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WO2025108561A1 - Dispositif d'analyses électrophysiologiques in vitro d'entités biologiques - Google Patents

Dispositif d'analyses électrophysiologiques in vitro d'entités biologiques Download PDF

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Publication number
WO2025108561A1
WO2025108561A1 PCT/EP2023/083056 EP2023083056W WO2025108561A1 WO 2025108561 A1 WO2025108561 A1 WO 2025108561A1 EP 2023083056 W EP2023083056 W EP 2023083056W WO 2025108561 A1 WO2025108561 A1 WO 2025108561A1
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Prior art keywords
layer
shape
hydrogel
passive layer
polymeric material
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PCT/EP2023/083056
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English (en)
Inventor
Akouissi OUTMAN
Eleonora MARTINELLI
Stephanie P. Lacour
Luca LIEBI
Ivan FURFARO
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Ecole Polytechnique Federale de Lausanne EPFL
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Ecole Polytechnique Federale de Lausanne EPFL
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Priority to PCT/EP2023/083056 priority Critical patent/WO2025108561A1/fr
Publication of WO2025108561A1 publication Critical patent/WO2025108561A1/fr
Pending legal-status Critical Current
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
    • G01N33/4836—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures using multielectrode arrays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells

Definitions

  • the present invention generally belongs to the fields of electronics, material science, biotechnology and laboratory instruments.
  • the invention concerns a device configured for electrophysiological analyses of biological entities such as for instance organoids and spheroids.
  • the brain is one of the most complex organs of the human body which makes it difficult to study. Most of the brain analysis is currently done on animals or on postmortem specimens. An in vitro specimen representative of the brain is needed to allow more accessible studies. First, studies were carried out on traditional two-dimensional (2D) cell cultures. However, due to the incomplete representation of a real brain inherent to the 2D nature of a cell culture, a better model is required to perform more advanced studies.
  • the cerebral spheroid which is an aggregate of neuron cells derived from human stem cell, has drawn the attention because it is viable specimen between the 2D cell culture and the in vivo animal brain. Indeed, since it has a 3D structure, its behavior is closer to the human brain compared to 2D cell cultures.
  • Neural organoids and spheroids offer exciting opportunities for understanding neuronal connectivity and exploring neurological disorders. Electrophysiological recordings play a crucial role in assessing these parameters.
  • the cerebral spheroid has a different geometry compared to a functional human brain, a different technology must be used in order to record its electrophysiological parameters.
  • MEA 2D multi-electrode array
  • the present inventors developed a device configured for monitoring and analysing electrophysiological parameters of biological entities such as embryoids, organoids or spheroids having improved features and capabilities.
  • a first purpose of the present invention is that of providing a device optimally configured for accommodating, culturing and electrophysiologically analysing over time biological entities such as for instance organoids and spheroids.
  • Another purpose of the present invention is that of providing a device having morphing structure robustly anchored to an electrically patterned support, while being fully immersed into a receptacle for long-term cell culture.
  • Still another purpose of the present invention is that of providing a device which can be manufactured through state-of-the-art cleanroom procedures (thereby assuring repeatability and reliability of the system), adaptable to various design and variation of elements, reusable and compatible with microfluidic chip designs.
  • one object of the present invention relates to a device comprising:
  • a responsive layer on the passive layer composed of a second polymeric material, the second polymeric material being a mechanically adaptive material capable of changing shape upon a trigger stimulus
  • the at least one shape changing unit including a base portion on said plane and a raising portion adapted to raise from the base portion to contact a biological entity arranged on the plane to raise, the raising portion raising as a consequence of a trigger stimulus changing the shape of the shape changing unit.
  • the devices further comprises an array of conductive tracks in or on the passive layer, and the shape changing unit further including electrical pads, each pad being connected to an end of a track of said conductive tracks.
  • the at least one shape changing unit may be a peninsula surrounded by the at least one cutting line, except in a region where the peninsula is continuous with the remaining portion of the passive layer and the responsive layer (the portion not affected by the cut). Therefore, the raising portion may deflect from the plane where the remaining portion of the passive layer and the responsive layer lay.
  • the responsive layer is arranged only in correspondence of the at least one shape changing unit.
  • Raising means that the raising portion no more lays on the plane when it raises. Different degrees of raising may bring the raising portion to different shapes, including curvatures.
  • the at least one shape changing unit may have the shape at rest of a flat leaf or petal which curves upright after application of the trigger stimulus. The idea is that the raising portion changes shapes until contacting a surface of the biological entity, where it assumes the shape of the entity.
  • raising at least in some possible embodiments of the device, has not to be interpreted in limiting terms, meaning that raising may be towards one direction or towards the opposite direction. In other words, if a reference system is given, the raising portion may raise towards the one direction and/or lower down towards the opposite direction.
  • the raising portion may have a same shape of the at least one cutting line.
  • a border or peripheral portion of the raising portion may have the same shape of the at least one cutting line.
  • the raising portion may have different shapes than the shape of the at least one cutting line, and the shape of the cutting delimits at least an opening through the passive layer and the responsive layer.
  • This configuration is preferable in case the biological material has solid or semisolid consistency or when it is preferably to allow its growing, at least partially, in both directions, i.e., upwards but also downwards, through the opening.
  • a plurality of cutting lines is provided.
  • At least two cutting lines delimit corresponding at least two shape changing units.
  • the base portions of the at least two shape changing units are distanced on the plane to delimit a portion on the passive layer for locating the biological entity, and the at least two cutting lines have shapes symmetrical with respect to a line passing through a centre of the portion so as, when the raising portions raise from the plane, as a consequence of a same trigger stimulus, the shape changing units are subject to a same shape change and contact, with same contact areas, the biological entity from opposite sides thereof.
  • the biological material is therefore embraced from opposite sides in equal measure by the raising portions curved on it.
  • a grater plurality of cutting lines is provided, as a plurality of petals originating from the portion P on the passive layer, symmetrical two by two.
  • a plurality of shape changing units forms a shape changing module 650, as indicated in fig. A.
  • the device may preferably include a first rigid support for supporting the passive layer on the plane.
  • a second rigid support is provided for protection.
  • the passive layer, the responsive layer and the array of conductive tracks in or on the passive layer are also said below “intermediate layer”, and the second rigid support is placed on top of the intermediate layer for protection thereof.
  • the second rigid support delimits a chamber with an opening.
  • the opening is over the intermediate layer.
  • the intermediate layer is arranged between the second rigid support and the first rigid layer so as the at least one shape changing unit is located inside the chamber, with the raising portion allowed to raise towards the opening (or downwards), i.e., it is arranged to float in the chamber.
  • the opening is suitable to deposit a cell culture or other biological entities (e.g. organoids or embryoids) onto the intermediate layer to grow or culture the biological entity thereon.
  • a cell culture or other biological entities e.g. organoids or embryoids
  • the first rigid support preferably includes a hole in correspondence to the shape changing unit, so as the raising portion is floating in the chamber in both directions, towards the opening and in the opposite direction towards the hole of the first rigid support.
  • At least one anchoring element interconnects with no discontinuities the portion of the passive layer for locating the biological entity with a portion of the passive layer where the at least one shape changing unit is not delimited.
  • the at least one anchoring element is not interested by the cutting lines and provides continuity to the passive layer (and to the responsive layer, if coupled on the whole surface of the passive layer).
  • the at least one shape changing unit is attached to the anchoring element through the portion of the passive layer for locating the biological entity.
  • the device includes for instance:
  • a second rigid support placed on top of said first rigid support, the second rigid support comprising a hole and defining a volume adapted to be used as a cell culture chamber;
  • said intermediate layer comprises
  • the shape changing units stemming from said anchoring element and floating inside said void portion, the shape changing units further comprising a responsive layer composed of a second polymeric material, facing and in contact with the passive layer, said second polymeric material being a mechanically adaptive material capable of changing shape upon a trigger stimulus, thereby allowing the shape changing units to change their shape upon a trigger stimulus.
  • said mechanically adaptive material is capable of changing shape upon a trigger stimulus selected from one or more of temperature change, electric field change, magnetic field change, light change, pressure change, pH change, ionic strength change and swelling by liquid absorption.
  • said mechanically adaptive material is substantially composed of a hydrophilic material, particularly a hydrogel.
