MANIPULATION AND TRANSFER OF OBJECTS BY PROPULSION

Abstract

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

Description

Title of the invention: MANIPULATION AND TRANSFER OF OBJECTS BY PROPULSION Scope of the 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 liquid carrier film containing the particles to be transferred being deposited on the donor substrate, and in particular to the field of bioprinting. The specific field of bioprinting concerns the use of digital manufacturing processes enabling the organization and assembly in 2D and 3D of the constituents of biological tissues for the purpose 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 (cells, for example), organic, or mineral objects from a carrier liquid deposited on a substrate in the form of a film and applying an energy pulse to form a cavitation bubble, either directly by vaporizing the portion of the liquid in the focal field of the energy source—generally a laser—or via a thin metallic coating forming a sacrificial layer on the donor substrate. This cavitation bubble carries the particle(s) located along the firing axis toward a receiving substrate, on which the transferred particles accumulate as the firings are repeated. State of the art

[0003] One of the solutions adapted to bioprinting is known by the acronym LIFT (Laser Induced Forward Transfer). The transfer of material is caused by

[0004] Focusing a laser onto a surface. This transfer can occur in two directions: either by ablating a surface from above and then allowing it to fall by capillary action, or by pulling the surface downwards and using the released energy to transfer material to a receiver positioned above the surface. LIFT consists of projecting a bio-ink spread on a donor substrate onto a receiver substrate. The donor substrate is composed of a glass slide coated with a thin layer of gold onto which the bio-ink is spread. The receiver substrate can be, for example, a 6-well cell culture plate, a Petri dish, or a wellless plate for printing large areas.

[0005] A laser delivers short pulses (from a few hundred femtoseconds up to several tens of nanoseconds). The beam is magnified and directed by a set of mirrors towards a scanner. This scanner directs the beam towards an F-Theta lens, which allows it to be focused on the donor blade. The coordinates of the laser ablation points on the donor blade are controlled by the angle applied by the scanner.

[0006] When the focused laser pulse reaches the gold layer, the electrons within it absorb the laser photons. A plasma is created, and a vapor bubble forms on the glass surface, called a cavitation bubble. This bubble, located between the glass and the bio-ink, projects an ink droplet containing one or more cells onto the receiving substrate. The distance between the donor gold layer and the receiver surface is defined as the Donor-Receiver Distance (DRD). This distance is one of the main parameters influencing print quality.

[0007] For example, we know of patent EP3941712 describing a printing process implementing equipment comprising a means of orientable energy excitation to produce a point interaction with at least one ink which may contain inhomogeneities deposited on a printing support having a transparent interaction surface, in order to cause the transfer of a targeted part of said ink to a receiver, characterized in that it comprises a step of generating a wetting film covering at least partially said transparent interaction surface, followed by a step of depositing said ink on the surface of said wetting film and transfer steps.

[0008] We also know of patent application EP22181067.4. Indeed, this is a patent protecting a process for manufacturing three-dimensional cartilage tissue by laser-assisted bioprinting, comprising the following steps:

[0009] a) provide a donor bio-ink comprising cartilage-forming cell aggregates,

[0010] b) transfer a structured layer of cell aggregates onto a receiving substrate by pulsed laser energy focused on the bio-ink.

[0011] c) repeat steps a) and b) to obtain additional patterned aggregate layers

[0012] on the previously deposited layers, thus obtaining said three-dimensional layer

[0013] with a minimum compactness factor of 30%.

[0014] Among these technologies is patent EP2873751A1, which describes a process for the direct deposition of a solid material onto a target substrate by passing a burst of ultrafast laser pulses from a focused laser beam below the diffraction limit through a carrier substrate that is transparent to the laser beam. The carrier substrate is coated with the solid material to be transferred on its underside. Electrons on the back of this transparent, material-coated support are excited by the first sub-pulses of the laser beam, which lift the material from the carrier substrate, and subsequent sub-pulses of the laser beam send the material into space. with hypersonic speed via a shock wave that propels the material forward with momentum through a narrow gap between the carrier substrate and the target substrate. The process described in this patent is based on melting the material to be transferred by laser absorption, and the shock waves required for the transfer are not compatible with use in the biological field.

