Device for in vitro electrophysiological analyses of biological entities

Abstract

The invention concerns a device configured for electrophysiological analyses of biological entities such as for instance organoids and spheroids. The device comprises a passive first polymer layer (301), a hydrogel layer (302) capable of changing shape upon a trigger stimulus. An array of conductive tracks (400) and contact pads (401) may be used to contact the biological entity by triggering shape change of the hydrogel layer (302). The device may be arranged in a sample well.

Description

Device for in vitro electrophysiological analyses of biological entitiesTechnical Field

[0001] The present invention generally belongs to the fields of electronics, material science, biotechnology and laboratory instruments. In particular, the invention concerns a device configured for electrophysiological analyses of biological entities such as for instance organoids and spheroids.Background Art

[0002] 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.

[0003] Recently, the development of 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. However since 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.

[0004] Currently the most used technology in this field is the 2D multi-electrode array (MEA). While commercially available MEAs are commonly used for such measurements, they are mostly limited to planar configurations, which do not fully capture the complexity of three-dimensional in vitro brain models.

[0005] To overcome this issue, disruptive sample processing techniques such as tissue slicing and penetrating probes have been used. In an effort to preserve the three-dimensional integrity of the targeted tissues, recent efforts have focused on the development of flexible 3D arrays specifically designed for organoids and spheroids.

[0006] An attempt made by Soscia et al. (Lab Chip, 2020,20, 901-911 ) has been done with the use of a 3D MEA that records the signal inside of the spheroid with the use of polyimide (PI) as substrate. However, their device only record the activity in a localized area that is inside of the spheroid.

[0007] Park et al. (Sci. Adv. 7, eabf9153 (2021 )) made a device that record the electrophysiological activity of the brain spheroid around its surface. In their design the spheroid is trapped in the device where it is in contact with a multitude of electrode. However, this technology needs to apply a stress on the device that will stretch this latter in order to place the spheroid.

[0008] In another study by Huang et al. (Sci. Adv. 8, eabq5031 (2022)), it was developed a flexible, reversible, 3D MEA in the shape of a flower, folding around a brain spheroid. The device uses two SU8 layers that encapsulate the tracks. Upon differential exposure to UV, the SU8 layers will induce the bending of the device. Depending on the parameters of the SU8 layers (thicknesses of the bilayer or differential exposure between the two SU8 layers for instance), the device can accommodate bigger or smaller spheroids / organoids. Yet, it comprises only a small number of electrodes (up to 9), and involves the use of toxic solvents such as acetone.

[0009] Finally, a last approach consists in using cyborg organoids such as those used by Liu et al. (Nano Lett. 2019 Aug 14;19(8):5781 -5789) with stretchable mesh nanoelectronics that grows within a 2D stem cell layer and which will be folded with the appropriated 3D structure during the endogenous 2D to 3D reconfiguration during the organoid development. After the 2D to 3D reconfiguration, the stretchable mesh is uniformly distributed across the brain organoid. However, this approach requires extensive amounts of time for the integration of the MEA with the growing tissue.Summary of invention

[0010] In order to address and overcome at least some of the above-mentioned drawbacks of the prior art solutions, 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.

[0011] In particular, 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.

[0012] 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.

[0013] 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.

[0014] All these aims have been accomplished with the present invention, as described herein and in the appended claims.

[0015] In view of the above-summarized drawbacks and / or problems affecting devices of the prior art, according to the present invention there is therefore provided a device according to claim 1 .

[0016] Particularly, one object of the present invention relates to a device comprising:

[0017] -a passive layer composed of a first polymeric material, the passive layer extending on a plane,

[0018] -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,

[0019] -at least one cutting line through the passive layer and the responsive layer delimiting at least one shape changing unit,

[0020] 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.

[0021] In preferred embodiments, 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.

[0022] 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. In one embodiment, moreover, the responsive layer is arranged only in correspondence of the at least one shape changing unit.

[0023] "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. Moreover, “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.

[0024] The raising portion may have a same shape of the at least one cutting line. In particular, a border or peripheral portion of the raising portion may have the same shape of the at least one cutting line. When the raising portion is not subject to the trigger stimulus, the raising portion lays on the plane inadherence with the remaining portion of the passive layer, i.e. adhered to regions thereof not forming the shape changing unit. In other words, the peninsula formed by the at least one shape changing unit is formed by a thin channel following the peninsula, wherein a width of the channel may be unperceivable at sight when the raising portion is not stimulated. This is advantageous for improving support of the biological entity and preventing flows or falls.

[0025] The raising portion, however, 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.

[0026] Preferably, a plurality of cutting lines is provided.

[0027] For instance, at least two cutting lines delimit corresponding at least two shape changing units.

[0028] 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.

[0029] Preferably, 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.

[0030] The device may preferably include a first rigid support for supporting the passive layer on the plane.

[0031] More preferably, a second rigid support is provided for protection.

[0032] 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.

[0033] The second rigid support delimits a chamber with an opening.

[0034] The opening is over the intermediate layer.

[0035] In particular, 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.

[0036] 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.

[0037] 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.

[0038] 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).

[0039] 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.

[0040] Based on the solution idea given above, the invention may be embodied with some changes. In the following description, an embodiment is disclosed wherein, however, not all the features already disclosed above are repeated, for convenience. The skilled person may easily understand, for the features already disclosed above and not explicitly repeated in the following embodiment, if same are applicable in the embodiment as follows.

[0041] The device includes for instance:

[0042] A first rigid support;

[0043] 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;

[0044] An intermediate layer located between said first and said second rigid supports, said intermediate layer comprising:

[0045] i) a passive layer composed of a first polymeric material, and

[0046] ii) an array of conductive tracks located onto said first polymeric material

[0047] wherein said intermediate layer comprises

[0048] - a void portion located in correspondence of the second rigid support hole,

[0049] - at least one anchoring element, composed of at least the first polymeric material, stemming from said intermediate layer and entering inside said void portion, and

[0050] - at least two 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.

[0051] In embodiments, 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.

[0052] In embodiments, said mechanically adaptive material is substantially composed of a hydrophilic material, particularly a hydrogel.

[0053] In embodiments, said hydrogel is selected from a non-limiting list comprising Polyacrylic acid, Polyacrylamide, Polyhydroxyethilmethacrylate.

