IT202100004313A1 - BIOCOMPATIBLE APPARATUS AND BIOCOMPATIBLE SYSTEM FOR THE CREATION OF IN VITRO CELL CULTURES - Google Patents

Description

Biocompatible Apparatus and System for Creating Cell Cultures in Vitro The invention relates to a biocompatible apparatus and system for creating interconnected 3D cell cultures in vitro.

The invention aimed at a biocompatible apparatus and system for creating 3D cell cultures in vitro, in particular human brain cell cultures.

Cell cultures have been widely used for many years to study the behavior and response of groups of cells to a particular electrical stimulus or drug, or in general to a perturbation that modifies the condition of the cell groups. The first models were 2D cell cultures, in which cells were grown on a rigid support, usually a petri dish.

? important that the in vitro systems resemble the pi? possible to the response of the real in vivo system to the perturbation. In many cases, 2D cell cultures are unable to provide a response close to the response of real biological systems.

The limits of 2D cell cultures are even more? evident when complex organs or cellular systems have to be studied.

Organs are structured collections of different cell types, where each cell type has a specific function, and the different cell types communicate with each other in the same organ to perform proper organ behavior. Therefore, different cell types are involved in an organ's response to an external stimulus.

Known 2D cell cultures are unable to provide reliable in vitro systems.

To overcome some of the above limitations of 2D cell cultures and to obtain a more accurate response? reliable from in vitro systems, 3D in vitro models have been proposed to more closely mimic the response of cellular systems to an external perturbation is reliable.

However, as noted above, in a complex cellular structure, such as an organ, or a cellular structure involving different cell types, many different cell types are included. For this reason, realistic in vitro cell culture models should account for the heterogeneity of cells and the different behavior of different cells in response to a perturbation.

Furthermore, the different cells in real cellular systems are spatially connected according to well-defined topological principles: in other words, they are not all arranged on the same level, and this spatial distribution strongly influences the behavior of the different cells and their response to an external perturbation. The in vitro cell model should mimic the pi? the complex 3D structure of in vivo systems possible. To mimic the 3D structures of cellular systems in vitro, some solutions have been proposed, using, for example, glass beads, or other biocompatible 3D scaffolds, which promotes higher levels of high levels of cell differentiation and tissue organization in relation to 2D cell culture systems.

A 3D cell culture allows you to obtain a more natural of the cells.

However, known 3D cell culture models fail to mimic many aspects of cell culture properties. cells of an organ, including, for example, tissue-tissue interfaces (e.g., vascular epithelium and endothelium), spatiotemporal gradients of chemicals, and mechanically active microenvironments (e.g., vasoconstriction of arteries and vasodilatory responses to temperature differentials). Traditional 3D cell culture models have all the above drawbacks.

Furthermore, the type of cells and the interactions between the different cells vary from organ to organ. Therefore, so-called "organ on a chip" (OOC) systems were developed. The OOC ? a microfluidic-based device fabricated ad hoc, containing sub-structures of engineered organs living in a controlled micro- or nano-environment. The OOC ? suitable for simulating one or more? aspects of the dynamics, of the functionality? and the (patho)physiological response of an organ in vivo, such as the activities, mechanics and physiological response of an organ and organ systems. OOCs are organ specific, and the structure of the holder and the device must be adapted to the different organs.

For example, in 2012 Kanagasabapathi and co-workers presented a dual compartment coupled microelectrode array (MEA) system to co-cultivate heterogeneous neuronal subpopulations, composed of cortical and thalamic neurons, and to record their activity. spontaneous. The system ? composed of two interconnected polydimethylsiloxane (PDMS) chambers that allow for both compartmentation and control of the fluidic microenvironment. An inconvenience related to the double compartment device (Kanagasabapathi) ? that does not ? capable of creating three-dimensional structures and does not allow spatial communication in 3D between the different modules, such as for example the cortical module and the thalamic module.

Furthermore, OOCs are highly specific, and models must be tailored to the specific organ being studied.

Therefore, the need remains of a reliable in vitro model that allows the preparation of cell cultures, which mimics more? real cellular systems and therefore allows for a better understanding of the basic physiology of cellular systems.

Furthermore, the need remains of a cell culture system that is suitable for simultaneously reproducing many characteristics of real cell systems.

In particular, ? A reliable in vitro model is needed to better understand the physiology of the neuronal system.

A goal of the invention ? provide an apparatus for in vitro models that reliably mimic the behavior of a cellular system such as, for example, neuronal cell assemblies.

Another goal of the invention ? provide an apparatus for in vitro models that reliably mimic the behavior of a complex cellular system, such as the neuronal system.

Another goal of the invention ? to provide an apparatus for an in vitro model that reproduces the physical and functional connections between the different cell types in a cellular system.

A goal of the invention ? to provide an apparatus for in vitro models that reproduces the physical and functional connection between different cell types, preferably the physical and functional connection between different cell types in the brain system, or in the neuronal system.

The purpose of the invention? to provide a cell culture apparatus that is suitable for obtaining a cell culture, which mimics the behavior of a complex cell system such as neuronal assemblies and brain system taking into account the complexity of the cellular system.

A further object of the invention ? provide a cell culture system that is suitable for obtaining a cell culture that is suitable for mimicking the behavior of a complex cell system such as neuronal assemblies and brain system taking into account the complexity of the cellular system.

According to one aspect of the invention, a biocompatible apparatus is provided for creating cell cultures in vitro.

The apparatus preferably comprises a support structure which defines a culture chamber.

Preferably, the support structure ? provided with at least one dividing device for dividing the culture chamber into at least two different culture regions in the culture chamber.

Preferably, the at least one dividing device ? configured to seal the at least two growing regions to prevent any passage between them.

Preferably, the at least one dividing device ? equipped with a sealing device for holding a separating membrane.

Preferably, the sealing device ? configured so that the separation membrane is interposed between the at least two different culture regions.

Preferably, the apparatus comprises at least one separation membrane coupled to the sealing device and arranged to separate the at least two different culture regions. Preferably, the separation membrane ? made with a biocompatible material.

Preferably, the separating membrane has pores whose dimensions allow the passage of fluids and/or cellular connections through the separating membrane and prevent the passage of cell bodies through the separating membrane and between the at least two culture regions.

The separating membrane places the two different growing regions in fluid connection by allowing the passage of material between them. At the same time, the separation membrane prevents the passage of material having certain dimensions between the at least two growing regions.

The separation membrane permits selective passage of material between the at least two culture regions.

Thanks to this aspect of the invention an apparatus is obtained suitable for obtaining 3D cell cultures in vitro with different types of cells in which the different cells are mutually interconnected.

Thanks to this aspect of the invention, an apparatus suitable for obtaining heterogeneous cell cultures is obtained.

Thanks to this aspect of the invention, an apparatus suitable for obtaining modular cell cultures is obtained.

Thanks to this aspect of the invention, an apparatus suitable for obtaining cell cultures interconnected at different levels along the z axis is obtained.

Thanks to this aspect of the invention an apparatus is obtained which is suitable for obtaining cell cultures belonging to the same mutually interconnected organ or cell system.

The apparatus of the invention ? also suitable for mimicking the reaction of a cell culture to an external stimulus or perturbation.

The apparatus of the invention ? suitable for mimicking the natural physiology of a cell culture, formed by both homogeneous and heterogeneous cells.

The cell culture obtained with the apparatus of the invention allows to imitate the modularity, the 3D structure of the cellular subsystem, the 3D morphology of the connections between the different cellular types of a system.

The apparatus of the invention ? suitable for mimicking the modularity, 3D structure of cell subsystem, 3D morphology of connections between cells of the same type in a homogeneous cell culture.

Why?, ? possible to mimic the behavior of a complex cellular system, such as an organ. For example, the behavior of the brain or neuronal system can be reliably created with the apparatus of the invention.

In particular, ? It is possible to grow different cell types in the different culture regions of the apparatus.

The apparatus of the invention allows to construct 3D modular interacting sub-populations of heterogeneous cells, such as for example neurons, and also promotes their connection in the three dimensions, XYZ.

Thanks to the apparatus of the invention, ? It is possible to obtain models to test the response of a cell culture, such as neuronal assemblies, to different external perturbations such as, for example, electrical stimulations, acoustic manipulations, drugs, etc.

Thanks to the apparatus of the invention, ? possible to reduce the number of experiments in vivo since? very reliable results are obtained through in vitro experiments conducted with the apparatus of the invention.

There? significantly reduces the costs associated with pharmaceutical research. There? it also greatly reduces the ethical problems associated with pharmaceutical research.

Furthermore, the apparatus of the invention can be used successfully to study some diseases affecting large-scale interconnected cell populations. For example, the apparatus of the invention can be used successfully to study neurological diseases such as schizophrenia, Alzheimer's disease, epilepsy, by examining the behavior of the complex neurological network. It could also be used to examine the evolution of a traumatic brain injury.

Furthermore, the apparatus of the invention can be used successfully to study the effects of drug delivery, even at different concentrations, on an organ, by testing the effects of the drug on all cells of the organ or system involved.

The separating membrane connects the at least two culture regions by allowing the passage of fluids, such as for example a solution, preferably a cellular medium, and cellular connections across the separating membrane. At the same time, the separating membrane prevents the passage of cell bodies through the separating membrane and between the at least two culture regions.