[0015] Kenzan bioprinting technology [Yurie et al. (2017) PLoS ONE 12(2), e0171448] is a method for assembling 3D cell aggregates without the aid of a scaffold made of collagen or hydrogel materials. Spheroids are arranged in a network of fine needles where they can fuse with adjacent spheroids to form a connected structure. By using appropriate needle alignment, the spheroids can be positioned in specific 3D arrangements, particularly for hollow constructs. This technology is limited in terms of production throughput because the spheroids must be handled individually using forceps or a syringe. Furthermore, the spheroids are generally large enough to accommodate needle insertion.Kenzan technology cannot produce dense tissues (with a high compactness factor) composed of spheroids smaller than 500 sq m in diameter. Furthermore, this technology is limited in its applications because it relies on the direct manipulation of objects. It is poorly suited to manipulating deformable objects, cells sensitive to environmental variations, or GMP conditions.

[0016] Suction-assisted bioprinting is another technique particularly suited to the manipulation and shaping of spheroids as described in the publication:

[0017] “Aspiration-assisted bioprinting for precise positioning of biologics”

[0018] Ayan B, Heo DN, Zhang Z, Dey M, Povilianskas A, Drapaca C and Ozbolat IT

[0019] 2020 Sci. Adv. 6 1-17

[0020] This technology enables the 3D harvesting and positioning of aggregates by harnessing the power of suction forces. It operates a pipette, which is used to "harvest" spheroids from a gel or bio-ink and "3D bioprint" them into or onto a gel substrate (receptor). This technology claims high-precision positioning and high viability, but the aspiration needle is in contact with the spheroid, with the potential for contamination and mechanical deformation. Furthermore, to ensure good aspiration, this solution is limited to handling large spheroids and is therefore limited in terms of production throughput. As with the Kenzan technology, the aspiration technology requires handling the spheroids individually at a slow speed and does not allow the production of dense tissues (high compactness factor) composed of Spheroids less than 500µm in diameter. This technology is limited in its applications because it relies on manipulating objects by contact. It is poorly suited to manipulating deformable objects, cells fragile to environmental variations, or GMP conditions.

[0021] Disadvantages of the prior art

[0022] Prior art solutions are suitable for the transfer of small particles, with dimensions significantly smaller than the thickness of the carrier liquid film when 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 disadvantages.

[0023] First, the focus of each shot must be adjusted to take into account the plane in which the particle within the firing field is positioned, in order to deliver the appropriate energy to the plane directly beneath the particle, so that the energy pulse causes cavitation of the liquid under the particle. This focus adjustment step before each shot significantly slows the rate of fire.

[0024] Secondly, the transfer of the particle is accompanied by a transfer of a non-negligible part of the carrier liquid, which leads to a material of low volume density.

[0025] Thirdly, the particles are relatively mobile in the liquid vector film, and the accuracy of the shot is disturbed by the displacements between the time of the analysis of the image of the firing field, and the triggering of the shot.

[0026] Solution provided by the invention

[0027] In order to remedy these drawbacks, the present invention relates in its most general sense to a method of manufacturing a material by transferring at least one object, in particular a particle from a donor substrate towards a target substrate, said donor substrate forming a surface on which is deposited a film of carrier liquid of a thickness ez in a vertical direction Z, said carrier liquid containing the objects to be transferred of dimension (Dx, Dy, Dz), said transfer being ensured by local energetic excitation of said liquid to form a cavitation bubble localized at the level of an object, characterized in that the ratio Dz / ez is greater than 1, and preferably greater than 0.5.

[0028] According to one variant, said objects are spheroids made up of an aggregation of elementary biological cells.

