WO2025233916A1

WO2025233916A1 - Optimized support device for test samples in a bioreactor

Info

Publication number: WO2025233916A1
Application number: PCT/IB2025/054914
Authority: WO
Inventors: Ileana ARMANDO; Stefano GABETTI; Beatrice MASANTE; Giovanni PUTAME; Diana Nadia Caterina MASSAI; Mauro SERPELLONI
Original assignee: Universita degli Studi di Brescia; Politecnico di Torino
Current assignee: Universita degli Studi di Brescia; Politecnico di Torino
Priority date: 2024-05-09
Filing date: 2025-05-09
Publication date: 2025-11-13
Anticipated expiration: 2026-11-09

Abstract

A support device for a test sample mountable in a culture unit of a bioreactor comprising a first and a second crosspiece arranged parallel and facing each other with respect to a median plane, and comprising respectively at least a first and a second pillar facing each other and extending transversely with respect to the direction of extension of the crosspieces, such pillars having longitudinal ends in use in contact with the test sample, a spacer arranged between the first and the second crosspiece to keep said crosspieces transversely spaced, and the spacer being further extendable in a direction transverse to said direction of extension of the crosspieces, and one of the first and the second crosspiece being releasably engageable on a first frame of the mobile culture unit transversely to said direction of extension of the crosspieces while the other of the first and the second crosspiece being releasably engageable on a second fixed frame of said culture unit, so that a movement of the first frame defines a change in the relative position between the first and the second crosspiece causing through the pillars a deformation of the test sample detectable by a sensor; and the spacer having a rigidity such as to keep the first and second crosspieces substantially parallel to each other when the support device is dismantled from the cultivation unit.

Description

OPTIMIZED SUPPORT DEVICE FOR TEST SAMPLES IN A BIOREACTOR

DESCRIPTION

TECHNICAL FIELD

The present invention concerns the field of bioreactors for the in vitro culture of test samples, preferably of biological material, in particular a support device for test samples that can be releasably mounted in a bioreactor.

STATE OF THE ART

In the field of bioreactors for the in vitro culture of biological tissue samples, solutions are widely used through which the biological sample receives a chemical and/or physical stimulation, e.g., electrical and/ or mechanical, aimed at mimicking the conditions found in vivo. Furthermore, some solutions provide real-time monitoring of the culture process as well as the possible characterization of samples useful for drug testing, pathology modeling and tissue engineering for clinical application and for cellular agriculture. However, the current commercially available solutions involve damaging the test sample during its extraction from the bioreactor, thus making it necessary to create a new sample of material to perform further tests or continue with the cultivation process.

Therefore, it is always a felt need to create solutions through which the manipulation of one or more test samples is facilitated during the insertion and extraction step from the bioreactor, thus also minimizing the risk of destruction of the test sample so that it can be reused for subsequent experiments. SCOPE AND SUMMARY OF THE INVENTION

The present invention has the purpose of satisfying at least in part the needs indicated above, wherein this purpose is achieved by means of a support device for one or more test samples that can be placed in a culture unit of a bioreactor according to claim 1.

According to a preferred embodiment of the present invention, a support device for test samples, preferably biological, in a bioreactor is presented with a simple construction configuration and through which it is possible to facilitate the handling of the test samples both during insertion and extraction from the culture unit of the bioreactor, thus minimizing the risk of damaging or destroying the test sample during an experiment. Advantageously, this device has a construction configuration that provides for easy assembly/ disassembly from the culture unit of a bioreactor, without having to require fastening elements such as screws or bolts. To achieve this result, this device comprises a first and a second crosspiece arranged parallel to and facing a median plane, and comprising at least a first and a second pillar respectively facing and extending transversely to the direction of extension of the crosspieces. In use, these crosspieces are arranged respectively on a fixed frame and on a movable frame of the culture unit, in particular the frame is movable to the translation transversely to the direction of extension of the crosspieces. In this way, a variation of the relative position between the frames causes a variation of the transverse distance between the first and the second crosspiece, one being arranged on the fixed frame and one on the movable frame. In this configuration, the pillars extend towards a tray of the culture unit on which there are culture wells, and the longitudinal ends of these pillars reach the wells retaining the test sample on board. It should be noted that the test sample is fixed to the pillars outside the bioreactor, i.e., when the support device is disassembled from the culture unit. When the test sample is fixed to the pillars, the support device is mounted on board the cultivation unit by arranging the first and second pillars inside a well, preferably spaced from the surface of the well. According to one aspect of the invention, this device also comprises a spacer arranged between the first and second crosspiece configured to keep such crosspieces transversely spaced, and this spacer is also extendable in a direction transverse to the direction of extension of the crosspieces. In this way, when the movable frame is actuated, the variation of the relative position between the first and second crosspiece causes a variation of the extension of the spacer, but above all a deformation of the test sample, detectable by means of a sensor, e.g., strain gauge, on board at least one of the pillars, due to the movement of the pillars on which this sample is fixed. Furthermore, the spacer has a rigidity such as to keep the first and second crosspieces substantially parallel to each other at a predefined distance when the support device is disassembled from the cultivation unit. Advantageously, such feature allows the pillars to be kept transversely spaced at any stage of an experiment, while at the same time ensuring easier sample management, e.g., insertion and removal of samples from the culture wells, without having to be detached from the pillars when they need to be extracted from the bioreactor, as they can in fact be removed from the same while remaining on board the support device.

