Object manipulation and transfer by propulsion

Abstract

The present invention relates to a method and equipment for transferring at least one object from a donor substrate (30) towards a target substrate (40), the donor substrate (30) forming a surface on which a film of carrier liquid having a thickness ez is deposited in a vertical direction Z, the carrier liquid containing the objects that are to be transferred and have dimensions (Dx, Dy, Dz), the transfer being ensured by local energy excitation of the liquid to form a local cavitation bubble at an object, characterized in that the ratio Dz / ez is greater than 1, preferably greater than 0.5.

Description

MANIPULATION AND TRANSFER OF OBJECTS BY PROPULSION Field of invention

[0001] The present invention relates to the field of additive manufacturing of a material by repeated transfers of particles between a donor substrate and a receiving substrate, a film of vector liquid containing the particles to be transferred being deposited on the donor substrate, and in particular the field of bioprinting. The particular field of bioprinting concerns the use of digital manufacturing processes making it possible to organize and assemble in 2D and 3D the constituents of biological tissues with the aim of producing grafts for regenerative medicine or physiological models for biomedical and pharmaceutical research.

[0002] The general principle of the invention consists of transferring biological objects (cells for example), organic or mineral, from a vector liquid deposited on a substrate in the form of a film and providing an energy pulse to form a cavitation bubble, directly by vaporization of the liquid part in the focal field of the energy source - generally a laser - or by means of a thin metallic coating forming a sacrificial layer on the donor substrate. This cavitation bubble carries the particle(s) located in the firing axis towards a receiving substrate, on which the transferred particles accumulate, as the firings are repeated. State of the art

[0003] Known in the state of the art is patent application US2020 / 009877 describing equipment for depositing particles on a target from a transparent slide carrying a film formed by a fluid containing suspended particles, by local excitation of the film by a laser beam oriented by a controlled optical deflection means, the equipment comprising means for observing said local activation zone by an optical imaging system comprising a sensor and a lighting source whose optical axes are substantially common in the part between an optical separator and the film. The optical beam of the imaging system and the optical beam of the laser are coaxial in the part between said controlled optical deflection means and the film.

[0004] The equipment comprises a first optical focusing unit arranged between said controlled optical deflection means and the film and a second optical image conjugation unit placed between said sensor and said separator. The sensor is placed in the focal plane of this second optical unit.

[0005] Patent CN113021874 discloses a single-cell printing method based on annular laser spot-induced transfer is characterized in that it comprises the following steps: step 1, after pulsed laser beams which are uniformly distributed are incident on a member of the annular light spot forming system, the emerging light is focused to form an annular light spot on a sacrificial layer through a transparent constraint layer; step 2, the sacrificial layer generates ablation under the action of laser light and heat and forms a plasma annular cavitation bubble; step 3, rapidly expanding the annular cavitation bubbles and pushing the cell solution away from the rings, and at the same time, pushing down the target transfer cells in the center of the rings under the expansion of the cavitation bubbles;step 4, collapse of annular cavitation bubbles and completely expel target transfer cells by collapsing shock waves generated in the vertical direction relative to the central position of the ring; step 5, under the action of gravity and self-impulse, print target transfer cells and transported solution onto a receiving plate.;

[0006] Patent application US2022152925 describes a printing method using equipment comprising an exciter delivering orientable energy to produce a point interaction with at least one ink which may contain non-uniformities and deposited on a printing medium having a transparent interaction zone, in order to cause a transfer of a targeted portion of said ink to a receiver, the method comprising the generation of a wetting film at least partially covering said transparent interaction zone, followed by the deposition of said ink on the surface of said wetting film and the transfer steps.

[0007] Patent application US2020102529 relates to equipment and a method for additive manufacturing, comprising an orientable energy excitation means for generating an intermittent interaction with a fluid covering a blade in order to trigger a jet oriented towards a target, the fluid consisting of a liquid vector containing inhomogeneities, in which: the fluid forms a liquid film with a thickness of less than 500 µm on a blade having at least one zone allowing interaction with the laser, into which at least one inlet opens, the interaction zone opening onto at least one outlet, the equipment also comprising means for circulating the fluid between the inlet and the outlet.

