Manipulation and transfer of objects by propulsion

Abstract

The present invention provides a method and apparatus for transferring at least one object from a donor substrate (30) to a target substrate (40), the donor substrate (30) having a thickness e z The carrier liquid forms a surface on which a film of the carrier liquid is deposited in the vertical direction Z, and the carrier liquid is transferred to the surface. x , D y , D z ), the transfer being ensured by local energy excitation of the liquid to form local cavitation bubbles at the object, D z / e z The method and the device are characterized in that the ratio is greater than 1, preferably greater than 1, preferably greater than 0.5.

Description

[Technical Field]

[0001] The present invention relates to the field of additive manufacturing of materials by repeatedly transferring particles between a donor substrate and a recipient substrate, whereby a film of a carrier liquid containing the transferred particles is deposited on the donor substrate, and in particular to the field of bioprinting. The particular field of bioprinting relates to the use of digital manufacturing methods to organize and assemble living tissue components in 2D and 3D, with the aim of producing grafts for regenerative medicine or physiological models for biomedical and pharmaceutical research.

[0002] The general principle of the invention is to transfer biological (e.g., cells), organic, or inorganic objects from a carrier liquid deposited on a substrate in the form of a film, and to deliver an energy pulse to form a cavitation bubble either directly by evaporation of a portion of the liquid in the focal field of the energy source (usually a laser) or through a thin metal coating that forms a sacrificial layer on the donor substrate. This cavitation bubble carries the particles in its targeting axis toward the receiving substrate, where the transferred particles accumulate as the pulses are repeated. [Background technology]

[0003] prior art In the state of the art, US Patent Application Publication No. 2020 / 009877 is known, which describes an apparatus for depositing particles onto a target from a transparent slide carrying a film formed by a fluid containing particles in suspension by locally exciting the film using a laser beam directed by controlled optical deflection means, the apparatus including means for observing the local excitation area by an optical imaging system including a sensor and a light source, the optical axis of the optical imaging system being substantially aligned in the section between the optical splitter and the film, the optical beam of the imaging system and the optical beam of the laser being coaxial in the section between the controlled optical deflection means and the film.

[0004] The device comprises a first focusing optical unit located between the controlled light deflection means and the film, and a second image-bearing optical unit located between the sensor and the separator, the sensor being positioned in the focal plane of the second optical unit.

[0005] Chinese Patent No. 113021874 describes a single cell printing method based on annular laser induced transfer, comprising the following steps: Step 1—after a uniformly distributed pulsed laser beam is incident on an element of an annular light spot forming system, the emerging light is focused to form an annular light spot on the sacrificial layer through the transparent stress layer; Step 2—Ablating the sacrificial layer with laser light and heat to form an annular plasma cavitation bubble; Step 3: Rapidly expand the annular cavitation bubble, remove the cell solution outside the ring, and thrust the target transcription cells downward parallel to the center of the ring under the expansion of the cavitation bubble. Step 4 - Collapse of the annular cavitation bubble, completely expelling the target transcription cells by the collapse of the shock wave generated perpendicular to the center position of the ring; Step 5—printing the target transfer cells and transport solution onto the receptor plate under the action of gravity and a naturally occurring impulse.

[0006] US Patent Application Publication No. 2022152925 describes a printing method that uses a portion of an instrument comprising an energy-delivering exciter that can be directed to effect point-like interaction with at least one ink deposited on a print medium, possibly including non-uniformities, and including a transparent interaction area, to transfer targeted portions of the ink to a receptor, the method comprising the steps of generating a wet film that at least partially covers the transparent interaction area, followed by depositing and transferring ink onto the surface of the wet film.

[0007] US Patent Application Publication No. 2020102529 relates to an additive manufacturing apparatus and method comprising a directable energy excitation means for generating an intermittent interaction with a fluid coating a blade to trigger a jet directed in a targeted direction, wherein the fluid comprises a liquid vector including an inhomogeneity, the fluid forming a liquid film having a measured thickness of less than 500 μm on the blade having at least one area allowing interaction with a laser, the area of ​​interaction being open to at least one outlet, the apparatus also comprising a means for circulating the fluid between the inlet and the outlet.