[0029] Advantageously, said values ​​Dx, Dy, Dz are greater than 100 pm and preferably greater than 200 pm.

[0030] According to a second variant, said energy excitation is achieved by focusing a laser at the interface between the surface of said substrate and said liquid vector film.

[0031] According to a second variant, said energy excitation is achieved by applying an electric field.

[0032] According to a third variant, the energy deposition is achieved by focusing an acoustic wave at the interface between the surface of said substrate and said liquid carrier film.

[0033] Advantageously, the level of energy applied to each shot is a function of the size of the object located in the firing axis.

[0034] According to one variant, said receiving substrate (40) is elastically deformable about the Z axis.

[0035] 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.

[0036] Advantageously, the objects to be transferred are spaced apart by a characteristic distance (d) of value d > 0.5Dxy.

[0037] According to one variant, the objects are of a biological nature comprising cellular aggregates, spheroids, organoids, expiants, expiants (islets of Langherans), polymer particles encapsulating cells (organoid covered with a layer of biomaterial), cell-seeded micro-carriers, biomaterial beads.

[0038] Preferably, the volume fraction or volume density of the biological objects transferred by propulsion within the printed tissue or organ is greater than 30%.

[0039] According to one variant, the transfer is repeated to manufacture a material, tissue or organ and the volume fraction or volume density of the biological objects transferred by propulsion in said printed material, tissue or organ is greater than 30%.

[0040] According to 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.

[0041] According to another variant, the process is carried out by using several simultaneous laser beams when the object has a non-isotropic shape in order to guarantee its transfer along a homogeneous trajectory.

[0042] The invention also relates to equipment for manipulating and transferring pulsed energy by deposition, comprising: - a power source directed towards the material to be transferred, - a donor substrate from which said material is manipulated and transferred by energetic pulses and, - a target receiving substrate that collects the transferred material

[0043] Said donor substrate comprises a blade coated with 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.

[0044] Preferably, the energy source is a laser.

[0045] According to one variant, the donor substrate blade is transparent or weakly absorbing at the wavelength of said laser beam.

[0046] According to a particular embodiment, it includes an opto-mechanical system for directing the laser spot relative to the centroid or center of mass of the object.

[0047] 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 said object in order to make it take off homogeneously.

[0048] According to a first variant, the energy source is constituted by a generator of an electric field.

[0049] According to a second variant, the energy source is constituted by a generator of an acoustic wave.

[0050] According to one variant, it includes a control and servo system for the energy value deposited as a function of the size of the particle to be transferred.

[0051] Advantageously, it integrates an intelligent object detection system, means for automating the material placement steps on the donor substrate and the handling / transfer steps.

[0052] According to one variant, the donor substrate is covered by a sacrificial layer having strong absorption / conduction properties for energy deposition by laser or by electric field.

[0053] According to one variant, the receiving substrate is not covered by a layer having strong absorption properties, this being made directly in the liquid.

[0054] According to one variant, it includes 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.

[0055] Preferably, said donor substrate is disposed below said receiving substrate.

[0056] According to another variant, it includes at least one other printing means including extrusion, inkjet, LIFT in order to manufacture complex materials or fabrics comprising different components.

[0057] According to another variant, it includes 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.

[0058] According to one variant, the equipment includes a control and servo system for 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 burst, several bursts repeated in time on the same point or several bursts sent simultaneously according to an XY pattern linked to the shape of the object.

[0059] According to one 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 by the liquid layer located between the object and the donor substrate.

[0060] According to another variant, the donor substrate consists of micro-wells in which the objects to be transferred are arranged.