[0054] In advantageous embodiments, said hydrogel is composed of Polyacrylic acid.

[0055] In embodiments, the second polymeric material has a monomer concentration comprised between 1 and 50 wt%.

[0056] In embodiments, 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.

[0057] In embodiments, the second polymeric material is manufactured by monomers crosslinking, with a crosslinker concentration comprised between 0.01 wt% and 10 wt%.

[0058] In embodiments, the crosslinker is selected from a non-limiting list comprising MBAA, PEGDA, EGDMA.

[0059] In embodiments, the first polymeric material has a Young’s modulus comprised between 1 kPa and 10 GPa.

[0060] In embodiments, when fully hydrated or fully swollen, the second polymeric material has a Young’s modulus comprised between 100 Pa and 1 MPa.

[0061] In embodiments, the first polymeric material is selected from a non-limiting list comprising Parylene, Polyimide, Sll-8, PDMS, Polyurethane, SEBS.

[0062] In embodiments, the thickness ratio between said passive layer and said responsive layer is comprised between 0.1 and 100 when dry.

[0063] In embodiments, 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.

[0064] In embodiments, the elongated conductive paths are passivated with a passivating layer.

[0065] 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.

[0066] Preferably, said biological entities selected from, but not limited to, egg cells, zygotes, embryos, animal tissues or a portion thereof, organoids, spheroids and larvae.

[0067] The above and other objects, features and advantages of the herein presented subject-matter will become more apparent from a study of the following description with reference to the attached figures showing some preferred aspects of said subject-matter.Brief description of drawings

[0068] Fig. A schematically represents a top view of a device according to the present invention;

[0069] Fig. B is a cross section of a detail of the device according to an embodiment of the present invention;

[0070] Fig. 1 A and 1 B are, respectively, magnified view of fig. A and B;

[0071] 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.

[0072] 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’;

[0073] Fig. 1 shows an exploded view according to one embodiment of the device of the invention;

[0074] Fig. 2 shows a top view of the intermediate layer according to one embodiment of the device of the invention;

[0075] Fig. 3 shows a top view of the shape changing units and anchoring elements according to one embodiment of the device of the invention;

[0076] 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;

[0077] 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. (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 theelectrode-bearing PI layer remains unaltered. This produces the 2D-to-3D reconfiguration of the system: the flower closes, the petals bend and bring the electrodes in close contact with the outer surface of the brain spheroid. (C) The e-Flower seamlessly interfaces with the pogo-pins of the MEA-2100 electrophysiological system. (D) Bright field image of an actuated e-Flower wrapping around the surface of a brain spheroid. (E) Representative neural event detected by one of the electrodes’ of the e-Flower during a 5 min electrophysiological recording;

[0078] 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). Mass ratio re-swelling curves (D), area (E) and thickness (F) re-swelling ratios of de-hydrated PAA hydrogel samples synthesized with various crosslinker concentrations and re-swollen in CCM at 37°C (n = 6 per condition). (G) Rheological properties of PAA hydrogels synthesized with 1X crosslinker concentration and re-swollen in CCM at 37°C (n = 6 per condition). (H) Elastic moduli of PAA hydrogels synthesized with 1X crosslinker concentration and re-swollen in various media (n = 6 per condition). (I) Elastic moduli of PAA hydrogels synthesized with various crosslinker concentrations and swollen in CCM at 37°C (n = 6 per condition). Scanning electron micrograph showing the morphology of the PAA hydrogel when reswollen in DIW (J) and CCM (K);

[0079] Fig. 7 shows PAA / PI hybrid bilayers. (A) Bright field image of a PAA / PI bilayer strip synthesized with 4X crosslinker concentration and swollen at equilibrium in CCM at 37°C. The dotted line represents the circumference whose radius of curvature was manually extracted from the image. Scale bar = 500 pm. (B) Radius of curvature of PAA / PI bilayer strips synthesized with 1X crosslinker concentration and swollen in various media (n = 6 per condition). (C) Radius of curvature of PAA / PI bilayer strips synthesized with various crosslinker concentrations and swollen in CCM at 37°C (n = 6 per condition). (D) Scanning electron micrograph of a PAA / PI bilayer strip reswollen in DIW. False colors are used to indicate the PI layer (yellow) andthe PAA layer (blue). (E) 3D CAD of the PAA / PI bilayer with a flower shape and supporting bridges. Orange: PI, blue: PAA. A quarter of the structure is cut for easier visualization. (F) FEM simulation of the closing dynamics of the PAA / PI flower-shaped bilayer at different swelling levels. Color legend representing the strain distribution on the PI layer along the midline of a petal at different hydrogel swelling levels. (G) Bright field image of a flowershaped PAA / PI bilayer (1X crosslinker concentration) in dry state and (H) when hydrated in PBS at different time points at times. (I) Time required by the de-hydrated flower-shaped PAA / PI bilayer to reach its equilibrium 3D configuration when re-swollen in various media (n = 6 per condition);

[0080] 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. (D) Electrochemical impedance modulus and phase spectra as a function of frequency for the platinum electrodes of the e-Flower in closed configuration in PBS (n = 3 e- Flower devices for a total of nrec_eiec = 95 and ngrn_eiect = 3). Arec_eiect = 707 pm2and Agm_eiect = 640000 pm2. (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;

[0081] 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 closedaround 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.Detailed description of the invention

[0082] The subject-matter described in the following will be clarified by means of a description of those aspects which are depicted in the drawings. It is however to be understood that the scope of protection of the invention is not limited to those aspects described in the following and depicted in the drawings; to the contrary, the scope of protection of the invention is defined by the claims. Moreover, it is to be understood that the specific conditions or parameters described and / or shown in the following are not limiting of the scope of protection of the invention, and that the terminology used herein is for the purpose of describing particular aspects by way of example only and is not intended to be limiting.

[0083] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, unless otherwise required by the context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Further, for the sake of clarity, the use of the term “about” is herein intended to encompass a variation of + / - 10% of a given value.

[0084] Non-limiting aspects of the subject-matter of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. For purposes of clarity, not every component is labelled in every figure, nor is every component of each aspect of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0085] The following description will be better understood by means of the following definitions.

