Printing objects from a well
Patent Information
- Application Number
- EP2023794289
- Authority / Receiving Office
- EP · EP
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2022-10-20
- Filing Date
- 2023-10-20
- Publication Date
- 2025-08-27
Smart Images
Figure 1.1
Abstract
Description
PRINTING OBJECTS FROM A WELL 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 consists of transferring biological objects (e.g. cells), organic or mineral, from a carrier liquid deposited on a substrate in the form of a film and providing an energy pulse to form a cavitation bubble, either directly by vaporization of the liquid portion in the focal field of the energy source - generally a laser - or via a thin metal 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 prior art is patent application CN115198376 which relates to a micropore array chip and a single cell sorting method for laser-induced direct transfer, wherein a metal-coated glass sheet is covered with a microporous membrane layer; the microporous membrane is a biocompatible film and is covered on the metal-coated glass sheet by adopting micro-nano processing technology; the single cell forms a single-cell micro-liquid droplet due to the hydrophobic characteristic of the micropore array chip material, and the single-cell micro-liquid droplet is fixed in the micropore, so that single-cell capture is realized, and subsequent single-cell sorting is facilitated.
[0004] Patent application US2020009877 proposes a solution for depositing particles onto a target from a transparent slide having a film formed by a fluid containing suspended particles, by locally exciting the film using a laser, comprising means for observing the local excitation region. The observation means comprise a sensor and a light source whose optical axes are substantially shared in a space between an optical separator and that of the film. The optical beam of the imaging system and the optical beam of the laser are arranged coaxially in the space between the controlled optical deflection means and the film. The device comprises a first optical focusing unit arranged between the controlled optical deflection means and the film.The device comprises a second optical image combining unit positioned between the sensor and the splitter, the sensor being positioned in the focal plane of the second optical unit.
[0005] Patent application 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 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.
[0007] Patent application US2016259250 relates to a method for providing a patterned structure on a substrate, the method comprisingproviding a donor substrate disposed between a light source and an acceptor substrate, the donor substrate comprising a donor material;providing a mask disposed between the light source and the donor substrate, the mask comprising a mask pattern for shaping light from the light source striking the donor substrate, the shaped light corresponding to the patterned structure to be created, the shaped light striking the donor substrate causes the donor material to be released from the donor substrate and transferred to the acceptor substrate to form the patterned structure therein;wherein the structured light is split into a plurality of separate beams of homogeneous size simultaneously striking the donor substrate to cause the donor material to be released from the donor substrate in the form of separate droplets of homogeneous size.;
[0008] Patent application US2018090314 relates to a method for arranging a material comprising positioning a donor film comprising a donor material at a predefined distance from an acceptor substrate, the donor film facing the acceptor substrate. One or more pulses of laser radiation are directed towards the donor film at a given location so as to induce the formation of a protrusion consisting of the donor material. A distal tip of the protrusion touches the acceptor substrate and is deposited thereon while the protrusion is still in contact with the donor film. A spot of the donor material is formed on the acceptor substrate by increasing the separation between the donor film and the acceptor substrate so as to detach the distal tip of the protrusion from the donor film. Disadvantages of the prior art
[0009] The solutions of the prior art are poorly suited to particles with sizes greater than 100 µm and also pose difficulties with regard to centering the shot relative to the particle to be transferred.
[0010] Furthermore, the solutions of the prior art involve operations of handling objects presenting risks of loss of viability, in particular for fragile biological objects.