  • said hydrogel is selected from a non-limiting list comprising Polyacrylic acid, Polyacrylamide, Polyhydroxyethilmethacrylate.
  • said hydrogel is composed of Polyacrylic acid.
  • the second polymeric material has a monomer concentration comprised between 1 and 50 wt%.
  • the second polymeric material has an expansion strain coefficient upon liquid absorption, defined as the ratio of the difference in area between dried and swollen states, comprised between 1.1 and 100.
  • the second polymeric material is manufactured by monomers crosslinking, with a crosslinker concentration comprised between 0.01 wt% and 10 wt%.
  • the crosslinker is selected from a non-limiting list comprising MBAA, PEGDA, EGDMA.
  • the first polymeric material has a Young’s modulus comprised between 1 kPa and 10 GPa.
  • the second polymeric material when fully hydrated or fully swollen, has a Young’s modulus comprised between 100 Pa and 1 MPa.
  • the first polymeric material is selected from a non-limiting list comprising Parylene, Polyimide, Sll-8, PDMS, Polyurethane, SEBS.
  • the thickness ratio between said passive layer and said responsive layer is comprised between 0.1 and 100 when dry.
  • said conductive tracks comprise: i) a plurality of contact pads for electrical contact with an external device, ii) elongated conductive paths, each departing at least from one of said contact pads, and running along the passive layer and the at least one anchoring element to arrive to at least one shape changing unit, and ii) a plurality of electrodes, each located at the distal end of at least one conductive track, the electrodes being configured to electrically interface the external environment, particularly biological material.
  • the elongated conductive paths are passivated with a passivating layer.
  • Another object of the present invention relates to the use of the device according to the invention for electrophysiological analyses of biological entities as per claim 16.
  • said biological entities selected from, but not limited to, egg cells, zygotes, embryos, animal tissues or a portion thereof, organoids, spheroids and larvae.
  • FIG. A schematically represents a top view of a device according to the present invention.
  • Fig. B is a cross section of a detail of the device according to an embodiment of the present invention.
  • FIG. 1 A and 1 B are, respectively, magnified view of fig. A and B;
  • Fig. B’ is a cross section of a detail of the device according to fig. A, in an embodiment in which a first and second rigid support are provided.
  • Fig. B is a cross section of a detail of the device according to fig. A, in an embodiment in which the first rigid support has a different arrangement than the one represented in fig. B’;
  • FIG. 1 shows an exploded view according to one embodiment of the device of the invention
  • FIG. 2 shows a top view of the intermediate layer according to one embodiment of the device of the invention
  • FIG. 3 shows a top view of the shape changing units and anchoring elements according to one embodiment of the device of the invention
  • Fig. 4 shows unactuated and actuated configurations of the shape changing units according to one embodiment of the device of the invention, the actuation being triggered upon application a trigger stimulus;
  • Fig. 5 shows an implemented exemplary embodiment of the device of the invention, named e-Flower.
  • A Optical image of the e-Flower platform.
  • the e-Flower includes a flower-shaped electrode-carrying polyimide (PI) foil accompanied by a backing layer of polyacrylic acid (PAA) hydrogel, encased within a PMMA fluidic channel.
  • PPA polyacrylic acid
  • B Schematic illustration of the 2D- to-3D shape reconfiguration of the e-Flower driven by the differential swelling properties of the PAA/PI layers.
  • the hydrogel layer swells when immersed in solution, such as water or cell culture media, while the electrode-bearing PI layer remains unaltered.
  • Fig. 6 shows PAA hydrogel re-swelling.
  • Mass ratio re-swelling curves as a function of time (A), area (B) and thickness (C) re-swelling ratios of dehydrated PAA hydrogel samples synthesized with 1X crosslinker concentration and re- swollen in various media (n 6 per condition).
  • Fig. 7 shows PAA/PI hybrid bilayers.
  • Fig. 8 shows the design and electrochemical characterization of the e- Flower.
  • A Left: optical photograph of the complete e-Flower device. The device includes an inlet, an outlet and the main well where the electrodes are positioned. Right: schematics of electrode distribution on the the e- Flower. Inlet: scanning electron micrograph of one electrode.
  • B Exploded view of the PMMA components forming the e-Flower platform.
  • C Bright field image of the e-Flower in closed configuration when immersed in DIW. The arrowhead indicates the swollen PAA layer.
  • (E) Top: electrochemical impedance modulus at 1 kHz for ground and recording electrodes over three swelling and drying cycles (n 3 e-Flower devices). The total number of electrodes considered is indicated for each swelling cycle. Bottom: electrochemical impedance modulus at 1 kHz over the same three swelling and drying cycles as a function of the position of the recording electrodes along the e- Flower petals;
  • FIG. 9 shows the three-dimensional spatiotemporal recordings of spontaneous neural activity of brain spheroids.
  • A Optical photographs of a brain spheroid while being entrapped by the e-Flower: (i) e-Flower in dry state; (ii) e-Flower partially closed around the spheroid (green dotted line) due to a drop of cell culture media (red dotted line); (iii) e-Flower fully closed around the brain spheroid and surrounded by cell culture media.
  • B Overlaid field potential waveforms detected by the 32 electrodes along the e-Flower in a 5 min recording.
  • the expression “operatively connected” and similar reflects a functional relationship between the several components of the device or a system among them, that is, the term means that the components are correlated in a way to perform a designated function.
  • the “designated function” can change depending on the different components involved in the connection.
  • any two components capable of being associated can also be viewed as being “operably couplable,” to each other to achieve the desired functionality.
  • a person skilled in the art would easily understand and figure out what are the designated functions of each and every component of the device or the system of the invention, as well as their correlations, on the basis of the present disclosure.
  • a “soft” material is any material that is either compressible, reversibly compressible, elastic, viscoelastic stretchable or any combination thereof.
  • stretchable refers to the elastic behaviour of an item and is herein used to mean an intrinsic or engineered property of a material or structure that allows such material or structure to withstand a large elongation or multidirectional strain upon a mechanical stress, upon a single or multiple cycles, comprised between 1 and 500%, typically of >5% of the elongation of a soft structure at rest, such as for instance more than about 10%, more than about 20%, more than about 50%, more than about 100% or even more than about 200% of a soft structure at rest without cracking or loss of its physical and/or mechanical properties, which represents an advantage in those contexts and/or body structures in which several cycles of mechanical stresses over time can be foreseen.
  • Suitable polymers according to the present disclosure may comprise one or more compounds selected from a non-exhaustive list comprising thermosets or thermoplastics such as styrene butadiene, styrene (SBS) or styrene ethylene butylene styrene (SEBS), alkyds, epoxies, phenolics (e.g., Bakelite), polyimides, formaldehyde resins (e.g., urea formaldehyde or melamine formaldehyde), polyester thermosets, unsaturated polyesters, polyurethane, bis-maleimides (BMI); polyvinyl chloride (PVC), neoprene, uncrosslinked neoprene, polyethylene (PE), cross-linked polyethylene (PEX), polyether, ethylene-vinyl acetate (EVA), polyethylene-vinyl acetate (PEVA), polypropylene glycol (PPG), latex; e
  • polydimethylsiloxane PDMS polydimethylsiloxane PDMS) or fluorosilicone rubber
  • thermoplastic elastomers such as styrenic block copolymer (SBC), ethylene propylene diene monomer (EDPM) rubber, butyl rubber, nitrile rubber
  • poly(lactic-co-glycolic acid) (PLGA) lactide and glycolide polymers, caprolactone polymers, hydroxybutyric acid, polyanhydrides, polyesters, polyphosphazenes, polyphosphoesters and poly(glycerol sebacate acrylate), polypropylene, polypropylenoxide or their derivatives, polymethylenoxide or its derivatives, polyethylene or its derivatives such as polyethylene glycole (PEG), polyethylenoxide or their derivatives, polyacrylate or its derivatives such as poly(2-hydroxyethylmethacrylate) (PHEMA), poly(vinyl alcohol) (PVA),
  • Further polymers according to the present disclosure may comprise one or more compounds selected from a non-exhaustive list comprising natural polymeric material (i.e. , non-synthetic polymers, polymers that can be found in nature) and/or polymers derived from Extra Cellular Matrix (ECM) such as gelatin, elastin, collagen, agar/agarose, chitosan, fibrin, proteoglycans, a polyamino-acid or its derivatives, preferably polylysin or gelatin methyl cellulose, carbomethyl cellulose, polysaccharides and their derivatives, preferably glycosaminoglycanes such as hyaluronic acid, chondroitinsulfate, dermatansulfate, heparansulfate, heparine, keratansulfate or alginate, as well as any derivative thereof, fragment thereof and any combination thereof.