[0086] As used in the following and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Also, the use of "or" means "and / or" unless stated otherwise. Similarly, "comprise", "comprises", "comprising", "include", "includes" and "including" are interchangeable and not intended to be limiting. It is to be further understood that where for the description of various embodiments use is made of the term "comprising", those skilled in the art will understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of" or "consisting of."

[0087] In the frame of the present disclosure, 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. Likewise, 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.

[0088] Some of the materials used in accordance with the present invention are soft polymers. In the frame of the present disclosure, a “soft” material is any material that is either compressible, reversibly compressible, elastic, viscoelastic stretchable or any combination thereof. The term “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 thanabout 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.

[0089] 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; elastomeric materials such as silicone rubber (e.g. 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), poly(lactic acid), poly(methacrylic acid), and copolymers, poly(vinylpyrrolidone) (PVP), Polyacrylamide (pAAm) and combinations thereof; as well as any combination of the foregoing.

[0090] 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) suchas 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. These further polymers are particularly suitable for hydrogel manufacturing, in view of their biocompatibility and Young’s modulus, among other.

[0091] As used herein, the term "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.

[0092] For "mechanically adaptive material" is herein meant a material, such as a polymeric material, which changes its mechanical properties on demand, and in particular upon activation and / or in presence of at least one trigger stimulus. These materials morph upon exposure to a predefined stimulus in a highly selective and possibly reversible manner and are attractive for many technologically relevant applications. In the frame of the present invention, 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. Accordingly, in preferred and non-limiting embodiments of the invention, 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.

[0093] The expressions “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 thedevice. Generally speaking, 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. Typically, 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. In some embodiments, 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.

[0094] The expression “conductive track” refers to any film, path, stripe, strand, wire or the like which is electrically conductive in nature. For the sake of clarity, the word “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.

[0095] 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 withoutsubstantially 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. In one embodiment, stretchable electrodes as the one described in International Patent Application WO 2004 / 095536, incorporated herein in its entirety by reference, can be used. Alternatively or additionally, 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.

[0096] 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. In an embodiment, the electrode can be formed by depositing an electrically conductive coating or layer by spraying a preselected onto the designated surface region. Alternatively, and preferably, 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.

[0097] The term “flexible” is used herein to refer to elements of the device of the present invention that can perform a deflection. Generally speaking, 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.

[0098] As used herein, 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. Furthermore, in the frame of the present disclosure, 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.

[0099] 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.

[0100] 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, 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, aneurotransmitter, 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. For example, 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.

[0101] 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.

[0102] With this in mind, 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

[0103] 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);

[0104] 2) obtaining a cell culture platform optimally adapted for long-term culture and analysis;

[0105] 3) obtaining an electrophysiology-analysis device capable of maintaining the structural integrity of the sample to be analysed;

[0106] 4) obtaining a universal device adapted for multi-use, while keeping performances unaltered.

[0107] 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.

[0108] Presented herein, in non-limiting embodiments of the invention, is a 3D MEA platform based on shape-morphing bilayers to engulf brain spheroids and enable recordings and stimulations from their entire surface. Inspired by soft grippers and hybrid hydrogel-polymer bilayers, the inventors developed an MEA that can wrap around the 3D surface of brain spheroids while being actuated by the simple addition of cell culture media, the trigger stimulus for responsive layer deflection. Fig. 5 shows an implemented exemplary embodiment of the device of the invention, named e-Flower. The proposed self-folding MEA, named e-Flower, includes a flower-shaped electrodecarrying polyimide (PI) foil accompanied by a backing layer of polyacrylic acid (PAA) hydrogel. 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.

[0109] Programmable shape-morphing materials are gaining interest in many fields, going from drug delivery to biomedical devices. In particular, a commonly used method to obtain bending / folding functional structures is to combine a functional material layer to a structural one, creating hybrid bilayers. Among all stimuli-responsive materials, 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.

[0110] In non-limiting exemplary embodiments, by precisely tuning the hydrogel formulation, bilayer thicknesses, and MEA geometry, inventors successfully designed a 3D MEA platform with a radius of closure of 300 pm, therefore compatible with targeted brain spheroids. To expedite and guide the design and microfabrication process, comprehensive mechanical and electrical characterization was conducted of each component of the device, and developed accurate in silico finite element models. Finally, it wasdemonstrated a proof of concept of the capability of the MEA to record the electrophysiological activity of brain spheroids in three-dimension.

[0111] However, in certain embodiments and generally speaking, 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. In this frame, 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.

[0112] With reference to Figures A, 1A and B, 1 B one embodiment of a device according to the invention is disclosed.

[0113] 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.

[0114] 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.

[0115] 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. In Fig. A, 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.

[0116] According to the invention, 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 cellculture which has grown over time, after being deposited onto the device, as will be explained.

[0117] The raising portion 640 raises as a consequence of a trigger stimulus changing the shape of the shape changing unit 600. In particular, 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.

[0118] In an embodiment, the raising portion 640 has a same shape of the at least one cutting line 950. In particular, 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. When the raising portion 640 is not subject to the trigger stimulus, the raising portion 640 lays on the plane in adherence with the other regions of the passive layer not delimiting the shape changing units. Advantageously, 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.

[0119] In another embodiment, 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.

[0120] In an embodiment, 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.

[0121] In a further embodiment, 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). Also in this embodiment,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.

[0122] Still with reference to fig. A, 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.

[0123] 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.

[0124] This behaviour and this arrangement is possible also when the cutting lines 950 are extended in openings 900 (as in fig. 3) and the description is not repeated.

[0125] 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.

[0126] In a preferred embodiment a first rigid support 100 supports the intermediate layer 300 on the plane PL (see fig. B’).

[0127] In a preferred embodiment, a second rigid support 200 is provided.

[0128] The second rigid support 200 is placed on top of the intermediate layer 300 for protection (fig. B’)

[0129] The second rigid support 200 delimits a chamber 350 with an opening 201 over the intermediate layer 300.

[0130] 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 cellculture onto the intermediate layer 300 to grow the biological entity 800 thereon.

[0131] In fig. B’, the responsive layer 302 is coupled to the passive layer 301 also in areas not forming the shape changing units 600.

[0132] However, in another possible embodiment (fig. B”), 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.

[0133] 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.

[0134] The shape changing unit(s) are anchored to the passive layer 301. In particular, 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.

[0135] In the embodiment of fig. 3 anchoring elements are delimited by cutting lines also delimiting the shape changing units. In the embodiment of fig. A, instead, 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.