[0061] Detailed description of a non-limiting example of embodiment

[0062] The present invention will be better understood upon reading the following description, concerning a non-limiting example of an embodiment illustrated by the accompanying drawings where:

[0063] [Fig. 1] Fig. 1 represents a schematic view of the transfer system

[0064] [Fig.2] Fig.2 represents an example of the chronology of the different components of the TRI for taking an image of the jet at a time T

[0065] [Fig.3] Fig.3 represents an exploded view of an example of a donor substrate

[0066] [Fig.4] Fig.4 shows a perspective view of an example connector fluidic.

[0067] General presentation of the transfer system

[0068] The transfer system comprises several parts: - an optical part comprising a pulsed laser (10) to produce the cavitation energy of the carrier liquid and a camera (20) for observing the donor substrate (30) and the receiving substrate (40) - An automated section, with a robotic 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 carrier liquid and the particles to be transferred

[0069] and optionally, the system may include an extruder to add on the receiving substrate (40) a link, for example a hydrogel-type biomaterial between the layers of transferred particles.

[0070] More generally, the equipment can combine several 3D printing, bio-printing, and photo-polymerization technologies.

[0071] 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 energetic pulses in a plane of the donor substrate (30) where there is a particle to be transferred.

[0072] 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, substantially higher than the energy usually used for LIFT processes.

[0073] According to 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.

[0074] The lens (16) is typically an F-Theta lens with a focal length of 100 mm suitable for laser scanning. The typical size of the spot at the focal plane is on the order of 30 to 35 pm in diameter.

[0075] The laser beam (14) passes through a shaping optic (13) and is then directed via a set of mirrors (11, 12) to a scanner (15) which will then send the beam vertically towards the donor substrate (30), via the F-Theta lens (16).

[0076] The scanner (15) consists of two automated mirrors that redirect the beam horizontally at a certain angle towards the objective lens (16). The objective lens (16) then straightens the beam (14) so ​​that it arrives perpendicular to the donor substrate (30) and focuses it. The mirrors of the scanner (15) 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 steerable along the X and Y axes defining the horizontal plane.

[0077] For a donor substrate (30), the beam (14) is focused on the sacrificial layer coating the surface of the substrate, for example a 20 nanometer gold layer deposited on a transparent optical window.

[0078] The second optical part, comprising the camera (20), is the aiming part. Indeed, the particles in the case of the present invention are relatively large objects, for example, spheroids formed by an aggregate of cells, with a diameter of more than 100 pm, typically 200 pm to 300 pm. These objects are randomly distributed on the donor substrate (30); it is therefore necessary to know the position and size of the objects in order to target them with a laser beam. For this purpose, a visible light source, 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 reaches a semi-reflective mirror (12) which allows visible light directed towards the camera (20) to pass through, but reflects The infrared light is directed towards the laser (10). The beam then passes through a lens (23), an iris (24) and an objective lens (25) before arriving at the camera (20). Everything is aligned so that the focus of the laser beam (10) is at the center of the image captured by the camera (20).

[0079] Nature of the transferred objects

[0080] The installation implemented by the invention is similar to an installation intended for LIFT bioprinting, except that the energy of the pulses is at least 2 or even 5 times greater, 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 bio-ink where the particles are much smaller than the thickness of the film deposited on the donor substrate (30).

[0081] In the context of the present invention, the objects have a size of more than 100 pm, typically 200 to 400 pm 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 of 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 not covering them totally.

[0082] The invention relates, without limitation, 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.

[0083] In order to produce the spheroids, stem, progenitor or differentiated cells are cultured by conventional methods and seeded in micro-wells in order to produce aggregates which are then manipulated according to the process of the present invention.

[0084] Firing sequences

[0085] The LIFT phenomenon is a process which takes place over very short times, on the order of hundreds of microseconds, and can be observed by a TRI device.

[0086] Time-Resolved Imaging (TRI) involves capturing images of a phenomenon at a precise time by synchronizing a camera with a laser (10) and an LED to take a picture of the jet at a specific moment. The three components are synchronized via an electronic board to precisely control the activation of the different components over time. To obtain an image of the jet at a precise instant, the camera is activated for several hundred microseconds, while the LED emits only a 2-second flash of light at the desired time. The LED is positioned in front of the camera so as to illuminate the jet.

