Printing object from aperture

Abstract

The invention relates to a method and a device for transferring an object from a donor substrate (30) containing an object to be transferred and having a size (Dx, Dy, Dz) towards a target substrate (40), the transfer being ensured by local energy excitation of a liquid for forming local cavitation bubbles at the object, characterized in that: a. The substrate consists of a microwell plate having a plurality of pores, each hole forms a container having an open upper base tapering towards the bottom, the width (L) of the base of the hole being Lgt; 2DxDy, and the height (h) of the hole is hgt; 2Dz; and b. The transfer is carried out in such a way that:-if the ratio of the size (Dz) of the object to the thickness (e) of the liquid film is less than 1, the transfer is carried out at a first energy level E1,-if the ratio of the size (Dz) of the object to the thickness (e) of the liquid film is greater than 1, the transfer is carried out at a second energy level E2gt; and the E1 is transferred.

Description

Technical Field

[0001] The present invention relates to the field of additive manufacturing of materials by repeatedly transferring particles between a donor substrate, on which a carrier film containing the particles to be transferred is deposited, and in particular to the field of bioprinting. Specifically, the field of bioprinting involves the use of digital fabrication methods to organize and assemble biological tissue components in 2D and 3D, with the goal of creating implants for regenerative medicine or physiological models for biomedical and pharmaceutical research.

[0002] The general principle is to transfer organisms (e.g., cells), organic objects, or inorganic objects from a carrier liquid deposited in the form of a film on a substrate, and to deliver energy pulses to form cavitation bubbles, either directly by evaporating a portion of the liquid in the focused field of an energy source (usually a laser) or via a thin metal coating forming a sacrificial layer on the donor substrate. The cavitation bubbles carry particles along the firing axis to a receiving substrate, where the transferred particles accumulate as the shots are repeated. Background Art

[0003] In the prior art, patent application CN115198376 is known, which relates to a microporous array chip and a single-cell sorting method for laser-induced direct transfer, wherein a metal-coated glass sheet is coated with a microporous membrane layer; the microporous membrane is a biocompatible membrane and is coated on the metal-coated glass sheet by adopting micro-nano processing technology; due to the hydrophobic properties of the microporous array chip material, single cells form single-cell microdroplets, and the single-cell microdroplets are fixed in the micropores, thereby achieving single-cell capture and facilitating subsequent single-cell sorting.

[0004] Patent application US2020009877 proposes a solution for depositing particles from a transparent glass slide having a film formed by a fluid containing suspended particles onto a target by locally exciting the film with a laser; the solution includes a device for observing the local excitation area. The observation device includes a sensor and a light source, whose optical axes are essentially shared in the space between the optical axis of the beam splitter and the film. The light beam of the imaging system and the light beam of the laser are coaxial in the portion between the controlled optical deflection device and the film. The device includes a first focusing optical unit arranged between the controlled optical deflection device and the film. The device includes a second optical image combination unit located between the sensor and the separator, wherein the sensor is located in the focal plane of the second optical unit.

[0005] Patent application CN113021874 describes a single cell printing method based on transfer induced by an annular laser spot, characterized in that the method comprises the following steps:

[0006] ●Step 1: After the uniformly distributed pulsed laser beam is incident on the components of the annular spot forming system,

[0007] The incident light passes through the transparent constraining layer and is focused on the sacrificial layer to form a ring-shaped light spot;

[0008] Step 2: ablating the sacrificial layer using laser and heat to form a ring-shaped plasma cavitation bubble;

[0009] ●Step 3: The annular cavitation bubble expands rapidly to remove the cell solution outside the ring and

[0010] Under the action of expansion, the target metastatic cells in the center of the ring are pushed downward in parallel;

[0011] ● Step 4, the ring is broken by the shock wave generated in the direction perpendicular to the center position of the ring.

[0012] The cavitation bubble collapses and the target metastatic cell is completely expelled;

[0013] ●Step 5, under the action of gravity and thrust, the target transfer cells and transfer solution are printed onto the receiving plate.

