EP4731746A1 - Sensorized multifunctional device for biological cultivations - Google Patents

Abstract

A sensorized multifunctional device (1) comprising: an upper chamber (13) and a lower chamber (14); said upper chamber (13) and said lower chamber (14) being separated by a membrane (31); a first pair of tubes (18) connected to said upper chamber (13); a second pair of tubes (20) connected to said lower chamber (14); an upper glass element (28) which delimits said upper chamber (13) at the top; a lower glass element (35) delimiting said lower chamber (14) at the bottom; characterized by comprising: a first and a second conductive electrode (27) arranged inside said upper chamber (13) electrically connected to a respective conductive track (26) arranged outside said upper chamber (13); a third and fourth conductive electrodes (39) arranged inside said lower chamber (14) electrically connected to a respective conductive track arranged outside said lower chamber (14); said first, second, third and fourth electrodes (27, 39) each being connected to a respective conductive structure (25, 44) adapted to receive measurement electrodes; said device further comprising a first upper body (10); a second intermediate body (11); a third lower body (12); said first (10), second (11) and third (12) body being assemblable with one another; said second intermediate body (11) has a hollow shape on the inside, open at the top and closed at the bottom by said membrane (31), which divides said upper chamber (13) from said lower chamber (14); said third lower body (12) is designed to be inserted into a lower housing (40) which comprises two conductive structures (44) adapted to receive the measurement electrodes.

Description

“SENSORIZED MULTIFU NCTIONAL DEVICE FOR

BIOLOGICAL CULTIVATIONS” DESCRIPTION

The present invention refers to a multifunction device of the sensorized m illifluidic type having optical accessibility, respective manufacturing method and respective multifunctional multi-device platform .

The m illifluidic device is a bioreactor formed by two chambers divided by a sem ipermeable membrane. Both chambers are provided with an inlet tube and an outlet tube to form two separate fluid circuits.

A device referred to above is described in patent application WO2021053458 in the name of the same Applicant.

The most widespread technique applied to evaluate the integrity of epithel ial/endothel ial barriers is the measurement of transepithelial/transendothelial electrical resistance (TEER).

Transendothelial/epithelial electrical resistance (TEER) is one of the widely used tests to confirm barrier integrity based on the physiological state of cell junctions (TJs).

Several strategies have been undertaken to perform TEER measurement.

The most popular TEER measurement systems use electrodes in a four-point measurement configuration.

These are a pair of electrodes that inject current and a pair of electrodes that measure voltage.

The object of the present invention is to provide a multifunctional device designed to allow electrical measurements to be carried out in a simple way, even in real time during use, without opening the device and without disturbing the biological cultures housed therein.

Another object is to provide a removable multifunctional device.

A further object is to provide a sensorized multifunctional device that can also be obtained in a multifunctional multi-device platform configuration.

Another object is to provide a multifunctional sensorized device having optical accessibility.

According to the present invention, said objects and others are achieved by a multifunctional device according to claim 1 .

Said objects and others are also achieved by a multifunctional platform suitable for supporting/integrating a plurality of multifunctional devices according to claim 1 .

Said objects and others are furthermore achieved by a method for making a device according to claim 1 .

Further features of the invention are described in the dependent claims. The advantages of this solution compared to prior art solutions are numerous.

The invention consists of a leak-proof device for the perfused culture of cells or bacteria both in suspension and in adhesion, in 2D or 3D, for obtaining perfused solutions containing cell-derived parts (e.g. , m icro/nanovesicles), or biological molecules (e.g. , proteins or isolated DNA) or even bioactive chem ical compounds (e.g. , drugs).

The device is optically accessible with standard and phase contrast optical m icroscopy, both upright and inverted, in both white light and fluorescence, and with confocal m icroscopy. The device therefore allows the inspection of the biological culture, of both chambers, by means of the aforementioned m icroscopy techniques without interrupting the culture itself. Optical accessibility allows the device to be used with any optical sensor, for example for measuring pH, oxygen, carbon dioxide, and allows measuring the concentration of solutes in a perfused solvent.

The device is preferably made by stereolithographic (SLA) 3D printing, due to its fine details, smooth finish, absolute precision, and accuracy of the parts.

The conductive tracks are made using Physical Vapor Deposition (PVD).