Preferably, the separation membrane ? equipped with a plurality? of pores.

Does the separation membrane have pores having an average nominal size which is ? small enough to make s? that during a plating the cells do not pass to adjacent culture regions. There? would allow for the desired selective placement of cells into a culture region of the culture chamber. There? it also prevents the passage of cells through the separation membrane.

According to a further aspect of the invention there is provided a biocompatible system for creating cell cultures in vitro comprising a support substrate and a biocompatible apparatus.

Preferably the biocompatible apparatus? placed on the support substrate to define a culture chamber between the support substrate and the apparatus.

The biocompatible apparatus preferably comprises a support structure defining a culture chamber and provided with at least one dividing device for dividing the culture chamber into at least two different culture regions in the culture chamber. The at least one dividing device ? preferably configured to seal the at least two growing regions to prevent any passage between them.

The at least one dividing device ? preferably provided with a sealing device for holding a separating membrane.

Preferably, the sealing device ? configured so that the separation membrane is interposed between the at least two different culture regions.

The biocompatible apparatus preferably comprises at least one separation membrane coupled to the sealing device and arranged to define at least two different culture regions in the culture chamber.

The separating membrane? preferably made from a biocompatible material.

The separating membrane? provided with pores sized so as to allow the passage of fluids and/or cellular connections through the separating membrane and to avoid the passage of cell bodies through the separating membrane and between the at least two culture regions.

The separating membrane places the two different growing regions in fluid connection by allowing the passage of material between them. At the same time, the separation membrane prevents the passage of material having certain dimensions between the two growing regions.

The separation membrane permits selective passage of material between the at least two culture regions.

Preferably, the apparatus of the invention ? configured to be placed on a supporting substrate such that the culture chamber is defined between the supporting substrate and the apparatus.

Preferably, the apparatus ? configured to adhere to the support substrate to define a sealed culture chamber.

In another version, ? provided a seal between the apparatus and the supporting substrate to seal the culture chamber and to define a sealed culture chamber which avoids leakage.

The sealing? preferably provided to seal the support structure to the support substrate so as to define a sealed culture chamber.

The sealing? preferably made with PDMS or other biocompatible and sticky materials.

In one version, the support substrate comprises an active portion made with MEA (microelectrode array).

Preferably the active portion of MEA ? provided on a portion of the support substrate.

Preferably, the support substrate is made with MEA.

The support substrate can be a Petri dish, or a coverslip.

The type of substrate support can? be chosen according to the test to be performed and/or the cell culture to be prepared.

Preferably, ? Also provided is a sealing element between the separation membrane and the support substrate.

The sealing element ? configured to allow the passage of fluids, and solutions through the two culture regions and/or the passage of cellular connections between the at least two culture regions and to avoid the passage of cell bodies between the at least two culture regions.

The sealing element ? preferably a sealing strip applied between the separation membrane and a support substrate.

Preferably, ? provided a further sealing element coupled to the dividing device and arranged to seal the at least two different growing regions.

Preferably, the further sealing element is attached to the dividing device. The additional sealing element? arranged to be interposed between the dividing device and the support substrate to effectively seal the at least two different growing regions.

The additional sealing element? configured to avoid any passage of material and fluids between the two cultivation regions.

The presence of the further sealing element allows to seal the at least two culture regions and to avoid the passage of the cell bodies between them.

In this way any fluid connection between the at least two culture regions ? only permitted at the separating membrane. Cell cultures could therefore be controlled and manipulated.

The additional sealing element? preferably a sealing layer disposed between the dividing element and the support substrate.

In one version of the apparatus the separation membrane ? made with a flexible polymer, such as PDMS.

In this case, sealed culture regions are obtained even without the provision of the sealing member. In this case the separating membrane acts as a sealing element.

The supply of the separating membrane made with PDMS also makes the supply of additional sealing elements superfluous.

Preferably, the separation membrane has pores having an average nominal size which is between about 0.1?m and about 1 mm.

Choosing the desired mean nominal pore size ? It is possible to selectively prevent/allow the passage of fluids and/or material between the culture regions.

A suitable nominal mean pore size can be chosen on the basis of specific characteristics of the system, for example the nature of the cells to be cultivated inside the chamber/s. Does this determination fall within the capacity? of an ordinary skilled technician in the field and therefore it is not discussed at length in this document.

Preferably, the separation membrane has pores having an average nominal size which is between about 2?m and about 1 mm, preferably about 5?m and about 200?m.

The choice of pores having an average nominal size in the ranges indicated above allows the passage of fluids between the at least two different culture regions and also the passage of cellular connections through the separating membrane and the two culture regions.

Preferably, the separation membrane has pores having an average nominal size which is between about 0.1?m and about 5?m, preferably between about 0.2?m and 2?m.

The choice of pores having an average nominal size in the range indicated above allows the passage of fluids between the at least two different culture regions.

In the above range the pores are preferably arranged randomly in the separation membrane.

A porosity? suitable for a membrane pu? be determined on the basis of specific characteristics of the cell system, for example the nature of the cells to be cultured within the culture chamber and/or within the at least two culture regions. Does this determination fall within the capacity? of an ordinary skilled technician in the field and therefore it is not discussed at length in this document.

Preferably the separation membrane has a density? of pores between about 150 pores/mm<2> and about 0.2 pores/cm<2>, preferably between about 800 pores/mm<2> and about 0.1 pores/cm<2>.

Adjust the density? of pores in the separation membrane allows you to regulate the type and/or composition of material that flows between the culture regions.

The pores of the separating membrane are preferably arranged homogeneously on the separating membrane.

There? allows you to establish homogeneous connections between the growing regions.

Preferably the separation membrane has pores of almost equal average nominal size.

There? allows cellular connections to be established homogeneously between the culture regions through the separation membrane.

Preferably, the pores are arranged evenly on the separation membrane.

Preferably, ? expected to obtain the pores in the separation membrane by means of a LASER radiation.

There? allows you to fine-tune the size of the pores and/or the location of the pores and/or the density? of the pores.

? what? Is it possible to define the properties? of the separation membrane and get cos? a particularly adapted and optimized separation membrane for a desired cell culture.

? Is it possible to obtain a membrane that allows control and repeatability? of cell cultures.

In this way ? Is it possible to precisely define the size of the pores in order to define the functionality? of cell cultures and of the connections to be obtained through the membrane.

Preferably the pores of the separation membrane are at least partially filled with ECM.

The provision of a filling made with ECM ? particularly preferred when the pores have dimensions greater than 5 µm.

There? further increases the properties? of the cell cultures to be obtained. For example, the ECM pu? allow growth of the cell connection while preventing the flow of cell bodies across the separating membrane.

In this way ? It is possible to obtain controlled connections between culture regions, for example assemblies of axons.

Preferably, the separating membrane has a thickness of between 50?m and 800?m, preferably a thickness of about 80-350?m, more? preferably between about 90-200 µm.

Thanks to this feature? a separation membrane is obtained which allows the growth of axonal processes and at the same time provides adequate separation of the different growing regions of the cell culture.

Preferably, the separation membrane ? a hydrophilic membrane.

There? allows you to get a better cell culture. Does the hydrophilicity of the membrane increase the affinity? of cells to the separating membrane and thus promote cell connections in at least two culture regions.

Preferably, the separation membrane has properties optics. There? allows a visual study of the properties? morphological connections, both to ascertain the validity? of the apparatus, and to have a visual confirmation during the experiments carried out with the apparatus of the invention.

How property? optics, it is understood that the separation membrane can? be studied by optical means of study including, but not limited to, fluorescence microscopy techniques, differential interference contrast microscopy techniques, laser confocal microscopy, multiphoton microscopy, optical coherence tomography, and nuclear magnetic resonance.

Preferably, the separation membrane ? made with a material that can? undergo a sterilization process.

The sterilization process ? preferably selected from a group comprising UV sterilization, gamma ray sterilization, autoclave sterilization, dry oven sterilization, chemical sterilization.

The separating membrane? preferably made with a biocompatible material selected from a group comprising cellulose acetate, polydimethylsiloxane (PDMS), polycarbonate, acrylic polymer, polyethersulfone, Hydrosart, etc.

In another version, the separation membrane can? include a biodegradable material. Through the use of a biodegradable material for the membrane, a porosity? of the separation membrane pu? be electively increased during the use of the separation membrane.

For example, can A non-porous membrane made of biodegradable material that prevents exchange of culture conditions, or that allows only very limited fluid passage between culture regions, should be used. During one use, since? the material ? biodegradable, the separation membrane degrades allowing the exchange of biochemical materials and cellular connections and the increase of fluid passage between the culture regions.

In a further alternative, the membrane can? comprising biodegradable and non-biodegradable material such as a non-biodegradable porous membrane in which the pores are sealed with a biodegradable material or coating. As the biodegradable material or coating dissolves, the non-degradable porous membrane is revealed.

In this way, the quantity of fluid exchange between the growing regions is changed during one use.

There? can? allow to obtain a system that allows improved cell culture studies.