[0014] Patent application US2017225390 relates to a method for additive manufacturing of a three-dimensional object. The method involves sequentially forming a plurality of layers, each layer being 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 distribute at least a first building material component, and performing a vector scan to distribute at least a second building material component. The vector scan optionally occurs along a path selected to form at least one structure, the at least one structure being selected from the group consisting of: (i) an elongated structure, (ii) a boundary structure at least partially surrounding an area filled with a first building material, and (iii) an interlayer connecting structure.

[0015] Patent application US2016259250 relates to a method for providing a patterned structure on a substrate, the method comprising the following steps:

[0016] • providing a donor substrate disposed between the light source and the receptor substrate, the donor substrate comprising a donor material;

[0017] ● providing a mask disposed between a light source and a donor substrate, the mask comprising a mask pattern for shaping light from the light source that is irradiated on the donor substrate, the shaped light corresponding to a patterned structure to be produced, the shaped light irradiated on the donor substrate causing the donor material to be released from the donor substrate and transferred to the receptor substrate to form a patterned structure thereon; wherein,

[0018] • The structured light is split into multiple separate beams of uniform size, which are simultaneously irradiated onto the donor substrate to release the donor material from the donor substrate in the form of separate droplets of uniform size.

[0019] Patent application US2018090314 relates to a material deposition method comprising the following steps: positioning a donor film comprising a donor material at a predetermined distance from a receptor substrate, with the donor film facing the receptor substrate. Directing one or more laser radiation pulses at a given location on the donor film to induce the formation of a protrusion made of the donor material. The distal tip of the protrusion contacts the receptor substrate and deposits thereon while the protrusion remains in contact with the donor film. A dot of donor material is formed on the receptor substrate by increasing the separation between the donor film and the receptor substrate so that the distal tip is separated from the donor film protrusion.

[0020] Disadvantages of existing technology

[0021] Prior art solutions are not well suited to particle sizes above 100 μm and also pose difficulties in centering the emission with respect to the particle to be transferred.

[0022] Furthermore, prior art solutions involve object handling operations that carry a risk of loss of viability, especially for fragile biological objects.

[0023] The present 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 have different sizes. Summary of the Invention

[0024] 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, the method having the technical features set forth in claim 1. More particularly, the invention relates to a method for transferring an object from a donor substrate towards a target substrate, a carrier liquid film containing the particles to be transferred being deposited on a substrate containing a particle having a size (D x , D y , D z ) onto the donor substrate of the object to be transferred, the transfer being achieved by local energy excitation for forming cavitation bubbles positioned at the object, characterized in that,

[0025] a. The substrate consists of a microplate having a plurality of wells, each well forming a container having an open upper base that tapers toward the bottom, the width (L) of the base of the well being L>2D x D y , and the height (h) of the hole is h>2D z ,and

[0026] b. Transfer as follows:

[0027] - If the size of the object (D z) to the thickness (e) of the liquid film is less than 1, the transfer is performed at a first energy level E1 according to a first transfer mode,

[0028] If the ratio of the size of the object to the thickness (e) of the liquid film is greater than 1, the transfer is carried out according to a second transfer mode at a second energy level E2>E1.

[0029] In a first variant, energy deposition is achieved by focusing the laser for both propulsion and LIFT transfer modes.

[0030] In a second variant, for the propulsion mode, energy is deposited via an electric field.

[0031] Generating cavitation bubbles using an electric field involves using electrolysis to generate bubbles in a liquid. Cavitation is a physical phenomenon in which bubbles form and implode in a liquid under variable pressure, generating force and shock waves. The application of an electric field can promote the formation of these bubbles. To generate cavitation bubbles using an electric field, it is known to those skilled in the art to use an electrolytic cell containing a conductive liquid and equipped with two electrodes connected to a direct current source.

[0032] In a third variant, for the propulsion mode, energy deposition is achieved by focusing the acoustic waves.

[0033] The sound waves are generated by an ultrasonic transducer designed to focus the sound energy at a precise point and create an area of low pressure in the liquid, causing bubbles to form.

[0034] In a particular embodiment, the receiving substrate comprises damping means along the object translation axis.