[0011] The invention aims to solve the problem of prior art solutions in order to improve the quality and precision of transferring objects onto the receiving substrate when the objects to be transferred have different sizes. Solution provided by the invention
[0012] In order to overcome these drawbacks, the present invention relates, in its most general sense, to a method of manufacturing a material by transferring at least one particle from a donor substrate to a target substrate, having the technical characteristics set out in claim 1. It relates more particularly to a method of transferring an object from a donor substrate to a target substrate, a film of vector liquid containing the particles to be transferred being deposited on said donor substrate containing the objects to be transferred of dimension (D x , D y , D z), said transfer being ensured by local energy excitation to form a cavitation bubble located at the level of an object, characterized in that said substrate is constituted by a micro-well plate having a plurality of wells each forming a receptacle having an open upper base narrowing towards the bottom, the width (L) of the base of said wells being L > 2D x D y and the height (h) of said well h > 2D z , and the transfer is doneAccording to a first transfer mode with a first energy level E1if the ratio between the dimension (D z ) of said object and thickness (e) of said liquid film is less than 1According to a second transfer mode with a second energy level E2> E1if the ratio between the dimension (D z ) of said object and thickness (e) of said liquid film is greater than 1.
[0013] According to a first variant, the energy deposition is carried out by focusing a laser for the two transfer modes, propulsion and LIFT.
[0014] According to a second variant, the energy deposition is carried out via an electric field for the propulsion mode.
[0015] Creating a cavitation bubble with an electric field involves using electrolysis to generate gas bubbles in a liquid. Cavitation is the physical phenomenon where gas bubbles form and implode in a liquid subjected to varying pressure, thus creating forces and shock waves. Applying an electric field can facilitate the formation of these bubbles. To create a cavitation bubble with an electric field, it is known to those skilled in the art to use an electrolytic cell containing the conductive liquid and equipped with two electrodes connected to a direct current source.
[0016] According to a third variant, the energy deposition is achieved by focusing an acoustic wave for the propulsion mode.
[0017] An acoustic wave produced by an ultrasonic transducer designed to focus acoustic energy at a specific point and generate low-pressure areas in a liquid, causing gas bubbles to form.
[0018] According to a particular embodiment, the receiving substrate comprises a damping means along the axis of transfer of the objects.
[0019] According to a variant, the transfer of biological objects is carried out without contact by the generation of a cavitation bubble in the liquid present between the object and the bottom of the well, the transfer being ensured: either by the conversion of the deposited energy into kinetic energy of the object which takes off and leaves the liquid layer with a strong directivity in the propulsion mode or by the conversion of the deposited energy into a movement of the liquid which carries the objects via a jet in the LIFT mode.
[0020] Contactless transfer guarantees the absence of plastic deformation of the object.
[0021] Advantageously, the objects to be transferred are chosen from the category of cellular aggregates, spheroids, organoids, explants (islets of Langherans), polymer particles encapsulating cells (organoid covered with a layer of biomaterial), micro-carriers seeded with cells, biomaterial beads.
[0022] 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%.
[0023] According to another variant, the transfer is carried out only once in order to precisely characterize a single object.
[0024] 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.
[0025] The invention also relates to equipment for handling and transferring by energy deposition comprising: an energy source directed towards the material to be transferred, at least one well from which said material is handled and transferred, a target receiving substrate which collects the transferred material
[0026] Said donor being constituted by a plate comprising wells containing the transferable objects arranged in a liquid with an orientation D x ,D y in the plane of the liquid film and D z perpendicular to said film, characterized in that the transfer is made
[0027] - either by LIFT if the ratio between the dimension (D z ) of the object and the thickness (e) of the liquid film is less than 1
[0028] - either by propulsion if the ratio between the dimension (D z ) of the object and the thickness (e) of the liquid film is greater than 1
[0029] Alternatively, energy deposition is achieved by focusing a laser for both transfer modes, propulsion and LIFT.
[0030] Advantageously, the substrate of the plate comprising the wells is transparent or weakly absorbent at the wavelength of said laser beam.
[0031] According to one variant, it includes a scanner allowing the laser beam to be precisely positioned on the centroid or center of mass of each object, thus ensuring strong directionality in the transfer of said object.
[0032] According to another variant, the energy deposition is carried out via an electric field.
[0033] According to another variant, the energy deposition is achieved by focusing an acoustic wave.
[0034] According to another variant, it includes a system for controlling and controlling the energy value deposited in order to transfer the object optimally depending on its size.