[0008] Patent application US2017225390 relates to a method for additively manufacturing a three-dimensional object. The method comprises sequentially forming a plurality of layers, each of which is structured according to the shape of a cross-section of the object. In some embodiments, forming at least one of the layers comprises performing a raster scan to dispense at least one first build material composition, and a vector scan to dispense at least one second build material composition. The vector scan is optionally performed along a selected path 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 build material, and (iii) an inter-layer connection structure. Disadvantages of the prior art

[0009] The prior art solutions are suitable for the transfer of small particles, with dimensions significantly smaller than the thickness of the carrier liquid film when they are based on LIFT, or suitable for the handling / transfer of large particles by contact when it comes to non-LIFT technologies such as Kenzan or suction. However, they have several drawbacks.

[0010] First, the focus of each shot must be adjusted to take into account the plane in which the particle is positioned within the firing range, to bring the appropriate energy into the plane directly below the particle, so that the energy pulse ensures cavitation of the liquid below the particle. This step of adjusting the focus before each shot greatly slows down the rate of fire.

[0011] Secondly, the transfer of the particle is accompanied by a transfer of a significant part of the carrier liquid, which leads to a material with low volume density.

[0012] Third, the particles are relatively mobile in the carrier liquid film, and the accuracy of the shot is disturbed by the movements between the time of analysis of the image of the shooting field and the triggering of the shot. Solution provided by the invention

[0013] In order to overcome these drawbacks, the present invention relates, in its most general sense, to a method of manufacturing a material having the characteristics set out in claim 1.

[0014] A method of manufacturing by transfer of at least one object, in particular a particle from a donor substrate towards a target substrate, according to the invention provides in particular a donor substrate forming a surface on which is deposited a film of vector liquid of a thickness e z along a vertical direction Z. A vector liquid contains the objects to be transferred of dimension (D x , D y , D z ). The transfer is ensured by local energetic excitation of said liquid to form a cavitation bubble located at the level of an object, characterized in that the ratio D z / e z is greater than 1, and preferably greater than 0.5.

[0015] According to a variant, said objects are spheroids constituted by an aggregation of elementary biological cells.

[0016] Advantageously, said values ​​D x , D y , D zare greater than 100 µm and preferably greater than 200 µm.

[0017] According to a second variant, said energy excitation is carried out by focusing a laser at the interface between the surface of said substrate and said film of vector liquid.

[0018] According to a second variant, said energy excitation is carried out by applying an electric field.

[0019] According to a third variant, the energy deposition is carried out by focusing an acoustic wave at the interface between the surface of said substrate and said film of vector liquid.

[0020] Advantageously, the energy level applied to each shot is a function of the size of the object located in the firing axis.

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

[0022] According to a particular embodiment, the method comprises steps of moving an object in the XOY plane by applying a shot with an energy at least 2 times lower than the energy required for a transfer of the object to the receiving substrate.

[0023] Advantageously, the objects to be transferred are spaced apart by a characteristic distance (d) of value d > 0.5D xy .

[0024] According to one variant, the objects are of a biological nature including cell aggregates, spheroids, organoids, explants, explants (islets of Langherans), polymer particles encapsulating cells (organoid covered with a layer of biomaterial), micro-carriers seeded with cells, biomaterial beads.

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

[0026] Alternatively, the transfer is repeated to fabricate a material, tissue, or organ and the volume fraction or volume density of the biological objects transferred by propulsion into said printed material, tissue, or organ is greater than 30%.

[0027] In another variant, the process is combined with other printing technologies such as extrusion, inkjet, LIFT in order to manufacture complex materials or fabrics comprising different components.

[0028] According to another variant, the method is carried out by the use of several simultaneous laser beams when the object has a non-isotropic shape in order to guarantee its transfer along a homogeneous trajectory.

[0029] The invention also relates to equipment for handling and transferring by pulsed energy deposition comprising: an energy source directed towards the material to be transferred, a donor substrate from which said material is manipulated and transferred by energy pulses and, a target receiving substrate which collects the transferred material

[0030] Said donor substrate comprises a slide covered by the material to be transferred, consisting of a liquid vector film intended to contain transferable objects of size DxDyDz, characterized in that said transferable objects have an orientation Dx,Dy in the plane of the film and Dz perpendicular to the film, the ratio between the dimension (Dz) of the object and the thickness (e) of the liquid film is greater than 1 and preferably greater than 0.5.

