Patent Description:
Devices for cell cultures exist, such as modular bioreactors having separate perfusion chambers where each chamber is independent and isolated from the others like a perfusion circuit, but at the same time, can be connected by bypass. These chambers are optically accessible and allow inspection of the cell culture by both standard and confocal optical microscopy, both in white light, in phase-contrast and fluorescent light.

A device, mentioned above, is described in the patent application <CIT> in the name of the same Applicant.

Another similar device is described in the document <CIT>.

The aim of the present invention is that of providing a millifluidic device for advanced cultures of biological agents which has a greater modularity than those of the known art.

Another aim is that of providing a device which enables a greater ease of use.

A further aim is that of providing a device that is simple to manufacture.

According to the present invention, these aims and others still are achieved by a millifluidic device for cultures of biological agents according to claim <NUM>.

Further characteristics of the invention are described in the dependent claims.

The advantages of this solution compared to the solutions of the known art are numerous.

The invention consists of a sealing device for the perfused culture of cells or bacteria both in suspension and adhesion, in 2D or 3D, for producing perfused solutions containing parts derived from cells (e.g. micro/nanovesicles), or biological molecules (e.g. protein or isolated DNA) or even bioactive chemical compounds (e.g. drugs).

The device consists of a host system formed of different culture/dilution chambers independent of one another, which are extractable and made with different diameters/measurements. The extractability enables an easy cell seeding on both sides of the membrane, and the possibility of reusing the body of the device by replacing the used culture chambers with new chambers and by sterilising the resulting device again.

The chambers host a membrane, which separates them into two half-chambers, of a permeable or, semi-permeable type with different porosity or non-permeable type, composed of different materials, such as polycarbonate, PET, PVC, TEFLON, PDMS, cellulose acetate, polyester, polystyrene, nylon and others.

The two half-chambers can be interconnected or independent of one another by appropriately choosing the material and the porosity of the membranes.

The cell cultures can be produced on one or both sides of the membrane, located in the culture chamber.

The device is optically accessible with standard optical microscopy and in phase-contrast, both straight and inverted, both in white light and fluorescent light, and with confocal microscopy. The device therefore allows inspection of the cell culture, in both chambers, by using the aforementioned microscopy techniques without interrupting the culture itself. The optical accessibility allows the device to be used with any type of optical sensor, for example a sensor for measuring pH, oxygen, carbon dioxide and allows measuring the concentration of solutes in a perfused solvent.

The device can be manufactured both in the absence and presence of electrodes, which are useful for measuring electrical parameters relevant to cellular and bacterial behaviour and for electrically stimulating cellular or bacterial cultures contained in each culture chamber, in a configuration that does not compromise the optical accessibility, thus guaranteeing a wide surface in contact with the content of the culture chamber. The electrodes can be conveniently and economically obtained by laser cutting from suitable metal sheets, for example, made of stainless steel or precious metals having a thickness from <NUM> to <NUM>.

The device is modular due to several aspects.

In particular, several devices can be connected to each other by creating a multi-device platform capable of simultaneously hosting and interfacing together different types of cell and bacterial cultures or types containing cell/bacteria derivatives, namely, chemically synthesised molecules, in the configurations described above, both for suspensions and with adhered cells grown in 2D or 3D.

The device and the resulting platform can be interfaced with liquid bacterial culture systems, either directly or through a dedicated system for producing bacterial cultures, in both standard liquid culture conditions, in suspension and in 2D, as well as cultivated in appropriate matrices in 3D, with a geometry similar to the culture chambers of the device.

d) Membrane support system, easily extractable from the device and manipulable. This characteristic makes it possible to carry out cell seeding operations and/or carry out assays and measurements outside the device, with established routine techniques. The extractable support system also makes it possible to quickly change the geometry of the perfusion chambers, by simply changing the type of membrane housing used, even by using commercially available inserts that are sterile and suitable for cell cultures. The advantage of this highly modular system is that it is possible to set different experimental conditions in each chamber, even by using the same base body of the device and/or without having to completely reassemble the entire experimental set-up.

e) System characterised by a double level of seals designed to isolate the two fluidic paths on both sides of the membrane and to prevent leakage of liquid outside of the chamber.

f) Anti-lifting safety system for the plugs, consisting of screws, or other fixing means, which make the system operable even at high pressure/high capacities. This system makes it safe to use the device even for long periods of time, and for frequent handling for carrying out microscopic inspections, namely, when stably mounted inside a microscope provided with a cell incubator.

