Printing objects from wells

Abstract

The present invention relates to a method and apparatus for transferring an object from a donor substrate (30) towards a target substrate (40), wherein the donor substrate (30) is transferred and dimensioned (D x , D y , D z ) and the transport is ensured by local energy excitation of the liquid to form local cavitation bubbles at the object, a. The substrate consists of a microwell plate having a plurality of wells, each forming a receptacle with an open upper base tapering towards the bottom, the width (L) of the base of the well being such that L>2D x D y and the height (h) of the well is h>2D z b.-Dimensions of the object (D z ) to the thickness of the liquid film (e) is less than 1, at the first energy level E1, - the object dimension (D z The second energy level E2>E1 is characterized by transport occurring when the ratio of the energy of the liquid to the thickness of the liquid film (e) is greater than 1.

Description

[Technical Field]

[0001] The present invention relates to the field of additive manufacturing of materials by repeatedly transferring particles between a donor substrate and a receiver substrate and depositing a film of a carrier liquid containing the transferred particles onto the donor substrate, and in particular to the field of bioprinting, which relates to the use of digital fabrication processes to organize and assemble biological tissue components in 2D and 3D for the purpose of generating implants for regenerative medicine or physiological models for biomedical and pharmaceutical research.

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

[0003] <Prior art> In the state of the art, Patent Document 1 is known, which relates to a micropore array chip and a single-cell sorting method for laser-induced direct transfer, in which a metal-coated glass sheet is coated with a microporous membrane layer, the microporous membrane being a biocompatible membrane, which is coated on the metal-coated glass plate by adopting micro-nano processing technology, and single cells form single-cell microdroplets due to the hydrophobic properties of the micropore array chip material, and the single-cell microdroplets are fixed in the micropores, so that single-cell capture is achieved and subsequent single-cell sorting is facilitated.

[0004] Patent Document 2 proposes a solution for depositing particles onto a target from a transparent slide having a film formed by a fluid containing particles in suspension by locally exciting the film with a laser, and includes a means for observing the local excitation region. The observation means includes a sensor and a light source whose optical axis is substantially shared in the space between an optical splitter and the optical axis of the film. The light beam of the imaging system and the light beam of the laser are coaxial in the portion between the controlled light deflection means and the film. The device includes a first focusing optical unit disposed between the controlled light deflection means and the film. The device includes a second optical image combining unit positioned between the sensor and a separator, and the sensor is positioned in the focal plane of the second optical unit.

[0005] Patent document 3 describes a single cell printing method based on transport induced by an annular laser spot, characterized in that it includes the following steps: Step 1: a uniformly distributed pulsed laser beam is incident on an element of an annular light spot forming system, and then the emitted light is focused to form an annular light spot on a sacrificial layer through a transparent constraining layer; Step 2, ablating the sacrificial layer with laser light and heat to form an annular plasma cavitation bubble; Step 3: Rapid expansion of the annular cavitation bubble and removal of the cell solution outside the ring, and parallel downward propulsion of the target transport cells in the center of the ring under the expansion of the cavitation bubble; Step 4: The collapse of the annular cavitation bubble due to the collapse of the shock wave generated in a direction perpendicular to the center position of the ring, and the complete expulsion of the target transport cell. - Step 5, under the action of gravity and momentum, the target transport cells and the transported solution are printed onto the receiver plate.

[0006] Patent Literature 4 relates to a method for additive manufacturing of three-dimensional objects. The method involves 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 includes performing a raster scan to dispense at least a first building material composition and performing a vector scan to dispense at least a second building material composition. The vector scan is optionally performed along a path selected to form at least one structure selected from the group consisting of: (i) an elongated structure; (ii) a boundary structure at least partially surrounding an area filled with the first building material; and (iii) an inter-layer connecting structure.