In one version ? a cellulose acetate separation membrane was used. In one version ? Was a Sartorius cellulose acetate membrane filter used?. This membrane? made with cellulose acetate, it has a pore size of about 5 ?m, a thickness of about 140 ?m and can? be subjected to some sterilization processes such as autoclaving at 121 ?C or 134 ?C, gamma radiation at 25 kGy, dry oven, ethylene oxide.

This membrane allows for flow between the growing regions and ? thermally stable. This membrane? hydrophilic and white in colour.

In another version, the separation membrane ? made with Versapor? acrylic copolymer.

Were Versapor hydrophilic acrylic polymer membranes used? on non-woven support. Versapor hydrophilic acrylic polymer membranes they are hydrophilic.

Versapor acrylic copolymer? ? made with a Versapor? hydrophilic acrylic polymer membrane, it has a pore size of about 5 ?m, a thickness of about 94 ?m. This membrane can undergo sterilization processes such as UV or gamma radiation.

Preferably, the separation membrane has a height of between 1 mm and 10 mm. Therefore, ? allowed both the separation of different culture regions and the development of 3D cell cultures more? realistic interconnected at different heights.

Preferably, the separation membrane has a length of between 1 mm and 10 mm.

The length and height of the separating membrane can be chosen as desired and are adjusted taking into account the functionalities? of the apparatus or cell culture to be prepared.

Preferably, the support substrate is provided with a coating to facilitate cell culture cell growth provided on at least a portion of the support substrate.

The provision of the coating allows for the enhancement of cell development in cell culture in certain areas of the culture chamber and/or culture regions.

Preferably, the coating? provided on at least a portion of the support substrate face facing the culture chamber.

Preferably, the coating? made with poly-L-ornithine.

Preferably, the support structure ? made from a biocompatible material, preferably a biocompatible polymer. The support structure? preferably made with a polymer that provides a solid support for cell culture, such as Formlabs Biocompatible Surgical Guide Photopolymer Resin, and Formlabs BioMed Clear Resin.

Preferably, the support structure comprises a support body defining a culture chamber having a circular shape.

Circular culture chambers have proven very effective in creating reliable cell culture models.

Preferably, the support structure comprises a support body defining a culture chamber having an oval shape. Oval culture chambers have proven effective in creating reliable cell culture templates for some cell types.

Preferably, the support structure comprises a support body defining a culture chamber having a quadrilateral shape, preferably a square culture chamber.

A support structure having a polygonal shape, for example, hexagonal, heptagonal, octagonal, nonagonal, decagonal, hendecagonal, dodecagonal, or polygonal with a desired number of sides could be selected.

The shape of the culture chamber can be selected on the basis of the particular characteristics that a person skilled in the art wishes to replicate.

L?at least a separation membrane ? preferably provided on the at least one dividing device. The at least one dividing device ? configured to avoid any cell connections between the culture regions in the culture chamber, so that the passage of cell connections between the two culture regions is only allowed through the separating membrane.

Thus, any unwanted exchange between the at least two growing regions is avoided. Preferably, the dividing device ? positioned in the apparatus such that the at least two different growing regions are nearly the same size with each other.

In a further version, ? provided a dividing device which defines at least two different growing regions having different dimensions from each other. There? it might be advantageous to run some cell or culture tests.

Preferably, if the culture chamber is circular, the dividing device ? arranged to define two semicircular cultivation regions, or radially to define a plurality? of growing regions having the shape of a circular sector.

By adjusting the size and/or shape of the culture chamber and/or culture regions ? Is it possible to adjust the properties? of the obtained cell cultures. ? It is possible to optimize culture conditions for a different type of cell culture.

Preferably, the apparatus comprises a cover lid arranged to be coupled to the support structure.

The support structure also allows the position of the cover lid to be fixed stably.

The cover lid preferably comprises the dividing device for dividing the culture chamber into at least two different culture regions. The dividing device ? configured so as to protrude from the cover lid so that when the cover lid ? coupled to the support structure, the division device ? placed in the culture chamber and defines the at least two culture regions in the culture chamber.

Therefore, the growing regions are effectively separated avoiding an unwanted exchange of material between them.

Preferably the dividing device ? formed integrally with the cover lid.

Preferably, the further sealing element is attached to the division device and cos? positioned to seal in use the at least two growing regions to avoid any unwanted exchange between them.

The dividing device preferably acts as a holding device for holding the separating membrane.

Preferably, the cover cap comprises a plurality of of cover elements, each of the cover elements being arranged to be coupled to a portion of the support structure.

The covering elements of the plurality? of cover elements are provided with coupling means for coupling the cover elements to the respective portion of the support structure.

Preferably, each cover element ? provided with a respective dividing element.

Preferably, the cover lid comprises two cover elements each arranged to be coupled to a portion of the support structure and provided with a dividing element.

Preferably, the apparatus includes a locking device for holding the separating membrane in the desired position on the dividing elements. The locking device? furthermore suitable for locking the covering elements together when they are coupled to the support structure.

There? does it increase the stability? of the apparatus of the invention.

The locking device? preferably configured to be coupled to the dividing elements such that the cover elements are mutually locked at the respective dividing element.

The locking device preferably comprises a clip to be applied to the dividing elements and defining a housing in which the dividing elements can be housed in order to be mutually locked.

The clip further comprises a head for being gripped by the user.

The locking device allows the separation membrane to be firmly positioned at the dividing device.

Preferably, at least one of the division elements ? provided with a guide to guide the insertion of the locking device.

Preferably, the locking device comprises a plurality of of locking elements to be positioned in a different region of the dividing elements to firmly lock the dividing elements. Preferably, the sealing device ? provided in a peripheral region of the dividing device so that the separating membrane is positioned in a central portion of the dividing device.

Preferably, the dividing device ? configured to define a window at which ? the separating membrane was placed.

Preferably, the dividing device ? configured in such a way that the window is arranged in a central position of the dividing device.

Preferably, the window ? positioned on the dividing device in such a way as to be arranged in a central position of the at least two growing regions.

Preferably, the apparatus further comprises a fastening device for fastening the cover elements together.

Preferably, the fastening device comprises two fastening elements provided at the peripheral region of the cover elements.

Preferably, each covering element of the plurality? of roofing elements? provided with coupling means for coupling the cover lid to the support structure.

Preferably, the coupling means is a form-fitting means, and preferably the cover lid has a body defining a housing in which the support structure can be inserted into a housing. be housed.

Preferably, the apparatus ? further provided with attachment means for attaching the support structure and the cover lid to each other.

Preferably, the attachment means comprises an attachment element and a further attachment element respectively provided in the support structure and in a cover lid and cooperating to engage the support structure and the cover element. The attachment element and the further fastening element are configured in such a way that relative rotation or sliding of the cover element with respect to the support structure is avoided.

The attachment element and the further attachment element are configured to allow the support structure and the cover element to be removably attached.

The support structure comprises a protrusion to be inserted into a corresponding hole provided in the cover cap, or vice versa, for attaching the cover cap and the support structure.

In this way a reciprocal movement between the cover lid and the support structure is avoided, also a relative rotation is prevented.

Alternatively, the coupling means provides a compression fitting coupling means.

Preferably attachment means can be used which allow the covering lid and the support structure to be attached by a pressing and/or twisting action.

In this version, a male compression fitting can be configured to sealingly engage the fitting to form a compression fitting.

Preferably, the attachment means comprises a male and female attachment element provided respectively on the supporting structure and on the cover lid, or vice versa.

The support structure and the covering lid can also comprise in a further version both the male and the female attachment element on the same faces or on opposite faces.

Preferably, the cover elements comprise coupling elements for mutually coupling the cover elements to each other.

Preferably, the coupling elements are provided at a cover portion of the cover elements.

Preferably, the coupling elements are form-fitting elements. Preferably, each cover element comprises ends complementary shapes arranged to be mutually coupled.

The coupling members are configured such that when fully engaged, the cover members can selectively, and optionally releasably, lock into place.

In one version the support structure comprises a peripheral body defining the culture chamber and the dividing device protruding from the peripheral body so as to be arranged internally in the culture chamber and defining at least two different culture regions configured so as to seal the at least two growing regions to avoid any passage between them.

Preferably the apparatus further comprises a limiting device to be inserted into the culture chamber and configured to define an active culture chamber in the culture chamber in which the cell culture is created.

The limiting device? configured to be crossed by the dividing device so that the separation membrane defines with the limiting device at least two different active culture regions in the culture chamber.

The limiting device limits the size of the culture chamber and thus of the culture regions. The limiting device limits the size of the culture chamber and therefore of the at least two culture regions. The limiting device cooperates with the dividing device and the separation membrane to define at least two active culture regions.

The limiting device? preferably made with PDMS or any other suitable biocompatible material.

The limiting device preferably comprises at least two limiting elements arranged to be each positioned in a growing region and defining an active growing region in each of the at least one growing region.

The limiting elements are preferably coupled to the dividing device so as to define at least two active crop regions in the corresponding at least two crop regions.

The limiting elements are preferably adapted to be coupled to the support substrate so as to define with the support substrate at least two sealed active culture regions.

The limiting elements preferably comprise two recesses to be coupled to the dipping device and arranged to house the dividing elements for coupling the limiting elements to the dividing device.

The limiting elements preferably comprise at least two central spaces which define at least two active growing regions.

The central spaces are preferably of limited size relative to the culture chamber so that culture regions of limited size are defined by the limiting device within the culture chamber.