[0136] With reference to Figures 1 to 9, the invention features a device 1 comprising:

[0137] A first rigid support 100;

[0138] A second rigid support 200 placed on top of said first rigid support 100, the second rigid support 200 comprising a hole 201 and defining a volume adapted to be used as a cell culture chamber;

[0139] An intermediate layer 300 located between said first 100 and said second 200 rigid supports, said intermediate layer 300 comprising:

[0140] i) a passive layer 301 composed of a first polymeric material, and

[0141] ii) an array of conductive tracks 400 located onto said first polymeric material

[0142] wherein said intermediate layer 300 comprises

[0143] - a void portion 310 located in correspondence of the second rigid support hole 201 ,

[0144] - 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

[0145] - at least two 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.

[0146] The void portion 310 of the intermediate layer 300 and the hole 201 in the second rigid support 200 may form the chamber 350.

[0147] 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. In certain embodiments, the passive layer 301 is reversibly stretchable (elastic). In particular, 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. In alternative or combination embodiments, 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.

[0148] 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. In the frame of the present invention, “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.

[0149] In preferred embodiments, the passive layer 301 is substantially composed of a first polymeric material having a Young’s modulus comprised between 1 kPa and 10 GPa. Non-limiting examples of suitable first polymeric materials include Parylene, Polyimide, Sll-8, PDMS, Polyurethane, SEBS.

[0150] In preferred embodiments, 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. In preferred embodiments, 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.

[0151] 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.

[0152] The device of the invention features 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 pins.

[0153] 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). In a preferred embodiment, the electrodes are made of non-toxic and biocompatible materials. Electrical components or portions thereof (e.g. 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. In embodiments, 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.

[0154] In embodiments according to the present disclosure, 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.

[0155] In another additional or alternative embodiment, 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.

[0156] One advantageous aspect of the device according to the present disclosure is relates to the design of the portion intended to accommodate the biological entity of interest. In fact, the present inventors tailored this device portion in order to overcome the drawbacks of the prior art devices and systems, which were not optimized for long run experiments. Accordingly,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. Preferably, a lid 700 is present to close the cell culture chamber and guarantee among others optimal sterility and humidity conditions.

[0157] While creating the second rigid support hole 201 , and therefore permitting the accommodation of a biological entity in a suitable volume for culture and analysis, 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. In advantageous embodiments, 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).

[0158] 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 shapeupon a trigger stimulus, thereby allowing the shape changing units 600 to change their shape, e.g. deflect, upon said trigger stimulus.

[0159] In embodiments where the trigger stimulus is water absorption from a surrounding aqueous solution or buffer, the second polymeric material constituting the responsive layer 302 is typically a hydrogel having a monomer concentration comprised between 1 and 50 wt%. Typically, 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. Additionally, 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. These features are selected to permit the necessary mechanical response of the responsive layer 302 to facilitate deflection of the shape changing units 600. In particular, 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.

[0160] In order to optimize the mechanical properties of the responsive layer 302, particularly the bending radius of the responsive layer 302 in the shape changing units 600 upon deflection following a trigger stimulus, in preferred embodiments - particularly embodiments in which the responsive layer 302 is in a hydrogel form - it is contemplated that 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.

[0161] It has to be noted that features or details disclosed with reference to the embodiments of fig. 1 to 9 may be applicable also to the embodiments offig. A, 1A, B, 1 B (B’, B”), as the skilled person can appreciate, and repetition of these details is avoided only for conciseness.

[0162] In preferred embodiments, the conductive tracks 400 are configured and designed to comprise:

[0163] i) a plurality of contact pads 410 for electrical contact with an external device,

[0164] ii) 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

[0165] ii) a plurality of electrode pads 401 , each located at the distal end of at least one conductive track 400, the electrode pads 401 being configured to electrically interface the external environment, particularly biological material.

[0166] As it will be evident, 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.

[0167] 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.

[0168] In one embodiment, 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. In other embodiments, communication device provides a wired network connection, such as an Ethernet connection or a modem.

[0169] A device according to this aspect of the invention 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.

[0170] In embodiments, the instructions contained by the non-transitory computer readable medium comprise, among others:

[0171] - 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;

[0172] - instructions for operating a power supply for providing electrophysiological electrical stimuli to the biological entity;

[0173] - instructions for operating sensors, as will be detailed hereinafter.

[0174] As anticipated, in some embodiments 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.

[0175] 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. The output is generally a signal that is converted to human-readable display at the sensor location or transmitted electronically over a network for reading or further processing. The specific input could be for instance light, heat, motion, moisture, pressure, or any one of a great number of other environmental phenomena. According to the invention, 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.

[0176] 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. In one embodiment, sensors further comprise means for wirelessly receiving a feedback input from a computer able to regulate the functioning of the system.

[0177] In additional embodiments of the invention, 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 rigidsupport 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.

[0178] In embodiments of the invention, 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. In some embodiments, 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. for delivering drugs, hormones, neurotransmitters or other compounds, to remove locally generated heat or unfavourable electrochemical products or waste products etc. Preferably, the channels can be operatively connected through their inlets to a fluidic pump or some other drug delivery device via fluidic connection means.

[0179] In embodiments, 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. In this latter mentioned embodiment, 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.

[0180] 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. For example, 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. Alternatively, covalent coupling, e.g. using alkylating or acylating agents, is used to provide a stable, long-term presentation of a bioactive substance on the responsive layer 302 in a defined conformation. Alternatively, non-covalent adsorption can be used, for example electrostatic, hydrophobic, dipole-dipole, hydrogen bonding, physisorption and the like. In an additional or alternative embodiment, 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.

[0181] EXAMPLES

[0182] To describe and illustrate more clearly the present invention, the following examples are provided in detail, which however are not intended to be limiting of the invention.

[0183] In the present example, 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.

[0184] 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. Moreover, such design is compatible with the integration of cell perfusion systems, which could be implemented to improve cell viability. Furthermore, the proposed deviceseamlessly integrates with commercially available electrophysiological readout systems, offering a plug-and-play solution without the necessity for custom-made wiring.

[0185] 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.

[0186] 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.

[0187] Through the device and system of the invention, 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.

[0188] In essence, 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.