[0008] US Patent Application Publication No. 2017225390 relates to a method for additive manufacturing of a three-dimensional object. The method includes sequentially forming a plurality of layers, each patterned according to a cross-sectional shape of the object. In some embodiments, forming at least one of the layers includes performing a raster scan to dispense at least a first building material composition and performing a vector scan to dispense at least a second building material composition. The vector scan is optionally along a path selected to form at least one structure selected from the group consisting of: (i) an elongated structure; (ii) a boundary structure at least partially surrounding an area filled with the first building material; and (iii) an interlayer connection structure.

[0009] Shortcomings of the prior art Prior art solutions are suitable for transferring fine particles with dimensions significantly smaller than the thickness of the carrier liquid film when based on LIFT, or for handling / transferring larger particles by contact when using non-LIFT techniques such as Kenzan or suction, however, they have several drawbacks.

[0010] First, the focus of each pulse must be adjusted to take into account the plane in which the particle is located within the target area to deliver the appropriate energy to the plane directly below the particle, thereby ensuring that the energy impulse cavitates the liquid below the particle. This step of adjusting the focus before each pulse significantly reduces cadence.

[0011] Second, the transfer of particles is accompanied by the transfer of a non-negligible amount of carrier liquid, resulting in a material with a low bulk density.

[0012] Third, particles are relatively mobile within the carrier liquid film, and the accuracy of the pulse is affected by the displacement between the time of analysis of the target area image and the triggering of the pulse. Summary of the Invention [Means for solving the problem]

[0013] To address these drawbacks, the present invention relates in its most general sense to a method for producing a material exhibiting the characteristics set forth in claim 1.

[0014] According to the invention, the method for producing by transferring at least one object, in particular particles, from a donor substrate to a target substrate is in particular a method for producing a target substrate having a thickness e z provides a donor substrate that forms a surface onto which a film of carrier liquid is deposited in the vertical direction Z. The liquid vector is x , D y , D z The object to be transferred has a liquid-like substance. The transfer is achieved by locally energetically exciting the liquid to form local cavitation bubbles on the object, and D z / e z The ratio is characterized by being greater than 1, preferably greater than 0.5.

[0015] According to one variant, the object is a spheroid consisting of an aggregate of elementary biological cells.

[0016] Advantageously, said Dx , D y , D z The value of is greater than 100 μm, preferably greater than 200 μm.

[0017] According to a second variant, the energy excitation is achieved by focusing a laser onto the interface between the surface of the substrate and the carrier liquid film.

[0018] According to a second variant, the energy excitation is performed by applying an electric field.

[0019] According to a third variant, energy deposition is achieved by focusing acoustic waves at the interface between the surface of the substrate and the carrier liquid film.

[0020] Advantageously, the energy level applied in each pulse is based on the size of the object located in the targeting axis.

[0021] According to one variant, the receiving substrate (40) is elastically deformable along the Z axis.

[0022] According to a particular embodiment, the method includes moving the object in the XOY plane by applying pulses having an energy at least half of the energy required to transfer the object to a receiving substrate.

[0023] Advantageously, the object to be transferred has a value d>0.5D xy are separated by a characteristic distance (d).

[0024] According to one embodiment, the object is a biological entity, including cell aggregates, spheroids, organoids, explants, explants (islets of Langerhans), cell-encapsulating polymer particles (organoids coated with biomaterials), cell-seeded microcarriers, biomaterial beads.

[0025] Preferably, the volume fraction or density of biological matter transferred by propulsion within the printed tissue or organ is greater than 30%.

[0026] According to one variation, the transfer is repeated to produce a material, tissue, or organ, and the volume fraction or volume density of biological matter transferred by propulsion of the printed material, tissue, or organ is greater than 30%.

[0027] According to another variant, the method is combined with other printing techniques such as extrusion, inkjet, LIFT, etc. to generate complex materials or tissues with different components.

[0028] According to another variant, the method is carried out using multiple laser beams simultaneously if the object has an anisotropic shape, in order to ensure the transfer of the object along a homogeneous trajectory.

[0029] The present invention provides an apparatus for handling and transferring pulsed energy by deposition, comprising: an energy source directed at the material to be transferred; a donor substrate onto which the material is manipulated and transferred by energy impulses; a target receiving substrate for collecting the transferred material.

[0030] the donor substrate is a slide coated with a material to be transferred, the slide consisting of a film of carrier liquid intended to contain a transferable object of size DxDyDz; the transferable object has an orientation Dx, Dy in the plane of the film and an orientation Dz perpendicular to the film, - comprising a slide characterized in that the ratio between the dimension of the object (Dz) and the thickness of the liquid film (e) is greater than 1, preferably greater than 0.5.