Therefore, in some cases where crop regions of limited size could be effectively used, ? is it possible to reduce the quantity? of cell material needed for cell culture and save the material needed to obtain cell culture. Preferably, the limiting elements are positioned such that at least two crop regions of the same or similar shape and/or size are defined by the dividing device.

Can the size and/or shape of the crop regions be chosen based on the functionality? of the cell culture to be obtained.

? is it also possible to better control the properties? of cell cultures.

The limiting device? preferably sized to define an active culture chamber having a circular shape.

The limiting device? preferably configured to define an active culture chamber having a shape and size selected to allow living cells to be grown within the chamber.

Preferably, the limiting device ? sized to define an active culture chamber of about 3 mm to about 10 mm in any cross-sectional direction.

Preferably, the active culture chamber has a circular or oval cross-sectional shape.

In further versions, active culture chambers could be obtained having a polygonal shape such as, for example, hexagonal, heptagonal, octagonal, nonagonal, decagonal, hendecagonal, dodecagonal.

The size and shape of the active culture chamber can be chosen by the user based on the particular characteristics one wishes to replicate.

The apparatus of the invention defines at least two culture regions and/or at least two active culture regions which are empty and in which a cell culture could be plated.

A scaffold could be inserted into the at least two culture regions and/or at least two active culture regions to enhance cell growth.

Preferably, the system further comprises a scaffold inserted into the growing region and/or active growing regions.

The scaffolding can be chosen based on functionality? of the cell culture to be obtained and/or the functionality? of the cells to be cultured.

Preferably, the apparatus comprises a plurality of of dividing elements to divide the culture chamber into more? of two different growing regions, at least one dividing member being provided with sealing means for holding a corresponding separating membrane.

In this way, models that mimic very complex structures can be efficiently obtained.

Preferably, at least two dividing elements are provided, preferably each provided with sealing means for holding a corresponding dividing membrane. In this way, the crop regions are connected at least in pairs.

In another version, the apparatus could be equipped with one or more? division elements configured to define three or more? culture regions in the culture chamber. The division elements are configured so that one or more? separating membranes are provided to connect at least two of the culture regions across the corresponding separating membrane.

Adjusting the number of culture regions defined in the culture chamber allows you to mimic cellular systems having a complex different.

Adjusting the number of dividing elements and/or the separating membrane allows to mimic different possible interactions between the different culture regions.

In the present description and in the attached claims, some terms and definitions are considered to have, unless explicitly indicated, the meaning indicated below.

A cell culture? considered here as the process by which cells are grown under controlled conditions, generally outside their natural environment and obtained system. The term cell culture herein refers to cultivating cells derived from multicellular eukaryotes, especially animal cells. A cell culture here means: primary cell culture, cell line culture, immortalized cell line, organoid culture, spheroid culture, tissue culture, ex vivo cell culture, etc.

The term "fluid" in the present document indicates material in fluidic form, such as for example a solution, a cellular solution, a cellular medium, a physiological solution, and solutes having dimensions which allow the passage in the pores of the separation membrane.

?Cellular system? ? here referred to as a system containing many cells, either of the same type or even of different types. The cellular system can comprise different cells of an organ or tissue.

"Cellular connection" herein means the means by which cells communicate. Cell connections are for example neurites for neurons.

"Cell body" herein means the cell body which contains the nucleus of the cell, cell body means the main part of the cell around the nucleus and which contains the nucleus excluding long processes (cell connections).

With the term ?separation membrane? the present document indicates a material having a thickness, which usually varies between 50 ?m and 800 ?m, pores with dimensions ranging between 0.1 ?m and 1 mm.

The term "perturbation" here indicates an event that modifies the condition of the cellular system. The disturbance can be an external perturbation from the external environment or an internal perturbation. A disturbance can be represented for example by an electrical stimulation, an acoustic manipulation, a drug.

The term "support substrate" in the present description indicates the support on which the cell culture is developed. The support substrate can be for example a Petri dish, coverslips, MEA, etc.

The definition "optical properties" indicates in this description that the material can be studied by optical study techniques including, but not limited to, fluorescence microscopy techniques, differential interference contrast microscopy techniques, laser confocal microscopy, multiphoton microscopy, optical coherence tomography, and nuclear magnetic resonance.

It is to be understood that the present invention does not limited to the specific devices, systems, and/or methods disclosed unless otherwise specified, since? these may, of course, vary. It must also be understood that the terminology used in this document is only intended to describe particular aspects and not ? intended to be limiting.

The following description of the invention is provided as an enabling teaching of the invention in its currently known best embodiment. To this end, those skilled in the art will recognize and appreciate that many changes can be made to the various aspects of the invention disclosed herein, while still obtaining the beneficial results of the present invention. will result? It is also evident that some of the desired benefits of the present invention can be obtained by selecting some of the functionalities of the present invention without using other functionalities?. Accordingly, those skilled in the art will recognize that many modifications and adaptations to the present invention are possible and may even be desirable under certain circumstances and form part of the present invention. Accordingly, the following description is provided as illustrative of the principles of the present invention and is not limited to.

As used herein, the singular forms "a/a", "a/one", and "the/the/the" include plural referents unless the context clearly indicates otherwise. So, for example, a reference to a "room" includes aspects having two or more of such rooms unless the context clearly indicates otherwise.

Ranges may be expressed herein as from "about" a particular value, and/or to "about" another particular value. When such an interval ? expressed, another aspect encompasses from one particular value and/or to the other particular value. Similarly, when the values are expressed as approximations, using the antecedent "about", it will be understood? that the particular value forms another aspect. Will you understand? moreover that the extremes of each of the intervals are significant both in relation to the other extreme and independently of the other extreme. As used herein, the terms "optional" or "optionally" mean that the event or circumstance described below may or may not occur, and that the description includes instances in which such event or circumstance occurs and cases where they do not.

The present invention can be understood more easily by referring to the following detailed description of some embodiments of the invention. Will the invention come? now further described with reference to the accompanying figures in which: Figure 1 is a schematic view of a system of the invention;

Figure 2 ? a schematic top view of the system of Figure 1 with some details removed for the sake of clarity;

Figure 2A ? an enlarged view of a detail of Figure 2

figure 3 ? a schematic perspective view of the apparatus used in the system of Figure 1 with some details removed for the sake of clarity;

Figures 3A and 3B are enlarged views of details of Figure 3 ;

figure 4 ? a side view of a detail of the system of Figure 1;

figure 5 ? an exploded view of the apparatus of the invention with some details removed for the sake of clarity;

Figures 5A and 5B are bottom views of covering elements of the support structure of Figure 4;

Figures 6A-6C represent an example of differential interference contrast microscopy images of a cell culture obtained with the system of the invention at DIV 8 (Day In Vitro (DIV), day in vitro); Figures 6D and 6E are enlarged views of Figure 6B;

Figure 7 ? a schematic representation of the activity? electric of an example of cell culture obtained with the system of the invention;

Figure 8 ? an exemplary fluorescence microscopy image of a cell culture obtained with the system of the invention at DIV 17.

In Figures 1-4, ? shown a biocompatible cell culture system 500 for creating in vitro 3D cell cultures 200 according to the invention. The biocompatible cell culture system 500 comprises a biocompatible cell culture apparatus 100, best seen in Figures 3-5 , which ? suitable for creating in vitro 3D cell cultures 200, best shown in Figures 6A-6C , and a support substrate 50. The apparatus 100 is arranged to be placed on the support substrate 50 to define a culture chamber 10 for culturing cells, as explained below.

The apparatus 100 of the invention ? suitable for obtaining modular, heterogeneous 3D cell cultures 200. The apparatus of the invention also allows the development of 3D connections between different cell culture populations.

The support substrate 50 ? made with MEA.

In a further version of the apparatus not visible in the Figures, the support substrate comprises at least one active portion of MEA arranged to be positioned in correspondence with the culture chamber 10 and in particular in correspondence with the active culture chamber 70, as better explained below .

The apparatus 100 comprises a support structure 1, better visible in Figures 3-5, made with a biocompatible material, preferably a biocompatible polymer. In the version shown, the support structure 1 ? made with Formlabs Dental Surgical Guide Resin.

The support structure 1 defines a culture chamber 10 in which cells can be grown, as better explained below. In the version shown, the support structure 1 ? a ring 11 with a shape such as to define a circular culture chamber 10.

In another version of the apparatus not shown in the figures, the support structure can having such a shape as to define a quadrilateral culture chamber, preferably a square culture chamber.

A culture chamber of any desired shape could also be provided. The 11 ring? arranged to be positioned on the support substrate 50 so that a bearing surface 11B of the ring 11 is positioned on the support substrate 50. In this way, a culture chamber 10 is positioned on the support substrate 50. defined between the ring 11 and the support substrate 50. The apparatus 100 further comprises a seal, not visible in the Figures, for sealing the support structure 1 to the support substrate 50 so as to define a sealed culture chamber 10 and avoid losses.

The sealing? usually made with PDMS (polydimethylsiloxane), a silicone elastomer suitable for biomedical applications. The PDMS ? thermally stable, chemically inert, permeable to gases. Also, PDMS ? a transparent, non-fluorescent, biocompatible and non-toxic material.