[0189] Characterization of the polyacrylic acid hydrogel

[0190] 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 PAAhydrogel depending on its crosslinker concentration and the re-swelling solution used. We synthesized bulk PAA hydrogels with three representativeN,N’methylenebisacrylamide (MBAA) crosslinker concentrations, namelyO.05 wt% (1X), 0.20 wt% (4X) and 0.80 wt% (16X). As the de-hydration process is known to influence the final hydrogel behavior, we mimicked the drying conditions of the hydrogel grafted on the polyimide substrate by performing an anisotropic drying process on the PAA. We then laser cut circular dry hydrogel samples which we re-swelled in four representative media formulations: de-ionized water (DIW), the typical swelling medium used in hydrogel characterization; phosphate-buffered saline (PBS), used during the electrochemical characterization of the e-Flower; cell culture media (CCM) at 25°C, to mimic the conditions during brain spheroid seeding; and the same media but at 37°C, representing the final working condition of the e-Flower inside the incubator. Finally we measured the hydrogels’ swelling behavior and storage moduli in each condition, as illustrated in Fig. 6. While the curvature of the e-Flower’s petals primarily originates from the lateral swelling of the PAA hydrogel, we measured not only the hydrogel’s mass and area swelling ratios, but also its variation in thickness.

[0191] As expected, the re-swelling medium strongly influenced the swelling behavior observed in the PAA hydrogels. Overall, re-swelling in CCM produced a lower level of swelling compared to DIW in terms of both mass, area, and thickness variations. As illustrated in Fig. 6A, 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. Moreover, 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). Similarly, considering the thickness swelling ratios, samples re-swollen in warm CCM were 33% thinner than the DIW equivalent, as visible in Fig. 6C. Such differences in swelling behavior were likely due to electrostatic interactions forming between the re-swelling medium and the hydrogel’s polymeric matrix, and further influenced by complex molecules present in the cell culture media, such as amino acids. To visualize possible morphological differences in the hydrogel depending on the re-swelling medium used, we obtained SEM images of freeze-dried samples previously soaked in either DIW (Fig. 6J) or CCM (Fig. 6K). Hydrogels re-swollen in DIW showed an open-cell foam morphology, with large (diameter ~ 50 pm) pores and thin cell walls. On the contrary, hydrogels re-swollen in CCM showed denser hydrogel cell walls, likely due to the presence of nutrients and other active ingredients in the CCM interacting with the hydrogel network.

[0192] The crosslinker concentration also influenced the extent of hydrogel swelling. As shown in Fig. 6D, 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. Also in this case, 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). In general, increasing crosslinker concentration made hydrogels swell more in area (0.27x, 1.85x and 2.79x) but less in thickness (23.61x, 8.87x and 4.87x). This behavior is likely attributed to the anisotropic drying procedure used to mimic the grafting condition of the hydrogel at the wafer level and is crucial for the design of bilayers with a tailored bending radius. Additionally, SEM observations revealed that higher crosslinker concentrations yieldedhydrogel networks with significantly smaller pore sizes and thicker polymeric structures

[0193] To evaluate the viscoelastic properties of the hydrogel samples, we performed rheological plate-plate oscillatory measurements with varying shear amplitudes, as represented in Fig. 6G, where the properties of a sample with 1X crosslinker concentration at equilibrium in warm CCM is reported. The hydrogels re-swollen in DIW showed a comparable elastic modulus compared to the ones re-swollen in PBS (2.95 kPa and 2.97 kPa respectively), while the CCM-swollen samples showed an up to 45% increase in elastic modulus (4.37 kPa and 3.56 kPa for RT and warm CCM, respectively), see Fig. 6H. As expected, 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). Overall, the hydrogels in all conditions showed an elastic behavior up until a 3% shear strain.

[0194] Hybrid polyacrylic acid / polyimide bilayers

[0195] To characterize the bending of the polyacrylic acid / polyimide bilayer constituting the e-Flower’s petal, we focused on the simplest bilayer shape, namely a rectangular strip. In this study, we aimed to obtain a customized radius of curvature suitable for the expected size of the neural spheroid at our disposal, between 500 and 1000 pm. The curvature of the bilayer is known to be influenced by the hydrogel / polymer thickness ratio. For consistency, we fixed the PI thickness at 6 pm and the PAA thickness at 150 pm as prepared. We then focused our investigation on the effect of the reswelling medium and crosslinker concentration on the bilayer’s curvature. The process involved fabricating PI layers via spin coating, followed by hydrogel’s grafting. To enable covalent PAA grafting onto the PI, we sputtered a 15 nm-thin layer of SiOx on the PI substrate, followed by silanization using 3-(trimethoxysilyl)propyl methacrylate (TMSPMA). We then dispensed and cured the PAA pre-gel solution directly on the TMSPMA- functionalized PI surface, while controlling its thickness with custom-made molds. Fig. 7A displays an optical microscope photograph of arepresentative bilayer strip swollen at equilibrium, that was used to manually evaluate the bilayer’s radius of curvature as shown by the dotted circle. The bilayer’s curvature depended on the re-swelling medium, as highlighted by Fig. 7B. The bilayer strips bent with a radius of curvature down to 99 pm in DIW, which doubled to 208 pm in PBS and almost tripled in CCM (285 pm and 295 pm at RT and 37°C, respectively). Equivalently, 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). This behavior is a consequence of the relative area’s swelling ratio and stiffness, both previously assessed within the bulk hydrogel. Notably, bilayers with a 1X crosslinker concentration reached a 300 pm curvature radius when swollen in CCM at 37°C, aligning with the our desired size range. We therefore kept this formulation throughout all the following experiments. 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.

[0196] Having confirmed our ability to match the radius of curvature of the spheroid, we devised a device geometry capable of engulfing the neural spheroid, while maintaining a stable position and facilitating the routing of metallic electrical interconnects. We envisioned a cup-like device resembling a flower, held in place by simple polyimide bridges. To predict the final shape of the hydrogel-actuated device and guide the design of the final 3D MEA, we developed a mechanical finite element model of the flower-shaped bilayer. To mimic the bilayer’s behavior when immersed in solution, we varied the hydrogel’s initial volumetric strain. We could then visualize the flower-shaped bilayer’s deformation from fully open to closed. Through thismodel, we assessed the strain experienced by the polyimide substrate during actuation. Fig. 7G illustrates the distribution of the volumetric strain in the polyimide layer along the midsection of a petal as the flower closed. When in closed configuration, 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.