[0031] Preferably, the energy source is a laser.

[0032] According to one variant, the donor substrate slide is transparent or has low absorption properties at the wavelength of the laser beam.

[0033] Certain embodiments include an optomechanical system for directing the laser spot relative to the center of gravity or mass of the object.

[0034] If the object has a particular anisotropic shape, the system can allow for the simultaneous use of multiple laser beams directed at different points on the object to separate the object uniformly.

[0035] According to a first variant, the energy source consists of an electric field generator.

[0036] According to a second variant, the energy source consists of an acoustic wave generator.

[0037] According to one variation, it includes a system for controlling and servo-controlling the amount of energy deposited based on the size of the particles being transferred.

[0038] Advantageously, it incorporates an intelligent object detection system, a means for automating the steps of placing material on the donor substrate and the handling / transfer steps.

[0039] According to one variant, the donor substrate is coated with a sacrificial layer with high absorption / conductivity properties for laser or field energy deposition.

[0040] According to one embodiment, the receiving substrate is not covered with a layer with highly absorbing properties that is produced directly in the liquid.

[0041] According to one embodiment, an optical viewing / detection system for locating and targeting objects to be transferred is included that is compatible with a random distribution of objects on the donor substrate.

[0042] Preferably, the donor substrate is disposed below the receiving substrate.

[0043] According to another embodiment, at least one other printing means including extrusion, inkjet, LIFT, etc. may be included to produce complex materials or tissues with different components.

[0044] According to another variant, it comprises means for simultaneously delivering multiple laser beams to the object when the object has an anisotropic shape in order to ensure transfer of the object along a homogeneous trajectory.

[0045] According to one variant, the equipment includes a system for controlling and servo-controlling the amount of energy deposited based on the size or shape of the object to be transferred, with this energy being deposited in the form of a single pulse, multiple pulses repeated over time on the same point, or multiple pulses sent simultaneously in an XY pattern linked to the shape of the object.

[0046] According to one variant, the receiving substrate is not coated with a layer with high laser absorption properties to initiate the propulsion process, and absorption is achieved directly by a liquid layer between the object and the donor substrate.

[0047] According to another variant, the donor substrate consists of microwells in which the objects to be transferred are disposed. [Brief explanation of the drawings]

[0048] The invention will be better understood on reading the following description of non-limiting exemplary embodiments illustrated by the accompanying drawings, in which: [Figure 1] 1 shows a schematic diagram of a transcription system. [Figure 2] 1 shows an exemplary chronology of various TRI (Time Resolved Imaging) components for imaging a jet at time T. [Figure 3] 1 illustrates an exploded view of an exemplary donor substrate. [Figure 4] 1 illustrates a perspective view of an exemplary fluid connector. DETAILED DESCRIPTION OF THE INVENTION

[0049] Schematic description of the transcription system The transcription system has several parts: an optical part comprising a pulsed laser (10) for generating cavitation energy in the carrier liquid and a camera (20) for observing the donor substrate (30) and the receiving substrate (40); an automation section comprising a robotic arm (50) used to automate the handling of the receptors (40); a microfluidic device (31) for providing a carrier liquid and particles to be transferred to a donor substrate (30), Optionally, the system can include an extruder for applying an adhesive, for example a hydrogel-type biomaterial, to the receiving substrate (40) between layers of transferred particles.

[0050] More generally, the device may combine multiple 3D printing, bioprinting, and photopolymerization technologies.

[0051] The optical part of the device consists of two parts: one part contains a camera (20) for aiming at the object, and one part contains a laser (10) that is used to "pulse", i.e. deliver an energy pulse in the plane of the donor substrate (30) where the particles to be transferred are located.

[0052] The laser (10) is a Nd-YAG laser, which emits, for example, 1 to 10 nanosecond pulses at 1064 nm with an energy of 15 to 60 microjoules, which is much higher than the energy typically used in the LIFT method.

[0053] Another example is a pulsed ytterbium fiber laser that emits shorter pulses at 1030 nm, from 350 femtoseconds to 10 picoseconds, with energy of tens of microjoules per pulse.

[0054] The lens (16) is typically an F-theta lens with a focal length of 100 mm, which is suitable for laser scanning. The typical spot size at the focal plane is approximately 30-35 μm in diameter.