DESCRIPTION OF THE DRAWINGS

The construction and functional features of the test sample support device may be better understood from the detailed description that follows, in which reference is made to the attached figures that represent a preferred and non-limiting embodiment thereof, in which:

• Fig. 1 shows an exploded view of a culture unit comprising a plurality of test sample support devices according to a preferred embodiment of the present invention;

• Fig. 2 shows a perspective view of the test sample support device that can be housed in the culture unit of Fig. 1; • Fig. 3 shows a schematic view of a bioreactor system comprising the culture unit with a plurality of test material support devices of Fig. 2 on board, a control unit, a monitoring unit and an electrical and/ or mechanical stimulation unit;

• Fig.4 shows a cross-sectional view of a pair of pillars of the test material support device according to a further preferred embodiment of the present invention, comprising a mechanism for adjusting the transverse distance between such pillars;

• Fig.5 shows a cross-sectional view of the pair of pillars of Fig.4 when the adjustment mechanism is actuated.

DETAILED DESCRIPTION OF THE INVENTION

According to a preferred embodiment of the present invention, Fig.l shows an exploded view of a culture unit C of a bioreactor through which it is possible to apply constant or adaptive chemical and/or physical stimulation protocols to a test sample, preferably biological, in an automated manner, but also to monitor in real time the conditions of the culture environment, and above all to characterize in a non-destructive manner the test samples during the culture, without interrupting the experiment. For example, the test sample may be in the form of biological tissues, cellularized constructs based on decellularized matrix or bioartificial scaffolds, cellularized constructs obtained by bioprinting, hydrogels with cells, scaffolds of biological origin not seeded with cells or scaffolds of synthetic origin whose degradation over time or the variation of mechanical properties under certain environmental conditions is to be evaluated. In particular, as shown in Fig. 1, the culture unit C comprises a base 1, preferably rectangular in plan view, having perimeter side walls 2 defining a housing seat 3, e.g., by means of shape coupling, for a tray 4 on which a plurality of culture wells 5 of the test samples has been created. In this way, the tray can house multiple samples in parallel immersed in the culture medium. Preferably, the culture wells are sensorized, and in each of them one or more test samples is housed, but lower cost solutions without sensors can be provided. The culture unit C also comprises a cover 6 comprising openings 7 shaped so as to define each one a seat for an electrode used in use for the electrical stimulation of the test samples. In use, each electrode (not shown in the figure) is housed in a respective seat for engagement extending towards the respective culture well in which the test material is placed. A port 7a is also provided which facilitates the exchange of gas with the environment outside the culture unit C, or to allow the replacement of the culture medium, for the injection of substances into the culture medium, etc. It should be noted, for example, that this port 7a is used when the culture unit on board a bioreactor B is placed inside an incubation chamber in which there is a controlled atmosphere, e.g. at predefined concentrations of CO2, humidity and temperature, so that through such port 7a the test material is subjected to the conditions of such controlled atmosphere. Preferably, to prevent the entry of unwanted material into the culture wells, filters may be provided in the port 7a. Furthermore, the culture unit C comprises a first frame 8 shaped so as to have perimeter walls that in use rest on and are rigidly fixed to the base 1 so as to surround the tray 4. For example, when the culture unit is assembled, the lid 6 is fixed to the base 1 and this configuration blocks the components placed between them, including the first frame 8. Furthermore, the culture unit comprises a second frame 9, arranged in use inside a housing compartment 10 made on the first frame 8 so that such second frame is arranged at a greater vertical height than that at which the culture wells of the tray 4 are located. To achieve this result, a support device D is provided for one or more test samples, preferably biological, which can be releasably mounted on board the culture unit, so as to facilitate the handling of the test samples both during insertion and extraction from the bioreactor, so that experiments are performed while minimizing the risk of destroying the test samples due to the difficulty of housing them on a support portion of the culture unit, as occurs in traditional solutions. In particular, as shown in more detail in Fig. 2, this support device comprises a first and a second crosspiece 13, 14 arranged parallel and facing each other with respect to a median plane included between these crosspieces. Furthermore, these crosspieces comprise at least a first and a second pillar 15, 16 respectively facing and extending transversally and preferably perpendicularly to the direction of extension of the crosspieces. It should be noted that the test sample is fixed to the pillars outside the bioreactor, i.e. when the support device D is removed from the culture unit. When the test sample is attached, the support device D is mounted on board the culture unit by arranging the first and second pillars inside a well, preferably spaced from the surface of the well. According to a first aspect of the present invention, the longitudinal ends of the first and second crosspieces 13, 14 are releasably engaged inside respectively first and second housings 17, 18 made on opposite perimeter edges of the first frame 8 and in a symmetrical manner with respect to the translation direction of the second frame 9. In particular, the first crosspiece 13 has longitudinal ends engaged in use inside the first housings 17 so as to define a coupling of such a shape as to keep the first crosspiece stationary in a predefined position. The second crosspiece 14 instead has longitudinal ends engaged in use inside the second housings 18, which have a greater longitudinal extension than the transverse extension of the second crosspiece, and this feature is exploited to make the second crosspiece movable in use. The first and second pillars 15, 16 in use extend towards the tray 4 so as to have longitudinal ends on which the sample of test material contained in the corresponding culture well is fixed. According to one aspect of the present invention, to make the second crosspiece 14 movable, the latter has a plurality of coupling seats 19, preferably holes, made transversely and preferably perpendicularly to the direction of longitudinal extension of the crosspiece, so as to releasably engage these seats on protrusions 20 present on longitudinal ribs 21 on the edge of the second frame 9, e.g., defining a shape coupling. Furthermore, the first and second crosspieces 13, 14 respectively have at least a first and a second projecting portion 23, 24 transversally to the direction of extension of such crosspieces. In particular, such first and second projecting portions face each other and the first and second pillars 15, 16 extend longitudinally respectively from such first and second projecting portion. Furthermore, such construction conformation defines respectively on the first and second crosspiece a first and a second recess 29, 30, facing each other and in an interposed position between two portions projecting on the respective crosspiece. In particular, each pair of first and second recesses 29, 30 defines in use an opening 30 in which an electrode extends towards the respective culture well, so that when the culture unit C is in operation with at least one support device D on board on which the test material is fixed, the latter is interposed between two electrodes (not shown in the figure) so that it can receive electrical stimulation during the test. In this way, the test material is interposed between the first and second electrode, held by the