[0035] According to a variant, the transfer of the biological object is carried out contactlessly by generating cavitation bubbles in the liquid present between the object and the bottom of the well, the transfer being ensured by:

[0036] ● Converting the deposited energy into kinetic energy of the object, which detaches and leaves the liquid layer with high directionality in propulsion mode,

[0037] • Or the deposited energy is converted into the movement of a liquid which carries the object away via a jet in LIFT transfer mode.

[0038] The contactless transfer ensures that the object does not deform plastically.

[0039] Advantageously, the objects to be transferred are selected from the class of: cell aggregates, spheroids, organoids, explants (islets of Langerhans), polymer particles encapsulating cells (organoids covered with a layer of biomaterial), cell-seeded microcarriers, biomaterial beads.

[0040] According to a variant, the transfer is repeated to produce a material, tissue or organ, and the volume fraction or density of the biological objects transferred into the printed material, tissue or organ by the advancement is greater than 30%.

[0041] In another variant, only one transfer is performed in order to accurately characterize a single object.

[0042] In another variation, the method is combined with other printing techniques (such as extrusion, inkjet, LIFT) to produce complex materials or tissues with different components.

[0043] The present invention also relates to a device for energy processing and transfer by deposition:

[0044] - an energy source, which is directed towards the material to be transferred,

[0045] - at least one aperture from which the material is processed and transferred,

[0046] - a target receiving substrate which collects the transferred material,

[0047] The donor consists of a plate comprising a well containing a transferable object arranged in a liquid, the transferable object being oriented D in the plane of the liquid film. x 、D y , perpendicular to the film orientation is D z , characterized in that the transfer occurs in the following ways:

[0048] - If the size of the object (D z ) to the thickness of the liquid film (e) is less than 1, the transfer is performed by LIFT,

[0049] - or if the object's dimensions (D z ) to the thickness of the liquid film (e) is greater than 1, the transfer is carried out by propulsion,

[0050] In one variation, energy deposition is achieved by focused laser light for both propulsion and LIFT transfer modes.

[0051] Advantageously, the plate substrate comprising the holes is transparent or weakly absorptive at the wavelength of said laser beam.

[0052] In one variant, a scanner is used to precisely position the laser beam on the centroid or center of mass of each object, thereby ensuring high directionality in the transfer of said objects.

[0053] In another variation, energy is deposited via an electric field.

[0054] In another variation, energy deposition is achieved by focusing the acoustic waves.

[0055] According to another variant, the device comprises a system for controlling and regulating in closed loop the amount of energy deposited so as to optimally transfer the object according to its size.

[0056] In another variant, the apparatus incorporates means for automating the movement of the substrate ( 30 ).

[0057] In another variation, the aperture plate substrate is covered by a sacrificial layer having high absorption / conduction properties for laser or electric field energy deposition.

[0058] Advantageously, the device comprises means for controlling the temporal sequence of several energy depositions on the same hole for the transfer of the object.

[0059] According to another variant, the device comprises means for controlling several spatially separated energy depositions in order to transfer several objects arranged in different holes in parallel.

[0060] According to another variant, the device also has at least one other printing technology to produce complex materials or tissues including different components, and the at least one other printing technology includes extrusion, inkjet, and LIFT.

[0061] According to another variant, the device incorporates a system for simultaneously transmitting several laser beams to the object in order to ensure its transfer along a uniform trajectory when the object has a non-isotropic shape.

[0062] Detailed Description of a Non-Limiting Exemplary Embodiment

[0063] The invention will be better understood after reading the following description, which relates to non-limiting exemplary embodiments illustrated by the accompanying drawings, in which:

[0064] [ Figure 1 ] Figure 1 shows a cross-sectional view of a microwell of an example donor substrate with small particles,

[0065] [ Figure 2 ] Figure 2 shows a cross-sectional view of a microwell of an example donor substrate with large particles,

[0066] [ Figure 3 ] Figure 3 shows a schematic view of the transfer system,

[0067] [ Figure 4 ] Figure 4 shows a cross-sectional view of a microporous variant,

[0068] [ Figure 5 ] Figure 5 A partial top view of a donor substrate is shown.