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

[0032] According to one variant, the blade of the donor substrate is transparent or weakly absorbent at the wavelength of said laser beam.

[0033] According to a particular embodiment, it comprises an opto-mechanical system making it possible to direct the laser spot relative to the centroid or center of mass of the object.

[0034] If the object has a particular non-isotropic shape, the system can allow the simultaneous use of several laser beams directed at different points of the object in order to make it take off in a homogeneous manner.

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

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

[0037] According to one variant, it includes a system for controlling and regulating the energy value deposited as a function of the size of the particle to be transferred.

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

[0039] Alternatively, the donor substrate is covered by a sacrificial layer having strong absorption / conduction properties of energy deposition by laser or electric field.

[0040] According to one variant, the receiving substrate is not covered by a layer having high absorption properties, this being produced directly in the liquid.

[0041] Alternatively, it includes an optical viewing / detection system for locating and targeting objects to be transferred, compatible with random distribution of objects on the donor substrate.

[0042] Preferably, said donor substrate is disposed below said recipient substrate.

[0043] According to another variant, it comprises at least one other printing means including extrusion, inkjet, LIFT in order to manufacture complex materials or fabrics comprising different components.

[0044] According to another variant, it comprises a means for delivering several laser beams simultaneously to the object when the latter has a non-isotropic shape in order to guarantee its transfer along a homogeneous trajectory.

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

[0046] According to a variant, the receiving substrate is not covered by a layer having strong laser absorption properties to initiate the propulsion process, the absorption then being carried out directly in the liquid layer located between the object and the donor substrate.

[0047] According to another variant, the donor substrate consists of micro-wells in which the objects to be transferred are arranged.

[0048] Detailed description of a non-limiting example of embodiment

[0049] The present invention will be better understood on reading the following description, concerning a non-limiting example of embodiment illustrated by the appended drawings where:

[0050] It represents a schematic view of the transfer system

[0051] This represents an example of the chronology of the different components of the TRI for taking an image of the jet at a time T

[0052] Shows an exploded view of an example donor substrate

[0053] It represents a perspective view of an example of a fluidic connector.

[0054] Overview of the transfer system

[0055] The transfer system comprises several parts: an optical part comprising a pulsed laser (10) to produce the cavitation energy of the vector liquid and a camera (20) for observing the donor substrate (30) and the receiver substrate (40) an automated part, with a robot arm (50) used for automating the handling of the receiver (40) a microfluidic device (31) ensuring the supply of the donor substrate (30) with the vector liquid and the particles to be transferred

[0056] and optionally, the system may comprise an extruder for adding to the receiving substrate (40) a link, for example a hydrogel-type biomaterial between the layers of transferred particles.

[0057] More generally, the equipment can combine several 3D printing, bioprinting and photopolymerization technologies.

[0058] The optical part of the device consists of two parts, one part comprising the camera (20) used to aim at objects, and one part comprising the laser (10) used to "shoot", i.e. deliver energy pulses into a plane of the donor substrate (30) where there is a particle to be transferred.

[0059] The laser (10) is for example an Nd-YAG laser which emits pulses of 1 to 10 ns at 1064 nm with an energy of 15 to 60 microjoules, significantly higher than the energy usually used for LIFT processes.

[0060] In another example, it consists of a pulsed Ytterbium fiber laser emitting at 1030nm, with shorter pulses, from 350 femtoseconds to 10 picoseconds, with several tens of microjoules of energy per pulse.

[0061] The lens (16) is typically an F-Theta lens with a focal length of 100mm suitable for laser scanning. The typical spot size at the focal plane is in the order of 30 to 35µm in diameter.

[0062] The laser beam (14) passes through a shaping optic (13) and is then directed via a set of mirrors (11, 12) towards a scanner (15) which will then send the beam vertically towards the donor substrate (30), via the F-Theta lens (16).

[0063] The scanner (15) is composed of two automated mirrors which will redirect the beam horizontally at a certain angle towards the objective (16). The objective (16) will then straighten the beam (14) so ​​that it arrives perpendicular to the donor substrate (30) and focus it. The mirrors of the scanner (15) make it possible to control the movement of the laser beam (14) along the horizontal axes on the donor substrate (30). The laser beam (14) is therefore focused on the donor substrate (30) and can be directed along the X and Y axes defining the horizontal plane.