respective ends of the first and second pillars 15, 16, thus avoiding contact interference during the mechanical stimulation movement. In this way, when the actuator of the second frame 9 is actuated, the second crosspiece 14 moves longitudinally together with the second frame 9, with the longitudinal ends sliding on the surface of the second housings 18. Therefore, the actuation of the movement of the second frame 9 defines a change in the relative position between the first and second crosspieces 13, 14 causing, via the pillars 15, 16, a deformation of the test sample, which can be detected via a sensor (not shown in the figure) preferably on board the support device D. Preferably, such a sensor is a deformation sensor, e.g., a strain gauge, and acts as a force sensor. Preferably, in each well there is a pair of electrodes to allow the samples to be subjected to controlled electrical stimulation. According to a further aspect of the present invention, the support device D comprises a spacer 22 arranged between the first and second crosspieces 13, 14 to keep such crosspieces transversally spaced. Furthermore, such spacer is extendable in a direction transverse to said direction of extension of the crosspieces, so that during an experiment it can extend longitudinally based on the variation of the relative position between the first and second crosspieces 13, 14. Preferably, to avoid an imbalance of forces when the crosspieces are spaced apart, the support device D comprises a pair of spacers 22, 22' arranged symmetrically with respect to a median plane transverse and preferably perpendicular to the direction of extension of the crosspieces 13, 14. For example, such spacer can be a spring having its ends connected to the first and second crosspieces, or it can be an elongated element having a first longitudinal end rigidly fixed and a second longitudinal end engaged in the other crosspiece so as to be relatively movable upon translation. In this way, at the end of the experiment, the support device D for the test material sample can be disassembled from the second frame 9 and the relative transverse position between the first and second crosspieces 13, 14 can be restored by exploiting the extensibility of the flexible spacer. In particular, the spacer 22 has a rigidity such as to keep the first and second crosspieces 13, 14 substantially parallel to each other even when the support device D is disassembled from the culture unit, while also keeping the respective longitudinal ends of the first and second pillars 15, 16 apart. It should also be noted that the culture unit C can be supplied to a user as a kit comprising the first and second frames 8, 9, the lid 6 releasably mountable on the first frame 8, and the support device D for the test materials described above, while for example the tray with the culture wells may already be available to the user in the test laboratory, e.g. a commercial tray used for this type of application. Preferably, the electromechanical actuator, together with its control unit, can be supplied in the kit when the test on the samples also involves performing a mechanical stimulation. Preferably, the first and second pillars 15, 16 can have a predefined geometry, such as circular, rectangular, square, etc., which is used depending on the characteristics of the test material, and more specifically depending on its stiffness. In fact, an increase in the length of the pillar and/or a decrease in the area of its section lead to a decrease in the bending stiffness, while a decrease in the length of the pillar and/or an increase in the area of its section lead to an increase in the bending stiffness. Based on the profile of the section, the moment of inertia around the axis of the section perpendicular to the translation direction of the pillar varies. For example, a rectangular section defines a preferential bending direction as well as a greater bending stiffness than a circular section. Furthermore, to process the acquired signals, the bioreactor may include an on-board electronic control unit configured to receive input signals from the sensors integrated into the wells of the culture unit and/ or those on board the pillars. For example, tray 4 may be sensorized, in which each well may be equipped with electrochemical sensors to measure the culture conditions (metabolite concentrations in the medium, pH, dissolved oxygen, etc.) and the properties of the sample (impedance). The sensor integrated into the pillars allows real-time monitoring of the force to which the sample is subjected and, at the same time, characterize the sample from a mechanical point of view (stiffness). The various sensors may already be integrated into the corresponding components described above. As shown in a schematic view in Fig.3, the culture unit C together with at least one support device D can be mounted on board a bioreactor B, and the latter can include on board one or more electronic control units connected to each other in data exchange to perform the test on the samples under examination. In particular, a monitoring unit U1 can be provided connected in data exchange to the sensors present in the culture unit, e.g. to the strain gauges on board the support device D or to the electrochemical sensors on board the culture wells, and such monitoring unit is configured to receive as input signals representing respectively a deformation or a culture condition detected of the test material under examination via such sensors. Furthermore, monitoring unit U1 is configured to process, e.g. condition, the input signals in order to be able to provide a user with the results obtained. To achieve this result, monitoring unit U1 is in turn connected in data exchange to a control unit U2 on board the bioreactor, which is configured to receive the processed signals from monitoring unit U1 and in turn process them to provide as an output one or more parameters representative of a detected deformation or culture condition of the test material under examination. For example, control unit U2 may comprise a graphical interface, e.g. a screen (not shown in the figure) for displaying the provided results. Control unit U2 is also configured to receive as an input control signals representative of an electrical and/ or mechanical stimulation to be applied to the samples under examination in culture unit C, e.g. set by a user via the screen. To achieve this result, the bioreactor comprises a stimulation unit U3 connected in data exchange at the input to control unit U2, and at the output to an electromechanical actuator and to electrical stimulation devices, e.g. electrodes. In particular, based on the stimulation control signal to be applied to the samples under test, stimulation unit U3 processes this signal to generate in turn an actuation signal for the electromechanical actuator and/or the electrical stimulation devices based on this input control signal, in particular for the application of mechanical stimulation and the generation of the electric field delivered to the samples via electrodes, and to report the results obtained to a user. For example, through integrated driving circuits and activation circuits, the stimulation unit provides as output the voltages and/or currents necessary for driving the actuator or activating the stimulation electrodes according to the predefined stimulation parameters. Therefore, based on this constructional configuration, it is possible to measure both an active twitch force, also known as ‘active twitch force’ of test materials, e.g. cellularized cardiac constructs, capable of contracting, spontaneously or following electrical stimulation, and a passive resistant force exerted by the test materials (even those not capable of contracting) subjected to mechanical stimulation, thus characterizing the stiffness of the test material. Furthermore, by implementing a specific control algorithm, the control unit provides a “smart” operating mode that provides the possibility of automatically modulating the stimulation parameters, based on the measurements carried out by the monitoring unit, in order to adapt the stimuli provided to the characteristics of the sample and its stage of maturation during the culture process.

