The present invention relates to a device for obtaining three-dimensional cell cultures, to a method for the implementation thereof, and to a use of such device for obtaining suspended cells not adherent to the substrate.

Propensity of a cell to adhere, grow, and differentiate on a compact substrate (or support) or on a scaffold having a supporting function is one of the most important aspect of tissue engineering and biomaterials characterization.

Most of the physiological and pathological cell processes, such as growth, adhesion, migration, secretion, and death are affected by the extracellular matrix three-dimensional organization. Cells respond to local concentrations of a great number of molecules that can be dissolved in extracellular media (gases, amino acids, ions, drugs), which are present on the growth surface and on the membrane of the adjacent cells (membrane receptors).

In three-dimensional cultures, the ratio between cells and substrate is controlled by the geometrical characteristics and the chemical and physical properties of the support, with the aim of producing cell cultures in vitro with an organization and functionality as near to those of native tissues as possible.

It is known that a cell behaviour can be controlled and guided through changes in the topographic characteristics of the support surface, on a micro- and nano-metric scale. It is exactly in this context that controlling the wettability may also have a very important role in optimizing the performance of biocompatible substrates.

By imposing specific geometries to the surface of a biocompatible solid device and changing the wettability characteristics thereof, it is possible to increase its surface-volume ratio, thus making, on a dimensional point of view, also the device geometric characteristics compatible with the morphologic characteristics of the cell type that is used. Hence, wettability and roughness of the growth supports are to be considered among the most important factors influencing cell spreading, alignment, and migration.

One of the theories validating these cell behaviours is the "contact guide effect" theory, according to which focal contacts mediated by integrins transfer variable degrees of tension and/or compression to the cytoskeleton elements by following the surface topographic variations. Such theory is described in the article by Tan, J. L. et al. Cells lying on a bed of microneedles: An approach to isolate mechanical force. Proceedings of the National Academy of Sciences of the United States of America 100, 1484-1489, doi:10.1073/pnas.0235407100 (2003).

The ability to grow by responding to the growth support topographic characteristics is well known for different cell types; they show interesting growth responses when plated on substrates characterized in surface by different geometries and different degrees of superhydrofobicity.

However, there are cell types that normally grow while being adherent to the substrate surface, such as neuronal cells. For such cell types, substrates capable of allowing a suspended growth thereof are not known.

Document WO 2010/023635 discloses a device in which, on a substrate, a plurality of micro-structures mutually spaced apart in a periodical manner are obtained, so as to make the substrate super hydrophobic, intended to the use for the concentration and localization of a solute in a reduced portion of space.

Among the systems used most recently for three-dimensional growing cells there are matrices of synthetic or natural biodegradable polymers. With regard to the various applications, polymers with different porosities can be obtained in devices such as membranes, films, and nano-tubes, as described in Langer, R. & Tirrell, D. A. Designing materials for biology and medicine. Nature 428, 487-492, doi: 10.1038/nature02388 (2004). For example, tubes made of a collagen matrix are used to promote nervous tissue regeneration, and materials in gel form are used mainly for isolation or as substrates for the connective tissue regeneration. In most cases, these devices are composed of a porous matrix on which the cells attach. Within the pores, in addition to the cells, the fluid acting as a nutrient also must fit in. Cell growth is strongly related to the amount of nutrients that are present in the growth medium and the motion type of the fluid. When a uniform culture inside the whole cellular construct is required, it will be necessary that the nutrient fluid succeeds in invading each pore of the matrix. On the other hand, the cell growth, in time, fills the voids that are present in the matrix, thus causing the nutrient flow to decrease.

The known supporting devices for the three-dimensional growth of suspended cells are not perfectly able to provide a sufficient amount of nutrients, while suitably removing waste products.

Furthermore, the limitations in standard treatment of peripheral nerve injuries, requiring the implantation of an autologous nerve, thus leading to a deficit at the donor, and requiring additional surgery, have driven the research for bioengineering solutions in manufacturing artificial implants allowing obtaining a good neural tissue regeneration.