Conductive parts are already provided in the device for the insertion of commercial plugs to carry out measurements with conventional instruments.

The device allows non-invasive real-time measurements to be carried out. Thanks to the electrodes already arranged on the device, the TEER measurement can be carried out simultaneously on all the wells of the multifunctional platform during the experiment without having to open the culture chambers or having to insert the probes in order to measure. This solves the problem of all dynam ic culture systems that use removable inserts and which need to disassemble the system to measure TEER.

To resolve the interference of metal electrodes with flows and biological culture, the electrodes have nanometric thicknesses (below 200 nm and typically 50 nm) and are directly deposited on the walls of the device.

In particular, in order to guarantee the creation of electrodes (continuous conductive tracks) on a three- dimensional device with complex geometries and shapes comprising edges and recesses and which is also optically accessible (transparent), the electrodes have specific thicknesses depending on the area of the device: up to 200 nm on the upper and lower body and in the angular areas to guarantee conductivity and up to a m inimum of 25 nm in the flat areas to ensure optical accessibility to the m icroscope. The very thin nanometric thickness allows the entire area of the biological culture to be covered, thus improving the uniform ity of the signal, but at the same time guaranteeing optical accessibility even with m icroscopes.

This is a multifunctional multifluidic sensorized platform that uses removable inserts for cell culture. Compared to already existing solutions of sensorized m icro-bioreactors (m icrofluidic), the present approach solves the problem of using complex microfabrication techniques using soft-lithography in special cleanrooms by depositing electrodes using a physical deposition technique on 3D pieces of macroscopic dimensions (in the order of centimetres).

The multifunctional platform has the size of a standard multi-well plate for standard biological cultures and fits perfectly into the housings of common m icroscopes.

The platform is compact and allows culture in perfusion, in a standard incubator for biological cultures, with the simultaneous detection of the signal generated by the electrodes outside the incubator itself, without therefore interfering with perfusion and culture.

The multifunctional platform allows to house/integrate individual sensorized devices of different sizes.

The conductive tracks deposited directly on the walls of the platform allow the electrical signal to be brought to the outside the device without providing electrode access points. This solves the problem of the risk of contam ination and the difficulty of assembly.

The characteristics and advantages of the present invention will be clear from the following detailed description of one of its practical embodiments, illustrated by way of non-lim iting example in the attached drawings, wherein:

Figure 1 schematically shows a multifunctional sensorized device having optical accessibility, according to the present invention;

Figure 2 schematically shows a multifunctional sensorized device having optical accessibility, shown in section, according to the present invention;

Figure 3 schematically shows a first upper body of a sensorized multifunctional device having optical accessibility, shown in perspective from above, according to the present invention;

Figure 4 schematically shows a first upper body of a sensorized multifunctional device having optical accessibility, shown in perspective from below, according to the present invention;

Figure 5 schematically shows a second intermediate body of a sensorized multifunctional device having optical accessibility, shown in perspective, according to the present invention;

Figure 6 schematically shows a lower third body of a sensorized multifunctional device having optical accessibility, shown in perspective from above, according to the present invention;

Figure 7 schematically shows a lower third body of a sensorized multifunctional device having optical accessibility, shown in perspective from below, according to the present invention;

Figure 8 schematically shows a lower housing for carrying out measurements, of a multifunctional device having optical accessibility, shown in perspective, according to the present invention;

Figure 9 schematically shows a multifunctional sensorized device having optical accessibility, comprising a lower housing for carrying out measurements, according to the present invention;

Figure 10 schematically shows a multifunctional multidevice platform that houses/integrates multiple sensorized multi-fluidic device units having optical accessibility, according to the present invention;

Figure 1 1 schematically shows a measurement platform of a sensorized multifunctional multi-device platform having optical accessibility, according to the present invention; Figure 12 schematically shows a sensorized multifunctional m ulti-device platform having complete optical accessibility according to the present invention;

Figure 13 schematically shows an alternative embodiment of a sensorized multifunctional multi-device platform having optical accessibility, as seen from above, according to the present invention;

Figure 14 schematically shows an alternative embodiment of a sensorized multifunctional multi-device platform having optical accessibility, as seen from below, according to the present invention.

Referring to the attached figures, a multifunctional sensorized device 1 having optical accessibility, according to the present invention, comprises a first upper body 10, a second intermediate body 1 1 and a third lower body 12.