[0007] Patent document 5 relates to a method for providing a patterned structure on a substrate, the method comprising: providing a donor substrate disposed between a light source and an acceptor substrate, the donor substrate comprising a donor material; providing a mask disposed between a light source and the donor substrate, the mask including a mask pattern for shaping light from the light source that impinges on the donor substrate, the shaped light corresponding to the patterned structure to be created, and wherein the shaped light impinging on the donor substrate causes donor material to be released from the donor substrate and transferred to the acceptor substrate to form the patterned structure on the acceptor substrate; The structured light is split into multiple separate beams of uniform size that simultaneously strike the donor substrate, causing the donor material to be ejected from the donor substrate in the form of separate droplets of uniform size.

[0008] Patent Document 6 relates to a material deposition method that involves positioning a donor film containing donor material at a predetermined distance from an acceptor substrate, with the donor film facing the acceptor substrate. One or more pulses of laser radiation are directed at a given location on the donor film to induce the formation of a protrusion of donor material. The distal tip of the protrusion contacts and is deposited on the acceptor substrate while the protrusion remains in contact with the donor film. A spot of donor material is formed on the acceptor substrate by increasing the separation between the donor film and the acceptor substrate to separate the distal tip from the donor film protrusion.

[0009] <Disadvantages of the prior art> Prior art solutions are not well suited for particle sizes above 100 μm and also present difficulties with regard to centering the shot relative to the particle being transported.

[0010] Furthermore, prior art solutions involve object handling operations that present a risk of loss of viability, especially for fragile biological objects. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Chinese Patent Application Publication No. 115198376 [Patent Document 2] US Patent Application Publication No. 2020 / 009877 [Patent Document 3] Chinese Patent Application Publication No. 113021874 [Patent Document 4] US Patent Application Publication No. 2017 / 225390 [Patent Document 5] US Patent Application Publication No. 2016 / 259250 [Patent Document 6] US Patent Application Publication No. 2018 / 090314 Summary of the Invention [Problem to be solved by the invention]

[0012] The invention aims to solve the problems of the prior art solutions in order to improve the quality and accuracy of transferring objects to a receiving substrate when the objects to be transferred are of different sizes. [Means for solving the problem]

[0013] In order to overcome these drawbacks, the present invention relates in its most general sense to a method for producing a material by transferring at least one particle from a donor substrate towards a target substrate, having the technical features set out in claim 1. More particularly, it is a method for transferring objects from a donor substrate towards a target substrate, in which a carrier liquid film containing the particles to be transferred has a size (D x , D y , D z ) is deposited on the donor substrate containing the object to be transferred, the transfer being caused by localized energy excitation to form localized cavitation bubbles in the object, a. The substrate comprises a microwell plate having a plurality of wells, each forming a receptacle having an open upper base tapering to the bottom, and the width (L) of the base of the well is such that L>2D x D y and the height (h) of the well is h>2D z and b. -Dimensions of the object (D z ) to the thickness (e) of the liquid film is less than 1, according to the first transport mode at the first energy level E1, -Dimensions of the object (D z ) to the thickness (e) of the liquid film is greater than 1, the transport occurs according to a second transport mode at a second energy level E2>E1.

[0014] In a first variant, the energy deposition is achieved by focusing a laser for both the propulsion and LIFT transport modes.

[0015] In a second variant, the energy is applied via an electric field for the propulsion mode.

[0016] Creating cavitation bubbles with an electric field involves generating gas bubbles in a liquid using electrolysis. Cavitation is a physical phenomenon in which gas bubbles form in a liquid under variable pressure and implode, creating force and shock waves. The application of an electric field can facilitate the formation of these bubbles. To create cavitation bubbles with an electric field, those skilled in the art are familiar with the use of an electrolytic cell containing a conductive liquid and equipped with two electrodes connected to a direct current source.

[0017] In a third variant, the energy deposition is achieved by focusing acoustic waves for the propulsion mode.

[0018] The sound waves are generated by an ultrasonic transducer designed to focus acoustic energy at a precise point, creating a low-pressure zone in the liquid and causing gas bubbles to form.

[0019] In certain embodiments, the receiving substrate includes a damping means along the object transport axis.

[0020] According to a variant, the transfer of the biological objects is carried out without contact by generating cavitation bubbles in the liquid present between the objects and the bottom of the well, the transfer being carried out by: • In propulsion mode, by converting the imparted energy into the kinetic energy of the object, which falls and leaves the liquid layer in a highly directional manner; - Or in LIFT mode, it is ensured either by carrying away the object via the jet, converting the applied energy into a liquid displacement.