Therefore, object of the present invention is to propose a device for obtaining three- dimensional cell cultures, which is capable of overcoming the above-mentioned problems, and allowing the growth of suspended cells also for cell types that usually grow while being adherent to the substrate, particularly for nervous tissue regeneration applications.

A further object of the present invention is to propose a method for manufacturing the above-mentioned device and a use of such device for obtaining suspended cells not adherent to the substrate.

These and other objects are achieved by a device, whose characteristics are defined in claim 1 , with a method for obtaining such device, whose characteristics are defined in claim 7, and with a use of such device for obtaining suspended cells not adherent to the substrate, as described in claim 13.

Particular embodiments are the subject of the dependent claims, the content of which is to be meant as an integral and integrating part of the present description.

Further characteristics and advantages of the invention will be apparent from the following detailed description, given by way of non-limiting example only, with reference to the appended drawings, in which:

- Fig. 1 is a top view of a device according to the invention,

Fig. 2 is a perspective enlargement of one of the nano-structures of the device of Fig. 1, and

Fig. 3 is a side view of the nano-structure of Fig. 2. In brief, the invention uses techniques of micro-fabrication for manufacturing a super hydrophobic device having structures with side walls at least partially nano-patterned, used in cell culture methods for obtaining cells that are suspended, or not completely adherent to the substrate, in particular for the three-dimensional growth of hippocampal neuronal cells and for the growth of hippocampal neurons and glial cells co-culture.

Such device combines two types of topographic details, having dimensions on a different scale, respectively, in particular micrometric structures conferring superhydrofobicity to the substrate surface, while nano-metric features of such micrometric structures make the stratification in three dimensions of the cell cultures possible.

The surface geometry of the supports is characterized by large free spaces between a micro-structure and the adjacent one, and this allows a better recirculation of the nutrient substances and easier gaseous exchanges between the cells and the growth medium. Compared to the conventional cell cultures in vitro, the device of the present invention allows achieving results that are qualitatively higher by providing to the cells the correct amount of nutrients and mechanical stimuli for the development of co-cultures and tissues for biomedical applications that are more reproducible and cost effective.

Therefore, the invention aims to manufacturing nano-patterned superhydrofobic devices for the three-dimensional growth of normal cells (also in co-culture), for the study of the spatial arrangement of tumoral cell cultures; for pharmacological and toxicological screening tests in vitro, aimed to cell therapy.

The invention allows performing tissue engineering applications, such as nervous regeneration.

In Fig. 1, the device according to the invention is illustrated, comprising a substrate 1 for example made of silicon or a biocompatible or bioerodible polymeric material, and a plurality of micro-structures 2, in particular micro-cylinders, extending from the substrate. Such micro-structures 2 can advantageously have a section in a circular, squared, rectangular, hexagonal shape, or any other shape.

Such micro-structures 2 are preferably arranged orthogonally to the surface of the substrate 1 and are located on the substrate 1 according to a periodical grid so as to make the substrate 1 super hydrophobic.

By the term super hydrophobic in the present description is meant a surface on which the contact angle that a drop of (distilled) water forms with the surface itself is above 150°. The contact angle is the angle formed by the tangent at the liquid-vapor interface with the tangent at the liquid-solid interface. Obtaining micro-patterned super hydrophobic surfaces is a function of the surface density and the transversal dimension of the micro-structures. The design principles for manufacturing the above-mentioned micro-patterned super hydrophobic surfaces are per se known and are described, for example, in de Angelis, F. et al. "Breaking the diffusion limit with super-hydrophobic delivery of molecules to plasmonic nanofocusing SERS structures", Nature Photonics, Volume 5, Issue 1 1, pp. 682-687 (201 1), incorporated herein for effect of such quote. Advantageously, the substrate 1 has a resistivity of about 5-10Ω/αη.

The micro-structures 2 are obtained by lithography and etching processes, or micromoulding processes, per se known, as described herein below.

The micro-structures 2 are spaced apart on the substrate 1 by a predetermined distance 4 preferably ranging within 15-30μηι.

The micro-structures 2 have preferably a base width ranging within 5-20μπι, and a height ranging within 5-30μηι.