[0064] For a donor substrate (30), the beam (14) is focused onto the sacrificial layer coating the surface of the substrate, for example a 20 nanometer gold layer deposited on a transparent optical window.

[0065] The second optical part comprising the camera (20) is the part used for aiming. Indeed, the particles are, in the case of the present invention, relatively large objects, for example spheroids formed by an aggregate of cells, with a diameter of more than 100µm, typically 200µm to 300µm. These objects are distributed randomly on the donor substrate (30); it is therefore necessary to know the position and size of the objects to aim them with a laser shot. For this, a light source in the visible range, typically an LED (21) is placed above the cartridge, and the light beam (22) passes through the scanner (15) following the reverse path of the laser beam. It then arrives at a semi-reflecting mirror (12) which lets through the visible light directed towards the camera (20), but reflects the infrared towards the laser (10). The beam then passes through a lens (23), an iris (24) and an objective (25) before arriving at the camera (20).The whole thing is aligned so that the focus of the laser beam (10) is in the center of the image recovered by the camera (20).

[0066] Nature of the objects transferred

[0067] The installation implemented by the invention is similar to an installation intended for LIFT bioprinting, with the difference that the energy of the pulses is at least 2 or even 5 times higher, and that the placement of the objects to be transferred differs fundamentally from the solutions used for LIFT bioprinting, where the particles to be transferred form a bioink where the particles are of sizes much smaller than the thickness of the film deposited on the donor substrate (30).

[0068] In the context of the present invention, the objects have a size of more than 100 µm, typically 200 to 400 µm or more, and are positioned in a liquid, typically water with added salts to adjust the density, or a 2% BSA (Bovine Serum Albumin) solution, forming a film with a thickness less than the size of the particles to be transferred, such that the donor substrate (30) no longer receives a bio-ink, but particles rest partially in an aqueous film which does not completely cover them.

[0069] The invention relates, but is not limited to, the transfer of spheroids formed by the aggregation of cells cultured in the laboratory and having the appearance of a small pearl made of cells and extracellular matrix.

[0070] In order to produce the spheroids, stem, progenitor or differentiated cells are cultured by conventional methods and seeded into microwells to produce aggregates which are then manipulated according to the method of the present invention.

[0071] Shooting Sequences

[0072] The LIFT phenomenon is a process that takes place over very short times, of the order of hundreds of microseconds, and can be observed by a TRI device.

[0073] TRI (Time Resolved Imaging) involves taking images of a phenomenon at a specific time by synchronizing a camera with the laser (10) and an LED in order to take a photo at a specific moment of the jet. The three components are synchronized via an electronic card in order to precisely control the activation of the different components over time. In order to obtain an image of the jet at a specific moment, the camera is switched on for several hundred microseconds, while the LED emits only a 2 µs flash of light at the desired moment. The LED is positioned in front of the camera, so as to illuminate the jet.

[0074] This shows the TRI timeline with a time scale in microseconds. First (100) the analyzer camera is turned on, which takes 60 µs to turn on. The laser (10) takes 4 µs to turn on (105), and another 5 µs to fire the laser. If the camera is on during the laser fire, the beam is visible on the image and makes it less readable, so it must be fired before the analyzer camera is turned on.

[0075] We therefore start to turn on (101) the laser (10) 50 µs after the analyzer camera, so that the shot (102) takes place at 59 µs, i.e. 1 µs before the analyzer camera is turned on (103). The camera then remains on for 500 µs. Finally, the LED will emit a sequence of light flashes (110, 111) of 2 µs from the desired time (106), T µs after the laser shot, i.e. 59+T µs after turning on the camera, to create a stroboscopic image with time intervals of 2 µs between two flashes.

[0076] The images thus obtained show a particle behavior very different from that observed in the state of the art with LIFT bioprinting. The large particle is propelled without carrying a part of the vector liquid by the formation of a cavity bubble in the liquid film at the interface between the particle and the bottom of the substrate, and not on an enveloping volume of the particle. The particle emerging from the liquid film, it pushes aside the liquid surrounding it, to emerge directly from the vector liquid and reach the receiving substrate with a very low liquid carryover.