[0035] According to another variant, it integrates means for automating the movement of the substrate (30).
[0036] According to another variant, the substrate of the well plate is covered by a sacrificial layer having strong absorption / conduction properties of energy deposition by laser or electric field.
[0037] Advantageously, it includes a means for controlling a temporal succession of several energy deposits on the same well for the transfer of an object.
[0038] According to another variant, it comprises a means for controlling several spatially separated energy depots in order to transfer several objects arranged in different wells in parallel.
[0039] According to another variant, it further comprises at least one other printing technology including extrusion, inkjet, LIFT for the manufacture of complex materials or fabrics comprising different components.
[0040] According to another variant, it integrates a system to deliver several laser beams simultaneously at the object level when it has a non-isotropic shape in order to guarantee its transfer along a homogeneous trajectory.
[0041] Detailed description of a non-limiting example of embodiment
[0042] 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:
[0043] Shows a cross-sectional view of a microwell of an example donor substrate with a small particle
[0044] Shows a cross-sectional view of a microwell of an example donor substrate with a large particle
[0045] It represents a schematic view of the transfer system
[0046] It represents a cross-sectional view of a variant of a micro-well
[0047] It represents a partial top view of a donor substrate.
[0048] General context of the invention
[0049] The invention particularly relates to the additive manufacturing of a biological graft by transferring biological objects with dimensions greater than 100 µm such as spheroids or organoids onto a target surface. Spheroids are three-dimensional (3D) cellular aggregates that can mimic tissues. Once seeded in a well of a pyramidal or truncated cone-bottomed microplate, these aggregates form a discrete spheroid.
[0050] Spheroids contain both deeply embedded and surface-exposed cells, proliferating and non-proliferating cells, and a center surrounded by a well-oxygenated outer layer of cells. Their assembly by transfer onto a target substrate allows the creation of three-dimensional tissues such as cartilage, for example, to reconstruct damaged cartilage.
[0051] Spheroids and organoids can be made up of different stem, progenitor and / or differentiated cells e.g. cardiac, brain, liver cells, etc.
[0052] The culture of spheroids and organoids can be carried out in single-well plates as well as in 12-, 24-, 48-, 96- or even 384-well plates containing several thousand to several tens of thousands of microwells.
[0053] General presentation of the donor substrate
[0054] Figures 1 and 2 show views of a microwell (31) of a donor substrate (30). The microwell (31) is made in a transparent plate comprising a matrix of microwells, and has an inverted pyramidal shape with an open square base (32) of width L typically between 200 µm and 800 µm and a lower bottom (33) which is pointed or preferably with a flat. It has a height h typically between 200 µm and 800 µm,
[0055] The spheroid (35) is composed of about 500 cells, and occupies only part of the height h of the micro-well,
[0056] The spheroid (36) is composed of about 2000 cells, and occupies the entire part of the micro-well of height h.
[0057] An aqueous liquid, e.g., water with added salts, or a culture medium, e.g., a 2% BSA (Bovine Serum Albumin) solution, at least partially fills the microwell (31).
[0058] Depending on the size of the spheroid (35, 36) that the microwell (31) contains, the liquid completely covers the spheroid, or the spheroid rests in a liquid background.
[0059] In the first case, the transfer will be done in LIFT mode, with a moderate power typically of 15 to 20 microjoules.
[0060] In the second case, the transfer will take place using a propulsion method, without the particle being carried in a liquid bubble, with a power 2 to 5 times greater, of the order of 30 to 60 microjoules.
[0061] The deposited energy value is minimized in order to transfer the object according to its size while ensuring its post-transfer integrity. Minimizing the deposited energy also ensures a low transfer speed allowing slow deposition on the receiving substrate, thus contributing to the integrity of the transferred object.
[0062] Optionally, the surface of the micro-well (31) is coated with a sacrificial layer, typically a layer of gold, to promote the formation of a cavitation bubble ejecting the particle contained in the micro-well. This sacrificial layer can also be constituted by a thin layer of metal, polymer, gel, etc.