[0021] The contactless transfer ensures that there is no plastic deformation of the object.

[0022] Advantageously, the objects to be transferred are selected from the categories of cell aggregates, spheroids, organoids, explants (islets of Langerhans), cell-encapsulating polymer particles (organoids covered with a layer of biomaterial), cell-seeded microcarriers, biomaterial beads.

[0023] According to a variant, the transfer is repeated to produce a material, tissue, or organ, wherein the volume fraction or volume density of biological matter transferred by propulsion in the printed material, tissue, or organ is greater than 30%.

[0024] In another variation, the transfer is performed only once to accurately characterize a single object.

[0025] In another variation, the process is combined with other printing techniques such as extrusion, inkjet, LIFT, etc. to produce composite materials or tissues with different components.

[0026] The invention also includes: - an energy source directed at the material being transported; at least one well into which the material is processed and transferred; a target receiving substrate for collecting the transferred material; The donor has an orientation D in the plane of the liquid film. x , D y and orientation D perpendicular to the film z 1. An apparatus for energy processing and transfer by application, comprising a plate including wells containing transferable objects disposed in a liquid by -Object size (D z If the ratio of the liquid film thickness (e) to the liquid film thickness (e) is less than 1, it is determined by LIFT. - or the size of the object (D z The invention relates to a device characterized in that the transport is carried out either by propulsion or by the ratio of the thickness of the liquid film (e) to the volume of the liquid (e) greater than 1.

[0027] In one variation, energy deposition is achieved by focusing a laser for both the propulsion and LIFT transport modes.

[0028] Advantageously, the plate substrate containing the wells is transparent or weakly absorbing at the wavelength of the laser beam.

[0029] In one variant, a scanner is used to position the laser beam precisely at the center of gravity or mass of each object, thus ensuring high directionality in the transfer of said objects.

[0030] In another variation, the energy is applied via an electric field.

[0031] In another variation, the energy deposition is achieved by focusing acoustic waves.

[0032] According to another variant, the device comprises a system for controlling and closed-loop regulating the amount of energy applied in order to optimally transport the objects according to their size.

[0033] In another variant, the device incorporates means for automating the movement of the substrate (30).

[0034] In another variation, the well plate substrate is covered with a sacrificial layer that has high absorption / conductivity properties for laser or electric field energy deposition.

[0035] Advantageously, the device comprises means for controlling the temporal succession of several energy depositions on the same well in order to transport the objects.

[0036] According to another variant, the device comprises means for controlling several spatially separated energy depositions in order to transport in parallel several objects placed in different wells.

[0037] According to another variant, the device also includes at least one other printing technique, including extrusion, inkjet, LIFT, for the production of complex materials or tissues containing different components.

[0038] According to another variant, the device incorporates a system for simultaneously delivering several laser beams to the object in order to ensure the transport of the object along a uniform trajectory when the object has an anisotropic shape. [Brief explanation of the drawings]

[0039] Detailed Description of Non-Limiting Exemplary Embodiments The invention will be better understood on reading the following description of non-limiting exemplary embodiments illustrated by the accompanying drawings, in which: [Figure 1] 1 shows a cross-sectional view of a microwell of an exemplary donor substrate with small particles. [Figure 2] 1 shows a cross-sectional view of a microwell of an exemplary donor substrate with large particles. [Figure 3] 1 shows a schematic diagram of a transfer system. [Figure 4] 1 shows a cross-sectional view of a variation of a microwell. [Figure 5] FIG. 1 shows a partial top view of a donor substrate. DETAILED DESCRIPTION OF THE INVENTION

[0040] <General Background of the Invention> The invention particularly relates to additive manufacturing of biological implants by transferring biological objects with dimensions greater than 100 μm, such as spheroids or organoids, onto a target surface. Spheroids are three-dimensional (3D) cell aggregates that can mimic tissue. When seeded into wells of a microplate with a pyramidal or frustoconical base, these aggregates form separate spheroids.