An upper chamber 13 is formed between the first upper body 10 and the second intermediate body 1 1 .

A lower chamber 14 is formed between the second intermediate body 1 1 and the third lower body 12.

Two holes 17 connected to the upper chamber 13 allow connection to external tubes 18 for perfusion.

Two inlet and outlet holes 19 connected to the lower chamber 14 allow connection to external tubes 20 for perfusion.

The first upper body 10 has a substantially cylindrical shape whose upper part 22 has a greater radius than its lower part 23.

The first body 10 has a central through hole 24.

On the upper part the two holes 17 are provided and two U-shaped structures 25 emerge, namely, a structure with three sides lying on the plane of the upper part 22, which will be made conductive for the insertion of the measurement electrodes (not shown).

A conductive track 26 will start from the structures 25 and continue on the remaining external portion of the upper part 22 and on the external portion of the lower part 23 until reaching the circular crown on the bottom of the first body 10, to form two conductive sem icircles 27. The circular crown 27 has a central hole in which a first circular transparent glass element 28 will be arranged.

The circular crown and the first glass element 28 will form the upper closure of the upper chamber 13.

Three L-shaped hooks 29 extend downwards from the upper part 22, oriented with the opening in a clockwise direction.

Externally to the lower part 23 of the first body 10 a gasket 30 is provided, which allows the holding of the second body 1 1 by interference.

The second intermediate body 1 1 has a substantially cylindrical shape hollow on the inside, open at the top and closed at the bottom by a membrane 31 , which divides the upper chamber 13 from the lower chamber 14.

Three offshoots 32 extend laterally above the second body 1 1 .

The third lower body 12 has a substantially cylindrical shape, hollow on the inside, open at the top and closed at the bottom by a second circular transparent glass element 35.

On the side of the third body 12 two holes 19 are provided, which allow connection to external tubes 20 for perfusion.

Three L-shaped hooks 37 extend laterally and above the third body 12, oriented with the opening in an anticlockwise direction, designed to cooperate with the three hooks 29 so as to close the device 1 .

Laterally to the third body 12 and interspersed with the three hooks 37, three recesses 38 are provided on which the three offshoots 32 of the second body 1 1 will rest.

Inside the third body 12 a gasket 34 is provided, which allows holding of the second body 1 1 by interference.

The second glass element 35 is covered on both sides by two conductive sem icircles 39. Each upper sem icircle is electrically connected to the corresponding lower sem icircle by means of a conductor arranged on the edge of the glass element. Each of the sem icircles, on both sides, are separated from one another to form two electrodes. The sem icircle shape can be substituted with a circular crown portion shape in order to leave a greater surface of the glass element free from the conductor.

For electrical measurements, use is made of a lower housing 40 which acts as a support for the device 1 and allows the measurement electrodes to be connected to the two conductive sem icircles 39.

The lower housing 40 supporting the device 1 has a substantially square external shape whereas on the inside has a circular seat 41 in order to support the device 1 and in particular the third body 12.

On the bottom of the housing 40 two conductive sem icircles 42 are provided adapted to cooperate with the two sem icircles 39 arranged below the glass element 35. A conductive track 43 connects each sem icircle 42 to respective two conductive U-shaped structures 44 and adapted to receive the measurement electrodes (not shown).

The seat 41 has two open lateral portions 45 so as to not interfere with the two holes 19 of the third body 12.

On the outside of the third body 12 a recess is provided where it is possible to insert one (or more) gaskets 46 which allows the holding of the housing 40 by interference. The upper chamber 13 is comprised at the top between the two conductive sem icircles 27 and by the glass element 28 and below by the membrane 31 . The inlet and outlet holes 17 serve to perfuse the upper chamber 13.

The lower chamber 14 is comprised above by the membrane 31 and below by the glass element 35 comprising the two conductive sem icircles 39. The inlet and outlet holes 19 serve to perfuse the lower chamber 14.

The device 1 can be integrated into a multifunctional multi-device platform 50, formed by a main rectangular body of standard dimensions (having typical dimensions 128x86 mm) in order to be housed in a m icroscope, which comprises four housings 51 designed to house four devices 1 .