According to a preferred embodiment of the present invention, on board the support device D it is possible to provide a mechanism for adjusting the transverse distance between the ends of the first and second pillars, i.e. those on which the sample to be examined is fixed. For example, as shown in Fig. 4, the first crosspiece 13 has a threaded hole 26 made on the protruding portion 23 in the transverse direction and preferably perpendicular to the direction of extension of such crosspiece. The corresponding first pillar 15 is instead shaped so that the longitudinal end 28 opposite to the one on which the test material is fixed, i.e. the one connected to the crosspiece, has a smaller cross -section than the remaining cross-section of the pillar. The hole 26 is placed between the longitudinal end with the smaller section 28 of the first pillar 15 and the second pillar 16, in particular it faces the surface of the first pillar with the larger section, and this feature is exploited to adjust the transverse distance LO between the first and the second pillar. In particular, it is possible to engage a threaded dowel 27 inside the hole 26, and this dowel in use is screwed inside the hole so as to advance axially towards the pillar until it comes into contact with the surface of the first pillar 15. In particular, as shown in Fig. 5, the dowel is mobile between a first position in which it is longitudinally spaced from the pillar, and a second position in which it is brought into contact with this pillar by exerting a force that causes a deformation of the reduced section end 28 and an increase in the transverse distance between the first and second pillar 15, 16. In particular, a further screwing of the dowel 27 generates a force on the surface of the first pillar causing an elastic deformation of the first pillar and a consequent separation of the end of the first pillar 15 from that of the second pillar 16 by a distance equal to L, this deformation being also favored thanks to the reduced flexural rigidity of the first pillar due to the reduced section longitudinal end 28. Alternatively, such a constructional measure may be provided on the second pillar 16 or on both. It should also be noted that the support device D may be supplied to a user separately from the other components forming the culture unit C, for example, sold to be used in combination with a tray 4 and a related lid already present, for example, in the user's laboratory.

