entering and leaving the base (s) 116 stored. According to one embodiment, this enclosure 124 has a parallelepipedal shape.
    [0044] Preferably, the enclosure 124 has dimensions adapted to be able to store several bases. Thus, the printing device can successively print several biological inks with the same laser-type printing head 102. The bases 116 are stored on a base 126 which comprises housings 128, a housing for each base 116. The base 126 has an elongate shape and comprises along its length slots 128 in U. According to a first variant illustrated in FIG. 8 , the length of the base 126 is oriented along the Y axis. According to a second preferred variant, the length of the base 126 is oriented along the X axis and the notches 128 are open in the direction of the printing heads. Advantageously, the enclosure 124 comprises, on a first face facing the printheads, a first opening 125 making it possible to extend the pedestals 116 and on another face a second opening 125 'making it possible to introduce the pedestals 116. According to one embodiment embodiment, the enclosure 124 comprises a guide system for positioning the base 126, for example a rail, the base 126 comprising in the lower part a groove whose section cooperates with that of the rail. This rail opens at the second opening 125 '. It is preferably oriented along the X axis. The enclosure 124 comprises confinement means for keeping inside the enclosure an atmosphere adapted to biological inks, in particular at temperature and / or hygrometry. These confinement means are provided in particular at each opening 125, 125 '. They can take the form of a barrier or an air curtain. In addition to the enclosure, the printing device comprises a mobile clamp 130 for moving the bases between the enclosure 124 and the laser-type printing head 102. According to a first variant, the mobile clamp 130 is secured to a movable carriage 132, independent of the mobile frame 106, which is configured to move in the X, Y, Z directions. According to another variant, the mobile clamp 130 is secured to the mobile frame 106.
    [0045] According to one embodiment, the printing device comprises a camera (not shown) whose line of sight is perpendicular to the vertical direction and oriented at the upper surface of the donor support. This apparatus can be used to calibrate the energy of the laser beam of the laser-type print head 102. Process for producing a biological tissue by bio-printing: The first step of said method consists in generating a three-dimensional digital representation of the biological tissue to be printed. In FIG. 12, there is shown at 140 a part of such a representation in the form of a cube which comprises a first volume zone 142 placed inside a second volume zone 144 itself disposed in a third volume zone 146.
    [0046] For the purposes of the description, the representation 140 is greatly simplified. Each volume zone 142, 144, 146 is colored or textured differently, each color or texture corresponding to a set of characteristics among the following characteristics (not limited) material, means of manufacture, trajectory, ... Preferably, each color or texture corresponds to a biological ink.
    [0047] All volume zones 142, 144, and 146 are closed. Advantageously, the representation comprises a plurality of small elementary volumes that have different colors or textures depending on the volume zone to which they belong. According to one embodiment, the representation 140 is derived from a PLY type computer file.
    [0048] The second step of the method consists in slicing the representation 140 in a succession of superimposed layers along an axis Z. In FIG. 13, a layer 148 of the representation 140 is isolated. When slicing the representation 140, the right to a volume zone change, each layer comprises an edge that corresponds to a zone change.
    [0049] As illustrated in FIG. 13, the layer 148 comprises a first zone 142 'which corresponds to the first volume zone 142, a second zone 144' which corresponds to the second volume zone 144 and a third zone 146 'which corresponds to the third zone 146. For each layer, the zones 142 ', 144', 146 'are colored or textured according to the color or texture of the volume zones 142, 144, 146. Each layer has a thickness E which is determined according to the height of the printed droplets. If the layer comprises only one material to be printed, the layer has a thickness substantially equal to the height of a droplet. When the layer comprises several printing materials, according to a first variant, the layer has a thickness equal to the least common multiple of the droplet heights associated with each material. This variant has the advantage of minimizing the offset in the entire height of the object to be printed and resulting in a fast printing. According to a second variant, the layer has a thickness equal to the greatest common divider of the heights of the droplets associated with each material. This variant has the advantage of increasing the resolution and the number of layers.
    [0050] By way of example, if the first material is printed by laser bio-printing, the printed droplets have a height of the order of 10 μm. If the second material is printed by bio-printing microvannes, the printed droplets have a height of the order of 100 microns. According to the first variant, the layers have a thickness of the order of 100 μm. According to the second variant, the layers have a thickness of the order of 10 μm.
    [0051] Preferably, each layer comprises a plurality of small elementary polygons, for example triangular, which have different colors depending on the zone to which they belong. Thus, the object to be printed corresponds to a set of layers each comprising a set of polygons each having an associated color or texture.