[0041] Spheroids contain both deeply embedded cells and cells with an exposed surface, proliferating and non-proliferating cells, and a core surrounded by a well-oxidized 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.

[0042] Spheroids and organoids can be composed of different stem cells, progenitor cells and / or differentiated cells, e.g., cardiac cells, brain cells, liver cells, etc.

[0043] Spheroids and organoids can be grown in single well plates or in 12, 24, 48, 96, or even 384 well plates containing thousands to tens of thousands of microwells.

[0044] <Outline of donor substrate> 1 and 2 show diagrams of microwells (31) on a donor substrate (30). The microwells (31) are made from a transparent plate containing a microwell matrix and typically have an inverted pyramidal shape with an open square base (32) of width L between 200 μm and 800 μm, and a pointed or preferably flat bottom (33). Their height h is typically between 200 μm and 800 μm.

[0045] The spheroid (35) consists of approximately 500 cells and occupies only a portion of the height h of the microwell. The spheroid (36) consists of approximately 2000 cells and occupies the entire portion of a microwell of height h.

[0046] An aqueous liquid, such as water with added salts, or a culture medium, such as a 2% BSA (Bovine Serum Albumin) solution, at least partially fills the microwells (31).

[0047] Depending on the size of the spheroids (35, 36) contained in the microwells (31), the liquid either completely covers the spheroids or the spheroids remain at the liquid base.

[0048] In the first case, the transfer is performed in LIFT mode at moderate power, typically 15-20 microjoules.

[0049] In the second case, the transport is carried out by propulsion with 2-5 times greater power, around 30-60 microjoules, without the particles being carried away in the liquid bubbles.

[0050] The amount of applied energy is minimized to transfer objects according to their size while ensuring their post-transfer integrity. Minimizing the applied energy also ensures low transfer speeds, allowing for slow deposition onto the receiving substrate, thus contributing to the integrity of the transferred objects.

[0051] Optionally, the surfaces of the microwells (31) are coated with a sacrificial layer, typically a layer of gold, to promote the formation of cavitation bubbles that release particles contained in the microwells. This sacrificial layer can also consist of a thin layer of metal, polymer, gel, etc.

[0052] <General description of the transport system> The transport system consists of several parts: - an optical component comprising a pulsed laser (10) for generating cavitation energy in the carrier liquid, and optionally a camera (20) for observing the donor substrate (30) and the receiver substrate (40). This camera and associated optical system is not required for imaging objects contained exclusively in the microwells, since the positioning of the imaged objects is constrained and does not require correction by optical observation. - an automation component with a robot arm (50) used to automate the handling of the receiver (40), which advantageously has mechanical properties that allow it to absorb shocks in order to guarantee the integrity of the transferred object, either by means of an elastically deformable coating or by means of supports mounted on a damping system along the transfer axis.

[0053] Optionally, the system can include an extruder for adding a binder, for example, collagen, to the receiving substrate (40) between the transferred particle layers.

[0054] More generally, the device may combine several 3D printing, bioprinting, and photopolymerization techniques.

[0055] The substrate (30) consists of the above-mentioned microwell plate held by a support that is advantageously moved by a motorized system (37) that positions the tip of one of the microwells in the optical axis, and knowledge of the geometry of the plate and the constrained positioning of the particles (35, 36) in the microwells (31) allows the transfer of the particles contained in the microwells with a high firing accuracy of less than 50 μm.

[0056] The optical components of the device are sometimes composed of two components: an optional component with a camera (20) for aiming at the object, and a component with a laser (10) for "taking the picture", i.e., delivering an energy pulse in the plane of the donor microwell (30) where the particle to be transferred is located.

[0057] The laser (10) is, for example, a Nd-YAG laser emitting 1-10 ns pulses at 1064 nm with an energy of 15-60 microjoules, which is substantially higher than the energy typically used in LIFT processes.

[0058] Another example is a pulsed ytterbium fiber laser emitting at 1030 nm with shorter pulses of 350 femtoseconds to 10 picoseconds, and energy of tens of microjoules per pulse.