[0069] Basic background of the invention

[0070] The present invention specifically relates to additive manufacturing of biografts by transferring biological objects larger than 100 μm in size, such as spheroids or organoids, onto a target surface. Spheroids are three-dimensional (3D) cell aggregates that can mimic tissue. When seeded in the wells of a microplate with a pyramidal or frustum-shaped base, these aggregates form discrete spheroids.

[0071] Spheroids contain deeply embedded cells and cells with exposed surfaces, both proliferating and non-proliferating, and are surrounded by a well-oxygenated outer layer of cells in the center. Their assembly by transfer onto a target substrate can generate three-dimensional tissues such as cartilage, for example, to reconstruct damaged cartilage.

[0072] Spheroids and organoids can be composed of different stem cells, progenitor cells, and / or differentiated cells (e.g., heart cells, brain cells, liver cells, etc.).

[0073] 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.

[0074] Overview of donor substrates

[0075] Figure 1 and Figure 2 A view of a microwell (31) on a donor substrate (30) is shown. The microwell (31) is made of a transparent plate comprising a microwell matrix and has an inverted pyramidal shape with an open square base (32) with a width L typically between 200 μm and 800 μm and a pointed or preferably flat bottom (33). Its height h is typically between 200 μm and 800 μm.

[0076] The spheroid (35) consists of approximately 500 cells and occupies only a portion of the height h of the microwell.

[0077] The spheroid (36) consists of approximately 2000 cells and occupies the entire portion of the microwell of height h.

[0078] An aqueous liquid (eg, water with added salt) or a culture medium (eg, a 2% BSA (bovine serum albumin) solution) at least partially fills the microwells (31).

[0079] Depending on the size of the spheroids (35, 36) contained in the microwells (31), the liquid completely covers the spheroids, or the spheroids are retained in the liquid matrix.

[0080] In the first case, the transfer is performed in LIFT mode at a medium power (typically 15 microjoules to 20 microjoules).

[0081] In the second case, where the transfer occurs by propulsion without the particles being carried away in the vacuole, the power is 2 to 5 times greater, at about 30 to 60 microjoules.

[0082] In order to transfer objects according to their size while ensuring their integrity after transfer, the amount of deposited energy is minimized. Minimizing the deposited energy also ensures a low transfer speed, thereby achieving slow deposition on the receiving substrate, thus contributing to the integrity of the transferred object.

[0083] Optionally, the surface of the micropores (31) is coated with a sacrificial layer (usually a gold layer) to promote the formation of cavitation bubbles, thereby expelling the particles contained in the micropores. The sacrificial layer can also be composed of a thin layer of metal, polymer, gel, etc.

[0084] General description of the transfer system

[0085] The transfer system consists of several components:

[0086] - an optical component comprising a pulsed laser (10) for generating cavitation energy of the carrier liquid and, optionally, a camera (20) for observing the donor substrate (30) and the receiving substrate (40). This camera and the associated optical system are not required to image an object completely located in the microwell, since the positioning of the object to be imaged is constrained and does not need to be corrected by optical observation,

[0087] - an automation component having a robot arm (50) for automating the handling of the recipient (40). The receiving substrate (40) advantageously has mechanical properties that allow shock absorption 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.

[0088] And optionally, the system may include an extruder for adding a binder (eg, collagen) to the receiving substrate (40) between the transferred particle layers.

[0089] More generally, the device can combine several 3D printing, bioprinting, and photopolymerization technologies.

[0090] The substrate (30) consists of a microwell plate as described above, held by a support advantageously moved by a motorized system (37) that positions the tip of one of the microwells on the optical axis so as to enable the transfer of the particles contained in the microwells with a high firing accuracy of less than 50 μm, thanks to knowledge of the geometry of the plate and the constrained positioning of the particles (35, 36) in the microwells (31).

[0091] The optical part of the device may consist of two parts: an optional part including a camera (20) for aiming the object, and a part including a laser (10) for "firing" (that is, delivering energy pulses in the plane of the donor microwell (30) where the particles to be transferred are located).

[0092] The laser (10) is, for example, an Nd-YAG laser which emits pulses of 1 ns to 10 ns at 1064 nm with an energy of 15 microjoules to 60 microjoules, which is significantly higher than the energy usually used in the LIFT method.