The device can be used for research and analysis of bioactive molecules within the biological, medical, biochemical and chemical, pharmacological and toxicological fields. In general, the device can be applied to all those situations, known or still unknown, characterised by the need to optically inspect and/or electrically stimulate and/or electrically measure biological parameters after interactions between two liquids (equal or different) that perfuse two sides of a membrane (or the surfaces of a non-porous septum), and that contain viable biological material, derived from vital systems or those of a chemical nature, on which membrane or septum, a third element (adhered or laid) may or may not be placed.

The characteristics and advantages of the present invention will become evident from the following detailed description of a practical embodiment thereof, illustrated by way of non-limiting example in the attached drawings, wherein:.

With reference to the attached figures, a device <NUM> for cell culture, according to the present invention, comprises a main rectangular-shaped body <NUM>, with plan overall dimensions so that it can be housed in a slide holder for standard and confocal microscopy (for example: length from <NUM> to <NUM>, width from <NUM> to <NUM>, preferably length <NUM> width <NUM>).

The main body <NUM>, as shown, comprises three chambers <NUM>, <NUM> and <NUM>, with an independent hydraulic circuit.

The number of chambers can be varied from one to multiple according to the needs.

The three chambers <NUM>, <NUM> and <NUM> are each provided with an oval-shaped and elongated plug <NUM> that reaches the edges of the body <NUM>. Recesses <NUM> are arranged on the edge of body <NUM> in which the ends of plug <NUM> are positioned in order to have a guide for the correct positioning of the plugs <NUM> themselves.

The plugs <NUM> preferably comprise two through-holes <NUM>, placed at the ends, in order to fix them firmly on the body <NUM> by means of screws. Alternatively, other means for fixing the plugs <NUM> to the body <NUM> can be used, such as, for example, hooks or pressure systems.

The plugs <NUM> have a substantially flat upper component <NUM> and a cylindrical-shaped lower component <NUM>.

A preferably circular-shaped slide <NUM> is fixed on the upper and central component to the plug <NUM> which allows a complete view of the inside of the chamber, for use with microscopes.

An O-ring seal <NUM> is arranged externally to the lower component <NUM>.

Below the body <NUM>, two pairs of tubes can be seen for each chamber, which protrude perpendicular to the body <NUM>, and more specifically, exit from a lower enlarging <NUM> of the body <NUM> which provides space for the chambers <NUM>-<NUM>.

A pair of internal tubes <NUM>, with an inlet tube and an outlet tube, for a first half-chamber (which we will define in the following) and a pair of external tubes <NUM>, with an inlet tube and an outlet tube, for a second half-chamber.

The threaded holes <NUM> can be seen on the body <NUM>, which correspond to the holes <NUM> for fixing the plugs <NUM> onto the body <NUM> by means of screws <NUM>.

Once the plugs <NUM> are removed the separators <NUM> can be seen which allow each of the chambers <NUM>-<NUM> to be divided into two half-chambers.

The separators <NUM> have a hollow cylindrical-shape open at the bottom and closed at the top by a membrane <NUM>, to form an overturned glass-shape, closed at the top by the membrane <NUM>.

The membrane <NUM> divides the upper half-chamber from the lower half-chamber.

The membrane <NUM> can be placed (glued) at the top of the separators <NUM> as shown in <FIG> and <FIG>, but can be placed in positions inside the separator <NUM> as shown in <FIG> and <FIG>, particularly at a predefined distance from the top of the separator <NUM>.