[0087] Figure 2 shows the chronology of the TRI with a time scale in microseconds. Initially (100), the analyzer camera is switched on, which takes 60 ps to turn on. The laser (10) takes 4 ps to turn on (105), and 5 ps more to fire the laser. If the camera is on during the laser firing, the beam is visible in the image and makes it less legible; therefore, the laser must be fired before the analyzer camera is switched on.

[0088] The laser (10) is therefore switched on (101) 50 ps after the analyzer camera, so that the shot (102) occurs at 59 ps, i.e., 1 ps before the analyzer camera (103) is switched on. The camera then remains on for 500 ps. Finally, the LED emits a sequence of 2 ps flashes (110, 111) starting at the desired time (106), T ps after the laser shot, i.e., 59 + T ps after the camera is switched on, to create a stroboscopic image with 2 ps intervals between two flashes.

[0089] The images thus obtained show a particle behavior very different from that observed in the prior art with LIFT bioprinting. The large particle is propelled without carrying away any of the carrier liquid by the formation of a cavity bubble in the liquid film at the interface between the particle and the substrate base, and not on a volume enveloping the particle. As the particle emerges from the liquid film, it displaces the surrounding liquid, emerging directly from the carrier liquid and reaching the receiving substrate with very little liquid carried.

[0090] Donor substrate

[0091] Figure 3 shows an exploded view of a donor substrate (30) receiving the technical liquid film and the ink containing the particles to be transferred, spread onto a cartridge that serves as a support for the laser beams. It is composed of an assembly of four layers (31, 32, 33, 34) of glass and PDMS. The technical liquid is intended 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.

[0092] The upper blade (31) of the cartridge is made of polydimethylsiloxane and has a window (35) surrounding the cylindrical volume receiving the technical fluid and the particles. It forms a hydrophobic layer acting as a barrier to prevent overflow of the technical fluid and the ink containing the particles. It has a thickness of 10 µm.

[0093] A first intermediate blade (32) also has a window (36) and serves to fix the upper blade (31). It is made of glass, 0.5 mm thick. It also serves to prevent overflow.

[0094] A second intermediate stage blade (33) has a window (37) and two microfluidic channels (38, 39), one for supplying the technical fluid and the other for drawing it out. It is made of polydimethylsiloxane and has a thickness of 160 µm.

[0095] The lower blade (34) is made of glass and is 0.5 mm thick. It has two holes (48, 49) at the point where the microfluidic channels of the blade (33) enter. These two holes (48, 49) serve as the inlet and outlet in the cartridge. The lower blade (34) is coated with a 20 nm layer of gold around the perimeter corresponding to the windows (35, 36, 37).

[0096] The assembly of these four stages (31 to 34) forms the donor substrate (30).

[0097] Fluidic connector

[0098] The donor substrate (30) is attached to a fluidic connector (50) shown in [Fig. 4], which has four channels (51 to 54) on its upper part. Seals or lip valves are fitted to these four inlets / outlets (51 to 54) to ensure a tight seal. When the donor substrate (30) is placed on the fluidic connector (50), the holes (51, 53) will align with the vents (48, 49) of the cartridge (50) and allow the entry and exit of the technical fluid. The inlets of these two channels are located on the side of the fluidic connector (50) and are connected, via white tubing, to pumps that circulate the technical fluid. The two pumps are connected to a reservoir containing the technical fluid. The particles to be transferred are then deposited onto the film using, for example, a pipette mounted on a robotic arm.The two other 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.

[0099] The fluidic connector (50) is mounted on a set of three micrometer screws along the three axes. The vertical micrometer screw is used to move the donor substrate (30) along the Z-axis to position it 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 to target different locations on the cartridge, as an alternative to using the scanner.