  • ECM Extra Cellular Matrix
  • hydrogel refers to a gel in which the swelling agent is water or other polar solvent.
  • a hydrogel is a macromolecular polymer gel constructed of a network of crosslinked polymer chains. It is synthesized from hydrophilic monomers, sometimes found as a colloidal gel in which water is the dispersion medium. Hydrogels are highly absorbent (they can contain over 90% water) natural or synthetic polymeric networks. As a result of their characteristics, hydrogels develop typical firm yet elastic mechanical properties.
  • a trigger stimulus comprises physical stimuli such as temperature, electric field, magnetic field, light, pressure and sound, as well as chemical stimuli such as pH, ionic strength, solvent composition and molecular species.
  • a mechanically adaptive material is capable of changing its mechanical properties upon one or more of temperature change, electric field change, magnetic field change, light change, pressure change, pH change, ionic strength change and swelling by liquid absorption.
  • film or “thin film” relate to the thin form factor of an element of the device of the invention such as the various layers of the device.
  • a “film” or “thin film” as used herein relates to a layer of a material having a thickness much smaller than the other dimensions, e.g. at least one fifth compared to the other dimensions.
  • a film is a solid layer having an upper surface and a bottom surface, with any suitable shape, and a thickness generally in the order of nanometres, micrometres or millimetres, depending on the needs and circumstances, e.g. the manufacturing steps used to produce it.
  • films according to the invention have a thickness comprised between 0.01 pm and 1 mm, such as between 5 pm and 1 mm, between 10 pm and 1 mm, between 5 pm and 500 pm, between 50 pm and 500 pm between, between 50 pm and 150 pm, 100 pm and 500 pm or between 200 pm and 500 pm.
  • conductive track refers to any film, path, stripe, strand, wire or the like which is electrically conductive in nature.
  • electrode is herein used to mean the distal part of a conductive track which is in direct contact with the external environment under analysis (i.e. from which one wants for instance to record/stimulate), such as for instance a subject’s tissue or a biological sample.
  • Conductive tracks according to the present disclosure are used to connect and/or close an electrical circuit, and are thus usually electrical connectors or “interconnects”.
  • a conductive track is generally a metallic element that conducts an electric current toward or away from an electric circuit, but can be made of any suitable electrically conductive material, including but not limited to metals such as Au, Pt, Al, Cu and the like, as well as any alloy, oxides and/or combinations thereof; conductive polymeric materials; composite material such as polymeric materials embedding metal particles and/or metal strands or stripes, including insulating materials functionalized with electrically conductive flakes or fibers, for example carbon-filled polymers; liquid metals, including alloys or oxides thereof, such as gallium; electrically conductive inks; as well as any suitable combination thereof.
  • any suitable electrically conductive material including but not limited to metals such as Au, Pt, Al, Cu and the like, as well as any alloy, oxides and/or combinations thereof; conductive polymeric materials; composite material such as polymeric materials embedding metal particles and/or metal strands or stripes, including insulating materials functionalized with electrically conductive flakes or fibers, for example carbon-filled
  • a “compliant electrode” is any structure or element able to mono- or bidirectionally transfer an electric current, and adapted to change its shape according to the shape change of the support it adheres to without substantially compromising mechanical or electrical performance.
  • Examples of complaint electrodes known in the art include metal thin-films (including patterned electrodes, of out-of-plane buckled electrodes, and corrugated membranes), metal-polymer nano-composites, carbon powder, carbon grease, conductive rubbers or paints, a review of which is provided by Rosset and Shea (Applied Physics A, February 2013, Volume 110, Issue 2, pp 281-307), incorporated herein in its entirety by reference.
  • stretchable electrodes as the one described in International Patent Application WO 2004/095536, incorporated herein in its entirety by reference, can be used.
  • tubular or plain elements filled with a ionic liquid, a hydrogel or with liquid metals such as mercury or gallium, or even alloys, oxides or combinations thereof, can be used.
  • the electrode material can be deposited on a film or layer according to the disclosure using a variety of techniques known in the art including, but not limited to, printing, pad printing, screen printing, silk screening, flexography, gravure, offset lithography, inkjet, painting, spraying, soldering.
  • the electrode can be formed by depositing an electrically conductive coating or layer by spraying a preselected onto the designated surface region.
  • the electrode can be formed by depositing the electrically-conductive material onto a region of a film or layer by vacuum deposition or printing the electrically conductive material on a designated surface region. This provides an electrically conductive coating of a desired thickness and a relatively uniform electrode through the desired area.
  • Printing processes can include pad printing, screen printing and the like.
  • Touch-free technologies such as positive material deposition of ink such as from a syringe or similar devices can also be used to transfer conductive film or ink onto the membrane or substrates that are sensitive to pressure.
  • the term “flexible” is used herein to refer to elements of the device of the present invention that can perform a deflection.
  • the term “deflection” refers to any displacement, expansion, contraction, bending, torsion, twist, linear or area strain, or any other kind of deformation, of at least a portion of the device structure.
  • the word “sensing” relates to the ability of the device of the invention to detect the presence of e.g. an electrophysiological signal in a sample through for instance electrophysiological analysis.
  • the word “measuring” relates to the ability of the device of the invention to estimate one or more of the properties of a sample, such as its biochemical or electrical activity, through for instance electrophysiological analysis.
  • the word “monitoring” relates to the ability of the device of the invention to control and/or record the trend of one or more variable properties of a sample, such as its developmental stage, over time, through for instance electrophysiological analysis.
  • a “bioactive agent”, as well as “bioactive molecule” or “bioactive compound”, is any compound or agent that is biologically active, i.e. having an effect upon a living organism, tissue, or cell.
  • the expression is used herein to refer to a compound or entity that alters, inhibits, activates, or otherwise affects biological or chemical events.
  • Bioactive compounds according to the present disclosure can be small molecules or macromolecules, including recombinant ones.
  • One skilled in the art will appreciate that a variety of bioactive compounds can be used depending upon the needs, e.g. a condition to be induced to a biological entity according to the invention.
  • a non-exhaustive list of suitable bioactive agents includes pharmacologically active substances, drugs such as antibiotics or chemotherapeutics, peptides, enzymes, antibodies, vitamins and the like.
  • Exemplary bioactive agents further include, but are not limited to, a growth factor, a protein, a peptide, an enzyme, an antibody or any derivative thereof (such as e.g. multivalent antibodies, multispecific antibodies, scFvs, bivalent or trivalent scFvs, triabodies, minibodies, nanobodies, diabodies etc.), an antigen, any type of nucleic acid, such as e.g.
  • DNA DNA, RNA, siRNA, miRNA and the like, a hormone, an anti-inflammatory agent, an anti-viral agent, an anti-bacterial agent, a cytokine, a transmembrane receptor, a protein receptor, a serum protein, an adhesion molecule, a lipid molecule, a neurotransmitter, a morphogenetic protein, a differentiation factor, an analgesic, organic molecules, polysaccharides, a matrix protein, a spore, a cell, and any functional fragment or derivative of the foregoing, as well as any combinations thereof.
  • a “functional fragment” is herein meant any portion of an active agent able to exert its physiological/pharmacological activity.
  • a functional fragment of an antibody could be an Fc region, an Fv region, a Fab/F(ab’)/F(ab’)2 region and so forth.
  • the main aim of one aspect of the present invention was the development of a non-invasive 3D platform to record the electrophysiological activity of biological entities, including but not limited to egg cells, zygotes, embryos, animal tissues or a portion thereof, organoids, spheroids and larvae.
  • the inventors proposed a flower-shaped 3D morphing platform based on a microarray of electrodes deposited on first passive layer.
  • the considerations leading to this design are several fold, and mainly relate to the aims of
  • [00103] 1 obtaining a maximum of physical and/or electrical contacts with a target biological entity, independent of the shape and the size of said biological entity, and/or proximity to the target biological entity to enable for instance optical, thermal, electrical, physical and/or chemical interfacing (recording and stimulation);
  • the 3D actuation of the device is allowed by the presence of a bilayer that composes a portion of the intermediate layer, the so-called shape changing units.