The sealing can be continuous, ie a layer of PDMS is applied over the entire support surface 11B of the ring 11, or discontinuous.

In one version, ring 11 ? made with material adhering to the surface of the support substrate 50, so as to define with the support substrate 50 a sealed culture chamber 10. In this case the sealing is not provided.

The 11 ring? provided with a pair of pins 12 protruding from an upper surface 11A of the ring 11, i.e. the surface opposite the support surface 11B, and arranged to turn, in use, towards a covering lid 3 of the apparatus 1. The pins 12 are positioned in diametrically opposite positions on the upper surface 11A. The pins 12 are arranged to attach the support structure 1 to the cover lid 3, as better explained below.

In the version shown, the ring 11 has a thickness d1 of about 2.5 mm, a height h1 of about 6.00 mm and a diameter D1 of about 25.5 mm.

The support structure 1 has dimensions which allow a user to handle and manipulate it as desired.

The dimensions of the culture chamber 10 and of the active culture chamber 70 defined by the apparatus 100 can be chosen so as to be suitable for the desired task and/or the properties? to be studied with the apparatus of the invention.

The apparatus 100 ? further provided with a sealing device 2 for holding a separating membrane 20, as better explained below.

The separating membrane 20 ? positioned in the sealing device 2 so as to define two different culture regions 10A, 10B in the culture chamber 10, as explained below.

The apparatus 100 further comprises a cover lid 3 arranged to be coupled to the support structure 1 to define a plurality of of culture regions 10A, 10B in the culture chamber 10.

The cover lid 3, better visible in Figure 5, comprises a dividing device 4 which protrudes from the cover lid 3 so that when the cover lid 3 is ? coupled to the support structure 1, the division device 4 ? positioned in the culture chamber 10 and defines the two culture regions 10A, 10B. The dividing device 4 acts as a sealing device 2 for holding the separating membrane 20, as further explained below.

The covering lid 3 comprises two covering elements 3A, 3B each being configured to be coupled to a respective portion of the ring 11. The covering elements 3A, 3B are semicircular. Each cover element 3A, 3B ? provided with an attachment hole 31 arranged to house a corresponding pin 12 provided on the ring 11 for attaching the cover elements 3A, 3B to the support structure 1. The attachment hole 31 and the pin 12 act respectively as an attachment element and further attachment elements by sealingly engaging the support structure 1 and the cover lid 3.

In another version not shown, the cover elements are provided with different attachment elements arranged to cooperate with corresponding further attachment elements provided in the support structure for attaching the cover elements to the support structure.

In another embodiment not visible in the Figures, the support structure and the cover lid are selectively coupled via a compression fitting.

Each covering element 3A, 3B, has a "C"-shaped body, better visible in Figures 5A and 5B, defining a respective housing 32A, 32B in which it can the ring 11 can be housed. The covering elements 3A, 3B and the ring 11 have a complementary shape so as to define coupling means for coupling the covering element 3A, 3B and the ring 11 with each other.

Each cover element 3A, 3B ? equipped with coupling elements 33, 34 for mutually coupling the covering elements 3A, 3B with each other.

The coupling elements 33, 34 are provided at the respective peripheral portion 3', 3" of the cover elements 3A, 3B so that they face each other and are used for coupling the cover elements 3A, 3B.

The coupling elements 33, 34 are shape coupling elements.

In the version shown, the peripheral portions 3" of a second covering element 3B have such a shape as to define a coupling housing 35 in which the first peripheral portion 3' of the first covering element 3A is housed, so as to define a coupling shaped between the covering elements 3A, 3B.

Each covering element 3A, 3B has an extension greater than the semicircle so that in correspondence with the respective peripheral portions 3', 3" the first and second covering elements 3A, 3B overlap each other in a corresponding coupling portion 39, as best seen in Figures 3 and 5.

The apparatus 100 further comprises a pair of fastening elements 37, one of which ? better visible in Figure 3B, arranged to be inserted on the covering elements 3A, 3B to mutually fix the covering elements 3A, 3B in the desired position. Each fastener 37 ? in the shape of a "C" and ? configured to be fixed at the mating portion 39 of the cover elements 3A, 3B so as to fix the cover elements 3A, 3B together in the desired position and avoid any reciprocal movement between the cover elements 3A, 3B, as well visible in Figure 3. The fixing elements 37 define a fixing housing 38 configured to house the coupling portion 39. The fixing elements 37 are arranged to be fixed to the apparatus 100 from the external portion, i.e. from the side opposite the chamber culture 10. This avoids any interference between the fixing elements 37 and the culture chamber 10.

Each cover element 3A, 3B ? equipped with a dividing element 5, 6 extending from the cover elements 3A, 3B configured to project into the culture chamber 10. The dividing elements 5, 6 extend almost diametrically into the culture chamber 10.

The dividing elements 5, 6 are positioned so that they are placed side by side when the cover elements 3A, 3B are coupled to the support structure 1.

The dividing elements 5, 6 cooperate with each other to form the dividing device 4 of the apparatus 100 which protrudes from the cover elements 3A, 3B and which defines two distinct culture regions 10A, 10B in the culture chamber 10.

The dividing elements 5, 6 are configured to extend up to the support substrate 50 on which ? positioned the apparatus 100, so that no exchange of fluid between the two cultivation regions 10A, 10B is allowed in correspondence with the division elements 5, 6, as better explained below. The dividing elements 5, 6 are configured so as to seal the two cultivation regions 10A, 10B to avoid any passage between them.

The division elements 5, 6 define a window 40 at which it can? the separating membrane 20 be positioned so that the separating membrane 20 is interposed between the two growing regions 10A, 10B and separates the two growing regions 10A, 10B. Thanks to the presence of the separation membrane 20, the culture regions 10A, 10B are mutually connected, as explained below.

Window 40? provided in a central position of the dividing elements 5, 6.

In a further version of the apparatus of the invention, not shown in the Figures, the window 40 is positioned peripherally in the division elements 5, 6.

In the illustrated version, the window 40 has a length L2 of about 8 mm and a height H2 of about 2 mm. The length L2 and the height H2 of the window 40 can be adjusted according to the wishes of the user.

The dividing elements 5, 6 are provided with the sealing means 2 for holding the separating membrane 20. The separating membrane 20 is positioned between the two dividing elements 5, 6, as better explained below.

The dividing elements 5, 6 are also provided with guides 90 arranged to guide the insertion of the locking device 7.

The apparatus 100 comprises a locking device 7 for locking the cover elements 3A, 3B together when they are attached to the support structure 1.

The locking device 7 ? configured to hold the separating membrane 20 in the desired position in the dividing elements 5, 6.

In the version shown, the locking device 7 comprises a pair of clips 8, one of which ? best seen in Figure 3A. Each clip 8 has a body 8A which defines a locking housing 8B in which the dividing elements 5, 6 can be housed to be mutually locked. Each clip 8 further comprises a head 9 to be gripped by the user to position the clip 8 on the dividing elements 5, 6.

The clips 8 allow to hold the separating membrane 20 firmly in the desired position in the dividing device 4.

In the illustrated versions, the clips 8 are arranged to be positioned on the division elements 5, 6 peripherally with respect to the window 40 so that the window 40 is interposed between the two clips 8. In this case, the window 40 is positioned in a central portion of the dividing elements 5, 6 and the clips 8 are interposed between the ring 11 and the window 40. In this way, the separating membrane 20 is held stably in the window 40.

Furthermore, the clips 8 are positioned close to the window 40 so as to increase the stability? of the separating membrane 20 in the apparatus 100.

The separating membrane 20 ? made from a biocompatible material, for example cellulose acetate, polydimethylsiloxane (PDMS), polycarbonate, acrylic polymer, polyethersulfone, Hydrosart, etc.

For the separation membrane 20 a biocompatible material must be used which can undergo sterilization processes in order to obtain sterile cell cultures 200 with the system 500 of the invention.

The separating membrane 20 ? provided with pores to allow fluid connection between the two culture regions 10A, 10B.

The pores of the separation membranes are sized to allow the selective passage of material between the two culture regions.

The separating membrane 20 puts the two culture regions in fluid connection allowing the passage of fluid and the supply of nutrients and allows a biochemical communication between the two culture regions 10A, 10B.

The separating membrane 20 ? configured to connect the growing regions 10A, 10B to allow only the desired material to pass between the growing regions 10A, 10B. In particular, the separation membrane 20 ? configured to allow the passage of cellular connections 22 through the separating membrane 20, as best discussed with reference to Figure 8 , and to prevent the passage of cell bodies 23 through the separating membrane.

The separating membrane 20 ? equipped with pores sized so as to allow the passage of cellular connections 22 through the pores and therefore through the separation membrane 20 and to avoid the passage of cell bodies 23 through the pores and the separation membrane 20 and therefore between the at least two regions of culture 10A, 10B.

The pores have an average nominal size of about 5?m.

The separation membrane 20 has a density? of pores indicated as the number of pores per square millimeter between 150 pores/mm<2 > and 0.2 pores/cm<2>.

The separating membrane 20 has a thickness ?d? between 50?m and 800?m, preferably a thickness "d" of about 100?m, and a height H of about 2.00 mm and a length L of about 8.00 mm. Preferably the separating membrane 20 has a height H > = 1 mm. Preferably the separating membrane 20 has a length L comprised between 1 mm and 10 mm. Can the effective length and height of the separation membrane be chosen by the user according to the functionalities? of the apparatus or of the assigned task.