[0055] The laser beam (14) passes through shaping optics (13) and is then directed via a set of mirrors (11, 12) to a scanner (15), which then transmits the beam perpendicular to the donor substrate (30) through an F-theta lens (16).

[0056] The scanner (15) includes two automatic mirrors that redirect the beam horizontally at an angle toward the objective lens (16). The objective lens (16) then straightens and focuses the beam (14) so ​​that it strikes the donor substrate (30) perpendicularly. The mirrors of the scanner (15) control the movement of the laser beam (14) along a horizontal axis above the donor substrate (30). Thus, the laser beam (14) is focused on the donor substrate (30) and is steerable along the X and Y axes that define a horizontal plane.

[0057] For the donor substrate (30), the beam (14) is focused onto a sacrificial layer coating the substrate surface, for example a 20 nanometer gold layer deposited on a transparent optical window.

[0058] The second optical section, including the camera (20), is the targeting section. In this case, the particles are relatively large objects, such as spheroids formed by cell aggregates, with diameters exceeding 100 μm, typically between 200 μm and 300 μm. These objects are randomly distributed on the donor substrate (30), so knowing their location and size is necessary to target them with laser pulses. To achieve this, a visible light source (typically an LED (21)) is placed on the cartridge, and a light beam (22) passes through the scanner (15) along the reverse path of the laser beam. The beam then reaches a semi-reflective mirror (12), which transmits visible light to the camera (20) but reflects infrared light back to the laser (10). The beam then passes through a lens (23), an iris (24), and an objective lens (25) before reaching the camera (20). All of these are aligned so that the laser beam (10) is focused at the centre of the image captured by the camera (20).

[0059] The nature of the item being transcribed The system implemented by the present invention is similar to the system designed for LIFT bioprinting, except that the impulse energy is at least two or even five times greater, and the geometry of the transferred object is fundamentally different from the solution used for LIFT bioprinting, forming a bioink in which the particle size is much smaller than the thickness of the film deposited on the donor substrate (30).

[0060] In the context of the present invention, the objects have a size greater than 100 μm, typically 200-400 μm or more, and are placed in a liquid, typically water with salt added to adjust the density, or a 2% BSA (Bovine Serum Albumin) solution, to form a film with a thickness less than the size of the particles to be transferred, so that the donor substrate (30) no longer receives the bioink, but the particles remain partially within the aqueous film that does not completely cover the particles.

[0061] The present invention relates to, but is not limited to, the transfer of spheroids, which are formed by the aggregation of cells cultured in the laboratory and have the appearance of small pearls consisting of cells and extracellular matrix.

[0062] To generate spheroids, stem cells, progenitor cells, or differentiated cells are cultured by conventional methods and seeded into microwells to generate aggregates, which are then manipulated according to the methods of the present invention.

[0063] Pulse sequence The LIFT phenomenon is a process that takes place over a very short time period, on the order of 100 microseconds, and can be observed by a TRI device.

[0064] TRI (Time-Resolved Imaging) involves imaging a phenomenon at a specific time by synchronizing a camera with a laser (10) and an LED to capture the jet at a precise moment. The three components are synchronized via an electronic board to control the precise activation of the various components over time. To capture an image of the jet at a precise moment, the camera is switched on for several hundred microseconds, while the LED flashes for 2 μs at the desired moment. The LED is positioned in front of the camera so that it illuminates the jet.

[0065] Figure 2 shows the TRI chronology, which has a microsecond time scale. The first step (100) is to switch on the analyzer camera, which takes 60 μs to light up. The laser (10) takes 4 μs to light up (105) and another 5 μs to perform the laser pulse. If the camera is switched on during the laser pulse, the beam will be visible on the image, making it difficult to read. Therefore, the pulse must be performed before the analyzer camera is switched on.

[0066] Therefore, the laser (10) is switched on (101) 50 μs after the scanner camera, so that the pulse (102) occurs at 59 μs, i.e., 1 μs before the scanner camera is switched on (103). The camera then remains on for 500 μs. Finally, the LED emits a series of 2 μs light flashes (110, 111) at the desired time (106) T μs after the laser pulse, i.e., 59 + T μs after the camera was switched on, to create a stroboscopic image with a 2 μs time interval between two flashes.