In particular, if the membrane <NUM> is placed at the top of the separators <NUM>, the upper half-chamber is <NUM> high. In the case of other positioning of the membrane <NUM>, a separator <NUM> is made formed by two parts having pre-set heights. The membrane <NUM> is glued onto the lower part of the separator and then the upper part of the separator is glued, thus positioning the membrane at the desired height and obtaining the two half-chambers having predefined heights.

The positioning of the membrane <NUM> allows to increase or decrease the volume of the upper half-chamber and consequently decrease or increase the volume of the lower half-chamber.

An O-ring seal <NUM> is placed inside the separators <NUM>, preferably in a position spaced from the membrane <NUM>.

When the separators <NUM> are removed, the prearranged areas for the chambers can be seen in body <NUM>.

For each chamber <NUM>-<NUM> there is a hole <NUM> in the body <NUM> closed at the bottom. At the centre of the hole <NUM>, and coaxially thereto, a cylindrical body <NUM> protrudes. A preferably circular-shaped slide <NUM> is placed at the top of the cylindrical body <NUM>, which allows a complete view of the inside of the chamber, for use with microscopes.

The slide <NUM> can be omitted if the materials used to make the device are transparent.

The inside of the cylindrical body <NUM> is hollow and forms a hole <NUM>, coaxial to the cylindrical body <NUM>, which is open at the bottom and closed at the top by the slide <NUM>.

The hole <NUM> allows there to be less material in the optical path of the microscopes, thus allowing a better view of the inside of the half-chambers.

The separator <NUM>, including the membrane <NUM>, is set in its place above the cylindrical body <NUM>.

The separator <NUM> can only be extracted from its place by opening the plug <NUM> and pulling it out, and it can be set again in its place by inserting it on top of the cylindrical body <NUM>.

The pairs of tubes <NUM> and <NUM> are laterally aligned with the hole <NUM> and transversely to the body <NUM>.

The device preferably comprises two electrically conductive electrodes, one for each half-chamber.

The electrodes can be used both for measuring electrical parameters and for electrically stimulating the cultures contained in each culture chamber, such as, for example, for measuring the transepithelial/transendothelial resistance, for determining the onset of ionic currents passing through cell membranes, useful for physiological and neurobiological studies or for inducing tissue contraction, useful for simulating muscle contractions such as peristalsis.

An electrode <NUM> for the lower half-chamber has a main circular crown-shaped body <NUM> with two bars <NUM> which extend from the circular body <NUM> and are facing downwards, for electrical connection.

The electrode <NUM> is placed above the cylindrical body <NUM> and the circular body <NUM> is external to the slide <NUM> and in the area of the lower half-chamber. The two bars <NUM> protrude at the bottom from the body <NUM> (enlargement <NUM>).

An electrode <NUM> for the upper half-chamber has a main circular crown-shaped body <NUM>, and has some bars <NUM>, to facilitate the positioning, and a bar <NUM>, longer than the previous ones for electrical connection, which extend from the circular body.

The electrode <NUM> is placed in a housing on the plug <NUM>, and the circular body <NUM> is external to and below the slide <NUM>, so that it is positioned in the area of the upper half-chamber.

The electrodes <NUM> and <NUM> have a main circular crown-shaped body, therefore with a central hole, which does not compromise the optical accessibility.

Each half-chamber is sealed and isolated and communicates with the outside only with the pairs of tubes <NUM> and <NUM>. Only the type of membrane <NUM> determines the permeability between the two half-chambers. For example, it is possible to insert membranes made of polycarbonate, PET, PVC, TEFLON, PDMS, cellulose acetate, polyester, polystyrene, nylon, with a pre-defined porosity.

The internal seal <NUM> of the separators <NUM> interferes with the external lateral surface of the cylindrical body <NUM>, and closes the lower half-chamber, delimited at the top by the membrane <NUM>, at the bottom by the slide <NUM> and at the sides by the separator <NUM>.