The separation membrane 20 has properties optics so that it can be studied through optical study techniques. The separation membrane 20 can? be transparent, or it could be made of a material that does not interrupt the optical path. Furthermore, the separation membrane 20 can understand a material that affects the transmission of physical parameters, but also the optical transmission.

Thus, separation membranes are contemplated herein wherein the separation membrane only allows the transmission of certain wavelengths of light to pass from one side of the membrane to the other or excludes specific wavelengths of light.

The apparatus 100 further comprises a limiting device 60, better visible in Figure 2A , to be inserted into the culture chamber 10 and configured to define in the culture chamber 10 an active culture chamber 70 in which the cell culture 200 is created. The limiting device 60 limits the size of the culture chamber 10 and therefore of the culture regions, as explained below.

The limiting device 60 ? made with PDMS or any other suitable biocompatible material.

The limiting device 60 comprises two limiting elements 60A, 60B arranged to be each positioned in a growing region 10A, 10B.

The limiting elements 60A, 60B are adapted to be coupled to the support substrate 50.

The limiting elements 60A, 60B are adapted to be coupled to the division device 4 so as to define therewith two active culture regions 70A, 70B, as explained below.

The limiting elements 60A, 60B comprise two recesses 61A, 61B each to be coupled to the immersion elements 5, 6 and arranged to house the dividing elements 5, 6 and two central spaces 62A, 62B which define two active culture regions 70A, 70B of the system 500. The two recesses 61A, 61B are arranged to mate with the dividing elements 5, 6 and/or with the support membrane 20 and also provide for a further anchorage so as to increase the stability? of the apparatus 100 of the invention.

The central spaces 62A, 62B have limited dimensions with respect to the culture chamber 10 so that the culture regions 70A, 70B of limited dimensions are defined by the limiting device 60 within the culture chamber 10.

Therefore, in some cases where crop regions of limited size could be effectively used, ? is it possible to reduce the quantity? of cellular material needed and save the material needed to obtain cell culture 200.

The limiting device 60 ? sized so as to define an active culture chamber 70 having a circular shape and a diameter D* of about 5 mm.

The active culture chamber 70 can? generally have a shape and size chosen to allow living cells to be grown within the chamber.

The shape and size of the active culture chamber are chosen to maintain a flow of material through a cellular construct held in the active culture chamber.

In a preferred embodiment, the active culture chamber 70 may be designed to accommodate a biomaterial scaffold to create a 3D environment while ensuring adequate nutrient flow through a cellular construct held in the active culture chamber 70.

The 500 system can? comprising a biomaterial scaffold, not visible in the Figures, positioned in the active culture chamber 70 arranged to facilitate the formation of a 3D environment and ensure adequate nutrient flow through a cellular construct held in the active culture chamber 70.

The limiting elements 60A, 60B are sized so that the central spaces 62A, 62B are defined in correspondence with the separating membrane 20.

The active culture regions 70A, 70B are mutually interconnected by the separation membrane 20 so that a selective exchange of material is permitted between the two active culture regions 70A, 70B.

The cell culture system 500 further comprises a sealing element, not visible in the Figures, disposed between the separation membrane 20 and the support substrate 50. The sealing element is chosen according to the properties? of the separating membrane so that the same material flows between the separating membrane and the sealing element.

The sealing element can? allow the passage of fluids through it, the passage of solutions between the two culture regions and/or the two active culture regions, the passage of cellular connections between the two culture regions and/or the two active culture regions and avoid the passage of cell bodies between the two culture regions.

The cell culture system 500 also comprises a further sealing element, not visible in the Figures, arranged between the dividing element 4 and the support substrate 50 and arranged to seal the two culture regions avoiding any passage of fluid between them .

To assemble the 500 system of the invention ? provided to place the ring 11 on a support substrate 50 and seal the ring 11 and the support substrate 50 by means of a gasket.

A little PDMS is applied between the ring 11 and the support substrate 50 and then cured in an oven to achieve the seal. For example, can be hardened in the oven at 120 ?C for at least 5 minutes or at a lower temperature for a longer period of time? prolonged, for example 70-80 ?C for at least 10 minutes.

A second layer of PDMS can? be applied on an external side of the ring 11, i.e. on the face opposite the culture chamber 10, to obtain a more effective sealing. strong.

The second layer of PDMS is also hardened in an oven, for example at 120 ?C for at least 5 minutes or at a lower temperature for a longer period of time. prolonged, for example 70-80 ?C for at least 10 minutes.

Once the ring 11 ? firmly coupled to the support substrate 50, the apparatus 100 is assembled as described below.

? a separating membrane 20 is provided. Preferably the separating membrane 20 is obtained by cutting a membrane having desired dimensions from a sheet of film material. ? a separating membrane 20 having dimensions greater than the window 40 is provided. allows to facilitate the positioning of the separation membrane 20 and to avoid an incorrect positioning of the separation membrane 20 which would imply that a portion of the window 40? without the separating membrane 20.

The separating membrane 20 ? inserted between the dividing elements 5, 6. The cover elements 3A, 3B are assembled and placed on the ring 11. Then the fastening means 37 are placed on the cover elements 3A, 3B and the clips 8 are inserted on the dividing elements 5, 6 in correspondence with guide 90.

A sealing layer is applied under the dividing elements 5, 6 to seal the dividing element at the support substrate 50, so as to seal the two growing regions 10A, 10B and avoid leakage between them.

The sealing layer can be formed in PDMS.

The sealing layer is placed as described below.

The cover elements 3A, 3B are assembled, then the fastening means 37 are placed on the cover elements 3A, 3B and the clips 8 are inserted on the division elements 5, 6. The sealing layer is applied under the division elements 5 , 6, and then the cover members 3A, 3B are attached to the ring 11 and the divider members 5, 6 are attached to the support substrate 50. The sealing layer is then cured, for example in a dry oven at 120 ?C for at least 5 minutes, or at 70-80 ?C for 10 minutes or more. The sealing layer then acts as a further sealing element for sealing the dividing elements 5, 6 to the support substrate 50

Subsequently, the clips 8 and the fastening means 37 are removed from the division members 5, 6 in order to insert the separation membrane 20 as explained below. A separating membrane 20 having a larger size than the window 40 is provided, preferably cut from a film material. There? allows to facilitate the positioning of the separation membrane 20 and to avoid an incorrect positioning which would imply that a portion of the window ? without the separating membrane 20. The membrane is inserted between the dividing elements 5, 6. The covering elements 3A, 3B, are thus? assembled, then the fastening means 37 are positioned on the covering elements 3A, 3B and the clips 8 are inserted on the dividing elements 5, 6.

Once the separation membrane 20 has been inserted and the covering lid 3 has been assembled, the limiting elements 60A and 60B are inserted into the culture regions 10A and 10B to form the active culture regions 70A and 70B and subsequently the apparatus 100 is sterilized .

The support substrate 50, the ring 11, the limiting device 60 and the sealing layer if present are sterilized, for example in a dry oven at 120°C for 2 hours. The cover lid 3 and the separation membrane 20 are also sterilized, for example in an autoclave or by exposure to UV irradiation for 20 minutes on each side, depending on the characteristics of the membrane.

After a sterilization, a coating is applied to the support substrate 50 at the active culture chamber 70. The coating is made with poly-L-ornithine, an adhesion factor that facilitates neuronal growth, which is been kept overnight and then washed before proceeding with the next steps.

Subsequently, the cover cap 3 is placed on the ring 11.

If necessary, before applying the cover cap 3 to the ring 11, a sealing strip, not visible in the Figures, is applied under the separating membrane 20 to form a sealing element for sealing the separating membrane 20 and the substrate supportive 50.

The sealing element allows to avoid any leakage under the membrane 20 between the two growing regions 10A, 10B.

The sealing strip? preferably made with ECM gel. The separating membrane 20 ? fixed to the support substrate 50 by means of the sealing strip made with ECM gel. ECM gel? a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells that mimics the extracellular environment and is used as a basement membrane matrix to culture cells.

Providing the ECM gel should also prevent movement of the soma to some extent, so placing a sealing strip of ECM gel on the support substrate 50 ? a tunnel was created where only neurites could pass. After applying the sealing strip, the cover cap 3 is applied to the ring 11, and the 500 system is placed in an incubator for 5 minutes to allow the ECM gel curing process to occur. From this stage on, the 500 system is stored in the incubator at temperature (37 ?C), humidity, and humidity. (95%) and CO2 level (5%) constant and ? ready to plate cells.

In other versions, not shown in the Figures, the apparatus can? include several dividing elements that divide the culture chamber into more? of two different growing regions. In this case, at least one element of division ? provided with sealing means for holding a corresponding separating membrane. In this case, the pluralities of regions of the culture chamber can all be coupled to each other through a separating membrane, or at least two of the culture regions are coupled to each other through a separating membrane, or each culture region ? coupled through a separation membrane to adjacent culture regions.

In this way, in vitro models of very complex assemblies can be obtained efficiently.