[0100] The two fluidic pumps are computer-controlled via software. Several routines are recorded, including the one for pre-wetting the cartridge. The preparation of the printing fluid is carried out in two stages. First, a pre-wetting film is applied to the bottom of the donor substrate (30). To do this, a volume of printing fluid is sent through the first channel, while the suction channel pump is not activated. The printing fluid accumulates on the surface of the donor substrate (30), covering the entire surface. Then, the suction channel is activated while the flow of printing fluid is stopped. The thick film thins, but the printing fluid remains spread over the entire surface of the donor substrate (30), ensuring the bottom is properly pre-wetted.Once this film is ready, a precise volume of ink containing particles to be transferred is pipetted onto the donor substrate (30).

[0101] Transfer of a particle

[0102] The particle is ejected from the first cavitation bubble, which is much larger than in known LIFT solutions, due to the greater power and the small volume of liquid between the underside of the particle and the bottom of the donor substrate. The particle is ejected alone from the liquid by a ballistic propulsion phenomenon. The cavitation bubble bursts and expels the particle upwards at a high speed. After 150 ps, ​​a polyethylene particle is approximately 3 mm high, corresponding to a speed of 20 m / s. It reaches the receiving substrate (40) almost dry and surrounded by residual liquid splashes.

[0103] The ejection of 250 pm collagen spheres is slower than for polyethylene beads. The particle reaches a height of 3 mm in 800 ps, ​​compared to 100 ps for polyethylene. The transfer of cell spheroids exhibits similar behavior to that described for collagen because their shape and density are very close. For example, the transfer of an IPSC (stem cell) spheroid also reaches a height of 3 mm in 700 to 800 ps.

[0104] The base of the jet takes 100 ps to form with a collagen sphere or one made of IPSC. Furthermore, it can be seen that there is only one jet. Indeed, the characteristic crown of the second jet is not present. Between 200 ps and 600 ps, ​​the jet is formed by the IPSC spheroid at the apex, and the carrier liquid forms the rest of the jet, still attached to the base. However, after 700 ps, ​​the IPSC spheroid detaches from the jet, and the liquid falls back down. The spheroid then continues its flight with little or no liquid, as with polyethylene. The jet breaks up at a similar time to that of the bio-ink jet, that is, around 700 ps.

[0105] Unlike known LIFT solutions, all the cavitation energy propels the particle, without loss, to enable it to pass through and leave the surrounding liquid layer, which can have a mass greater than that of the particle itself. Furthermore, with the process according to the invention, the particle experiences very little fluid friction during its ejection from the carrier liquid due to the viscosity of the latter.

[0106] Transfer onto the receiving substrate

[0107] The transfer takes place 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.

[0108] 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 its initial path. Indeed, due to their porosity and / or hydrophilic nature, the particles can carry liquid that forms a kind of bond (capillary bridge) as long as the particle remains close to the surface of the film. The liquid filament breaks down at a certain distance, depending on the characteristics of both the liquid and the particle. Typically, this distance is a few millimeters, generally between 3 and 5 mm. Furthermore, the distance must be limited to ensure sufficient kinetic energy to guarantee that most particles reach the receiving substrate. A distance between 3 and 10 mm is usually appropriate, but those skilled in the art can determine the optimal distance by observing, for example, using the aforementioned TRI process, the formation and rupture of the liquid filament as the particles travel. This distance can also be chosen to minimize the impact velocity of the object on the receiver.

[0109] 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 collagen film at 4 mg / mL. Collagen, like any other hydrogel, is more elastic than glass and will deform upon impact with a particle, thus reducing the shock for the transferred particle, which retains its original shape.

[0110] Displacement of a particle

[0111] 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 pm from the centroid (or center of mass) of the particle, the percentage of particles transferred decreases rapidly, and the accuracy of the localization decreases and beyond 80 pm of offset of the shot from the center of the particle, for particles of 200 pm average diameter, the transfer fails.