[0059] The power is determined for each shot according to the size of the particles present in the microwells of the optical axis and the appropriate transport mode.

[0060] Lens (16) is typically an F-theta lens with a focal length of 100 mm, suitable for laser scanning. The typical spot size at the focal plane is approximately 30-35 μm in diameter.

[0061] The laser beam (14) passes through shaping optics (13) and is then directed via a set of mirrors (11, 12) to a scanner (15), which then directs the beam perpendicular to the donor substrate (30) via an F-theta lens (16).

[0062] The scanner (15) includes two automatic mirrors that redirect the beam horizontally at an angle toward the objective lens (16). The lens (16) then straightens and focuses the beam (14) so ​​that it strikes and focuses perpendicularly on the donor substrate (30). The scanner mirrors (15) control the movement of the laser beam (14) along a horizontal axis above the donor substrate (30). Thus, the laser beam (14) is focused on the donor substrate (30) and is steerable along the X and Y axes that define a horizontal plane.

[0063] In the case of the donor substrate (30), the beam (14) is focused onto a sacrificial layer coating the substrate surface, for example a 20 nanometer gold layer deposited on a transparent optical window.

[0064] A second optional optical component, including a camera (20), is an aiming component. If the particles are smaller than the size of the microwells used, for example, spheroids formed by cell aggregation with a diameter of 100 μm, it may be necessary to use a visualization system.

[0065] A visible light source, typically an LED (21), is positioned above the cartridge, and a 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 passes the visible light to the camera (20) but reflects the infrared light back to the laser (10). The beam then passes through a lens (23), an aperture (24), and an objective lens (25) before reaching the camera (20). Everything is aligned so that the laser beam (10) is focused at the center of the image captured by the camera (20).

[0066] This image can be used to determine the size of the particles present in the microwells of the firing axis and therefore the power of the pulse required.

[0067] The invention relates to, but is not limited to, the transfer of spheroids, which are formed by the aggregation of cells cultured in the laboratory and have the appearance of small pearls made of cells and extracellular matrix.

[0068] To generate spheroids, stem cells, progenitor cells, or differentiated cells are cultured by conventional methods and seeded into microwells to generate aggregates, which are then manipulated according to the methods of the present invention.

[0069] As mentioned above, the "Imaging the Image Region" section is optional. It is useful when the microwell can accommodate small particles whose positioning is not constrained by the microwell walls. It is then necessary to use the information provided by the imaging system to accurately center the laser beam axis on the particle's center to within 50 µm.

[0070] On the other hand, if the particles are systematically large, larger than the cross section at mid-height of the microwell, this imaging subsystem is optional and can be omitted, as the accuracy of the shot will result from the constrained positioning of the particle in the microwell and the precise positioning of the substrate with the microwell relative to the framework of the device.

[0071] <Flat-bottom microwell> According to the variant shown in Figure 4, the microwell has a truncated inverted pyramidal cavity to limit reflection and diffraction phenomena of the laser shot centered on the microwell. The surface area of ​​the microwell base is larger than the width of the laser beam, typically 50-100 µm.

Claims

1. A method for transferring objects from a donor substrate (30) towards a target substrate (40), comprising: forming a film of carrier liquid containing particles to be transferred, the film having a dimension (D ｘ , D ｙ , D ｚ ) is deposited on the donor substrate (30) containing the object to be transferred, the transfer being caused by localized energy excitation of the liquid to form localized cavitation bubbles in the object, a. The donor substrate (30) comprises a microwell plate having a plurality of wells, each forming a receptacle having an open top base that tapers to a bottom, and the width (L) of the base of the well is such that L>2D ｘ D ｙ and the height (h) of the well is h>2D ｚ and b. The dimension (D ｚ When the ratio of the thickness (e) of the liquid film to the first energy level E １ According to the first transport mode, The dimension (D ｚ ) to the thickness (e) of the liquid film is greater than 1, the second energy level E ２ >E １ wherein the transfer is performed according to a second transfer mode.

2. 10. The method of claim 1, wherein the energy deposition is achieved by focusing a laser for propulsive or LIFT transport modes.