Claims

1. Support device (D) for a test sample mountable in a culture unit (C) of a bioreactor (B) comprising:

- A first and a second crossbar (13, 14) arranged parallel and facing in relation to a median plane, and respectively comprising at least a first and a second pillar (15, 16) facing and extending transversely with respect to the direction of extension of the crossbars, such pillars having longitudinal ends in use in contact with the test sample;

- A spacer (22) arranged between the first and second crossbars (13, 14) to keep said crossbars transversally spaced apart, and the spacer also being extensible in a direction transverse to said direction of extension of the crossbars to cause a deformation of the test sample attached to the first and second pillar; And one of the first and the second crossbar (13, 14) being releasably engageable on a first frame (9) of the movable culture unit transversally to the said direction of extension of the crossbars while the other between the first and the second crossbar being releasably engageable to a second fixed frame (8) of the culture unit, so that a movement of the first frame defines a change in the relative position between the first and second crossbars causing, via the pillars, a deformation of the test sample detectable by a sensor; and the spacer (22) having such a rigidity as to keep the first and second crossbars substantially parallel to each other when the support device (D) is dismantled from the culture unit.

2. Device (D) according to claim 1, wherein the sensor is located on board at least one of the first and second pillars (15, 16).

3. Device (D) according to claim 2, wherein the sensor is a strain gauge integrated on board one of the first and second pillars (15, 16).