  • Said intermediate layer is constituted, at the level of the shape changing units, of the previously mentioned electrode-containing passive layer located on top of a mechanically-adaptive responsive layer, capable of deflecting upon application of one or more trigger stimuli.
  • a 3D MEA platform based on shape-morphing bilayers to engulf brain spheroids and enable recordings and stimulations from their entire surface.
  • e-Flower an implemented exemplary embodiment of the device of the invention, named e-Flower.
  • the proposed self-folding MEA includes a flower-shaped electrodecarrying polyimide (PI) foil accompanied by a backing layer of polyacrylic acid (PAA) hydrogel.
  • PI flower-shaped electrodecarrying polyimide
  • PAA polyacrylic acid
  • the hydrogel layer swells when immersed in solution, be it water or cell culture media, while the electrode-bearing PI layer remains unaltered. This produces the 2D-to-3D reconfiguration of the system: the flower closes, the petals (shape changing, reconfigurable units) bend and bring the electrodes in close contact with the outer surface of the brain spheroid.
  • Programmable shape-morphing materials are gaining interest in many fields, going from drug delivery to biomedical devices.
  • a commonly used method to obtain bending/folding functional structures is to combine a functional material layer to a structural one, creating hybrid bilayers.
  • hydrogels are promising materials for designing structures as they can respond to a wide range of external or internal stimuli, such as chemical composition, temperature, pH, and electric field.
  • Hydrogel bilayers consisting of two types of materials with different volume expansion properties go through differential deformation upon swelling, results in a unique 3D structure.
  • the device of the invention can be devoid of electrical, electronic or sensor elements, thereby providing a platform for locking in place certain biological entities.
  • the device may represent an advanced culture solution in context such as long-term, dynamically-adaptable and possibly microfluidic-based cell culture, targeting mainly solid biological entities generally in the range of 100 to 1000 pm in diameter.
  • the device comprises a passive layer 301 composed of a first polymeric material, the passive layer 301 extending on a plane PL; a responsive layer 302 on the passive layer 301 , composed of a second polymeric material, the second polymeric material being a mechanically adaptive material capable of changing shape upon a trigger stimulus.
  • the responsive layer 302 is represented in figure B as arranged below a part or region of the passive layer 301 , i.e., the part or region forming the shape changing unit 600. However, according to a different embodiment, the responsive layer 302 may be attached below the passive layer 301 at any region where the passive layer 301 is extended.
  • An array of conductive tracks 400 is in or on the passive layer 301.
  • the arrangement of the responsive layer 302 and the passive layer 301 (with the array of conductive tracks 400) is also said below as intermediate layer 300.
  • the array of conductive tracks 400 is represented only partially. However, the array may be, for instance, as the one represented in more detail in Fig. 2.
  • At least one cutting line 950 pass through the passive layer 301 and the responsive layer 302 delimiting at least one shape changing unit 600.
  • the at least one shape changing unit 600 includes a base portion 630 on said plane PL and a raising portion 640 adapted to raise from the base portion 630 to contact a biological entity 800 arranged on the plane PL to raise.
  • the biological entity may for instance derive from a cell culture which has grown over time, after being deposited onto the device, as will be explained.
  • the raising portion 640 raises as a consequence of a trigger stimulus changing the shape of the shape changing unit 600.
  • raising is indicated with an arrow upward in the schematic drawing of fig. B, but this is only an example.
  • the shape changing unit 600 further includes electrical pads 401 , each pad being connected to an end of a track of said conductive tracks 400.
  • the raising portion 640 has a same shape of the at least one cutting line 950.
  • the raising portion 640 has a border or peripheral portion and the cutting line 950 follows said border or peripheral portion.
  • the raising portion has a surface area delimited by the border and the surface area of the raising portion has a width (much more) greater than the cutting line 950.
  • the raising portion 640 lays on the plane in adherence with the other regions of the passive layer not delimiting the shape changing units.
  • the shape changing units are so close to the other regions (i.e.
  • the cutting lines are so thin) that the cell culture deposited onto the passive layer 301 , especially a cell culture in solid, semisolid (such as gel) state, is prevented from entering the cutting lines.
  • the cutting lines in this case are thin channels, for instance in the order of nanometres or microns.
  • the cutting lines 950 has a greater size but a same shape of the shape changing unit 600; in this case, the cutting lines 950 are larger channels, for instance in the order of micrometres or millimetres, following the border of the shape changing unit 600.
  • the cutting line 950 is coupled one by one to the shape changing unit. In another embodiment, more cutting lines are coupled to a same shape changing unit.
  • the raising portion 640 has a different shape than the shape of the at least one cutting line 950.
  • the shape of the cutting lines 950 delimits at least an opening 900 through the passive layer 301 and the responsive layer 302.
  • An embodiment with openings 900 is for instance disclosed at fig. 3 (and described further below).
  • the raising portion 640 has a border or peripheral portion but, in this case, the cutting lines 950 follows the border of the raising portion and then distance from it to delimit the opening 900.
  • the surface area of the raising portion delimited by the border may have a width greater or lower than the openings 900.
  • At least two cutting lines 950 delimit corresponding at least two shape changing units 600, wherein the raising portions 640 of the at least two shape changing units 600 are distanced on the plane PL to delimit a portion P on the passive layer 301 for locating the biological entity 800.
  • the two cutting lines 950 have shapes symmetrical with respect to a line B passing through a centre of the portion P so as, when the raising portions 640 raise from the plane PL, as a consequence of a same trigger stimulus, the shape changing units 600 are subject to a same shape change to contact with same contact areas the biological entity from opposite sides thereof.
  • the passive layer 301 , the responsive layer 302 and the array of conductive tracks 400 in the passive layer 301 are an intermediate layer 300.
  • a first rigid support 100 supports the intermediate layer 300 on the plane PL (see fig. B’).
  • a second rigid support 200 is provided.
  • the second rigid support 200 is placed on top of the intermediate layer 300 for protection (fig. B’)
  • the second rigid support 200 delimits a chamber 350 with an opening 201 over the intermediate layer 300.
  • the intermediate layer is arranged between the second rigid support 200 and the first rigid layer 100 so as the at least one shape changing unit 600 is located inside the chamber 350, with the raising portion 640 allowed to raise towards the opening 201 .
  • the opening 201 is suitable to deposit a cell culture onto the intermediate layer 300 to grow the biological entity 800 thereon.
  • the responsive layer 302 is coupled to the passive layer 301 also in areas not forming the shape changing units 600.
  • the responsive layer 302 is coupled to the passive layer 301 only in areas forming the shape changing units 600; in this case, the first rigid support 100 is in direct contact with the passive layer 301 in areas not forming the shape changing units 600.
  • the raising portion 640 is floating in the chamber 350. Floating may be downward or upwards, as indicated in fig. B”. Downward and upwards movements is also possible in all the embodiments where the rigid support is not provided directly below the responsive layer.
  • the shape changing unit(s) are anchored to the passive layer 301.
  • at least one anchoring element 500 (indicated for instance in fig. 3) interconnects with no discontinuities the portion P of the passive layer 301 for locating the biological entity 800 with a portion of the passive layer 301 and the responsive layer 301 where the at least one shape changing unit 600 is not delimited.
  • the at least one shape changing unit 600 is attached to the anchoring element 500 through the portion P of the passive layer 301 for locating the biological entity 800.
  • anchoring elements are delimited by cutting lines also delimiting the shape changing units.
  • the anchoring elements are formed by the whole surface of the passive layer 201 not interested by cutting lines. Narrow cutting lines as those disclosed in embodiment A result in strong anchoring.
  • the invention features a device 1 comprising:
  • a passive layer 301 composed of a first polymeric material
  • At least one anchoring element 500 composed of at least the first polymeric material, stemming from said intermediate layer 300 and entering inside said void portion 310, and
  • the shape changing units 600 stemming from said anchoring element 500 and floating inside said void portion 310, the shape changing units 600 further comprising a responsive layer 302 composed of a second polymeric material, facing and in contact with the passive layer 301 , said second polymeric material being a mechanically adaptive material capable of changing shape upon a trigger stimulus, thereby allowing the shape changing units 600 to change their shape upon a trigger stimulus.
  • the void portion 310 of the intermediate layer 300 and the hole 201 in the second rigid support 200 may form the chamber 350.