[0067] The acquired images therefore show particle behavior that is very different from that observed in the state-of-the-art LIFT bioprinting. Large particles are propelled without any carrier liquid by forming a cavity bubble in the liquid film at the interface between the particle and the bottom of the substrate, rather than in the volume surrounding the particle. As the particles emerge from the liquid film, they displace the surrounding liquid and emerge directly from the carrier liquid, reaching the receiving substrate with almost no liquid behind.

[0068] Donor substrate Figure 3 shows an exploded view of the donor substrate (30) that receives the ink containing the particles to be transferred and a film of technical liquid dispersed on a cartridge that serves as a support for the laser pulses. It consists of an assembly of four stages (31, 32, 33, 34) of glass and PDMS (polydimethylsiloxane). The purpose of the technical liquid is to clean the central area of ​​the donor substrate and pre-wet the surface to facilitate the dispersion of the ink containing the particles.

[0069] The upper slide (31) of the cartridge is made of polydimethylsiloxane and has a window (35) surrounding a cylindrical volume that receives the industrial liquid and particles. To prevent overflow of the ink containing the industrial liquid and particles, a hydrophobic barrier layer is formed. Its thickness is 10 μm.

[0070] The first intermediate slide (32) also has a window (36) and serves to secure the upper slide (31). It is made of 0.5 mm thick glass. It also acts as an overflow barrier.

[0071] The second intermediate slide (33) features a window (37) and two microfluidic channels (38, 39), one for supplying the industrial fluid and the other for drawing it in. It is made of polydimethylsiloxane and is 160 μm thick.

[0072] The bottom slide (34) is made of glass and is 0.5 mm thick. The slide (33) has two holes (48, 49) at the entrance to the microfluidic channels. These two holes (48, 49) serve as the inlet and outlet for the cartridge. The bottom slide (34) is coated with a 20 nm layer of gold around its periphery, corresponding to the windows (35, 36, 37).

[0073] The assembly of these four stages (31-34) forms the donor substrate (30).

[0074] Fluid Connector The donor substrate (30) is hooked onto the fluidic connector (50) shown in Figure 4, which features four channels (51-54) on its top. These four inlet / outlet ports (51-54) are fitted with lip seals or valves to ensure a secure fit. When the donor substrate (30) is placed on the fluidic connector (50), the holes (51, 53) align with the recesses (48, 49) in the cartridge (50), allowing the industrial fluids to flow in and out. These two channels enter from the side of the fluidic connector (50) and are connected by white pipes to pumps for circulating the industrial fluids. Both pumps are connected to reservoirs containing the industrial fluids. The particles to be transferred are then deposited onto the membrane using, for example, a pipette carried by a robotic arm. The other two holes (51, 54) of the fluidic connector (50) are connected to a vacuum pump, which presses the donor substrate (30) onto the fluidic connector (50) by suction.

[0075] The fluidic connector (50) is attached to a set of three micrometer screws along three axes. A vertical micrometer screw is used to move the donor substrate (30) along the Z axis to position it on the laser focal plane. Instead of using a scanner, two horizontal screws are used to move the donor substrate (30) along the X and Y axes to target different areas of the cartridge.

[0076] Both fluid pumps are computer-controlled via software. Several routines are recorded, including one for pre-wetting the cartridge. Preparing the industrial liquid for printing is a two-step process. First, a pre-wetting film is applied to the bottom of the donor substrate (30). To achieve this, a certain volume of industrial liquid is delivered through the first channel, but the pump in the suction channel is not switched on. In this way, the industrial liquid accumulates on the surface of the donor substrate (30), covering the entire surface. Then, while the supply of industrial liquid is stopped, the suction channel is activated. In this way, the thick film becomes thinner, but the industrial liquid remains dispersed across the entire surface of the donor substrate (30), leaving the bottom sufficiently pre-wet. Once this film is ready, a precise volume of ink containing the particles to be transferred is pipetted onto the donor substrate (30).

[0077] Particle transfer Due to the higher power and lower liquid volume between the underside of the particle and the bottom of the donor substrate, particles much larger than those from known LIFT solutions are ejected from the first cavitation bubble. The particles are spontaneously ejected from the liquid using impact propulsion. The cavitation bubble bursts, expelling the particles upward at high speed. The polyethylene particles reach a height of approximately 3 mm, i.e., a velocity of 20 m / s, after 150 μs. They arrive at the receiving substrate (40) in a nearly dry state and are surrounded by residual liquid droplets.