Changing the number of growing regions or the connections between growing regions allows you to mimic different complex structures

In another version, the apparatus could be provided with division elements configured in such a way as to define three or more? culture regions in the culture chamber. The sealing means can be positioned in the dividing element so that one or more Separation membranes are provided to connect at least two of the culture regions across the membrane. The dividing elements may extend radially into the culture chamber.

In further versions of the apparatus not shown in the Figures, the sealing device can be provided on the ring and protruding into the culture chamber.

EXAMPLES

Example 1

? a system 500 was prepared comprising a support structure 1 and a separation membrane 20 and a support substrate 50 made with MEA.

The separating membrane? It was made from cellulose acetate, available as a Sartorius<TM> cellulose acetate membrane. The membrane has pores having an average nominal size of about 5?m, a thickness D of about 140?m. ? hydrophilic and white in colour.

The support structure 1 ? a ring 11 made with a biocompatible resin having a height of 6.00 mm, a thickness of 2.25 mm, and an internal diameter of 25.50 mm and an external diameter of 30.00 mm. The 11 ring? provided with a pair of pins having a diameter of 1.2 mm and a height of 1.00 mm.

Cover lid 3 ? made with a biocompatible resin and consists of two distinct cover elements 3A, 3B with extremities? complementary peripherals 3, 3", to form a fixed joint, preventing its movement to some extent and ensuring vertical alignment.

The partition elements 5, 6 define a window 40 having a height of 2 mm and a length L2 of 8 mm and are configured to be positioned so that the respective windows correspond to each other. The dividing elements 5, 6 have a maximum height of 7 mm and a thickness of 1 mm. The dividing members 5, 6 are provided with guides 90 arranged for inserting and holding the separating membrane 20. The guides have a width of 1.2 mm.

The separating membrane 20 ? seal between the two dividing elements 5, 6.

Example 2

? Was a system set up as in Example 1, except that ? a hydrophilic acrylic polymer separation membrane 20 was used, available as Versapor<R >on a non-woven support. The separation membrane has pores having an average nominal size of about 5?m, a thickness D of about 94?m. ? hydrophilic and white in colour.

Once inserted, the membrane ? been sterilized with UV radiation together with the upper part of the apparatus.

Cell cultures

Cell cultures were prepared using the procedures for preparing experimental cell cultures in accordance with the European Animal Care Legislation (2010/63/EU), and in accordance with with the legislative decree of the Ministry of Health (DL 116/1992) and the guidelines of the University? degli Studi di Genova (Prot. 75F11.N.6JI, 08/08/18), in order to reduce the number of animals needed for testing and their suffering.

Cell cultures of dissociated cortical and hippocampal neurons from embryonic rats at day 18 of gestation were used. Briefly, cerebral cortices/hippocampi of embryos were dissected and subjected to a first step of dissociation by enzymatic digestion in 0.125% trypsin solution and DNAse solution for 20 minutes at 37°C. The action of the trypsin enzyme is then blocked by washing in culture medium containing fetal bovine serum (FBS). The enzymatic dissociation ? was followed by a mechanical one using a fine tip Pasteur pipette. The resulting fabric? was resuspended in Neurobasal medium supplemented with a solution of 2% B-27, 1% Glutamax-I and 1% Pen-Strep.

Neurons were plated on the apparatus prepared as in Example 1 or 2. Neurons are plated on the active portion of the support substrate (coverslip or MEA depending on the experiment) at one of the two active culture regions 70A , 70B.

The electrophysiological results for n = 5 2D hippocampal networks and n = 1 3D culture are presented below, where n represents the number of cell cultures.

In the 2D configuration, the plating procedure for plating the neuronal cells onto the system consisted of placing the cells on one of the active culture regions 70A, 70B defined in the culture chamber 10.

To sow, a drop of 20 ?l of the cell suspension ? been placed in an active culture region 70A. Considering 1500 cells/mm<2 > and a total surface area of 30 mm<2 > (area of each active culture region 70A, 70B plus area of the separation membrane 20 in contact with the cells and lateral surface of the limiting device 60 ), cells were suspended at a concentration of 2250 cells/?l.

The Neurobasal medium? was added 1 hour after the plating procedure and the devices were stored in the incubator. A DIV 5, half? of the land ? was replaced with BrainPhys medium, supplemented with a solution of 2% NeuroClut SM1, 1% Glutamax and 1% Pen-Strep. For the maintenance of cell cultures, once a week ? a partial terrain change was performed (50%). The proliferation of glia is not been prevented by adding anti-mitotic drugs, since? plays an essential role in the nervous system.

In the 3D configuration, a first layer of neurons, which acts as an interface between the MEA recording electrodes and the 3D population, ? been plated directly onto the support substrate area 50 within a culture region 70A or 70B. For the realization of the first cell monolayer the cell plating protocols, described above for the 2D configuration, were followed, with the addition of the following procedure to create the 3D culture before adding the culture medium (Neurobasal). The 3D assembly ? was built on the monolayer, 1 hour after its plating using ECM gel, following the protocol described below.

Considering that the ECM gel will undergo? a thermally activated polymerization when brought to 20-40°C, the following steps were followed.

The day before cell plating, a set of sterile vials, a set of sterile gloves, some pipettes and vial holders were placed in the refrigerator and ? ice was also prepared.

On the day of the experiment, the ECM gel ? was kept below 10 ?C using the above pre-chilled materials and working on ice. ECM gel? was diluted with Dulbecco's modified Eagle's medium (1:2 ratio) and cells were added to the solution at the desired concentration. Then, cells were plated on the monolayer and stored in the incubator at temperature (37 ?C), humidity? (95%) and CO2 level (5%) constant.

Different experiments were carried out to confirm the effectiveness of the apparatus 100 in creating a cell culture system 500 which allows to create 3D, modular and heterogeneous cell cultures and which supports the development of connections between the different subpopulations in 3 dimensions.

First, the apparatus 100 ? been coupled to coverslips as a support substrate 50 to enable visual inspection by microscopy techniques. Following the procedures described in Examples 1 and 2, cell cultures 200 were plated in one of the active culture regions 70A or 70B, to test whether the cell bodies 23 were held in the designed active culture region 70A or 70B.

Figure 6 shows some images obtained with DIC microscopy techniques. Neuronal cells were plated in active culture region 70A. An enlargement of the active culture region 70A ? shown in Figure 6E , and a close-up of cell-free, i.e., not plated with cells, active culture region 70B ? shown in Figure 6D. In Figure 6E , the cell bodies 23 are clearly visible in the plated active culture region 70A, while Figure 6D clearly shows that the active culture region 70B ? empty. Figures 6A-C , show different magnifications of both active culture chambers 70A and 70B. In particular, they show a clear division between the plated active culture region 70A and the empty one 70B. This test demonstrates that the cell culture system 500 was able to prevent a migration of the cell bodies 23 from the active culture region 70A in which they had been plated to the adjacent active culture region 70B. This result is maintained for extended periods of time (more than 3 weeks).

To further support these observations, ? Has the activity been registered? electrophysiological analysis of n = 6 hippocampal cultures (of which n = 5 were in 2D and n = 1 in a 3D configuration) plated on the support substrate 50 made with MEA coupled to the apparatus 100.

The MEAs featured 120 electrodes for recording the electrophysiological signal. The cell cultures 200 were plated in the active culture region 70A, to verify if the cell bodies 23 were held in the active culture region 70A in which the cells are plated. 200 cell cultures were left in the incubator to mature and recorded at DIV (Day In Vitro) 17 for 15 minutes.

Figure 7 shows a representative example of raw data recorded by MEAs. Each rectangle shows the signal recorded by one of the electrodes. The position of the electrode in the active culture chamber 70 is maintained in the grid where the recorded data is displayed. There? means that the data shown in the upper left rectangle was recorded from the upper left electrode in the active culture chamber 70. In each rectangle, the x-axis shows the time, while the y-axis shows the recorded potential at the site.

In the graph of Figure 7 ? reported a plurality? of boxes 700, the boxes to the right of the graph correspond to the electrodes positioned in the active culture region 70A in which ? plated the cell culture, while the boxes on the left of the graph correspond to the electrodes placed in the active culture region 70B without cell culture.

In figure 7, ? clearly shown that only the electrodes on the right have registered an activity? electrophysiology, while the electrodes in the active culture region 70B do not register any activity. There? confirms from a functional point of view that the cultured cells remained confined only in the active culture region 70A subjected to plating.

There? also demonstrates that the apparatus 100 ? able to support vitality? of 200 cell cultures for extended periods of time and that the cells are able to show activity? strong and sustained. This feature? ? was obtained for both 2D and 3D crops.

In the end, ? It has been verified that the separation membranes 20 promote an outgrowth of neurites, i.e. cellular connections 22, through their pores. Following the procedures described in Examples 1 and 2, 200 cell cultures were plated in active culture region 70A, leaving active culture region 70B empty as a control. Upon reaching a mature stage (DIV 17), the established cellular network is ? been inspected using fluorescence microscopy techniques. Specifically, cells were stained with NeuroFluor, a membrane-permeable fluorescent probe that selectively labels primary and hPSC-derived neural progenitor cells without fixation. In this way, ? it was possible to verify whether the neurites (cell connections 22) were able to pass to the adjacent control active culture region 70B. Figure 8 shows a close-up of the neurite arborization (cell connections 22) of a neuronal cell. Can you? clearly see that the cell connections 22 start from the cell body 23 in an active cell culture region 70A. The cellular connections 22 pass through the separation membrane 20, represented with dashed lines in Figure 8 , and reach the empty control active culture region 70B.