  • the intermediate layer 300, passive layer 301 and responsive layer 302 are typically configured as flexible thin films, having thickness in the nanometres to micrometres range, preferably between 0.01 and 500 pm.
  • the passive layer 301 is reversibly stretchable (elastic).
  • passive layer 301 can withstand an elongation or multidirectional strain, upon a single or multiple cycles, comprised between 1 and 500%, preferably at least 5%, such as about 50%, about 100% or about 200%, of its size at rest without cracking or loss of its mechanical properties.
  • the responsive layer 302 is reversibly stretchable (elastic).
  • the flexible/elastic behavior of the passive layer 301 and responsive layer 302 is provided by the materials they are substantially composed of.
  • the passive layer 301 and/or the responsive layer 302 have typically a Young's modulus comprised between about 100 Pa and 10 GPa, such as for instance between about 100 kPa to about 1 GPa, between about 100 kPa to about 1 GPa, between about 5 MPa to about 1 GPa, between about 100 kPa to about 100 MPa, between about 100 kPa to about 5 MPa, between about 10 kPa to about 300 kPa or between about 10 kPa to about 10 MPa, preferably between about 1 MPa to about 10 MPa, which are suitable ranges of values matching the Young's modulus of many biological tissues and surfaces to avoid mechanical mismatches between said tissues and a biomedical device, and/or for mimicking physical and/or mechanical properties of bodily tissues.
  • “physical and/or mechanical properties” means, by way of examples, stress-strain behaviour, elastic modulus, fracture strain, conform ability to curvilinear surfaces, thickness, area and shape which have to be as similar as possible to those to be found in biological tissues.
  • the passive layer 301 is substantially composed of a first polymeric material having a Young’s modulus comprised between 1 kPa and 10 GPa.
  • suitable first polymeric materials include Parylene, Polyimide, Sll-8, PDMS, Polyurethane, SEBS.
  • the responsive layer 302 is substantially composed of a second polymeric material having a Young’s modulus comprised between 100 Pa and 1 MPa.
  • the responsive layer 302 is constituted by a mechanically adaptive material capable of changing shape upon a trigger stimulus selected from one or more of temperature change, electric field change, magnetic field change, light change, pressure change, pH change, ionic strength change and swelling by liquid absorption, preferably swelling by liquid absorption.
  • a mechanically adaptive material is substantially composed of a hydrogel, preferably selected from a non-limiting list comprising Polyacrylic acid hydrogel, Polyacrylamide hydrogel, Polyhydroxyethilmethacrylate hydrogel, most preferably Polyacrylic acid hydrogel.
  • the MEA structure particularly the electrically conductive tracks 400, can be rationally designed to have many different shapes, such as branches, tentacles, round or squared patches and so forth, typically depending on the application or its purpose. Therefore, one advantage of the device according to the invention is its universality of design and tailored structure based on the purposed application and the sample it is used on.
  • connection means 410 for electrical and/or magnetic connection with external devices, located on the intermediate layer 300.
  • Connection means 410 can be implemented for instance as contact pad(s) for establishing an electrical connection between electrodes interconnects 400 and one or more electrical and/or electronic device, such as for instance with an electrophysiological recording system or a power supply, via direcct physical connection means such as wired connections or pogo pins; additionally or alternatively, connection means 410 can be implemented as wireless means for coupling the device of the invention with external receivers in a wireless mode, for instance through (resonant) inductive coupling, (resonant) capacitive coupling, magnetodynamic coupling, ultrasounds and/or infrared radiation, by the simple implementation of solenoid antennas or coils in any suitable position within the device. Accordingly, the connection can be operatively established in any suitable way; with reference for instance to the embodiment shown in Fig. 2, connection means 410 configured as metallic pads are shown, preferably for wired electrical connection or connection with pogo pin
  • the array of conductive tracks 400 located onto the first polymeric material of the passive layer 301 , act as interconnects between a biological entity sample and the connection means 410.
  • Electrical conductive tracks 400 preferably comprise a distal, end electrode portion, such as an electrode pad, configured to directly interface a target biological entity either directly or through an electronic component.
  • Conductive tracks and/or electrode pads of the electrode and/or electronic component can be made of any suitable electrical conductive material, including but not limited to metals such as Au, Pt, Al, Cu, Pt — Ir, Ir, and the like, as well as any alloy thereof, oxide thereof and combinations thereof, composite metal-polymer materials, such as Pt-PDMS composites or Pt — Ir-PDMS composites or Ir- PDMS composites and so forth, as well as conductive polymers such as poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) or polypyrrole (Ppy).
  • the electrodes are made of non-toxic and biocompatible materials.
  • conductive tracks 400 or contact pads 410 can be placed on or within the support 301 with any suitable means such as for instance photolithography, electron beam evaporation, thermal evaporation, sputter deposition, chemical vapour deposition (CVD), electro-plating, molecular beam epitaxy (MBE), inkjet printing, stencil printing, contact printing, transfer printing or any other conventional means known in the art.
  • conductive tracks 400 are encapsulated with an encapsulation/passivation layer 402 later on, to avoid short circuits and failure thereof, i.e. passivated whilst leaving the electrode pads 401 exposed through connecting vias.
  • the conductive tracks 400 are compliant interconnects, facilitating the establishment and maintenance of electrical current passage from external devices up to the distal electrode pads 401 or vice-versa during e.g. electrophysiological experiments at the time of shape changing, deflection of the shape changing units 600.
  • the MEA structure comprises conductive tracks 400 that are operatively connected with elements such as LEDs.
  • LEDs can be positioned in the device to be at least partially in contact with, or close to, a surface of a biological entity of interest, and can be switched on and off by providing an electrical current through the tracks 400.
  • An advantage of this embodiment relies in the possibility to operate LEDs for e.g. optogenetic experiments.
  • the intermediate layer 300 has been adapted to comprise a void portion 310 located in correspondence of the second rigid support hole 201.
  • This arrangement is expedient to create a volume usable as a cell culture chamber, said chamber therefore comprising a bottom part given by the first rigid support 100, a top part defining the chamber itself given by the second rigid support 200 thickness, and its hole 201 , and the void portion 310 of the intermediate layer 300.
  • a lid 700 is present to close the cell culture chamber and guarantee among others optimal sterility and humidity conditions.
  • the problem remained of properly fitting the sample of interest in the middle of said volume.
  • This problem was solved by including at least one anchoring element 500, composed of at least the first polymeric material, stemming from the intermediate layer 300 and entering inside the void portion 310.
  • the at least one, preferably several, anchoring element(s) 500 are sufficiently rigid to position and lock at least two shape changing units 600, stemming from said anchoring element(s) 500 and floating inside said void portion 310.
  • the shape changing units 600 representing the “nest” for housing the biological entity of interest, are in such a way located within the void portion 310 in the middle of the cell culture chamber.
  • the anchoring element(s) 500 can be stretchable, as a result of a material choice or by design (by e.g. adding serpentine or accordeon-like elements), to enable additional degrees of freedom and adaptability of the system to various conditions (for instance, physical or mechanical stabilisation of the shape changing units 600 during fluid flow supply inside the culture chamber).
  • the shape changing units 600 are so called because of their capacity of morphing; in fact, while being composed of the first polymeric material of the passive layer 301 (being part of the intermediate layer 300), they further comprise a responsive layer 302 composed of a second polymeric material, facing and in contact with the passive layer 301 , said second polymeric material being a mechanically adaptive material capable of changing shape upon a trigger stimulus, thereby allowing the shape changing units 600 to change their shape, e.g. deflect, upon said trigger stimulus.
  • the second polymeric material constituting the responsive layer 302 is typically a hydrogel having a monomer concentration comprised between 1 and 50 wt%.
  • the second polymeric material is manufactured by monomers crosslinking via method readily available to a skilled person, with a crosslinker concentration comprised between 0.01 wt% and 10 wt%.
  • the crosslinker(s) can be selected from a non-limiting list comprising MBAA, PEGDA, EGDMA.
  • the thickness ratio between the passive layer 301 and the responsive layer 302 is generally comprised between 0.1 and 100 when the responsive layer is dry.
  • the above features are selected to permit the necessary mechanical response of the responsive layer 302 to facilitate deflection of the shape changing units 600.