By the term base width is meant in the present description the minimum dimension among those defining the section shape. The base width and the distance 4 between the micro-structures 2 are selected according to a per se known criterion from the above-mentioned article.

In this manner, a small fraction of solid material of the overall device is obtained, which, in the case of micro-cylinders, is of:

where 1 is the distance 4 and d is the diameter of the micro-cylinders.

Such fraction f allows increasing the hydrofobicity of the substrate 1, which therefore becomes able to reach contact angles approaching to 170°. However, the parameter f is still sufficiently large to prevent the collapse of the drop at the early stages of evaporation.

In order to use the device for the objects that are the subject-matter of the invention, it is preferred to apply on the device surface coatings suitable to promote the growth of cell cultures while maintaining or imposing the superhydrofobicity properties. To this aim, the application of a biocompatible coating (where the substrate is not per se biocompatible) can be contemplated, for example, a metallic, glass, or glass-ceramic coating, and an optional adhesion layer for said biocompatible coating, applied on the substrate surface. A thin hydrophobic polymeric coating, for example, a fluorinated polymer, as obtained by the polymerisation of octafluorocyclobutane, can finally be applied to the biocompatible coating, having thicknesses such as not to change the device superficial biocompatibility.

In Fig. 2, a perspective enlargement is illustrated of one of the micro-structures of the device of Fig. 1.

The side walls of the micro-structure 2 are not smooth, but they are patterned, i.e., they have alternated projections 14 and recesses 16, with a predetermined pitch (or spatial distance) depending on the duration of the plasma isotropic dry etching step and the deposition step of a passivation layer, described herein below.

The height of the projections 14 or recesses 16 can be constant along the whole wall of a micro-structure, or it can vary, as well as the height between adjacent projections 14 and recesses 16 can be the same or different.

In the present description by the term smooth is meant that the side wall of a micro- structure, as seen in section of a plane parallel to the main extension axis of the micro- structure, has a rectilinear profile r and it does not have projections and recesses.

In Fig. 3, a side view of the micro-structure 2 of Fig. 2 is shown, in which the height h of the micro-structure 2 and the height i separating two adjacent projections 14 are highlighted, respectively.

In a variant of the invention, the micro-structures 2 do not have completely patterned side walls, but they have an alternation of patterned zones (bands) and smooth zones (bands) without projections 14 and recesses 16. Preferably, the device of the present invention also comprises a plurality of electrodes, each associated to a micro-structure 2 (whether it is of the type with completely patterned side walls, or of the type with side walls with alternation of smooth zones and patterned zones), connected to conductive tracks manufactured in a per se known manner in the substrate 1. In such a manner, it is possible to apply voltages to such electrodes, therefore to such micro-structures 2, thus obtaining an electric device. Advantageously, the device of the present invention also comprises a plurality of micro- channels, which can be manufactured in a per se known manner, allowing controlled changes of medium or cytotoxicity tests.

Advantageously, the device of the present invention comprises an alphanumerical grid, located in the peripheral zone of the substrate 1 , so as to easily allow counting and localizing the cells, to allow an analysis by different techniques, such as confocal optical microscope, or atomic force microscope, Raman spectroscopy, or also SEM.

The manufacturing method of the above-described device will be now described, respectively for the case where the substrate is made of silicon or a bioerodible and biocompatible polymeric material.

In the case that the substrate 1 is made of silicon, it is first cleaned in a per se known manner.

Subsequently, a per se known optic lithography step is performed, by using an optical mask obtained by a laser writing process.

Particularly, a regular pattern of disks (or other polygons) is obtained within a covering layer that had previously deposited by spin coating on the cleaned substrate 1.

Then a covering mask is obtained, comprising a plurality of disks or polygons by carrying out an optic lithography on a uniform covering layer deposited on the substrate 1, for example, a resist, using in a known manner a light beam having a predetermined wavelength λ ranging within between 350 and 450nm.

Subsequently, in a known manner, the covering layer externally to the disks (or polygons) is removed, and in the following step a dry etching step of the DRIE type is carried out, of the substrate 1 , by using the pre-prepared covering mask.