    [0052] A third step of the method consists in determining for each layer the position of the droplets to be printed of each biological ink as a function of the zones 142 ', 144', 146 'which are colored or textured and the expected volume of each of the droplets. For this purpose, each zone 142 ', 144', 146 'of each layer is filled with ellipses 142 ", 144", 146 "whose dimensions are a function of the size of the droplets of the biological ink to be printed in said zone, as illustrated in Figure 14. For each zone, the ellipses have the same dimensions All ellipses have parallel focal axes.
    [0053] The elliptical shape makes it possible to adapt the distances between the droplets in two directions (a first direction parallel to the focal axes and a second direction perpendicular to the first direction). The center of each ellipse corresponds to the position of the center of a droplet.
    [0054] The positioning of the ellipses is zone by zone, in decreasing order of size. Thus, the largest ellipses arranged in the zone 146 'are positioned first and the smallest ellipses arranged in the zone 142' are positioned last. Preferably, at the level of a zone change, optimization of the positioning is done according to two criteria: maximum ratio of elementary polygons having the right color or texture within an ellipse, of the order of 75% for example , Minimum ratio of elementary polygons having the wrong color or texture within an ellipse, of the order of 5% for example. Overlaps between ellipses can be tolerated.
    [0055] A fourth step of the method is to synchronize the displacement of the dispense surface 56 on which the organic ink droplets are printed and the different print heads. For laser bio-printing, the focus area of the laser is the center of each laser-printed ellipse, and each ellipse is laser pulsed. In this case, the removal surface is fixed, it is the laser that sweeps the entire removal surface. For a larger dispensing surface than the donor support, it is possible to also move the substrate (on which the dispensing surfaces are reported) synchronously with the scanning of the laser. For a nozzle bio-printing, the center of each ellipse corresponds to the supposed point of impact of a droplet on the dispense surface 56. In this case, the printing nozzle is fixed, it is the substrate which is moves. However, the print nozzle could be mobile. Applications: The bio-printing according to the invention can be used to produce: - Implantable tissues for regenerative medicine, individualized tissues, made from the patient's cells, to select in vitro treatments and develop therapeutic solutions Predictive models that reproduce the physiology of healthy human tissues or pathologically-affected tissues to predictively test the efficacy or toxicity of molecules, ingredients, and drug candidates. By way of example and without limitation, the biological tissue is bone tissue.
    权利要求:
    Claims (4)
    [0001]
    REVENDICATIONS1. A method of printing at least one biological ink, said method using at least one laser-type printing head (102) for depositing at least one droplet (82) of at least one biological ink onto a dispensing surface ( 56) of a receiving substrate (58), characterized in that the printing method uses at least one nozzle-printing head (104, 104 ', 104') for depositing at least one droplet of at least one biological ink on a dispensing surface (56) of the same receiving substrate (58) as the laser-like printing head (102).
    [0002]
    2. A method of printing according to claim 1, said method being used to obtain a biological tissue comprising cells and an extracellular matrix, characterized in that the materials constituting the extracellular matrix are deposited by the head (s) of printing (104, 104 ', 104 ") nozzle and that the cells are deposited by the laser-type printing head (s) (102).
    [0003]
    3. Process for producing a biological tissue using the printing method according to claim 1 or 2, characterized in that it comprises the steps of: Generating a three-dimensional digital representation (140) of the biological tissue to performing, said representation comprising a plurality of colored or textured volume zones (142, 144, 146), each color or texture being associated with a biological ink, slicing the representation (140) into a succession of superimposed layers (148), each layer ( 148) comprising colored or textured zones (142 ', 144', 146 ') corresponding to the volume zones (142, 144, 146) of the representation (140), - For each layer, determining the position of the droplets to be printed. each biological ink according to the areas (142 ', 144', 146 ') colored or textured and the expected volume of each of the droplets, 25 - Print the different droplets.