[0093] Another example is the pulsed ytterbium fiber laser, which emits shorter pulses from 350 femtoseconds to 10 picoseconds at 1030 nm, with energy of tens of microjoules per pulse.

[0094] The power will be determined for each shot based on the size of the particles present in the microwell on the optical axis and the appropriate transfer mode.

[0095] The 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 about 30 μm to 35 μm in diameter.

[0096] 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 sends the beam perpendicularly to the donor substrate (30) via an F-Theta lens (16).

[0097] The scanner (15) includes two automatic mirrors that redirect the light beam horizontally at a certain angle to the objective lens (16). The objective lens (16) then straightens and focuses the light beam (14) so that it arrives perpendicular to the donor substrate (30) and is focused on it. The mirrors of the scanner (15) control the movement of the laser beam (14) along a horizontal axis on the donor substrate (30). As a result, the laser beam (14) is focused on the donor substrate (30) and can be steered along the X-axis and the Y-axis that define the horizontal plane.

[0098] For a donor substrate (30), the light beam (14) is focused on a sacrificial layer coating the substrate surface, such as a 20 nm layer of gold deposited on a transparent optical window.

[0099] A second optional optical component comprising a camera (20) is a sighting component. If the particles are smaller than the size of the microwell used, for example a spheroid formed by an aggregation of cells with a diameter of 100 μm, a visualization system may need to be used.

[0100] A visible light source (typically an LED (21)) is placed above the magazine, and a light beam (22) follows the reverse path of the laser beam through the scanner (15). The light beam then reaches a half-mirror (12), which allows the visible light to pass through the camera (20) but reflects the infrared light back to the laser (10). The light beam then passes through a lens (23), an aperture (24), and an objective lens (25) before reaching the camera (20). All components are aligned so that the laser beam (10) is focused in the center of the image captured by the camera (20).

[0101] This image can be used to determine the size of the particles present in the micropores along the firing axis, and thus determine the required pulse power.

[0102] The present invention relates, in a non-limiting manner, to the transfer of spheroids, which are formed from aggregates of cells cultured in the laboratory and have the appearance of small pearls composed of cells and extracellular matrix.

[0103] To generate spheroids, stem cells, progenitor cells, or differentiated cells are cultured by conventional methods and seeded in microwells to generate aggregates, which are then manipulated according to the methods of the present invention.

[0104] As explained above, the "Imaging the Shot Area" section is optional. This is useful when the microwell can accommodate small particles that are 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 about the particle to within 50 μm.

[0105] On the other hand, when the particles are systematically large, larger than the cross-section at the mid-height of the microwell, this imaging subsystem is optional and can be omitted, since the accuracy of the emission is caused by the constrained positioning of the particles in the microwell and the precise positioning of the substrate with the microwell relative to the device frame.

[0106] Microwells with flat bottom

[0107] according to Figure 4 In the variant shown in , the microwell has an inverted pyramidal cavity with a truncated tip to limit reflection and diffraction phenomena of the laser emission centered on the microwell. The surface area of the microwell base is larger than the width of the laser beam, typically between 50 μm and 100 μm.

Claims

1. A method for transferring an object from a donor substrate (30) toward a target substrate (40), wherein a carrier liquid film containing particles to be transferred is deposited on a substrate having a size (D x , D y , D z ) onto the donor substrate (30) of the object to be transferred, the transfer being achieved by local energy excitation of the liquid for forming cavitation bubbles positioned at the object, characterized in that a. The donor substrate (30) is composed of a microplate having a plurality of holes, each hole forming a container having an open upper base that tapers toward the bottom, and the width (L) of the base of the hole is L>2D x D y , and the height (h) of the hole is h>2D z ,and b. The transfer is carried out in the following manner: - If the size of the object (D z ) to the thickness (e) of the liquid film is less than 1, Then according to the first transfer mode, the transfer is performed at the first energy level E1, - If the size of the object (D z ) to the thickness (e) of the liquid film is greater than 1, the transfer is performed at a second energy level E2>E1 according to a second transfer mode.

2. The method according to claim 1, characterized in that For the propulsion transfer mode or for the LIFT transfer mode, energy deposition is achieved by focusing the laser.