[0078] The ejection of 250 μm collagen spheres is slower than that of polyethylene beads. The particles reach a height of 3 mm in 800 μs compared to 100 μs for polyethylene. The transfer of cell spheroids behaves similarly to that described for collagen, as their shape and density are very similar. For example, the transfer of IPSC spheroids (stem cells) also reaches a height of 3 mm in 700–800 μs.

[0079] The base of the jet takes 100 μs to form a sphere of collagen or IPSCs. Furthermore, a single jet is visible. The characteristic crown of a second jet is absent. Between 200 μs and 600 μs, the jet is formed by the IPSC spheroid at the top of the jet, and the carrier liquid forms the remainder of the jet, still attached to the base. However, after 700 μs, the IPSC spheroid detaches from the jet, and the liquid recedes. The spheroid then continues its flight with little or no liquid, similar to polyethylene. The jet ruptures in a time similar to that of bioinkjet, i.e., at approximately 700 μs.

[0080] Unlike known LIFT solutions, all of the cavitation energy is used to propel the particle, with no energy lost to propelling the particle through a layer of liquid around the particle, which may represent a mass greater than the particle's own mass. Additionally, with the method described in this invention, the particle experiences little fluid friction when ejected from the carrier liquid due to the viscosity of the carrier liquid.

[0081] Transfer to a recipient substrate The transfer is performed from a donor substrate (30) containing particles to which the cartridge is to be transferred to a receiving substrate (40) consisting of a cell culture plate or glass slide.

[0082] The distance between the donor substrates (30) is important. It must be longer than the time required to separate the liquid filaments connecting the particles during their initial movement. Due to their porous and / or hydrophilic nature, the particles can entrain the liquid. As long as the particles are near the surface of the liquid film, they form a kind of adhesive (capillary bridge) that breaks at a certain distance, depending on the properties of the liquid, on the one hand, and the particles, on the other. Typically, this distance is a few millimeters, usually 3-5 mm. This distance must also be limited so that the kinetic energy is sufficient to ensure that most particles reach the receiving substrate. A distance of 3-10 mm is usually appropriate, but those skilled in the art can also determine the optimal distance by observing the formation and rupture of liquid filaments that form during the particle trajectory, for example, in the aforementioned TRI method. This distance can also be selected to minimize the impact velocity of the object on the receiving substrate.

[0083] One solution to buffer particle impacts on the surface of the receiving substrate (40) is to deposit an elastic coating, such as a 4 mg / ml collagen film, on the receiving surface. Collagen, like any other hydrogel, is more elastic than glass and deforms upon impact, making the impact less severe for the transferred particles and maintaining their original shape.

[0084] Particle Displacement The precision of the laser pulse is a critical parameter. If the center of the pulse is offset from the particle's center of gravity (or center of mass) in the horizontal XOY plane by more than 50 μm, the rate at which particles are transferred drops off sharply, reducing localization accuracy. For particles with an average diameter of 200 μm, transfer fails if the pulse is offset from the particle's center by more than 80 μm.

[0085] The laser beam can be positioned near the particle moving in the horizontal XOY plane by a scanner (15) or by positioning the donor substrate (30), for example by acting on a micrometer screw that positions the fluidic connector (50), or by a low-power laser pulse, for example at less than half the power required for transfer.

[0086] Advantages and Disadvantages of Sacrificial Layers The sacrificial layer ensures a constant and reproducible absorption over the entire surface of the donor substrate. A visible ablation point on the sacrificial layer can further be used to check that the laser is functioning properly.

[0087] However, the use of sacrificial layers is costly and time-consuming, and among other things means that the donor substrate must be replaced as soon as the ablation rate becomes too high. However, when producing multiple and / or many sizes of tissues, the number of donor substrates required can quickly become large, increasing the production time and cost of the tissues.

[0088] Therefore, the benefits of using a sacrificial layer-free approach are crucial for mass production applications, especially in clinical and industrial fields. Therefore, printing large objects such as spheroids using a system that works without a sacrificial layer is particularly interesting for shortening production time and reducing costs.

[0089] For this purpose, solutions such as ultrashort impulse lasers (femtosecond / picosecond range) or lasers operating in the mid-infrared wavelength range (2.6-3.2 μm) are ideal.