  • the above features have been tailored so that the second polymeric material can have an expansion strain coefficient upon liquid absorption, defined as the ratio of the difference in area between dried and swollen states, comprised between 1.1 and 100, as measured by methods known in the art such as optical analyses via microscope picture gathering and image processing.
  • the responsive layer 302 comprises a final polymer weight in a dried form of at least the 1 % of the total responsive layer material weight (i.e. 1 % w/w, also called mass fraction, wherein the remaining 99% or less is substantially composed of water or of an aqueous solution).
  • a suitable polymer mass fraction depends on e.g. the molecular weight of the monomer, the nature of the monomer, the crosslinking strategy and the ratio of polymers.
  • the conductive tracks 400 are configured and designed to comprise:
  • elongated conductive paths 400 each departing at least from one of said contact pads 410, and running along the passive layer 301 and the at least one anchoring element 500 to arrive to at least one shape changing unit 600, and
  • Electrodes pads 401 are configured to electrically interface the external environment, particularly biological material.
  • a further aspect of the present invention relates to the use of the device according to the present disclosure for electrophysiological analyses of biological entities selected from, but not limited to, egg cells, zygotes, embryos, animal tissues or a portion thereof, organoids, spheroids and larvae.
  • the device of the invention may be part of a system comprising a data processing apparatus operatively connected thereto, the data processing apparatus having a processor comprising instructions configured to operate the system to perform a method according to the invention.
  • the data processing apparatus of the invention can comprise any suitable device such as computers, smartphones, tablets, voice-activated devices (i.e. smart speakers/voice assistants) and the like.
  • the data processing apparatus comprises memory storing software modules that provide functionality when executed by the processor.
  • the modules include an operating system that provides operating system functionality for the apparatus.
  • the system in embodiments that transmit and/or receive data from remote sources, may further include a communication device, such as a network interface card, to provide mobile wireless communication, such as Bluetooth, infrared, radio, Wi-Fi, cellular network, or other next-generation wireless-data network communication.
  • communication device provides a wired network connection, such as an Ethernet connection or a modem.
  • a device comprises a non-transitory computer readable medium containing a set of instructions that, when executed by data processing apparatus, cause said data processing apparatus to operate the system to perform a method according to the invention. Further, one aspect of the invention relates to a data processing apparatus comprising the non-transitory computer readable medium of the invention.
  • the instructions contained by the non-transitory computer readable medium comprise, among others:
  • [00171 ] - instructions for performing an electrophysiological analysis of a biological entity once located inside the device of the invention including at least one of providing electrophysiological electrical stimuli to the biological entity, receiving electrophysiological electrical stimuli from the biological entity, storing electrophysiological electrical stimuli obtained from the biological entity, and optionally comparing electrophysiological electrical stimuli obtained from the biological entity with a set of electrophysiological electrical stimuli of reference;
  • the system further comprises one or more sensors operatively connected with the data processing apparatus and with other portions of the system, in suitable positions thereof, such as first rigid support 100, second rigid support 200, intermediate layer 300, including in correspondence of void portion 310 (chamber 350), at least one anchoring element 500, at least one shape changing units 600, the sensors being configured for instance to measure, detect and/or analyse a parameter of the system and/or of the surrounding environment.
  • a “sensor” as used herein is a device that detects (and possibly responds to) signals, stimuli or changes in quantitative and/or qualitative features of a given system, or the environment in general, and provides a corresponding output.
  • a sensor preferably comprises means for detecting and possibly storing a system’s parameter, an environmental parameter or a combination thereof.
  • the sensors can therefore comprise a data storage device to hold information, process information, or both. Common used data storage devices include memory cards, disk drives, ROM cartridges, volatile and non-volatile RAMs, optical discs, hard disk drives, flash memories and the like.
  • a sensor according to the present disclosure may be for instance a position sensor, an optical sensor such as a light sensor, infrared sensor or a camera, a motion sensor, a velocity sensor, a touch sensor, a proximity sensor, a temperature sensor, a microphone, a force I torque sensor, as well as combinations thereof.
  • Sensors may further comprise means for transmitting the detected and possibly stored data concerning the above-mentioned parameters to the data processing apparatus, in some embodiments through a wireless connection.
  • “Wireless” refers herein to the transfer of information signals between two or more devices that are not connected by an electrical conductor, that is, without using wires. Some common means of wirelessly transferring signals includes, without limitations, WiFi, Bluetooth, magnetic, radio, telemetric, infrared, optical, ultrasonic connection and the like.
  • sensors further comprise means for wirelessly receiving a feedback input from a computer able to regulate the functioning of the system.
  • the device can be configured for fluidic connection with external devices.
  • Fluidic connection means can be implemented for instance as mL, pL or nL-scale reservoirs, tubes and/or channels embedded into, located onto or otherwise connected to, first rigid support 100, second rigid support 200, intermediate layer 300 in any suitable way as known in the art such as gluing, molding, scissor blading, (photo)lithography, etching, screen printing, riveting and the like.
  • Connection means ' can be implemented for instance as mL or 4-scale reservoirs and/or tubes for establishing a fluidic connection between channels and one or more external device such as syringes, external mechanical pumps, integrated mechanical micropumps, peristaltic pumps, haemostatic pumps and the like.
  • the device comprises at least one channel such as a microfluidic channel, configured to transport a fluid, rendering de facto the device of the invention a fluidic or microfluidic device, depending on the needs and circumstances.
  • the device comprises a plurality, such as an array, of channels or microchannels.
  • the fluidic channels may be used for instance to supply a compound, such as a pharmaceutical compound, a gas, a buffer medium and the like, into the culture chamber in order to maintain fluid circulation and thermo-regulation, and/or for pharmacologically stimulating a sample biological entity.
  • the fluidic channels of the device according to the invention can be used e.g.
  • the channels can be operatively connected through their inlets to a fluidic pump or some other drug delivery device via fluidic connection means.
  • the device can comprise small molecules or macromolecules, preferably bioactive compounds, as well as other substances such as nano/microparticles, embedded into more or more portions of the device, such as for instance and advantageously into the responsive layer 302.
  • bioactive agents could be for instance embedded inside a hydrogel-based responsive layer 302 during a drying phase thereof, in such a way to release on demand, for instance upon provision of a trigger stimulus to mechanically alter the layer 302, said bioactive agent inside the culture chamber, to influence one or more physiological parameters of the biological entity under analysis.
  • the bioactive compounds and/or particles can be added to the responsive layer 302 material by using any suitable method known in the art, such as surface absorption, physical immobilization, e.g., using a phase change to entrap the substance in the material, and the like.
  • a growth factor can be mixed with a polymeric composition while it is in an aqueous or liquid phase, and after a change in environmental conditions (e.g., pH, temperature, ion concentration), the liquid gels or solidifies, thereby entrapping the bioactive substance.
  • covalent coupling e.g.
  • bioactive molecules can be encapsulated within nano/micro spheres or beads included within the material of the responsive layer 302 and/or a polymeric gel during or after a manufacturing step.
  • a cell culture media actuated self-folding MEA that enables the monitoring of the functional electrical activity of brain spheroids in three dimensions is presented, and shown in an implemented embodiment in Fig. 5.
  • the unique feature of the system lies in the self-folding mechanism of its petals, which is driven by the swelling properties of soft hydrogels and occurs directly within standard cell culture media, thus enhancing its versatility and convenience.
  • the compact design of the device incorporates a PMMA-based fluidic channel and culture well, eliminating the need for additional plasticware such as Petri Dishes.
  • such design is compatible with the integration of cell perfusion systems, which could be implemented to improve cell viability.
  • the proposed device seamlessly integrates with commercially available electrophysiological readout systems, offering a plug-and-play solution without the necessity for custom-made wiring.
  • the optimized fabrication process flow includes standard microfabrication techniques and allows hydrogel grafting at the wafer level. This approach is compatible with various hydrogel formulations, thus paving the way for MEAs with micromechanical actuation driven by thermo-responsive and light-responsive hydrogels.
  • the developed MEA houses a total of 32 electrodes, demonstrating robustness in sustaining the small radius of curvature and the repetitive bending required during petal actuation.
  • the number of electrodes per petal and their arrangement can be tailored, with the possibility of incorporating additional components such as punctual heaters.
  • the design presented herein represents one feasible configuration; some alternative embodiments envisage the introduction of stretchable anchoring elements and customized 3D array geometries for diverse tissue and cell types.