3. 2. The method of claim 1, wherein the energy deposition is performed via an electric field for propulsive transport mode.

4. 10. The method of claim 1, wherein the energization is achieved by focusing acoustic waves for a propulsive transport mode.

5. 2. The method of claim 1, wherein the receiving substrate (40) is provided with damping means along the object transport axis.

6. The transfer of the object is performed without contact by generating cavitation bubbles in the liquid present between the object and the bottom of the well, and the transfer is performed by: In the propulsion mode, by converting the imparted energy into kinetic energy of the object, which falls and leaves the liquid layer in a highly directional manner; 2. A method according to claim 1, characterized in that the method is ensured either by carrying away the object via a jet in a LIFT transport mode, or by converting the applied energy into the movement of the liquid.

7. 2. The method of claim 1, characterized in that the objects to be transported are selected from the categories of cell aggregates, spheroids, organoids, explants (islets of Langerhans), cell-encapsulated polymer particles (organoids covered with a biomaterial layer), cell-seeded microcarriers, biomaterial beads.

8. 10. The method of claim 1, wherein the transport is repeated to generate a material, tissue, or organ, wherein the volume fraction or volume density of biological matter transported by propulsion in the printed material, tissue, or organ is greater than 30%.

9. 10. The method of claim 1, wherein the transfer is performed only once for the purpose of accurately characterizing a single object.

10. 10. The method of claim 1, characterized in that it can be combined with other printing techniques such as extrusion, inkjet, LIFT, etc., to produce complex materials or structures containing different components.

11. an energy source directed at the material being transferred; at least one well into which the material is processed and transferred; a target receiving substrate for collecting the transferred material; The donor has an orientation D in the plane of the liquid film. ｘ , D ｙ , and D perpendicular to the film ｚ 1. An apparatus for energy processing and transfer by application, comprising a plate including wells containing transferable objects disposed in a liquid by a. The substrate comprises a microwell plate having a plurality of wells, each forming a receptacle having an open top base that tapers to a bottom, and the width (L) of the base of the well is such that L>2D ｘ D ｙ and the height (h) of the well is h>2D ｚ and a. The device comprises: The size (D ｚ When the ratio of the thickness (e) of the liquid film to the first energy level E １ So, by LIFT, Or, the size (D ｚ ) to the thickness (e) of the liquid film is greater than 1, the second energy level E ２ >E １ and including means of transport, either by propulsion or 1. An apparatus, characterized in that it incorporates a system for simultaneously delivering several laser beams to an object for its transport along a uniform trajectory when said object has an anisotropic shape.

12. 12. The device of claim 11, wherein the energy deposition is achieved by focusing a laser for two transport modes: propulsion and LIFT.

13. 12. The apparatus of claim 11, wherein the substrate of the plate containing the wells is transparent or weakly absorbing at the wavelength of the laser beam.

14. 12. The device according to claim 11, characterized in that it comprises a scanner that enables the laser beam to be positioned precisely at the center of gravity or mass of each object, thus ensuring high directionality in the transfer of the objects.

15. 12. The device according to claim 11, characterized in that the energization is carried out via an electric field.

16. 12. The device of claim 11, wherein the energization is achieved by focusing acoustic waves.

17. 12. The device according to claim 11, characterized in that it comprises a system for controlling and closed-loop adjusting the value of the applied energy in order to move the object according to its size.

18. 12. Apparatus according to claim 11, characterized in that it incorporates means for automating the movement of the substrate (30).

19. 12. The apparatus of claim 11, wherein the well plate substrate (30) is covered with a sacrificial layer having laser or electric field energy deposition absorption / conductivity properties.

20. 12. Device according to claim 11, characterized in that it comprises means for controlling the temporal succession of several energy depositions on the same well for said transport of objects.

21. 12. The device of claim 11, wherein the device comprises means for controlling multiple spatially separated energy depositions for parallel transport of multiple objects located in different wells.

22. 12. The device according to claim 11, characterized in that the device further comprises at least one other printing technology including extrusion, inkjet, LIFT for the production of complex materials or tissues containing different components.

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