After the etching step, the above-described micro-structures 2 are obtained (thus having all the spatial disposition characteristics illustrated above) and having an aspect ratio, i.e., the ratio between the height of the micro-structure 2 and the area of the base thereof, higher than 2.

The DRIE process used repeatedly alternates three steps:

- deposition of a chemically inert passivation layer;

dry etching with isotropic plasma;

cleaning the substrate 1 and the chamber where the etching is performed.

By virtue of this alternation of steps, micro-structures 2 are obtained, having nano- patterned side walls as shown in Fig. 2.

The passivation, dry etching, and cleaning steps have respective time durations ti, t ₂ and t ₃ ; the ratio r=t[/t ₂ has a predetermined value, for example, ranging between 0.5 and 1.5, and it is from such value that the possibility to obtain micro-structures 2 with substantially vertical walls is derived.

Any deviation from r would unbalance the process: if tj/t ₂ is higher than r, passivation step would dominate over dry etching, and therefore the micro-structures 2 would be frustoconical shaped; if t _t /t ₂ is lesser than r, the dry etching would dominate over the passivation step, and the micro-structures 2 would be shaped into an upside-down frustum.

The total time t= ti+t ₂ +t ₃ defines the total duration of a cycle; provided that ti/t ₂ is held constant at the predetermined value as defined above, the total time t can be selected as required. The longer t, the longer the recesses 16 of the side walls will be.

Subsequently, the covering layer is removed in a per se known manner with a removal solution. Finally, the above-described coatings are deposited on the substrate.

In the case where the substrate 1 of the device of the invention is made of a bioerodible and biocompatible polymeric material, initially a mould (master) of a rigid and undeformable material is made, such as, for example, silicon. The mould is manufactured starting from a starting substrate, in a similar manner to what has been described above with reference to the implementations steps of the micro-structures 2. The parameters d, I, and / remain the same and are inspired to the same designing criteria. The mould manufacturing process provides for the use of the same micro-optic lithography techniques as described above. Particularly, the same covering layer is used to obtain a covering mask complementary to that described above.

On the starting substrate for manufacturing the mould a uniform covering layer is deposited, by spin coating, on which, in a per se known manner by a lithographic process, a regular pattern of disks (or other polygons) is obtained.

A covering mask is thus obtained. Subsequently, the covering layer within the disks (or other polygons) is removed in a known manner, and, in the following step, a dry etching step of the DRIE type is carried out, as described above, by virtue of which the starting substrate is eroded at the disks, thus obtaining a mould comprising a plurality of holes having the same characteristics of the micro-structures 2 described above.

Particularly, such holes, by virtue of the alternation of steps of the DRIE process, have nano-patterned side walls.

After the dry etching step, the covering mask is removed in a known manner and the mould is then further cleaned.

Preferably, the so-obtained mould is silanized in a vacuum reaction bell, and it is subsequently "baked" at temperatures ranging between 100° C and 120° C.

At this point, bioerodible and biocompatible polymeric material is brought to melt in a known manner on a support.

Subsequently, the mould is placed in contact with said molten bioerodible and biocompatible polymeric material, avoiding the formation of air bubbles at the interface between the mould and the biocompatible material, so as to carry out a micromoulding process as described herein.

A forming or moulding step of the bioerodible and biocompatible polymeric material layer is carried out in a known manner.

The forming or moulding process comprises two steps:

1 ) forming or moulding at a constant pressure;

2) solidification at a constant pressure until reaching room temperature.

Finally, the bioerodible and biocompatible polymeric material is released from the mould by first removing the support, then separating the mould and the biocompatible material substrate with a slight pulling action.

Two implementation examples of the methods described above are described herein below, such examples being given by way of illustrative example only, non-limiting to the scope of the invention.

Implementation example No. 1

A first implementation example of the present invention comprises the use of a P doped silicon substrate 1 and the micro-structures 2 are arranged on the substrate 1 according to a periodical hexagonal grid and are spaced apart by a distance 4 of 20μιη. The micro- structures 2 have a base width of ΙΟμιη and a height of ΙΟμιη. The micro-structure 2, having a P doped silicon core 6, are coated with a first titanium layer 8, completely covering the core 6, a second gold layer 10, covering the first layer 8, and a third layer 12, covering the second layer 10, of a hydrophobic polymer obtained by performing a polymerisation of octafluorocyclobutane (C ₄ Fg).