[0063] Overview of the transfer system
[0064] The transfer system comprises several parts: an optical part comprising a pulsed laser (10) to produce the cavitation energy of the vector liquid and optionally a camera (20) for observing the donor substrate (30) and the receiver substrate (40). This camera and the associated optical system are not necessary for shooting objects exclusively contained in micro-wells, because the positioning of the objects to be shot is constrained and does not require correction by optical observation. An automated part, with a robot arm (50) used for automating the handling of the receiver (40). The receiver substrate (40) advantageously has mechanical properties capable of absorbing shocks in order to guarantee the integrity of the transferred object, either by an elastically deformable coating, or by a support mounted on a damping system along the transfer axis.
[0065] and optionally, the system may comprise an extruder for adding to the receiving substrate (40) a link, for example collagen between the layers of transferred particles.
[0066] More generally, the equipment can combine several 3D printing, bioprinting and photopolymerization technologies.
[0067] The substrate (30) is constituted by a micro-well plate described above, held by a support advantageously moved by a motorized system (37) positioning the tip of one of the micro-wells in the optical axis, to allow the transfer of the particle contained in the micro-well with high firing precision, less than 50 µm, due to the knowledge of the geometry of the plate and the constrained positioning of the particle (35, 36) in the micro-well (31).
[0068] The optical part of the device is possibly made up of two parts, an optional part comprising the camera (20) used to aim at the objects, and a part comprising the laser (10) used to "shoot", i.e. deliver energy pulses in a plane of the donor micro-well (30) where there is a particle to be transferred.
[0069] 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.
[0070] 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.
[0071] The power will be determined for each shot based on the size of the particle present in the microwell in the optical axis, and the appropriate transfer mode.
[0072] 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.
[0073] 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).
[0074] 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.
[0075] 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.
[0076] The second optional optical part comprising the camera (20) is the aiming part. It may be necessary to use a visualization system if the particles are smaller than the size of the microwell used, for example spheroids formed by an aggregation of cells having a diameter of 100µm.
[0077] 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 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). Everything is aligned so that the focus of the laser beam (10) is at the center of the image recovered by the camera (20).
[0078] This image makes it possible to determine the size of the particle present in the micro-well located in the firing axis, and thus determine the power of the required pulse.
[0079] The invention relates, but is not limited to, the transfer of spheroids formed by an aggregate of cells cultured in the laboratory and having the appearance of a small pearl made of cells and extracellular matrix.
[0080] 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.
[0081] As explained above, the "shot zone imaging" part is optional. It is useful when the microwells can accommodate small particles, whose positioning will not be constrained by the walls of the microwell. It is then necessary to use the information provided by an imaging system to precisely center, to within 50 µm, the axis of the laser beam with the center of the particle.
[0082] On the other hand, when the particles are systematically of large size, greater than the mid-height section of the micro-well, this imaging subsystem is optional and can be omitted, because the precision of the shot results from the constrained positioning of the particle in the micro-well, and from the precise positioning of the substrate presenting the micro-wells relative to a reference of the equipment.
[0083] Flat-bottomed microwells
[0084] According to an embodiment variant illustrated by the, the micro-wells have an inverted pyramidal cavity with a truncated tip, to limit the phenomena of reflection and diffraction of the laser shot centered on the micro-well. The surface of the base of the micro-well is greater than the width of the laser beam, typically between 50 and 100 µm.