There? confirms that the established network of cellular connections 22 was capable of diffusing across the separating membrane 20 and reaching the empty active culture region 70B. There? it also confirms that on the contrary the cell body 23 does not spread through the active culture regions and remains in the active culture region in which they are plated.

Is it shown like this? that the invention achieves the proposed object and allows to obtain numerous advantages.

Claims (38)

1. Biocompatible apparatus (100) for creating cell cultures in vitro comprising: - a support structure (1) defining a culture chamber (10) and provided with a dividing device (4, 5, 6) which ? positioned to define at least two different culture regions (10A, 10B) in the culture chamber (10) and configured in such a way as to avoid any passage of fluid between the at least two culture regions (10A, 10B), the division device (4 , 5, 6) being provided with a sealing device (2) for holding a separation membrane (20) so that the separation membrane (20) is interposed between the at least two different growing regions (10A, 10B), - and at least one separation membrane (20) coupled to the sealing device (2) made with a biocompatible material and being provided with pores sized so as to allow the passage of fluids and/or cellular connections (22) through it and to avoid the passage of cell bodies (23) through the separation membrane (20) and between the at least two culture regions (10A, 10B). The apparatus according to claim 1, wherein the separating membrane (20) has pores having an average nominal size of between about 0.1?m, and about 1 mm, preferably between about 2?m and about 1 mm, plus? preferably between about 5?m and about 200?m. 3. Apparatus according to any one of claims 1-2, wherein the separation membrane (20) has a density? of pores between about 150 pores/mm<2 > and about 0.2 pores/cm<2>, preferably between about 800 pores/mm<2 > and about 0.1 pores/cm<2> 4. Apparatus according to any one of claims 1-3, wherein the pores are arranged regularly on the separation membrane (20). The apparatus according to any one of claims 1-4, wherein the separation membrane (20) has pores having substantially the same average nominal size. The apparatus according to claim 1, wherein the separation membrane (20) has pores having an average nominal size of between about 0.1 µm and about 5 µm, preferably between about 0.2 µm and 2 µm. The apparatus according to claim 6, wherein the pores are arranged randomly on the separation membrane (20). The apparatus according to any one of claims 1-5, wherein at least some of the pores of the separation membrane (20) are at least partially filled with ECM. 9. Apparatus according to any one of claims 1 to 8, wherein the separation membrane (20) has properties optics to allow the study of the biological component using microscopy tools. 10. Apparatus according to any one of claims 1 to 9, wherein the separating membrane (20) is made with a hydrophilic material, selected from a group comprising cellulose acetate, polydimethylsiloxane (PDMS), polycarbonate, acrylic polymer, polyethersulfone, Hydrosart. 11. Apparatus according to any one of claims 1 to 9, wherein the separating membrane (20) is made with PDMS. 12. Apparatus according to any one of claims from 1 to 11, wherein the separating membrane (20) has a thickness comprised between 50?m and 800?m, preferably a thickness of about 80-350?m, more? preferably between about 90-200 µm. 13. Apparatus according to any one of claims 1 to 12, wherein the support structure (1) is ? made of biocompatible material, preferably a biocompatible polymer. 14. Apparatus according to any of claims from 1 to 13, wherein the support structure (1) has a shape such as to define a culture chamber having a substantially circular shape, or an ovoid shape, or a polygonal culture chamber, preferably a square culture chamber. The apparatus according to any one of claims 1 to 14, and further comprising a cover lid (3) arranged to be coupled to the support structure (1). The apparatus according to claim 15, wherein the cover lid (3) comprises a dividing device (4) which protrudes from the cover lid (3) so that when the cover lid (3) is ? coupled to the support structure (1), the division device (4) ? positioned in the culture chamber (10) and defines the at least two culture regions (10A, 10B) in the culture chamber (10), the dividing device (4) preferably acting as a sealing device (2) to hold the dividing membrane separation (20). 17. Apparatus according to claim 16, wherein the dividing device (4) is configured so as to define a window (40) in which the separation membrane (20) ? positioned. 18. Apparatus according to any one of claims 15 to 17, wherein the cover lid (3) is? provided with coupling means (31) for coupling the cover lid (3) to the support structure (1), the support structure preferably being provided with further coupling means (12) configured to couple with the coupling means for coupling the support structure (1) and the cover (3) to each other. 19. Apparatus according to any one of claims 14 to 17, wherein the cover lid (3) comprises at least two cover elements (3A, 3B) each being configured to be coupled to a respective portion (1A, 1B) of the structure support (1). 20. Apparatus according to claim 19, wherein the at least two cover elements (3A, 3B) are provided with respective coupling means for coupling the cover elements (3A, 3B) to the respective portion of the support structure (1), the coupling means preferably comprise a housing (32A, 32b) defined in the corresponding cover elements (3A, 3B) and arranged to house the respective portion of the support structure (1). 21. Apparatus according to claim 19, or 20 in which the at least two covering elements (3A, 3B) are provided with a respective attachment element (31) the support structure (1) being provided with a further attachment element (12 ) cooperating with the attachment element (31) to attach the cover elements (3A, 3B) and the support structure (1). 22. Apparatus according to any one of claims 19 or 21, wherein the at least two cover elements (3A, 3B) are provided with coupling elements (33, 34) for mutually coupling the cover elements (3A, 3B) to each other , and in which ? a fixing device (37) is provided for fixing the covering elements (3A, 3B) together. 23. Apparatus according to any one of claims 19 to 22, wherein each cover member (3A, 3B) is preferably provided with a dividing element (4, 5, 6). The apparatus according to any one of claims 1 to 23 and further comprising a locking device (7, 8) for holding the separating membrane (20) in the desired position on the dividing device (4, 5, 6). The apparatus according to claim 24 and further comprising a guide element (9) defined on at least one dividing element (5, 6) of the dividing device (4) and arranged to guide the insertion of the locking device (7 ) on it. The apparatus according to any one of claims 1-25 and further comprising a limiting device (60) arranged to be inserted into the culture chamber (10) and configured to define an active culture chamber (70) in the culture chamber (10) where the cell culture is created. 27. Apparatus according to claim 26, wherein the limiting device (60) is provided with a connection element to be connected to the separation device (4) so that the latter defines at least two active culture regions (70A, 70B) in the active culture chamber (70), to hold a separation membrane ( 20) so that the separation membrane (20) is interposed between the at least two different active culture regions (70A, 70B). 28. Apparatus according to claim 26 or 27, wherein the limiting device preferably comprises at least two limiting elements (61, 62) arranged to be each positioned in a culture region (10A, 10B) and defining an active culture region ( 70A, 70B) in each of the at least one growing region (10A, 10B), wherein the limiting elements (61, 62) are preferably coupled to the dividing device (4, 5, 6) so as to define the at least two active culture regions (70A, 70B) in the corresponding at least two culture regions (10A, 10B). 29. Apparatus according to any one of claims 26 to 28, wherein the limiting device (60) is made with PDMS. 30. Apparatus according to any one of claims from 1 to 29, wherein the division device (4, 5, 6) has such a shape as to define two or more? different culture regions in the culture chamber (10). The biocompatible system (500) for creating 3D cell cultures in vitro comprising: an apparatus (100) according to any one of claims 1-29, a support substrate (50) arranged to support the apparatus (100), and a seal for sealing the apparatus (100) to the support substrate (50) in order to define, with the support substrate (50), a sealed culture chamber (10). The biocompatible system (500) according to claim 30 and wherein the sealing is applied at a support structure (1) of the apparatus (100) so that the seal is interposed between the support structure (1) and the support substrate (50) to seal the support structure (1) to the substrate support (50) to define a sealed culture chamber (10). The biocompatible system (500) according to claim 30 or 31, wherein the support substrate (50) comprises an active portion made with MEA, the support substrate (50) preferably being made with MEA. The biocompatible system (500) according to any one of claims 30 to 32, and further comprising a sealing element disposed between the separation membrane (20) and the support substrate (50) configured to allow the passage of fluids and/or or of cellular connections (22) therethrough and to avoid the passage of cell bodies (23) through the separating membrane (20) and between the at least two culture regions (10A, 10B). The biocompatible system (500) according to claim 33, wherein the sealing element is a sealing strip disposed between the separation membrane (20) and the support substrate (50). The biocompatible system (500) according to any one of claims 30 to 34, and further comprising a further sealing element disposed between the dividing device (4) and the support substrate (50) configured to seal the at least two different regions culture medium (10A, 10B), the further sealing element preferably being a sealing layer arranged between the dividing element (4, 5, 6) and the support substrate (50). The biocompatible system (500) according to any one of claims 30 to 35, wherein a support substrate (50) comprises a coating provided on at least a portion of one face (50A) of the support substrate (50) facing in use towards the culture chamber (10) and arranged to facilitate the growth of the cells of the cell culture, the coating preferably being made with poly-L-ornithine. The biocompatible system (500) according to any one of claims 30 to 36 and further comprising a scaffold arranged in at least one of the culture region to enhance the growth of the cell culture.