In particular, the housings 51 of the plate 50 comprise the third lower body 12 on which the second body 1 1 and the first body 10 will be mounted.

The various bodies 10-12 of the device can be assembled or disassembled by interlocking without using screws.

It also comprises as many supply tanks 52 each having an inlet hole 54 and an outlet hole 53 connectable by means of tubes (not shown) to the inlet hole 19 of the third lower body 12. The hole 19, in the multifunctional multi-device platform configuration, flows into m icrochannels printed inside the platform structure so as to allow perfusion.

The platform 50 also comprises a further housing 55 of larger dimensions than the housings 51 , arranged alongside the tanks 52. The housings 51 have a typical diameter of 20 mm , whereas the housing 55 has a typical diameter of 30 mm . The housing 55 also comprises the third lower body 12.

To carry out measurements on the devices provided on the platform 50, use is made of a further measurement platform 60 having substantially the same shape and size as the platform 50, which is applied under the platform 50.

The measuring platform 60 in the area of each of the housings 51 com prises a pair of sem icircular-shaped conductive tracks 61 suitable for being connected with the conductive tracks arranged on the second glass element 35. Two straight conductive tracks 62 extend from the sem icircular conductive tracks 61 , which end with two conductive U-shaped structures 63, arranged on the edge of the measurement platform 60 and adapted to receive the measurement electrodes (not shown).

Also in the area of the housing 55 a pair of conductive tracks 64 is provided in the shape of a sem icircle suitable for being connected with the conductive tracks arranged on the second glass element 35. From the sem icircular conductive tracks 64 extend two straight conductive tracks 65, which end with two conductive U-shaped structures 66, arranged on the edge of the measurement platform 60 and adapted to receive the measurement electrodes (not shown).

As an alternative to the lower housing 40 and the measuring platform 60, to carry out the measurements it is possible to provide that conductive tracks extend from the two conductive sem icircles 39 of the second glass element 35, which lead to conductive U-shaped structures adapted to receive the measuring electrodes arranged directly on the lower third body 12.

For example, a multifunctional multi-device platform 70 can be provided that incorporates both the multifunctional multi-device platform 50 and the measurement platform 60.

Within the same the two conductive tracks 71 are provided, in this case in the shape of a portion of a circular crown, designed to be connected with the two conductive tracks arranged on the second glass element 35, which extend below the platform 70 passing through the inner edge of the bottom hole of the third lower body 12, form ing the tracks 72, which extend recti linearly and end with two conductive U-shaped structures 73, arranged on the edge of the upper surface of the platform 70 and adapted to receive the measurement electrodes (not shown) .

It was chosen to use conductive U-shaped structures adapted to receive the measurement electrodes, but other structures adapted to receive the measurement electrodes can be used, for example cylindrical structures or shapes suitable for housing pins for electrical measurements can be used with commercial measurement systems. The device 1 , the plate 50 and the plate 60 are preferably made by stereolithographic (SLA) 3D printing. The material used to obtain the latter is preferably a photopolymerizing resin. It is also possible to produce the device by means of injection moulding by using plastic materials.

The membrane 31 , which separates the two chambers, can be of the permeable or sem i-permeable type with different porosities, or non-permeable.

The membrane 31 is formed by various materials, such as for example polycarbonate, PET, PVC, TEFLON, PDMS, cellulose acetate, polyester, polystyrene, nylon, and others.

By appropriately choosing the material and porosity of the membrane, the two chambers can be interconnected or independent.

The TEER measurement is carried out in the four-point measurement configuration, that is, one pair of electrodes injects current and one pair of electrodes measures voltage.

Therefore, two conductive electrodes 27 will be provided in the first chamber 13 and two conductive electrodes 39 will be provided in the second chamber 14.

The shape of the conductive electrodes 27 and 39 can be a sem icircle, a circular crown, as portions subtending a circumferential arc or a double circle or other shapes that can form two conductive electrodes.

For the measurements, rod electrodes were considered and the device was designed for this type of electrodes but other types of electrodes can be used, for example clamp electrodes, and the device can be modified for this purpose.

The conductive tracks are preferably made by gold plating with the physical vapor deposition (PVD) process.

The gold plating covers a good part of the area of the glass elements in order to cover as much surface as possible and to have the most uniform signal possible. Optical accessibility is however maintained thanks to the nanometric thickness of the layer of gold or other conductive material which is less than 200 nm and is transparent under the m icroscope.