    [0004]
    4. Process for producing a biological tissue according to claim 3, characterized in that a printing method according to claim 1 or 2 is used to print the different droplets. . A method of producing a biological tissue according to claim 3 or 4, characterized in that the layers (148) have a thickness depending on the size of the droplets. 6. Process for producing a biological tissue according to one of claims 3 to 5, characterized in that each layer comprises a set of small elementary polygons which have different colors or textures depending on the zone to which they belong . 7. A method of producing a biological tissue according to one of claims 3 to 6, characterized in that to determine the position of each droplet, each zone (142 ', 144', 146 ') is filled by ellipses ( 142 ", 144", 146 ") which have dimensions dependent on the size of the droplets of the biological ink to be printed in said area, the center of each ellipse corresponding to the position of the center of a droplet. Process for producing a biological tissue according to claim 7, characterized in that the positioning of the ellipses is carried out zone by zone, in descending order of size 9. Printing device comprising at least one receiving substrate (58) with a deposition surface (56) and at least one laser-type printing head (102) which comprises: - at least one pulsed laser source (64) configured to emit a laser beam (66), - an optical system (68) to focus and steer said laser beam (66), - at least one donor support (70) which comprises at least one biological ink, characterized in that the printing device comprises at least one printing head (104, 104 ', 104 ") to nozzle for printing at least one biological ink on the same receiving substrate (58) as the laser-like printing head (s) (102). 10. Printing device according to claim 9, characterized in that the printing device comprises an enclosure (124) configured to store at least one base (116) supporting a donor support (70), said enclosure (124) being equipped with containment means to keep inside an atmosphere suitable for biological inks. 11. Printing device according to claim 10, characterized in that the enclosure (124) has dimensions adapted to store a plurality of pedestals (116) and in that the printing device comprises at least one base (126) which comprises slots (128), a housing for each base (116), said base being configured to be stored within said enclosure (124). 12. Printing device according to claim 11, characterized in that the base (126) comprises a guide system for positioning in the enclosure (124). 13. Printing device according to one of claims 9 to 12, characterized in that the enclosure (124) comprises on a first face facing the printheads a first opening (125) for removing the bases (116) and on another side a second opening (125 ') for introducing the bases (116). 14. Printing device according to one of claims 9 to 13, characterized in that the printing device comprises a movable clamp (130) for moving the bases between the enclosure (124) and the print head of laser type (102). 15. Printing device according to one of claims 9 to 14, characterized in that the printing device comprises a movable frame (106) supporting at least one receiving substrate (58), a system for guiding and moving the movable frame (106) relative to a frame (100) in three directions and a control system for controlling the displacements of the movable frame (106), said guide and displacement system and said control system having a micrometer accuracy. 16. Printing device according to claims 14 and 15, characterized in that the movable clamp (130) is secured to the movable frame (106).
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US20130017564A1|2010-03-04|2013-01-17|Fabien Guillemot|Bioprinting station, assembly comprising such bioprinting station and bioprinting method|
    WO2014110590A1|2013-01-14|2014-07-17|Scripps Health|Tissue array printing|FR3093944A1|2019-03-22|2020-09-25|Poietis|CARTRIDGE FOR BIOIMPRESSION|DE3702643A1|1986-02-10|1987-08-13|Toshiba Kawasaki Kk|INK NIBLE PEN AND WRITING HEAD AND WRITING HEAD CASSETTE DAFUER|
    JPS62191157A|1986-02-17|1987-08-21|Toshiba Corp|Recording apparatus using thermal ink jet system|
    JPH07164656A|1993-10-22|1995-06-27|Sony Corp|Recording part structure and recording apparatus|
    CA2311622A1|2000-06-15|2001-12-15|Moussa Hoummady|Sub-nanoliter liquid drop dispensing system and method therefor|
    CN1275772C|2001-12-20|2006-09-20|明基电通股份有限公司|Fluid jetting head structure and its making process|