  • the inventors proved able to demonstrate the feasibility of spatiotemporal recordings of neural activity with the e-Flower by detecting and modulating spontaneous spiking patterns across the entire spheroid surface.
  • the presented self-folding MEA-based device offers a user- friendly and adaptable platform for three-dimensional electrophysiological monitoring, highlighting its practicality and ease of use in advancing neurophysiological research.
  • the e-Flower’s three-dimensional configuration relies on the mechanical and swelling properties of the polyacrylic acid hydrogel layer grafted to the electrodes’ substrate.
  • the hydrogel’s proprieties are known to depend on the crosslinker concentration and re-swelling medium. Therefore, to establish a predictive model of the hydrogel's behavior when grafted on the e-Flower and swollen in different media, we thoroughly characterized both the swelling behavior and the mechanical properties of the bulk PAA hydrogel depending on its crosslinker concentration and the re-swelling solution used.
  • N,N’methylenebisacrylamide (MBAA) crosslinker concentrations namely
  • the re-swelling medium strongly influenced the swelling behavior observed in the PAA hydrogels.
  • re-swelling in CCM produced a lower level of swelling compared to DIW in terms of both mass, area, and thickness variations.
  • hydrogels re-swollen in CCM swelled less than half in terms of mass compared to the ones reswollen in DIW (48x for CCM at 25°C and 45x at 37°C, vs. 102x for DIW), with the PBS condition lying in between these two values (76x).
  • the slope of the initial swelling phase also indicated that the swelling rate was medium dependent, with PBS being the slowest.
  • the PAA gels swollen in CCM showed an overshooting behavior after circa 60 minutes (66x at 25°C and 60x at 37°C), a behavior observed in previous literature. Importantly, this overshoot, though observed, is of minimal significance when compared to the equilibrium swelling value. As a result, we expect that it will not adversely impact the functionality of the e-Flower when immersed in CCM. Measuring the relative variation in area of fully swollen samples (Fig. 6B), re-swelling in warm CCM yielded an area increase that was circa 7 times lower than DIW (0.27, 0.4, 1 .00 and 1 .90 for warm CCM, RT CCM, PBS and DIW, respectively).
  • the crosslinker concentration also influenced the extent of hydrogel swelling.
  • the mass swelling was inversely related to the MBAA concentration, with equilibrium mass swelling ratios of 46x, 40x and 18x for samples with crosslinker concentrations of 1X, 4X, and 16X, respectively.
  • an overshooting swelling behavior was present and clearly dependent on the crosslinker concentration in the gel (60x, 49x, 22x with increasing crosslinker concentration).
  • a remarkable anisotropic swelling behavior was observed when varying the crosslinker concentration: samples with high crosslinker concentration (16X) swelled 10 times more in area but almost 5 times less in thickness compared to samples with standard crosslinker concentration (see Fig. 6E and Fig. 6F).
  • the crosslinker’s concentration strongly influenced the hydrogel’s stiffness, with the elastic modulus that increased with the crosslinker concentration (16.1 kPa for 4X and 44.6 kPa for 16X, see Fig. 6I).
  • the hydrogels in all conditions showed an elastic behavior up until a 3% shear strain.
  • PI layers via spin coating, followed by hydrogel’s grafting.
  • TMSPMA 3-(trimethoxysilyl)propyl methacrylate
  • Fig. 7A displays an optical microscope photograph of a representative bilayer strip swollen at equilibrium, that was used to manually evaluate the bilayer’s radius of curvature as shown by the dotted circle.
  • Fig. 7B The bilayer’s curvature depended on the re-swelling medium, as highlighted by Fig. 7B.
  • Fig. 7C shows the influence of the crosslinker concentration on the bending radius of the same strips swollen in warm CCM. In this case, the increasing the crosslinker concentration induced a less pronounced bending, as the 4X (257 pm) and 16X samples (221 pm) bent with radii 13% and 33% smaller than the base sample 1X (295 pm).
  • Fig. 7D shows a SEM micrograph of a freeze-dried bilayer strip swollen in DIW, showing the morphology of the hydrogel (blue) covalently grafted on the surface of the polyimide layer (yellow).
  • Freeze- dried samples further validate the robust molecular-level attachment of the hydrogel to the polyimide layer, as they endured rigorous treatments, including repeated drying-swelling cycles, flash freezing, and lyophilization. It is worth highlighting that the freeze-drying process altered the bilayer’s radius of curvature.
  • Fig. 7G illustrates the distribution of the volumetric strain in the polyimide layer along the midsection of a petal as the flower closed.
  • the tensile stresses at the interface with the hydrogel were slightly higher than the compressive ones measured on the opposite side, indicating a shift of the neutral plane towards the internal part of the device as the hydrogel swelled.
  • the polyimide experienced a maximum strain of 1.3% when in closed configuration, which is below its critical strain before plastic deformation. This enables the reversible actuation of the bilayer.
  • Fig. 7H To validate the FEM results, we built a PI/PAA bilayer with a geometry comparable to the FEM model, as shown in Fig. 7H. We subsequently immersed the device in PBS and tracked its 2D-to-3D reconfiguration. Fig. 7I shows snapshots of the shape morphing process at different time points. These time-lapse results validate the stability of the model, as well as the platform's mechanical stability. To understand the closure dynamics and the required time to reach a stable 3D geometry, we evaluated the time to final configuration, as shown in Fig. 7J. The devices reached equilibrium in approximately two minutes, with the closed shape forming in less than 109 s.
  • the final cross-section of the e-Flower included: (i) a 3 pm top polyimide encapsulation layer, (ii) a 150 nm platinum layer forming electrodes and interconnects, (iii) a 3 pm bottom polyimide encapsulation layer, (iv) a 15 nm layer of SiOx for the hydrogel grafting, (v) and a 150 pm layer of PAA (as prepared).
  • e-Flower within a poly(methyl methacrylate) (PMMA) fluidic channel, as illustrated in Fig. 8A and Fig. 8B.
  • the fluidic channel comprised essential components including an inlet, an outlet, a culture well, and a removable lid designed to protect the cultured tissue.
  • the e-Flower is positioned in the center of the culture chamber, ensuring its complete or partial submersion in cell culture media.
  • Fig. 8C shows the e-Flower mounted in the fluidic channel once having reached its closed configuration when immersed in solution.
  • the electrodes’ impedance values when in 3D configuration (327.7 kQ) was comparable with the average impedance amplitude measured for the electrodes in planar configuration, when in absence of mechanical actuation (251.7 kQ).
  • e-Flower a cell culture media actuated selffolding MEA designed to monitor the functional electrical activity of brain spheroids’ in three dimensions. Its three-dimensional actuation is driven by the swelling properties of polyacrylic acid.
  • the e-Flower’s petals reach a radius of curvature down to 300 pm in the presented configuration, which depends upon the re-swelling medium used and the crosslinker concentration adopted in synthesizing the polyacrylic hydrogel.
  • Our device brings several advantages to the field. It enables three- dimensional recordings of the neural activity of pre-formed three- dimensional in vitro brain models, without the need for disruptive sample processing, nor the necessity to culture the tissue from scratch. Thanks to its cell-friendly actuation mechanism, it mechanically actuates directly around the spheroid, without the need of potentially harmful solvents, nor complex mechanical actuators. Featuring 32 electrodes, our MEA offers a higher number of contacts compared to previous bilayers used in brain spheroid’s electrophysiology, as well as surpassing other examples of MEAs actuated by hydrogels, such as for retina and peripheral nerve applications.
  • the design allows for customization, for example in terms of electrode number and arrangement per petal, and with the possibility of incorporating supplementary components such as punctual heaters, microfluidic channels and light sources.
  • This adaptability paves the way for the development of MEAs with tailored curvatures to the targeted application, and where the micromechanical actuation is driven by specific stimuli, such as temperature, light and pH variations.
  • the device is suitable for adaptation to various tissue models, including assembloids, but also small organisms like larvae, and zebrafish, according to the needs. [00211 ] Beyond the MEA itself, our work features a comprehensive system design enabling in vitro signal recordings from pre-formed three-dimensional tissues.