However, the production method allows the dimensions and thicknesses of the track on the glass element to be easily increased or reduced depending on specific needs. Simply modify the masks for selective deposition of gold or other conductive material. The assembly of the device 1 is simple and is carried out as follows.

The second intermediate body 1 1 is inserted into the third lower body 12.

The three offshoots 32 of the second body 1 1 , which form abutment elements, abut against the three recesses 38. The dimensions, and in particular the distance between the membrane 31 and the glass element 35, are arranged so that the second body 1 1 remains raised from the bottom of the third lower body 12 so as to form the lower chamber 14.

The gasket 34 of the third lower body 12 holds the second body 1 1 .

In the device it is possible to house, instead of the second body 1 1 , also commercial inserts.

The first upper body 10 is inserted into the second body 1 1 and is rotated in an anti-clockwise direction so that the hooks 29 of the first body 10 fit with the respective hooks 37 of the third lower body 12, and the gasket 30 of the first body 10 holds the second body 1 1 .

The device as assembled without the use of screws or anything else can be disassembled in a sim ilar way.

The device 1 can be used on its own or integrated into the multifunctional multi-device platform 50.

Claims

1 . A sensorized multifunctional device (1 ) comprising: an upper chamber (13) and a lower chamber (14); said upper chamber (1 3) and said lower chamber (14) being separated by a membrane (31 ); a first pair of tubes (18) connected to said upper chamber (13); a second pair of tubes (20) connected to said lower chamber (14); an upper glass element (28) delim iting said upper chamber (13) at the top; a lower glass element (35) delim iting said lower chamber (14) at the bottom ; characterized by comprising: a first and a second conductive electrode (27) arranged inside said upper chamber (13) electrically connected to a respective conductive track (26) arranged outside said upper chamber (13); a third and fourth conductive electrodes (39) arranged inside said lower chamber (14) electrically connected to a respective conductive track arranged outside said lower chamber (14); said first, second, third and fourth electrodes (27, 39) each being connected to a respective conductive structure (25, 44) adapted to receive measurement electrodes; said device further comprises a first upper body (10); a second intermediate body (1 1 ); a third lower body (12); said first (10), second (1 1 ) and third (12) body being assemblable with one another; said second intermediate body (1 1 ) has a hollow shape on the inside, open at the top and closed at the bottom by said membrane (31 ), which divides said upper chamber (1 3) from said lower chamber (14); said third lower body (12) is adapted to be inserted into a lower housing (40) which comprises two conductive structures (44) adapted to receive the measurement electrodes.

2. The device (1 ) according to claim 1 , characterized in that said first, second, third and fourth electrodes (27, 39) have a thickness of less than 200 nm .

3. The device (1 ) according to claim 2, characterized by comprising: a first glass element (28) arranged below said first upper body (10); a second glass element (35) arranged below said third lower body (12).

4. The device (1 ) according to claim 2, characterized in that said first and second electrodes (27) are arranged on the bottom of said first upper body (10).

5. The device (1 ) according to claim 2, characterized in that said third and fourth electrodes (39) are arranged on both surfaces of said second glass element (35).

6. The device (1 ) according to claim 5, characterized in that said third and fourth electrodes (39) arranged on a first surface of said second glass element (35) are electrically connected to the corresponding said third and fourth electrodes (39) arranged on a second surface of said second glass element (35) opposite to said first surface by means of a corresponding conductor arranged on the edge of said second glass element (35).

7. The device (1 ) according to claim 2, characterized in that said first and second electrodes (27) are electrically connected to two structures (25) arranged on said first upper body (10) adapted to receive the measurement electrodes.

8. A multifunctional multi-device platform characterized by comprising a plurality of seats (51 , 55) suitable to contain a plurality of multifunctional devices (1 ) according to claim 1 , wherein each of said plurality of multifunctional devices (1 ) comprises a first upper body (10); a second intermediate body (1 1 ); a third lower body (12); and wherein said third lower body (12) is incorporated in said multifunctional multi-device platform .

9. A method for making a device (1 ) according to claim 1 characterized by making the first and second conductive electrodes (27) and said third and fourth conductive electrodes (39) by means of the physical vapor deposition (PVD) technique.