[0197] 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. Apart for the devices swollen in PBS that reached the desired 3D shape in 221 s, there was no significant effect of the swelling medium on the morphing dynamics (113 s in DIW and 130 s circa in CCM). This closing dynamics renders the device suitable for manual seeding spheroids within a typical cell culture laboratory environment.

[0198] Design and characterization of the e-Flower

[0199] After confirming the effective closure of the flower-shaped bilayer, we proceeded to the integration of platinum electrodes within its polyimide petals. We devised an e-Flower geometry including 32 platinum electrodes of 30 pm diameter distributed across four polyimide petals, as depicted in Fig. 8A. Additionally, we located a squared reference electrode with a side length of 800 pm on the non-actuated part of the microelectrode array, which will be used as reference in the electrophysiological recordings. As done forthe flower-shaped bilayers without metallization, we connected the e- Flower’s petals to the body of the array by four arms (or bridges). These arms serve two purposes: they secure the e-Flower in place facilitating its manipulation, and they host the electrical tracks connecting the active electrodes to the contact pads. Finally, we arranged the e-Flower’s pads in a rectangular array, to interface with the pogo pins of the commercially available MEA-2100 recording platform.

[0200] To graft the hydrogel on the back side of the microelectrode array, we developed a microfabrication process flow in which the electrodes were fabricated upside-down, and the hydrogel actuation layer was confined to the flower region. We fabricated the metallic tracks using standard thin-film technology and lithographic patterning. To ensure the continuity of the metallic layer in the upside-down processing, we performed thermal reflow (150°C for 30s) to smoothen the sidewalls of the photoresist to then define the electrode and pad openings. A representative resulting platinum electrode is visible in the SEM image of Fig. 8A (inlet). We then grafted the PAA hydrogel on the PI as described in the previous paragraphs. 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).

[0201] To facilitate the cell culture media replacement, we integrated the 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.

[0202] In light of the three-dimensional design of the proposed MEA, a crucial step in our investigation involved determining the electrodes' capacity to endure the small radius of curvature of the e-Flower’s petals. To do so, weperformed electrochemical impedance spectroscopy (EIS) in PBS, a standard methodology for assessing electrode electrochemical properties. The obtained results are illustrated in Fig. 8D, where we represent the impedance amplitude and phase spectra obtained for 3 e-Flowers. The three-dimensionally actuated electrodes showed impedance and phase spectra consistent with the anticipated behavior from thin-film platinum electrodes. The electrodes showed reproducible impedance spectra both within the same device and across different devices, with an average impedance amplitude at 1 kHz of 327.7 kQ (n = 3 devices, for a total of n = 95 recording electrodes and n = 3 ground electrodes). Moreover, the recording electrodes consistently displayed a higher impedance amplitude across all frequencies compared to the ground electrodes, as anticipated owing to the significant difference in surface area between the recording (Arec_eiect = 707 pm2) and ground electrodes (Agm_eiect = 640000 pm2). Notably, 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). Collectively these observations confirm the electrodes’ ability to withstand the small radius of curvature of the e-Flower’s petals.

[0203] To further assess the system’s reversibility and reusability, we evaluated whether repetitive bending of the petals affected the electrode behavior. We therefore conducted EIS measurements after three repetitive drying-swelling (opening-closing) cycles, as presented in Fig. 8E. Remarkably, the electrode impedance amplitude remained relatively stable throughout the cycles, regardless of the electrode’s position along the petal. This result suggests the electrodes’ capability to endure the repetitive bending required during petal actuation.

[0204] Integration of the neural spheroids in the e-Flower

[0205] Following the successful fabrication and electrochemical characterization of the e-Flower, our focus shifted towards achieving an initial proof of concept for the device's functionality with brain spheroids. Our primary objective was to conduct electrophysiological recordings to demonstrate the e-Flower'scapability to capture neural activity in brain spheroids. For this purpose, we selected a brain spheroid and delicately transferred it onto the open e- Flower, along with a small volume of cell culture media (< 200 pL), as illustrated in Fig. 9A. As the hydrogel started swelling, it triggered the reconfiguration of the e-Flower from 2D to 3D, enveloping the spheroid and bringing the electrodes into close proximity to its surface within minutes. As final step, we introduced additional cell culture media to fill the fluidic channel and transferred the e-Flower to the incubator.

[0206] After an 8h stabilization period, we extracted the e-Flower from the incubator and transferred it to the electrophysiological set-up. We could obtain a three-dimensional recording of spontaneous field potentials across the surface of the spheroid, as visually represented in Fig. 9B. The overlaid field potential waveforms exhibited distinct patterns, each with an average duration of approximately 2 ms. The recorded activity was comparable to prior studies.

[0207] To establish the neural origin of the recorded signal, we conducted a chemical manipulation of neural activity by elevating the extracellular K+concentration, a known method for suppressing spiking activity. Specifically, we increased the extracellular KCI concentration to 100 mM, a level that was twenty times higher than the K+concentration in the standard cell culture media formulation (5 mM). Moreover, the electrodes’ noise level remained stable throughout the experiment, highlighting the stability of the electrodes through the multiple change of media and washing steps.

[0208] In this study, we introduce the 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. The e- Flower hosts 32 thin-film electrodes embedded within its polyimide structure and demonstrated the ability to reversibly open and close at least 3 times.Finally, we demonstrated a proof of concept of the e-Flower’s capability to detect spontaneous neural patterns across the entire spheroid surface.

[0209] 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.

[0210] We proposed a fabrication process flow that integrates electrode and hydrogel processing, while being compatible with conventional thin-film microfabrication techniques and enabling hydrogel grafting at the wafer level. This process flow offers high versatility, accommodating various materials and device geometries. First, it allows the fabrication of bilayers with adjustable thicknesses both in the passive polymer component, and in the hydrogel component, dependent on the mold thickness. It is adaptable to a range of polymers, including Sll-8 and parylene, as well a spectrum of hydrogel formulations, including polyacrylamide and polyhydroxyethilmethacrylate (PHEMA), but also stimuli responsive hydrogels such as the thermo-responsive pNIPAm. Also 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.