[0077] Donor substrate

[0078] The depicts an exploded view of a donor substrate (30) receiving the film of technical liquid and the ink containing the particles to be transferred, spread on a cartridge which serves as a support for the laser shots. It is composed of an assembly of four stages (31, 32, 33, 34) of glass and PDMS. The purpose of the technical liquid is to clean the central area of ​​the donor substrate and to pre-wet the surface in order to facilitate the spreading of the ink to be transferred containing the particles.

[0079] The upper blade (31) of the cartridge is made of Polydimethylsiloxane and has a window (35) surrounding the cylindrical volume receiving the technical liquid and the particles. It constitutes a hydrophobic layer forming a barrier to prevent overflow of technical liquid and ink containing the particles. It has a thickness of 10 µm.

[0080] A first intermediate blade (32) also has a window (36) and is used to fix the upper blade (31). It is made of glass, 0.5 mm thick. It also serves to prevent overflow.

[0081] A second intermediate blade (33) stage has a window (37) and two microfluidic channels (38, 39), one for bringing the technical liquid and the other for sucking it up. It is made of Polydimethylsiloxane and has a thickness of 160 µm.

[0082] The lower blade (34) is made of glass and is 0.5 mm thick. It has two holes (48, 49) at the arrival of the microfluidic channels of the blade (33). These two holes (48, 49) serve as inlet and outlet in the cartridge. The lower blade (34) is coated with a 20 nm layer of gold in the perimeter corresponding to the windows (35, 36, 37).

[0083] The assembly of these four stages (31 to 34) forms the donor substrate (30).

[0084] Fluidic connector

[0085] The donor substrate (30) is attached to a fluidic connector (50) shown in, having four channels (51 to 54) on its upper part. On these four inlets / outlets (51 to 54) are placed lip seals or valves to ensure sealing. When the donor substrate (30) is placed on the fluidic connector (50), the holes (51, 53) will coincide with the recesses (48, 49) of the cartridge (50) and allow the entry and exit of the technical liquid. The entry of these two channels is on the side of the fluidic connector (50), and are connected, by white pipes to pumps allowing the circulation of the technical liquid. The two pumps are connected to a reservoir containing the technical liquid. The particles to be transferred are then deposited on the film with a pipette for example carried by a robotic arm.The other two holes (51, 54) of the fluidic connector (50) are connected to a vacuum pump and are used to press the donor substrate (30) onto the fluidic connector (50) by suction.

[0086] The fluidic connector (50) is mounted on a set of three micrometric screws along the three axes. The vertical micrometric screw is used to move the donor substrate (30) along the Z axis in order to position itself on the focal plane of the laser. The two horizontal screws are used to move the donor substrate (30) along the X and Y axes, in order to target different locations on the cartridge, alternatively to using the scanner.

[0087] The two fluidic pumps are controlled by computer via software. Several routines are recorded, including the one allowing the pre-wetting of the cartridge. Indeed, the preparation of the technical liquid for printing is done in two stages. Firstly, a pre-wetting film is made on the bottom of the donor substrate (30). For this, a volume of technical liquid is sent through the first channel, while the suction channel pump is not engaged. Thus, the technical liquid will accumulate on the surface of the donor substrate (30), and cover the entire surface, then the suction channel comes into play while the supply of technical liquid is stopped. Thus, the thick film will become thinner but the technical liquid will remain spread over the entire surface of the donor substrate (30), the bottom is well pre-wetted.Once this film is ready, a precise volume of ink containing particles to be transferred is deposited using a pipette, which will spread over the donor substrate (30).

[0088] Transfer of a particle

[0089] The particle is ejected from the first cavitation bubble, which is much larger than in known LIFT solutions, due to the higher power and the small volume of liquid between the lower face of the particle and the bottom of the donor substrate. The particle is ejected alone, out of the liquid with a ballistic propulsion phenomenon. The cavitation bubble explodes and expels the particle upwards at a high speed. After 150 µs a Polyethylene particle is about 3 mm high, i.e. a speed of 20 m / s. It reaches the receiving substrate (40) almost dry and surrounded by residual splashes of liquid.