Claims

1. A method for transferring at least one object from a donor substrate (30) towards a target substrate (40), wherein said donor substrate (30) has a thickness e ｚ forming a surface onto which a film of carrier liquid is deposited in a vertical direction Z, the carrier liquid having a dimension (D ｘ , D ｙ , D ｚ ) and the transfer is ensured by local energy excitation of the liquid to form local cavitation bubbles in the object, D ｚ / e ｚ The method is characterized in that the ratio is greater than 0.

5.

2. - A method according to claim 1, characterized in that said object is a spheroid constituted by an aggregate of elementary biological cells.

3. A method according to claim 1, characterized in that the Dx, Dy, Dz values ​​are greater than 100 μm, preferably greater than 200 μm.

4. A method according to claim 1, characterized in that said energy excitation is achieved by focusing a laser (10) on the interface between said surface of said substrate and said carrier liquid film.

5. The method of claim 1, characterized in that the energy level applied in each pulse is based on the size of the object located in the targeting axis.

6. A method according to claim 1, characterized in that said receiving substrate (40) is elastically deformable along said Z-axis.

7. 2. A method according to claim 1, characterized in that it comprises a step of moving the object in the XOY plane by applying pulses having an energy that is at least half of the energy required to transfer the object to the receiving substrate.

8. 2. A method according to claim 1, characterized in that the objects to be transferred are spaced apart by a characteristic distance (d) of value d>0.5Dxy.

9. The method of claim 1, characterized in that the objects are of biological nature, including cell aggregates, spheroids, organoids, explants, explants (islets of Langerhans), cell-encapsulating polymer particles (organoids coated with biomaterial), cell-seeded microcarriers, biomaterial beads.

10. The method of claim 1, wherein the transfer is repeated to produce a material, tissue, or organ, and the volume fraction or volume density of biological matter transferred by propulsion of the printed material, tissue, or organ is greater than 30%.

11. 2. A method according to claim 1, characterized in that the transfer is carried out using multiple laser beams simultaneously if the object has an anisotropic shape, in order to ensure the transfer of the object along a homogeneous trajectory.

12. - Part of a pulsed energy deposition and transfer device, an energy source (10) directed at the material to be transferred, and a donor substrate (30) on which said material is manipulated and transferred by the energy impulses; a target receiving substrate (40) for collecting the transferred material; 1. The apparatus according to claim 1, wherein the donor substrate (30) comprises a slide (34) coated with the material to be transferred, the slide (34) being constituted by a carrier liquid film intended to contain a transferable object of size DxDyDz, the slide (34) of the donor substrate (30) being coated with a liquid carrier film the thickness (e) of which is less than 2×Dz, Dz being the dimension of the transferable object in a direction perpendicular to the film, and Dx, Dy being the orientation of the transferable object in the plane of the film.

13. - Device according to claim 12, characterized in that said energy source (10) is a laser.

14. An apparatus according to claim 12, characterized in that the slide (34) of the donor substrate (30) is transparent or has low absorption properties at the wavelength of the laser beam (10).

15. An apparatus according to claim 12, characterized in that it comprises an optomechanical system (15) for directing the laser spot relative to the center of gravity or mass of the object.

16. An apparatus according to claim 12, characterized in that it incorporates an intelligent object detection system, means for automating the steps of placing and handling / transferring said material on said donor substrate.

17. - Device according to claim 12, characterized in that said donor substrate (30) is covered with a sacrificial layer with high absorption / conductivity properties for laser or field energy deposition.

18. - The device according to claim 12, characterized in that the receiving substrate (40) is not covered by a layer with high laser absorption properties to initiate the propulsion process, but absorption is achieved directly by a liquid layer between the object and the donor substrate.

19. 13. The apparatus of claim 12, comprising an optical viewing / detection system for locating and targeting the objects to be transferred that is compatible with a random distribution of objects on the donor substrate.

20. An apparatus according to claim 12, characterized in that the donor substrate (30) is arranged below the receiving substrate (40).

21. - Equipment according to claim 12, characterized in that it comprises at least one other printing means, including extrusion, inkjet, LIFT, for producing complex materials or structures with different components.

22. 13. The apparatus according to claim 12, characterized in that it comprises means for simultaneously delivering a plurality of laser beams to the object when the object has an anisotropic shape, in order to ensure the transfer of the object along a homogeneous trajectory.

23. - Instrument according to claim 12, characterized in that the donor substrate consists of microwells in which the objects to be transferred are arranged.

Images