[0211] Beyond the MEA itself, our work features a comprehensive system design enabling in vitro signal recordings from pre-formed three-dimensional tissues. We propose a device design that seamlessly integrates with commercially available electrophysiological readout systems, without the necessity for custom-made wiring. Inspired by prior studies, our compact device design incorporates a PMMA-based fluidic channel and culture well. This integration eliminates the requirement for additional plasticware such as Petri Dishes and well-plates. Consequently, it facilitates the transfer of the tissues between the incubator and the recording setup and it simplifies the process of changing the cell culture media. By doing so, this design potentially reduces the risk of contamination, a significant concern when culturing tissues alongside electronic devices. Moreover, such design is compatible with the integration of perfusion systems, which would ensure a continuous flow of cell culture media to the tissue, thereby enabling future pharmacological experiments.

[0212] To create a system fully compatible with fluorescence imaging, a substitution of polyimide with parylene is envisageable, and platinum with the transparent Indium-Tin-Oxide (ITO).

[0213] Materials and Methods

[0214] 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. Three crosslinker concentrations were used: 0.05 wt% (designated as 1X), 0.20 wt% (4X), and 0.80 wt% (16X). Hydrogel-precursor solutions were vortexed for 2 min, sonicated for 10 min, and finally subjected to 10 min of nitrogen bubbling to remove air and dissolved oxygen.

[0215] Bulk PAA Hydrogel Synthesis. The PAA hydrogel samples were synthesized through 20 min UV curing of gel-precursor solution dispensedbetween 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. This anisotropic drying procedure was adopted to prevent wrinkling and other deformations associated with stresses arising from water evaporation, and to mimic the conditions experienced by the hydrogel during the fabrication of the e-Flower. Lastly, hydrogel disks of 1 cm in diameter were machined using a femtosecond excimer laser (WS Turret, Optec Laser Systems), unless specified otherwise.

[0216] Swelling Tests. Fully de-hydrated PAA hydrogel samples were weighted to quantify their dry mass Mt=o and subsequently submerged either in DIW at 25°C, phosphate-buffered saline (PBS; 1X) at 25°C, or cell culture medium (CCM; 500 mL NeurobasalT M Plus + 10 mL B27 Plus Supplement + 1.25 mL GlutaMAXT M Supplement, Thermo Fisher Scientific) at 25°C or 37°C. The mass Mt of submerged samples was measured at multiple time points (5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h). The mass swelling ratio was calculated at every time point as RatiOMass=(Mt-Mt=o) / Mt=o. To quantify variations in area and thickness, the hydrogel samples were imaged with a digital microscope (DVM6, Leica Microsystems) in the fully dehydrated state and after overnight swelling in the different media mentioned above. Images were imported into Fiji and manually analyzed. The area and thickness swelling ratios were computed as RatiOA rea- (Aswollen- Adry) / Adry, and RatiOThickness=(tswollen—tdry) / tdry-

[0217] Rheological Characterization. The mechanical properties of the PAA hydrogels were measured on swollen hydrogel disks of 25 mm in diameter obtained with a puncher. All measurements were performed using a rheometer (MCR 302, Anton Paar) equipped with a Peltier heating system and an aluminum plate-plate geometry (PP25, 25 mm in diameter) at 25°C and 37°C. Storage G' and loss G" moduli were measured through amplitudesweep tests conducted in oscillatory mode with strain amplitudes ranging from 0.01 % to 250% at a frequency of 1 Hz. The elastic modulus E of the hydrogel samples was estimated as E = 2G'(1 + v), where G' was defined as the storage modulus value measured in the middle of the linear viscoelastic region, and the Poisson ratio v was assumed to be equal to 0.5.

[0218] 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. Under N2 environment, the PAA hydrogel-precursor solution prepared as previously described was pipetted on the TMSPMA-functionalized surface, covered with an HMDS-treated glass wafer, and polymerized via exposure to UV light for 20 min. 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.

[0219] Curvature Analysis of the PAA-Polyimide Bilayers. Dry PAA-PI bilayer strips of 1.2 mm or 1.5 mm length and 0.3 mm width were submerged in different swelling media at 25°C and 37°C for at least 30 min and then imaged with a digital microscope (DVM6, Leica Microsystems) while still submerged. After pre-processing images with brightness and contrastenhancement as well as noise removal algorithms, the average radius of curvature of the inner outline of bilayer splines was quantified in Fiji using the Kappa plugin.

[0220] Closing Dynamics Measurements. Flower-shaped PAA-PI bilayers were fabricated as described in the previous paragraphs. The fully dehydrated samples were submerged in different swelling media either at 25°C or at 37°C, and the time needed to reach the closed 3D configuration was measured. In the case of tests involving multiple closing and opening cycles, the flower-shaped PAA-PI bilayers were washed in DIW and dried overnight between cycles.

[0221] Scanning Electron Microscopy (SEM). Scanning electron micrographs of the PAA hydrogel cross-sections and the PAA-PI bilayers were acquired (SLI5000, Hitachi). Fully swollen samples were lyophilized overnight, followed by SEM imaging in low vacuum mode without any additional coating.

[0222] Finite Element Analysis (FEA). A finite element model of the flowershaped bilayer was created in COMSOL Multiphysics 6.1 (COMSOL Inc., Stockholm, Sweden). The geometry was built starting from original sketches of the fabricated devices, to then extrude two different layers: the passive polymer (polyimide in our case), included the main flower and the bridges and the hydrogel only below the flower part. The thickness of the active swelling layer is 10 times higher than the passive layer. To simplify the model and speed up the solving step, the 3D geometry was split and only an eight was kept, including therefore half a bridge and half a petal, to then use symmetry boundary conditions on the sides. The passive layer was set with a Young’s modulus of 1 GPa and a Poisson ratio of 0.35. 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 samestrain 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.