The external seal <NUM> of the plug <NUM> interferes with the internal lateral surface of the hole <NUM>, and closes the upper half-chamber, delimited at the top by the slide <NUM>, at the bottom by the membrane <NUM> and at the sides by the lower component <NUM> of the plug <NUM> or by the separator <NUM>.

The cylindrical body <NUM>, which emerges from the hole <NUM>, forms a base <NUM>, around the cylindrical body <NUM>, with a circular crown-shape that acts as the lower reference abutment for both the plug <NUM> (lower component <NUM>) and the base of the separator <NUM>.

Therefore, after the opening of the plug <NUM> and the extraction of the separator <NUM>, when they have to be inserted again, both are positioned correctly as in the original position.

The perfusion of the chambers takes place via the pairs of tubes <NUM> and <NUM>.

The upper half-chamber uses the outermost pair of tubes <NUM>. They are fixed in a hole <NUM> of the body <NUM> and reach the base <NUM> and are aligned with the line where the separator <NUM> and the plug <NUM> are arranged side-by-side.

Between the separator <NUM> and the plug <NUM> a passage is prearranged that reaches the upper half-chamber. This passage is a pair of grooves <NUM> prearranged on the internal lateral wall of the plug <NUM> and, to facilitate the inflow inside the half-chamber, the groove also partially continues on the upper internal wall of the plug <NUM> until it reaches the slide <NUM> and/or the electrode <NUM>.

This fluid path can also be conveniently made by means of metal tubes connected to the plug and integral thereto and which are close to or, better still, engage directly in the internal lumen of the tubes <NUM> once the device has been assembled.

The lower half-chamber uses the innermost pair of tubes <NUM>. They are fixed in a hole <NUM>, side-by-side to the internal wall of the separator <NUM> and reaches a through-hole <NUM>, between the external edge of the separator <NUM> and the slide <NUM>.

If the electrode <NUM> is present, it has a bevelling <NUM> at the hole <NUM> to allow the perfusion flow.

The electrodes <NUM> and <NUM>, made of an electrically conductive material, may be present or absent in the device according to the needs.

In one embodiment of the device, the body <NUM> has a size of 25x68 mm, the half-chambers (upper and lower) have an average diameter comprised between <NUM> and <NUM> (preferably <NUM>) and a depth comprised between <NUM> and <NUM> (preferably <NUM> for the upper half-chamber and <NUM> for the lower half-chamber). In this case the membrane <NUM> has been placed above the separator <NUM>. The device <NUM> can be used for the culture of cells derived from living organisms both of the immortalised type (i.e. able to replicate indefinitely in culture) and of the primary type (i.e. with a limited or no capacity to replicate in culture), both in suspension and adhesion, in 2D and hosted in 3D in appropriate polymeric mediums and matrices. It may also be adapted for incubating parts derived from cells (e.g. microvesicles or particular cellular organelles), as well as for bacteria or solutions containing chemical molecules, including drugs.

It can also be used to combine a culture, for example neurons or endothelial cells or specific bacteria, grown individually or in co-culture such as in the intestinal microbiota, in one half-chamber, and a culture in the other half-chamber, such as, for example, astrocytes or blood cells or endothelial cells of the intestine to analyse how their interaction is of physiological or pathological relevance.

According to a first variant of the present invention, the device is now shown as a single device but could be structured, as in the previous case, with two or more devices placed in a single body.

In this case the single device <NUM> has a substantially circular body <NUM>, and comprises an extractable separator <NUM>, formed by a circular disc to the bottom of which a membrane <NUM> is fixed, which divides the body <NUM> into two half-chambers, one upper and one lower. The separator <NUM>, when inserted in the body <NUM>, rests on an edge arranged in body <NUM> itself.

The perfusion channels <NUM> and <NUM>, created inside body <NUM>, access the two half-chambers at the sides and are parallel to the membrane. The connection tubes (not shown) can possibly be applied to the channels <NUM> and <NUM>.