[0112] 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 by low power laser shots, for example with a power less than half the power required for the transfer, in the vicinity of a particle to be moved in the horizontal XOY plane.

[0113] Advantages and disadvantages of a sacrificial layer

[0114] The sacrificial layer ensures constant and reproducible absorption over the entire surface of the donor substrate. It also allows verification of the laser's proper operation through the ablated spots visible on the sacrificial layer.

[0115] However, the use of a sacrificial layer is costly and time-consuming, but above all, it requires changing the donor substrate as soon as the ablation rate becomes too high. For the production of large quantities and / or large-sized tissues, the number of donor substrates required can very quickly become substantial, thereby increasing both the production time and cost of said tissues.

[0116] The advantage of using a sacrificial layer-free approach is therefore very significant for large-scale production applications, particularly in clinical and industrial fields. Printing large objects such as spheroids using a sacrificial layer-free system is thus particularly attractive for reducing manufacturing time and cost.

[0117] Ultra-short pulse laser solutions (femtosecond / picosecond regime) or lasers working in the Mid-IR wavelength range (2.6 to 3.2 pm) are perfectly usable in this context.

Claims

Demands

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

5.

2. - Method according to claim 1 characterized in that said objects are spheroids constituted by the aggregation of elementary biological cells.

3. - Method according to claim 1 characterized in that said values ​​Dx, Dy, Dz are greater than 100 pm and preferably greater than 200 pm.

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

5. 5 - Method according to claim 1, characterized in that the level of energy applied to each shot is a function of the size of the object located in the firing axis.

6. 6 - Method according to claim 1, characterized in that said receiving substrate (40) is elastically deformable about the Z axis.

7. 7 - Method according to claim 1, characterized in that it comprises steps of displacement in the XOY plane of an object by application of firing with an energy at least 2 times less than the energy required for a transfer of the object to the receiving substrate.

8. 8 - 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. 9 - A method according to claim 1, characterized in that the objects are of a biological nature comprising cell aggregates, spheroids, organoids, expiants, expiants (islets of Langerhans), and polymer particles encapsulating cells (organoid covered with a layer of biomaterial), cell-seeded microcarriers, biomaterial beads.

10. 10 - 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 volume density of the biological objects transferred by propulsion in said printed material, tissue or organ is greater than 30%.

11. 11 - 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.

12. 12 - Equipment for manipulating and transferring by pulsed energy deposition comprising: - an energy source (10) oriented towards the material to be transferred, - a donor substrate (30) from which said material is manipulated 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 (e) is less than 2xDz, where Dz is the dimension of the transferable objects along the direction perpendicular to said film, Dx,Dy being the orientation of said transferable objects in the plane of said film.

13. 13 - Equipment according to claim 12 characterized in that the energy source (10) is constituted by a laser.

14. 14 - Equipment according to claim 12 characterized in that the blade (34) of the donor substrate (30) is transparent or weakly absorbing at the wavelength of said laser beam (10).

15. 15 - Equipment according to claim 12 characterized in that it comprises an opto-mechanical system (15) allowing the laser spot to be directed relative to the centroid or center of mass of the object.

16. 16 - Equipment according to claim 12, characterized in that it integrates an intelligent object detection system, means for automating the material placement steps on the donor substrate and the handling / transfer steps.

17. 17 - Equipment according to claim 12, characterized in that the donor substrate (30) is covered by a sacrificial layer having strong absorption / conduction properties of energy deposition by laser or by electric field.

18. 18 - 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 on the liquid layer located between the object and the donor substrate.

19. 19 - Equipment according to claim 12, characterized in that it comprises an optical viewing / detection system for locating and targeting objects to be transferred, compatible with a random distribution of objects on the donor substrate.

20. 20 - Equipment according to claim 12, characterized in that said donor substrate (30) is disposed below said receiving substrate (40).

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

22. 22 - Equipment according to claim 12, characterized in that 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.

23. 23 - 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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