    GR1004059B|2001-12-31|2002-11-15|Ιωαννα Ζεργιωτη|Fabrication of biopolymer patterns by means of laser transfer|
    FR2881373B1|2005-02-01|2008-08-22|Centre Nat Rech Scient|DETECTION SYSTEM FOR OFFSET PRINTING AND MACHINE USING THE SAME|
    US7841700B2|2006-11-20|2010-11-30|Lexmark International, Inc.|Radiation activated micro-fluid ejection devices and methods for ejecting fluids|
    US8702216B2|2011-12-21|2014-04-22|Xerox Corporation|Polymer internal contamination filter for ink jet printhead|
    JP5483488B2|2012-03-02|2014-05-07|株式会社古河電工アドバンストエンジニアリング|Cell assembling apparatus and cell assembling method|
    EP2892577B1|2012-09-04|2019-01-30|Anthrogenesis Corporation|Methods of tissue generation|
    US9347037B2|2013-02-11|2016-05-24|Evan Masataka Masutani|Methods and apparatus for building complex 3D scaffolds and biomimetic scaffolds built therefrom|
    EP3065875A4|2013-11-04|2018-02-07|University of Iowa Research Foundation|Bioprinter and methods of using same|
    CN203752527U|2013-12-25|2014-08-06|杭州铭展网络科技有限公司|Probe-type 3D printer spray head device|
    CN104146794A|2014-08-24|2014-11-19|周惠兴|Blood vessel forming device and method for 3D bioprinting|
    FR3030360B1|2014-12-17|2018-07-13|Universite de Bordeaux|LASER PRINTING METHOD AND DEVICE FOR IMPLEMENTING SAME|
    US9968437B2|2015-06-22|2018-05-15|Bao Tran|3D printer|
    US20190275205A1|2016-05-26|2019-09-12|Scripps Health|Systems and methods to repair tissue defects|
    CN109641384A|2016-10-21|2019-04-16|惠普发展公司，有限责任合伙企业|Overabsorption technology 3D printing|
    SE542653C2|2017-01-13|2020-06-23|Cellink Ab|A 3D bioprinter and a 3D bioprinter system|JP6619335B2|2013-10-14|2019-12-11|オルボテック リミテッド|LIFT printing of multiple composition material structures|
    FR3030360B1|2014-12-17|2018-07-13|Universite de Bordeaux|LASER PRINTING METHOD AND DEVICE FOR IMPLEMENTING SAME|
    FR3063931B1|2017-03-15|2019-03-22|Poietis|EQUIPMENT AND METHOD FOR ADDITIVE PRINTING|
    FR3063929B1|2017-03-15|2019-03-22|Poietis|EQUIPMENT FOR BIO-INK TRANSFER|
    FR3082123B1|2018-06-07|2020-10-16|Urgo Rech Innovation Et Developpement|CELLULARIZED DRESSING AND ITS MANUFACTURING PROCESS|
    KR102115229B1|2018-06-20|2020-05-27|한국생산기술연구원|One-step manufacturing method of laminated molding porous component which has curved surface|
    FR3087702B1|2018-10-25|2021-03-19|Poietis|ISOLATED BIO-PRINTING DEVICE|
    FR3087703B1|2018-10-25|2020-12-04|Poietis|ROBOTIZED BIO-PRINTING SYSTEM|
    EP3659728B1|2018-11-29|2021-01-27|Ivoclar Vivadent AG|Method for layer-wise additive manufacturing of a shaped body|
    法律状态:
    2015-12-23| PLFP| Fee payment|Year of fee payment: 2 |
    2016-06-24| PLSC| Search report ready|Effective date: 20160624 |
    2016-12-22| PLFP| Fee payment|Year of fee payment: 3 |
    2017-11-27| PLFP| Fee payment|Year of fee payment: 4 |
    2018-12-21| PLFP| Fee payment|Year of fee payment: 5 |
    2020-10-16| ST| Notification of lapse|Effective date: 20200906 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1462570A|FR3030361B1|2014-12-17|2014-12-17|METHOD FOR PRINTING BIOLOGICAL ELEMENTS BY LASER AND DEVICE FOR IMPLEMENTING SAID METHOD|FR1462570A| FR3030361B1|2014-12-17|2014-12-17|METHOD FOR PRINTING BIOLOGICAL ELEMENTS BY LASER AND DEVICE FOR IMPLEMENTING SAID METHOD|
    EP15831060.7A| EP3234102B1|2014-12-17|2015-12-17|Method for laser printing biological components, and device for implementing said method|
    CN201580073864.9A| CN107208013B|2014-12-17|2015-12-17|Method for laser printing biological components and device for carrying out said method|
    ES15831060.7T| ES2685696T3|2014-12-17|2015-12-17|Method of printing biological elements with laser and device for its realization|
    US15/536,564| US11045996B2|2014-12-17|2015-12-17|Method for laser printing biological components, and device for implementing said method|
    PCT/FR2015/053570| WO2016097620A1|2014-12-17|2015-12-17|Method for laser printing biological components, and device for implementing said method|
    JP2017531540A| JP6814138B2|2014-12-17|2015-12-17|Laser printing method of biological components and equipment for implementing the method|
    KR1020177018694A| KR20170126441A|2014-12-17|2015-12-17|Method for laser printing biological components, and device for implementing said method|
    US17/229,058| US20210229342A1|2014-12-17|2021-04-13|Method for laser printing biological components, and device for implementing said method|
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