The device <NUM> can be used on its own or can be used in combination with the device <NUM>.

In particular it is possible to cultivate, in the device <NUM>, a bacterial culture placed in a half-chamber, fed by a fermenter or a bacterial culture in a suitable container, potentially also connected to a device that allows the mixing of bacteria in a polymeric matrix that simulates, for example, the intestinal mucus in the case of bacterial culture in 3D; the resulting secretome, namely, the set of molecules produced by the resulting culture, is transferred through the membrane into the other half-chamber and is sent to one or more chambers of the device <NUM> to verify the impact of the molecules produced on particular organs and biological systems, under physiological or pathological conditions including, by way of example but not limited to: intestinal endothelium: liver; immune system; blood-brain barrier; brain. In this case a porous membrane is used with a molecular cleaving that allows the passage of substances but not of cells (typically <NUM> micrometers between bacteria and secretome and <NUM> for the other half-chambers hosting cell cultures).

According to a second variant of the present invention, the perfusion of the lower half-chamber takes place as previously described from the tubes <NUM> coming from below and placed inside the device, while the perfusion of the upper half-chamber takes place through tubes <NUM> placed in the plug <NUM>. The half-chambers are separated by the membrane <NUM> supported by a small cylindrical element <NUM>, which may also be of the already commercially available type. For example, it is possible to use chambers compatible with Transwell type cell culture inserts produced by Greiner Bio-One International GmbH, both with translucent and transparent membranes.

According to a third variant of the present invention, perfusion of the lower half-chamber takes place by means of tubes <NUM> placed below the device, the perfusion of the upper half-chamber takes place by means of tubes <NUM> placed in the plug, and the two half-chambers are separated by a separator <NUM> supporting a membrane <NUM>.

All the components are made of plastic and elastomeric materials and are designed to be manufactured by injection moulding of plastic materials or other manufacturing method that can be applied on an industrial scale.

The materials as well as the dimensions and shapes used can be of any type according to the needs and the state of the art. For example, the chambers and all the connected elements have been made in a circular shape but nothing prevents them from being made in other shapes such as square or oval.

Claim 1:
A millifluidic device for cultures of biological agents comprising:
a main body (<NUM>) comprising at least a chamber (<NUM>-<NUM>) with an independent hydraulic circuit with a first hole (<NUM>) closed at the bottom and provided with a plug (<NUM>);
a separator (<NUM>) designed to be placed in said first hole (<NUM>) and which allow said chamber (<NUM>-<NUM>) to be divided into an upper half-chamber and a lower half-chamber;
a membrane (<NUM>) fixed to said separator (<NUM>) to divides said upper half-chamber from said lower half-chamber;
said separator (<NUM>) being extractable from said first hole (<NUM>) once the plug (<NUM>) is removed;
a pair of tubes (<NUM>) to perfuse said lower half-chamber;
a pair of tubes (<NUM>) to perfuse said upper half-chamber;
a first slide (<NUM>) placed concentrically on said plug (<NUM>);
a second slide (<NUM>) placed concentrically on said first hole (<NUM>);
a cylindrical body (<NUM>) rises coaxially from said first hole (<NUM>) toward said lower half-chamber;
said second slide (<NUM>) is placed on the top of said cylindrical body (<NUM>);
said first hole (<NUM>) is closed at the bottom by said second slide (<NUM>);
said cylindrical body (<NUM>) has a second hole (<NUM>), coaxial to said cylindrical body (<NUM>);
said pairs of tubes (<NUM>) and (<NUM>) are laterally aligned with said hole (<NUM>) and transversely to said body (<NUM>);
the lower half-chamber, is delimited at the top by the membrane (<NUM>), at the bottom by the slide (<NUM>) and at the sides by the separator (<NUM>);
the upper half-chamber, is delimited at the top by the slide (<NUM>), at the bottom by the membrane (<NUM>) and at the sides by a lower component (<NUM>) of the plug (<NUM>) or by the separator (<NUM>).