Claims
- Method for transferring an object from a donor substrate (30) towards a target substrate (40), a film of vector liquid containing the particles to be transferred being deposited on said donor substrate (30) 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 said donor substrate (30) is constituted by a micro-well plate having a plurality of wells each forming a receptacle having an open upper base narrowing towards the bottom, the width (L) of the base of said wells being L > 2D x D y and the height (h) of said well h > 2D z , and the transfer is doneAccording to a first transfer mode with a first energy level E1if the ratio between the dimension (D z) of said object and thickness (e) of said liquid film is less than 1According to a second transfer mode with a second energy level E2> E1if the ratio between the dimension (D z ) of said object and thickness (e) of said liquid film is greater than 1. - Method according to claim 1 characterized in that the energy deposition is carried out by focusing a laser for a propulsion transfer mode or for a LIFT transfer mode. Method according to claim 1 characterized in that the energy deposition is carried out via an electric field for a propulsion transfer mode. - Method according to claim 1 characterized in that the energy deposition is carried out by the focusing of an acoustic wave for a propulsion transfer mode. - Method according to claim 1, characterized in that the receiving substrate (40) comprises a damping means along the axis of transfer of the objects. - Method according to claim 1, characterized in that the transfer of the objects is carried out without contact by the generation of a cavitation bubble in the liquid present between the object and the bottom of the well, the transfer being ensured - either by the conversion of the deposited energy into kinetic energy of the object which takes off and leaves the liquid layer with a strong directivity in the propulsion mode - or by the conversion of the deposited energy into a movement of the liquid which carries the objects via a jet in a LIFT transfer mode - Method according to claim 1, characterized in that the objects to be transferred are chosen from the category of cellular aggregates, spheroids, organoids, 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 1 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 only once in order to precisely characterize a single object. - Method according to claim 1, characterized in that it can be combined with other printing technologies such as extrusion, inkjet, LIFT in order to manufacture complex materials or fabrics comprising different components. - Equipment for handling and transferring by energy deposition comprising:an energy source directed towards the material to be transferred,at least one well from which said material is handled and transferred,a target receiving substrate which collects the transferred material.Said donor being constituted by a plate comprising wells containing the transferable objects arranged in a liquid with an orientation D x ,D y in the plane of the liquid film and D z perpendicular to said film, characterized in that said substrate consists of a micro-well plate having a plurality of wells each forming a receptacle having an open upper base narrowing towards the bottom, the width (L) of the base of said wells being L > 2D x D y and the height (h) of said well h > 2D z , and in that it includes transfer means: - either by LIFT if the ratio between the dimension (D z) of the object and the thickness (e) of the liquid film is less than 1 with a first energy level E1- either by propulsion if the ratio between the dimension (D z ) of the object and the thickness (e) of the liquid film is greater than 1 with a second energy level E2> E1And in that it integrates a system for delivering several laser beams simultaneously at the level of the object when the latter has a non-isotropic shape for its transfer along a homogeneous trajectory. - Equipment according to claim 11 characterized in that the energy deposition is carried out by focusing a laser for said two transfer modes, propulsion and LIFT. - Equipment according to claim 11 characterized in that the substrate of the plate comprising the wells is transparent or weakly absorbent at the wavelength of said laser beam. - Equipment according to claim 11 characterized in that it comprises a scanner allowing the laser beam to be precisely positioned on the centroid or center of mass of each object, thus ensuring strong directionality in the transfer of said object. - Equipment according to claim 11 characterized in that the energy deposition is carried out via an electric field. - Equipment according to claim 11 characterized in that the energy deposition is carried out by the focusing of an acoustic wave. - Equipment according to claim 11, characterized in that it comprises a system for controlling and controlling the energy value deposited in order to transfer the object according to its size. - Equipment according to claim 11, characterized in that it integrates means for automating the movement of the substrate (30). - Equipment according to claim 11, characterized in that the substrate (30) of the well plate is covered by a sacrificial layer having properties of absorption / conduction of the deposition of energy by laser or by electric field. - Equipment according to claim 11, characterized in that it comprises means for controlling a temporal succession of several energy deposits on the same well for the transfer of an object. - Equipment according to claim 11, characterized in that it comprises means for controlling several spatially separated energy depots in order to transfer several objects arranged in different wells in parallel. - Equipment according to claim 11, characterized in that it further comprises at least one other printing technology including extrusion, inkjet, LIFT for the manufacture of complex materials or fabrics comprising different components.