To obtain such device, the silicon substrate is first cleaned with acetone and isopropanol, and, after immersion in a 4% hydrofluoric acid solution, it is cleaned with deionized water and dried with nitrogen. For the optic lithography, a Mask Aligner of the type MA6/BA6, SUSS MICROTEC is used, obtaining a regular pattern of disks (or other polygon) within a covering layer of AZ5214 negative resist.

Then the Mask Aligner produce a resist mask by carrying out an optic lithography on a uniform resist layer deposited on the substrate 1 , using in a known manner a light beam having a wavelength λ ranging between 350 and 450nm.

The dry etching step is carried out with a ICP-RIE, SI 500, Sentech Instrument GmbH instrument, and the used DRIE process repeatedly alternates three steps:

- deposition of a layer of a polymer obtained by polymerisation of octafluorocyclobutane (C ₄ F ₈ );

- dry etching with isotropic plasma with sulphur hexafluoride (SF ₆ );

- a cleaning of the substrate 1 and the chamber where the etching is performed. The value has a value ranging between 0.5 and 1.5 and with ti=8s, t ₂ =7s and t ₃ =4s recesses 16 having a spatial periodicity of 500nm are obtained.

Finally, the covering layer is removed with H ₂ S0 ₄ :H ₂ 0 ₂ =3 : 1 v/v. A this point, a first titanium layer having a thickness of 5nm and a second gold layer having a thickness of lOOnm are deposited on the substrate 1 by sputtering. The titanium layer improves adhesion of the gold layer. Finally, the second layer is coated with a 5nm third layer of a hydrophobic polymer obtained by polymerisation of octafluorocyclobutane (C ₄ F ₈ ).

Implementation example No. 2

A second implementation example of the present invention comprises the use of a substrate 1 of polycaprolactone (PLC), and the micro-structures 2 are arranged on the substrate 1 according to a periodical hexagonal grid and are spaced apart by a distance 4 of 20μιη. The micro-structures 2 have a base width of ΙΟμπι and a height of ΙΟμπι.

In this case, coating are not provided for.

A silicon master is obtained by using the same micro-optic lithography techniques described above. Particularly, negative resist layer of the AZ5214 type is used to obtain a covering mask complementary to that described above.

After DRIE etching and after obtaining a mould comprising a plurality of holes having the same characteristics of the micro-structures 2 as described above, the covering mask is removed by sonication in acetone at 55° C, and the mould is cleaned by a piranha solution.

The so-obtained mould is silanized in a vacuum reaction bell, and it is then "baked" at 112° C.

It is brought to melt on a silanized glass slide arranged on a Sigma type polycaprolactone plate at 133°, with M _w =77000 and T _me i _t = 65° C in pellets.

Then the mould is placed in contact with polycaprolactone as described before, the forming or moulding step is carried out by a Nanoimprinter type press, Obducat. The forming or moulding process comprises the steps of:

1) forming or moulding at a constant pressure at 5 bar at 68° C;

2) solidifying at a constant pressure a 5 bar until reaching room temperature. Finally, the polycaprolactone is released from the mould, by first removing the glass slide and then by separating in water the mould and the polycaprolactone substrate by a slight pulling action. The thus described process is optimized for small moulding surfaces (for example, about 1 cm ² ) and high final thicknesses of the bioerodible and biocompatible polymeric material layer (for example, about 500 μηι), but it can be further optimized for different moulding surfaces and thicknesses. A method to obtain three-dimensional cell cultures of suspended cells will be now described.

If a drop of growth medium, of the type described herein below, is deposited on the surface of the device of the present invention, it keeps an almost spherical shape with a contact angle at the air- water interface that can be theoretically predicted in a per se known manner, by using the Cassie and Baxter model. According to such model, the wettability behaviour of the device surface is described by the sole parameter f. When f tends to zero, at the interface with the device substrate 1, the liquid mostly "senses" air, and the drop would resemble a perfect sphere.