  • PAA Hydrogel-Precursor Solution Preparation Aqueous poly(acrylic acid) (PAA) hydrogel-precursor solutions were prepared by dissolving acrylic acid monomers (AA; Sigma-Aldrich) and N,N’methylenebisacrylamide crosslinkers (MBAA; Sigma-Aldrich) in deionized water (DIW) and subsequently adding a-ketoglutaric acid (Sigma- Aldrich) as photo-initiator. For all hydrogel-precursor solutions, the final AA and a-ketoglutaric acid concentrations were set to 20 wt% and 0.07 wt% respectively.
  • PAA Hydrogel Synthesis The PAA hydrogel samples were synthesized through 20 min UV curing of gel-precursor solution dispensed between two HMDS-treated glass slides spaced by three coverslips ( « 450 pm) and clamped by paper clips in a nitrogen-controlled environment. After UV exposure, hydrogels were swelled in DIW to remove non-crosslinked molecules. Subsequently, swollen hydrogels were laid on a polyethylene terephthalate sheet and secured to it along their edges using adhesive tape for overnight drying at room temperature.
  • DVM6 Digital microscope
  • RatiOThickness (tswollen — tdry)/tdry-
  • PAA-Polyimide Bilayer Microfabrication First, a 6-pm-thick polyimide (PI; PI-2611 , HD MicroSystems) layer was spin-coated and cured on a 4- inch silicon wafer previously coated with a Ti/AI (20/100 nm) sacrificial layer. To graft the PAA onto the polyimide, the PI surface was prepared by sputtering (AC 450, Alliance Concept) a Ti/SiOx layer (10/15 nm).
  • the SiOx surface was then activated by O2 plasma (Zepto, Diener Electronic) and subsequently functionalized by 10 min incubation at room temperature in a functionalization solution prepared by mixing 3-(trimethoxysilyl)propyl methacrylate (TMSPMA; Sigma-Aldrich), DIW and isopropyl alcohol (IPA; Sigma-Aldrich) in a 2:10:150 volume ratio.
  • TMSPMA 3-(trimethoxysilyl)propyl methacrylate
  • DIW isopropyl alcohol
  • IPA isopropyl alcohol
  • the thickness of the dispensed gel-precursor solution layer was set either by four 150-pm-thick glass coverslips placed at four different positions around the wafer’s perimeter or by 150-pm-thick films made out of polyethylene terephthalate and Elastosil ® (EL; EL Film 2030, Wacker) patterned with a femtosecond excimer laser (WS Turret, Optec Laser Systems) to obtain circular molds confining the hydrogel to specific locations. Dry PAA-PI bilayers were then micromachined (WS Turret, Optec Laser Systems) into the desired shapes, and finally detached from the underlying wafer by anodically dissolving the Al sacrificial layer in a saturated NaCI solution.
  • EL polyethylene terephthalate and Elastosil ®
  • WS Turret femtosecond excimer laser
  • FEA Finite Element Analysis
  • the hydrogel was set with a Young’s modulus of 100 kPa and a Poisson ratio of 0.49.
  • the Structural Mechanics module was chosen, to use the following boundary conditions: on the extremity of the bridge, a Fixed Constraint was set to immobilize the structure. On the lateral sides of the design, a Symmetry boundary condition was set to consider the symmetries of the design. Finally, to implement a simplified version of the hydrogel swelling, t an Initial Strain boundary condition was set on the hydrogel domain, setting the same strain values along the X and Y axes, keeping the strain along the Z axis null.
  • a Stationary Study was performed, including geometric nonlinearities and adjuvated by an auxiliary sweep increasing the strain in steps of 0.05, starting from 0. We visualized and exported the graphical results using the built-in tools in COMSOL.
  • the PI substrate was etched (21 OIL, Corial) to open the electrodes and pads, followed by photoresist removal in an ultrasonication bath in acetone. Subsequently, a second photolithography step was performed as described above to cover the electrodes and pads.
  • O2 plasma activation Ti and Pt were sputtered with thicknesses of 15 nm and 10 nm respectively (AC 450, Alliance Concept). Following photoresist lift-off, 150 nm of Pt and 10 nm of Ti were sputtered to form electrodes and pads. Tracks connecting electrodes to pads were defined by photolithography of 6-pm- thick AZ10XT and subsequent metal etching.
  • Electrochemical Characterization All measurements were performed in 1X PBS in a three-electrode setup with an Ag/AgCI reference electrode and a platinum wire as the counter electrode. The electrochemical impedance was recorded with an impedance analyzer (Autolab PGSTAT302N, Metrohm) for frequencies between 1 Hz and 10 5 Hz, measuring five points per decade. All electrodes were activated before impedance characterization by applying a train of 5 biphasic pulses of 50 pA amplitude and 250 ps duration. A custom-made adapter was used to interface the e- Flower with the impedance analyzer. The electrochemical measurements were performed in the closed 3D configuration, after inspecting that the equilibrium radius of curvature had been reached. In the case of tests involving multiple closing and opening cycles, each e-Flower was washed in DIW and dried between cycles.
  • Human brain spheroids were collected from the culture wells by gently pipetting them along with a little volume ( ⁇ 200 pL) of CCM and were subsequently released from the pipette at the center of the dry e-Flowers. After having reached almost complete actuation (app. 3 min), CCM at 37°C was added to fill the microfluidic chamber, thereby enabling e-Flowers to attain the equilibrium 3D configuration and to fully enclose brain spheroids. Before starting electrophysiological recordings, e- Flowers with seeded spheroids were transferred to an incubator (NU-5500, NuAire) and maintained at 37°C and 5% CO2 for at least 8 h to ensure environment stabilization.
  • an incubator NU-5500, NuAire
  • Electrophysiological Data Acquisition A commercial system (MEA- 2100, Multi Channel Systems) and its companion software (MC_Rack, Multi Channel Systems) were used for the collection and preliminary analysis of electrophysiological data. Recordings were acquired from all 32 channels simultaneously in a grounded Faraday cage with a sampling rate of 30 kHz. Raw signals were preprocessed with a band-pass filter featuring lower and upper cut-off frequencies of 200 Hz and 5000 Hz respectively using the MC_Rack software. Neuronal field potentials were detected for each channel using a threshold-based peak detection method, with the threshold set to be 6o n away from the mean of the filtered signal.
  • o n median(

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Abstract

L'invention concerne un dispositif conçu pour des analyses électrophysiologiques d'entités biologiques telles que par exemple des organoïdes et des sphéroïdes. Le dispositif comprend une première couche polymère passive (301), une couche d'hydrogel (302) capable de changer de forme lors d'un stimulus déclencheur. Un réseau de pistes conductrices (400) et de plots de contact (401) peuvent être utilisés pour entrer en contact avec l'entité biologique par déclenchement d'un changement de forme de la couche d'hydrogel (302). Le dispositif peut être disposé dans un puits d'échantillon.
PCT/EP2023/083056 2023-11-24 2023-11-24 Dispositif d'analyses électrophysiologiques in vitro d'entités biologiques Pending WO2025108561A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004095536A2 (fr) 2003-03-28 2004-11-04 Princeton University Interconnexions etirables et elastiques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004095536A2 (fr) 2003-03-28 2004-11-04 Princeton University Interconnexions etirables et elastiques

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HUANG ET AL., SCI. ADV., vol. 8, 2022, pages eabq5031
LIU ET AL., NANO LETT., vol. 19, no. 8, 14 August 2019 (2019-08-14), pages 5781 - 5789
LIU JIANZHI ET AL: "Programmable shape deformation actuated bilayer hydrogel based on mixed metal ions", EUROPEAN POLYMER JOURNAL, PERGAMON PRESS LTD OXFORD, GB, vol. 175, 18 June 2022 (2022-06-18), XP087116759, ISSN: 0014-3057, [retrieved on 20220618], DOI: 10.1016/J.EURPOLYMJ.2022.111375 *
MURU ZHOU: "Full-field, conformal epiretinal electrode array using hydrogel and polymer hybrid technology", SCIENTIFIC REPORTS, vol. 13, no. 1, 28 April 2023 (2023-04-28), US, XP093162132, ISSN: 2045-2322, DOI: 10.1038/s41598-023-32976-9 *
MURU ZHOU: "Shape Morphable Hydrogel/Elastomer Bilayer for Implanted Retinal Electronics", MICROMACHINES, vol. 11, no. 4, 9 April 2020 (2020-04-09), pages 392, XP093162398, ISSN: 2072-666X, DOI: 10.3390/mi11040392 *
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