[0223] e-Flower Microfabrication. First, a 3-pm-thick polyimide (PI; PI-2611 , HD MicroSystems) layer was spin-coated and baked on a 4-inch silicon wafer previously coated with a Ti / AI (20 / 100 nm) sacrificial layer. Secondly, 6 pm of photoresist (AZ10XT, Merck) were spin-coated on the wafer, baked, patterned by photolithography (MLA 150, Heidelberg Instruments), and developed to define the e-Flower electrodes and pads. Thereafter, photoresist reflow was induced by placing the wafer on a hotplate at 150°C for 30 s. 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. After 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. After surface preparation (V10-G, PINK), the aforementioned layers were covered by spin-coating and baking a 3-pm-thick PI superstrata. Following 10 min of dehydration at 110°C and O2 plasma activation, wafers were sputtered with 10 nm of Ti and 15 nm of SiOx. Surface functionalization and PAA hydrogel grafting were performed as elucidated in the previous paragraphs. Once the hydrogel dried, the flower-shaped outline of the microelectrode array was micromachined using a femtosecond excimer laser, as well as the outer outline of the entire device. The devices were released from the underlying wafer by anodically dissolving the Al sacrificial layer in a saturated NaCI solution. Finally, a custom-designed fluidic platform made of poly(methyl methacrylate) (PMMA) layers was assembled around the device.

[0224] 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 105Hz, 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.

[0225] Insertion of the Neural Spheroids in the e-Flower. Human brain spheroids were provided by the Tissue Engineering Laboratory, HEPIA HES-SO, and cultured as previously described. Once formed, the brain spheroids were maintained in cell culture media (500 mL NeurobasalTM Plus + 10 mL B27 Plus Supplement + 1 .25 mL GlutaMAXT M Supplement, Thermo Fisher Scientific) at 37°C and 5% CO2 under agitation. Before spheroid insertion into the e-Flower, the device was sterilized by UV light illumination for 30 min. 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.

[0226] 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 ofelectrophysiological 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 6onaway from the mean of the filtered signal. A robust estimation of the average noise level of each channel was obtained through on= median(|S(t)| / 0.6745), where |S(t)| represents the absolute amplitude of the filtered signal. Peaks less than 3.5 ms away were discarded, as well as peaks detected synchronously by more than one electrode. Detected events were isolated by windows of 5 ms (from 2 ms before to 3 ms after the detected event) and event times used for raster plot representation.

[0227] While the invention has been disclosed with reference to certain preferred embodiments, numerous modifications, alterations, and changes to the described embodiments, and equivalents thereof, are possible without departing from the sphere and scope of the invention. Accordingly, it is intended that the invention not be limited to the described embodiments, and be given the broadest reasonable interpretation in accordance with the language of the appended claims.

Claims

Claims1. A device comprising-a passive layer (301 ) composed of a first polymeric material, the passive layer (301 ) extending on a plane (PL);-a responsive layer (302) in contact with 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,-at least one cutting line (950) 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) including 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 to raise, the raising portion (640) raising as a consequence of a trigger stimulus changing the shape of the shape changing unit (600).

2. The device of claim 1 further comprising-an array of conductive tracks (400) in or on the passive layer (301 ), the shape changing unit (600) further including electrical pads (401 ), each pad being connected to an end of a track of said conductive tracks (400).

3. The device of claim 1 wherein the raising portion (640) has a same shape of the at least one cutting line (950) and wherein, when the raising portion (640) is not subject to the trigger stimulus, the raising portion (640) lays on the plane (PL) in close proximity with the base portion (630).

4. The device of claim 1 wherein the raising portion (640) has a different shape than the shape of the at least one cutting line (950), wherein said shape of the cutting delimits at least an opening (900) through the passive layer (301 ) and the responsive layer (302).

5. The device of claim 1 wherein at least two cutting lines (950) delimit corresponding at least two shape changing units (600), wherein the raising portionsto delimit a portion (P) on the passive layer (301 ) for locating the biological entity (800), and wherein the two cutting lines 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.

6. The device of claim 1 , wherein 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), the device further including a first rigid support (100) for supporting the intermediate layer (300) on the plane (PL) and a second rigid support (200), and wherein the second rigid support (200) is placed on top of the intermediate layer (300) for protection.

7. The device of claim 6 wherein the second rigid support (200) delimits a chamber (350) with an opening (201 ) over the intermediate layer (300), the intermediate layer being arranged between the second rigid support (200) and the first rigid support (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 ), and wherein the opening (201 ) is suitable to deposit a biological entity onto the intermediate layer (300).

8. The device of claim 7 wherein the raising portion (640) is floating in the chamber (350).

9. The device of claim 5 wherein at least one anchoring element (500) 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 (302) where the at least one shape changing unit (600) is not delimited, said at least one shape changing unit (600) being attached to the anchoring element (500) through the portion (P) of the passive layer (301 ) for locating the biological entity (800).

10. The device of claim 1 , wherein said mechanically adaptive material is capable of changing shape upon a trigger stimulus selected from one or more of temperaturechange, electric field change, magnetic field change, light change, pressure change, pH change, ionic strength change and swelling by liquid absorption.

11. The device of claim 1 , wherein said mechanically adaptive material is substantially composed of a hydrogel.

12. The device of claim 11 , wherein said hydrogel is selected from a non-limiting list comprising Polyacrylic acid, Polyacrylamide, Polyhydroxyethilmethacrylate, preferably Polyacrylic acid.

13. The device of any of preceding claims, wherein the second polymeric material has a monomer concentration comprised between 1 and 50 wt%.

14. The device of any of preceding claims, wherein 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.

15. The device of any of preceding claims, wherein the second polymeric material is manufactured by monomers crosslinking, with a crosslinker concentration comprised between 0.01 wt% and 10 wt%.

16. The device of any of preceding claims, wherein the first polymeric material has a Young’s modulus comprised between 1 kPa and 10 GPa.

17. The device of any of preceding claims, wherein the second polymeric material has a Young’s modulus comprised between 100 Pa and 1 MPa when fully swollen.

18. The device of any of preceding claims, wherein the first polymeric material is selected from a non-limiting list comprising Parylene, Polyimide, Sll-8, PDMS, Polyurethane, SEBS.

19. The device of any of preceding claims, wherein the thickness ratio between said passive layer and said responsive layer is comprised between 0.1 and 100 when dry.

20. The device of claim 2, wherein said conductive tracks (400) comprise: i) a plurality of contact pads (410) for electrical contact with an external device,ii) elongated conductive paths, each departing at least from one of said contact pads (410), and running along the passive layer and the at least one anchoring element to arrive to at least one shape changing unit (600).

21. The device of any of preceding claims, further comprising, operatively connected thereto, at least one of a sensor, an LED, a fluidic connection, a fluidic channel, a power supply, an electrophysiological system and a computer device.

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