3. The method according to claim 1, characterized in that For the push transfer mode, energy deposition is performed via an electric field.

4. The method according to claim 1, wherein For propulsion transfer mode, energy deposition is achieved by focusing the acoustic waves.

5. The method according to claim 1, wherein The receiving substrate (40) comprises damping means along the object transfer axis.

6. The method according to claim 1, characterized in that The transfer of the object is performed contactlessly by generating cavitation bubbles in the liquid present between the object and the bottom of the well, the transfer being ensured by: - converting the deposited energy into kinetic energy of the object, which in the propulsion mode breaks away from and leaves the liquid layer with high directionality, - or converting the deposited energy into a movement of the liquid, which carries the object away via a jet in LIFT transfer mode.

7. The method according to claim 1, characterized in that Said objects to be transferred are selected from the classes of: cell aggregates, spheroids, organoids, explants (islets of Langerhans), polymer particles encapsulating cells (organoids covered with a biomaterial layer), cell-seeded microcarriers, biomaterial beads.

8. The method according to claim 1, characterized in that The transferring is repeated to generate a material, tissue or organ, and the volume fraction or volume density of the biological object transferred into the printed material, tissue or organ by the advancing is greater than 30%.

9. The method according to claim 1, characterized in that In order to accurately characterize a single object, the transfer is performed only once.

10. The method according to claim 1, characterized in that The method can be combined with other printing techniques such as extrusion, inkjet, LIFT, in order to fabricate complex materials or tissues comprising different components.

11. An apparatus for energy processing and transfer by deposition, the apparatus comprising: - an energy source directed towards the material to be transferred, - at least one hole from which the material is processed and transferred, - a target receiving substrate that collects the transferred material, The donor consists of a plate comprising a well containing a transferable object arranged in a liquid with an orientation D in the plane of the liquid film. x 、D y , perpendicular to the film orientation is D z , characterized in that, a. The substrate consists of a microplate having a plurality of wells, each well forming a container having an open upper base that tapers toward the bottom, the width (L) of the base of the well being L>2D x D y , and the height (h) of the hole is h>2D z , and a. The device has the following transfer modes: - If the size of the object (D z ) to the thickness of the liquid film (e) is less than 1, the transfer is performed by LIFT at a first energy level E1, - or if the size of the object (D z ) to said thickness (e) of said liquid film is greater than 1, the transfer is performed by propulsion at a second energy level E2>E1, And the apparatus incorporates a system for simultaneously delivering several laser beams to the object so that the object is transferred along a uniform trajectory when the object has a non-isotropic shape.

12. The device according to claim 11, characterized in that For both propulsion and LIFT transfer modes, energy deposition is achieved by focusing the laser.

13. The device according to claim 11, characterized in that The substrate of the plate containing the holes is transparent or weakly absorptive at the wavelength of the laser beam.

14. The device according to claim 11, characterized in that The device comprises a scanner which enables the laser beam to be precisely positioned on the centroid or center of mass of each object, thereby ensuring high directionality in the transfer of the objects.

15. The device according to claim 11, characterized in that The energy deposition is performed via an electric field.

16. The device according to claim 11, characterized in that Energy deposition is achieved by focusing the sound waves.

17. The device according to claim 11, characterized in that The device comprises a system for controlling and closed-loop regulating the value of the deposited energy in order to displace the object according to its size.

18. The device according to claim 11, characterized in that The apparatus incorporates means for automating the movement of the substrate (30).

19. The device according to claim 11, characterized in that The aperture plate substrate (30) is covered by a sacrificial layer having laser or electric field energy deposition absorption / conduction properties.

20. The device according to claim 11, characterized in that The device comprises means for controlling the temporal sequence of several energy depositions on the same hole for said transfer of the object.

21. The device according to claim 11, characterized in that The apparatus comprises means for controlling a plurality of spatially separated energy depositions in order to transfer a plurality of objects arranged in different wells in parallel.

22. The device according to claim 11, characterized in that The device also has at least one other printing technology to produce complex materials or tissues including different components, and the at least one other printing technology includes extrusion, inkjet, and LIFT.

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