By virtue of this, the device can be suitably used to manipulate different samples of biomedical interest.

Ii is generally known that the hydro-repellent properties of the superhydrofobic materials reduce the water contact area on their surface, thereby minimizing the absortion of particles or molecules. The nano-patterned superhydrofobic surface of the device of the invention is used as a substrate for cell cultures.

In order to make so that on the device described above three-dimensional cell cultures can be obtained, it is essential to provide for seeding the cells in a superhydrofobicity condition. As the first step of the method of use of the nano-patterned super hydrophobic device (DSN) of the present invention, such device is coated with a material promoting the adhesion for the cells to be grown, such as, for example poly-D-lysine. Subsequently, cells to be grown are deposited on the device, preferably, a predetermined number of such cells is deposited in a single drop of a predetermined growth medium (Plating Medium -PM).

The drop deposited on the DSN surface takes an almost spherical shape, with a contact angle above 160°, which is maintained by the relative humidity that is present in the incubator of the cell culture. The high relative humidity rate prevents a rapid PM evaporation and a reduction in the diameter of the medium drop in which the cells are dispersed. At this point, the cells are left in incubation in superhydrofobicity for a predetermined time interval ranging between 4 and 6 hours, and then the PM is removed and replaced with maintenance medium.

The cells previously dispersed in PM, during their incubation in superhydrofobicity migrate towards the liquid-air-surface interface of the device. The cells build up in a very localized region, increasing the density thereof, strengthening cell adhesion and their ability to form extensions forming points extending from a micro-structure 2 to an adjacent one. An example of a method for obtaining three-dimensional cell cultures of suspended cells is described herein below, such an example being given by way of illustrative example, non- limiting to the scope of the invention.

Implementation example

A device having an area of 1cm ² is coated with poly-D-lysine (in a concentration of 1- 10(^g/ml). Subsequently, 50,000 hippocampal cells are deposited on the device in a single drop of ΙΟΟμΙ PM containing 10% horse serum and 2mM glutamine and antibiotics. The drop takes an almost spherical shape, with a contact angle above 160° kept by a relative humidity of more than 80% that is present in the incubator of the cell culture at 37° C.

At this point, the cells are left to incubate.

After 4 hours in superhydrofobicity culture, the growth medium is removed and replaced by 2ml Neurobasal maintenance medium containing 2% B27 2mM glutamine and antibiotics. By a confocal microscope and SEM analysis, it is noticed that starting from the first 4 hours of growth, neuritis adhere to the micro-patterned structures 2, and upon growing, they push neurons into a suspended bridge position.

Therefore, the device of the present invention is particularly suitable for nervous regeneration applications, when manufactured with biocompatible and biodegradable materials. Particularly, it can act as an aid by combining the principles of:

co-implantation of cell lines of nerves or glial cells by performing a crucial role in supporting axons regeneration after peripheral nerve injuries;

obtainment of large channels (10-20μιη) oriented towards the biological tissue to increase cell infiltration, migration and tissue interruption.

The type of cell growth described above allowed underlining the implications of these observations for designing future biomaterials applicable, besides to the nervous regeneration context, also in other tissue engineering fields, or for applications relating to cell toxicity and invasivity tests, as well as for optogenetics studies in which the integration between cells and optical fibros is improved by their arrangement along the axis z. One last application is related to the DNA manipulation on superhydrofobic scaffolds, for manipulating nucleic acids, and implementing transfection systems.

In summary, a vertically patterned silicon device with micro-structures 2 aligned and periodically spaced apart is able to control wettability and have a distinctive role in the three-dimensional culture of cells.

By adding a third dimension to the conventional bidimensional culture techniques, significant differences in the features of the thus-obtained cells are obtained, which cells show behaviours very similar to those of the cells within native tissues.

This new type of three-dimensional growth device can be easily seeded by an automatic process, making its high-throughput use possible for any laboratory.

Of course, without prejudice to the principle of the invention, the embodiments and implementation details will be able to be widely varied with respect to what has been described and illustrated by ways of non-limiting example only, without for this departing from the scope of the invention as defined in the appended claims.