[0090] The ejection of 250 µm collagen spheres is slower than for polyethylene beads. The particle reaches 3 mm in height in 800 µs, compared to 100 µs for polyethylene. The transfer of cell spheroids has a similar behavior to that described for collagen because their shape and density are very similar. For example, the transfer of an IPSC (stem cell) spheroid also reaches 3 mm in height in 700 to 800 µs.

[0091] The base of the jet takes 100 µs to form with a collagen sphere or made of IPSC. In addition, we can see that there is a single jet. Indeed, the characteristic crown of the second jet is not present. 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 rest of the jet, still attached to the base. However, after 700 µs, the IPSC spheroid detaches from the jet, and the liquid falls back. The spheroid then continues its flight without or with little liquid, as for polyethylene. The jet breaks at a similar time as for the bio-ink jet, i.e. around 700 µs.

[0092] Unlike known LIFT solutions, all the cavitation energy propels the particle, without loss in making it pass through and leave the layer of liquid around it, which can represent a mass greater than that of the particle itself. Furthermore, with the method according to the invention, the particle undergoes little fluid friction during its ejection from the carrier liquid due to the latter's viscosity.

[0093] Transfer to the receiving substrate

[0094] The transfer is made from the donor substrate (30), the cartridge containing the particles to be transferred, to a receiving substrate (40) consisting of a cell culture plate or a glass slide.

[0095] The distance between the donor substrate (30) and the donor substrate (30) is critical. It must be greater than the time required for the separation of the liquid filament connecting the particle during the initial journey. Indeed, the particles can, due to their porosity and / or their hydrophilic nature, carry liquid which forms a sort of bond (capillary bridge) as long as the particle remains close to the surface of the liquid film, and which breaks from a certain distance depending on the characteristics of the liquid on the one hand and the particle on the other hand. Typically, this distance is a few millimeters, generally between 3 and 5 mm. The distance must also be limited so that the kinetic energy is sufficient to ensure that most of the particles reach the receiving substrate.A distance of between 3 and 10 mm is usually appropriate, but the person skilled in the art will be able to determine the optimal distance by observing, for example with the above-mentioned TRI method, the formation and breakage of the liquid filament forming during the trajectory of the particles. This distance may also be chosen to minimize the impact speed of the object on the receiver.

[0096] To cushion the impact of particles on the surface of the receiving substrate (40), one solution is to deposit an elastic coating on its receiving surface, for example a 4 mg / ml collagen film. Collagen, like any other hydrogel, is more elastic than glass and will deform upon impact of a particle, which makes the shock less violent for the transferred particle which retains its original shape.

[0097] Displacement of a particle

[0098] The accuracy of the laser shot is an important parameter. When the center of the shot is offset in the horizontal XOY plane by more than 50 µm from the centroid (or center of mass) of the particle, the percentage of transferred particles decreases rapidly, and the accuracy of the localization decreases. Beyond 80 µm of offset of the shot from the center of the particle, for particles with an average diameter of 200 µm, the transfer fails.

[0099] The positioning of the laser beam can be ensured by the scanner (15) or by the positioning of the donor substrate (30) for example by an action on the micrometric screws ensuring the positioning of the fluidic connector (50) or even by low power laser shots, for example of a power less than half of the power required for the transfer, close to a particle to be moved in the horizontal XOY plane.

[0100] Advantages and disadvantages of a sacrificial layer

[0101] The sacrificial layer ensures constant and reproducible absorption across the entire surface of the donor substrate. It also allows the proper functioning of the laser to be verified thanks to the ablated spots visible on the sacrificial layer.

[0102] However, using a sacrificial layer is costly and time-consuming, but above all it involves having to change the donor substrate as soon as the ablation rate becomes too high. However, for the production of large numbers and / or large sizes of tissues, the number of donor substrates required can very quickly become very large, thereby increasing the time and cost of producing said tissues.

[0103] The interest in using a sacrificial layer-free approach is therefore very important for large-scale production applications, particularly for clinical and industrial fields. Printing large objects such as spheroids using a system working without sacrificial layers is therefore particularly interesting for reducing manufacturing time and cost.

[0104] Ultra-short pulse laser solutions (femtosecond / picosecond regime) or lasers working in the Mid-IR wavelength range (2.6 to 3.2 µm) are perfectly usable in this context.