4. Device (D) according to any one of the previous claims, wherein the spacer (22) comprises a spring.

5. Device (D) according to claim 4, wherein the spacer comprises an 'S' shaped leaf spring.

6. Device (D) according to any one of the previous claims, wherein the extendable spacer (22) is made integrally together with the first and second crossbars (13, 14).

7. Device (D) according to any of the previous claims, comprising a further spacer (22'), these spacers (22, 22') being arranged symmetrically with respect to a median plane transverse to the direction of extension of the crossbars (13, 14) .

8. Device (D) according to any one of the previous claims, wherein the first and second crossbars (13, 14) respectively have at least one first and second projecting portions (23, 24) transversely to the direction of extension of said crossbars, these first and second projecting portions facing each other and the first and second pillars (15, 16) extending longitudinally respectively from said first and second projecting portions.

9. Device (D) according to any of the previous claims, wherein one of the first and second crossbars (13, 14) has a plurality of coupling seats (19) arranged longitudinally along said crossbar to engage said crossbar to the first frame cabinet (9) of the culture unit.

10. Device (D) according to any of the previous claims, wherein the first and second crossbars (13, 14) respectively comprise a first and a second recess (28, 29) facing each other and each interposed between two respective first and second protruding portions placed side by side, such first and second recesses (28, 29) defining an opening (30) in which in use an electrode extends towards the ends of the pillars on which the test material is fixed.

11. Device (D) according to any of the previous claims, in which one of the first and second pillars (15, 16) has an elastically deformable and reduced-section longitudinal end (28) connected to the corresponding crossbar (13, 14), and this device also presents a threaded hole (26) made in the direction of extension of the pillars and placed between the end with a reduced section (28) and the other between the first and second pillars (15, 16), and a threaded dowel (27) engaged inside this threaded hole and mobile between a first position in which it is longitudinally spaced from the pillar, and a second position in which it is brought into contact with this pillar by exerting a force that causes a deformation of the ends with reduced section (28) and an increase in the transverse distance between the first and second pillars (15, 16).

12. Culture unit (C) releasably mountable in a bioreactor, comprising:

At least one support device (D) for a test sample according to any of claims 1 to 11, a first frame (8) comprising a seat in which a test tray (4) can be housed in use, having a plurality of culture wells (5) for test samples, this first frame further comprising first and second slots (17, 18) made in pairs on opposite perimeter edges, and the first crossbar (13) presenting a first and second opposite longitudinal ends that can be releasably engaged inside the first housings (17) so as to define a coupling of such a shape as to maintain the first crossbar (13) in use stops in a predefined position on board the first frame, a second frame (9) movable for translation with respect to the first frame and arranged for use inside a housing compartment (10) on the first frame (8) so that this second frame is arranged at a higher vertical height than to that in which the culture wells (5) are located, and the second crossbar (14) presenting a third and fourth opposite longitudinal ends that can be releasably engaged in the second slots (18), the latter having a greater longitudinal extension with respect to the transverse extent of the third and fourth longitudinal extremities, a cover (6) which can be releasably mounted on the first frame (8) in a position opposite to said first frame with respect to the second frame (9), and this cover has a plurality of openings (7) defining seats for housing electrodes which in use extend longitudinally towards the tray (4), e the first and second crossbars (13, 14), when engaged respectively on the edge of the first and second frames (8, 9), have the respective first and second pillars (15, 16) extended towards the tray (4) in so as to be at least partly arranged inside the culture wells (5).

13. Cultivation unit (C) according to claim 9, in which the second frame (9) has longitudinal ribs (21) including protrusions (20) on which each coupling seat (19) defined on one of the first and second crossbar (13, 14) is in use rigidly engaged in a releasable manner defining a form fit.

14. Cultivation unit (C) according to claim 9 or 10, wherein the first frame (8) has an opening (11) made on a perimeter wall, and the second frame (9) includes an arm (12) which extends in use out of the first frame through the opening (11), this arm being connectable to a linear actuator so that in use an actuation of this actuator causes a translation of the crossbar on board the second frame (9) and the movement of the third and fourth longitudinal ends inside the respective second housings (18).

15. Cultivation unit (C) according to any one of claims 12 to 14, further comprising an electromechanical actuator connectable in force transmission to the second mobile frame

(9) so that the actuation of said actuator causes a longitudinal movement of said second frame.