Claims

- Method for transferring at least one object from a donor substrate (30) towards a target substrate (40), said donor substrate (30) forming a surface on which is deposited a film of vector liquid of a thickness e z in a vertical direction Z, said vector liquid containing the objects to be transferred of dimension (D x , D y , D z ), said transfer being ensured by local energetic excitation of said liquid to form a cavitation bubble located at the level of an object, characterized in that the ratio D z / e z is greater than 0.

5. - Method according to claim 1 characterized in that said objects are spheroids constituted by the aggregation of elementary biological cells. - Method according to claim 1 characterized in that said values ​​Dx, Dy, Dz are greater than 100 µm and preferably greater than 200 µm. - Method according to claim 1 characterized in that said energy excitation is carried out by focusing a laser (10) at the interface between the surface of said substrate and said film of vector liquid. - Method according to claim 1, characterized in that the energy level applied to each shot is a function of the size of the object located in the firing axis. - Method according to claim 1, characterized in that said receiving substrate (40) is elastically deformable along the Z axis. - Method according to claim 1, characterized in that it comprises steps of moving an object in the XOY plane by applying a shot with an energy at least 2 times lower than the energy required for a transfer of the object to the receiving substrate. - 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. - Method according to claim 1, characterized in that the objects are of a biological nature comprising cellular aggregates, spheroids, organoids, explants, explants (islets of Langherans), polymer particles encapsulating the cells (organoid covered with a layer of biomaterial), micro-carriers seeded with cells, biomaterial beads. - Method according to claim 11 characterized in that the transfer is repeated to manufacture a material, tissue or organ and that the volume fraction or the volume density of the biological objects transferred by propulsion into said printed material, tissue or organ is greater than 30%. - Method according to claim 1, characterized in that the transfer is carried out by the use of several simultaneous laser beams when the object has a non-isotropic shape in order to guarantee its transfer along a homogeneous trajectory. - Equipment for handling and transferring by pulsed energy deposition comprising: an energy source (10) directed towards the material to be transferred, a donor substrate (30) from which said material is handled and transferred by energy pulses and, a target receiving substrate (40) which collects the transferred material. Said donor substrate (30) comprises a blade (34) covered by the material to be transferred, consisting of a liquid vector film intended to contain transferable objects of size DxDyDz, characterized in that said blade (34) of said donor substrate (30) is covered with a liquid vector film whose thickness is (e) less than 2xDz, where Dz is the dimension of the transferable objects in the direction perpendicular to said film, Dx,Dy being the orientation of said transferable objects in the plane of said film. - Equipment according to claim 12 characterized in that the energy source (10) consists of a laser. - Equipment according to claim 12 characterized in that the blade (34) of the donor substrate (30) is transparent or weakly absorbent at the wavelength of said laser beam (10). - Equipment according to claim 12 characterized in that it comprises an opto-mechanical system (15) making it possible to direct the laser spot relative to the centroid or center of mass of the object. - Equipment according to claim 12, characterized in that it integrates an intelligent object detection system, means for automating the steps of placing the material on the donor substrate and the handling / transfer steps. - Equipment according to claim 12, characterized in that the donor substrate (30) is covered by a sacrificial layer having properties of strong absorption / conduction of the energy deposition by laser or by electric field. - Equipment according to claim 12, characterized in that the receiving substrate (40) is not covered by a layer having strong laser absorption properties to initiate the propulsion process, the absorption then being carried out directly in the liquid layer located between the object and the donor substrate. - Equipment according to claim 12, characterized in that it comprises an optical visualization / detection system for locating and targeting the objects to be transferred, compatible with a random distribution of the objects on the donor substrate. - Equipment according to claim 12, characterized in that said donor substrate (30) is arranged below said receiving substrate (40). - Equipment according to claim 12, characterized in that it comprises at least one other printing means comprising extrusion, inkjet, LIFT in order to manufacture complex materials or fabrics comprising different components. - Equipment according to claim 12, characterized in that it comprises means for delivering several laser beams simultaneously to the object when the latter has a non-isotropic shape in order to guarantee its transfer along a homogeneous trajectory. - Equipment according to claim 12, characterized in that the donor substrate consists of micro-wells in which the objects to be transferred are arranged.

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