WO2019057686A1

WO2019057686A1 - Device for compressing a biological sample, system and bench for measuring the rigidity of a sample comprising such a device

Info

Publication number: WO2019057686A1
Application number: PCT/EP2018/075146
Authority: WO
Inventors: Ludivine GUILLAUME; Olivier GUENAT; Valérie LOBJOIS; Bernard Ducommun
Original assignee: Universite Toulouse Iii - Paul Sabatier; Université de Berne; Centre Hospitalier Universitaire De Toulouse; Centre National De La Recherche Scientifique
Priority date: 2017-09-21
Filing date: 2018-09-18
Publication date: 2019-03-28
Also published as: FR3071317B1; FR3071317A1

Abstract

Device (10) for compressing a sample, in particular a biological sample, which has a micrometric or millimetric size, characterised in that it comprises: a plate (1) made of a rigid, biocompatible and transparent material to which a sample plate (2) is rigidly connected, defining a receiving space (2a) for said sample; a compressive plate (3) receiving a compression pad (3a) centred with respect to the sample-receiving space (2a) and an elastic membrane (3b) rigidly connected to said compressive plate (3) and said compression pad (3a) and responsible for moving the compression pad (3a) vertically under the effect of a pressure; an intermediate plate (4) rigidly connected to the compressive plate (3) and defining a pressure chamber (4a); and a plate (5) provided with an opening (5a) for receiving means for applying pressure to said elastic membrane (3b).

Description

Device for compressing a biological sample, system and bench for measuring the rigidity of a sample comprising such a device

Field of the invention

The invention relates to the field of designing and manufacturing devices for measuring the mechanical properties of material samples such as biological tissue samples.

More specifically, the invention relates to the measurement of the rigidity of such samples of micrometric or millimeter size.

The invention finds application particularly in the medical and pharmaceutical field.

Many pathologies result in strong variations in the rigidity of the biological tissues concerned. For example, tumor tissue is often much harder than healthy tissue. A factor of 100 or more can thus be observed. The detection of these variations may constitute an aid in the diagnosis of these variations or in the evaluation of the effectiveness of the protocols of their treatment or in the evaluation of the effectiveness of pharmaceutical molecules for this purpose.

The invention may for example be used to measure the rigidity of very small samples of skin, blood vessels, heart valves, ligaments, sclera, etc. Such samples may be taken by biopsy, needle, etc.

The invention also finds application in the field of tissue engineering. In this field, it is important that the rigidity of a substitute material, for example a material intended to achieve a meniscus, best approaches that of the tissue to be replaced.

Prior art

Two types of devices are known in the prior art that make it possible to measure the mechanical properties of small samples, especially of biological tissues.

Extensometers are used to measure the tensile strength and elongation of such samples. The indentors make it possible for them to compress the tissue sample and to deduce the rigidity of the tested fabric. It is to this second category that the present invention relates.

The indentors have the disadvantage of not allowing to analyze samples with large differences in stiffness with the same equipment, without making any changes. Thus, the equipment marketed by the company Celiscale under the naming Microsquisher ^® allows to apply on the sample tested a pressure thanks to a lever arm. The lever arm can be changed to apply more and more pressure. A disadvantage of this device is that to offer a wide range of measurement, up to seven different lever arms can be used. The manipulations to make the changes of lever are delicate. In addition, they can alter the quality of measurements. For example, to apply a pressure of 40 kPa on a sample of healthy biological tissue at 180 kPa on a sample of diseased tissue, three leverage arms are required.

In practice, these devices are difficult to use for testing biological samples whose hardness can vary over wide ranges.

Objectives of the invention

An object of the invention is to propose a device for the compression of samples, in particular biological samples, which can be integrated in a system for measuring the rigidity of these, less expensive and less complicated to implement than the prior art.

An object of the invention is also to provide means for measuring the hardness of various samples that can have very variable hardness, without the need for delicate manipulations such as changes of lever arm.

Another object of the present invention is to provide such a device and such a system particularly suitable for small biological samples.

Yet another object of the present invention is to describe a measuring bench that can accommodate such a device for the compression of samples.

Presentation of the invention

All or part of these objectives are achieved thanks to the invention which relates to a device for compressing a sample, in particular a biological sample, of micrometric or millimetric dimensions, characterized in that it comprises

a plate made of a rigid, biocompatible and transparent material (such as for example glass, polymethyl methacrylate, polystyrene or a cycloolefin copolymer) to which is secured a sample-carrying plate delimiting a space for receiving said sample, a compression plate accommodating a compression pad centered with respect to said receiving space of said sample and an integral elastic membrane of said compressive plate and said compression pad adapted to organize the mobility of said compression pad in a vertical movement under the effect of a pressure, an intermediate plate secured to said compressive plate and delimiting a pressure chamber, and a plate provided an opening for accommodating means for applying a pressure on said elastic membrane.

Preferably, the device comprises said plate of rigid material, biocompatible and transparent, on the upper part of which is secured said sample holder plate delimiting said reception space of said sample, said compression plate placed on an upper face of said sample plate and accommodating said compression pad centered with respect to said receiving space of said sample and said elastic membrane integral with said compressive plate and said compression pad adapted to organize the mobility of said compression pad in a vertical movement under the effect of a pressure said intermediate plate secured to an upper portion of said compressive plate and delimiting a pressure chamber, and said plate provided with an opening for accommodating means for applying a pressure on said elastic membrane, attached to a face upper of said intermediate plate diary

The device thus comprises, on the one hand, a first assembly constituted by the plate of rigid, biocompatible and transparent material, and the sample-carrying plate secured thereto, and, on the other hand, a second assembly consisting of the compressive plate. secured to the intermediate plate, itself secured to the plate of rigid material, biocompatible and transparent. The joining of the plates may be made by gluing after a plasma treatment of the surfaces.

During its use, the first set may be placed on a support, for example the plate of a test bench, so that the glass plate is in contact with this support. The sample to be compressed can then be installed in the reception space of said sample plate. Once the sample is positioned, the second set can be placed on the first set so that the compression pad comes into contact with said sample by focusing on said receiving space.

The sample reception area makes it possible to receive samples of micrometric or millimeter size, in practice generally ranging from a few hundred microns to a few millimeters. These samples may in particular be cylindrical and for example from a sample taken by a needle or a trocar. Because of their very small size, their hardness can be considered homogeneous.

Preferably said compression pad, said elastic membrane and said compressive plate are integrated into a single element. Such a characteristic makes it possible to facilitate the centering of the compression pad on the reception space of the sample. Such an element can be easily manufactured by molding.

According to a preferred variant of the invention, said elastic membrane has a thickness of between 5 μιη and 200 μιη. Such small thicknesses make it possible to optimize the flexibility of the membrane, that is to say to obtain a deformation thereof in the pressure range that will be applied to the sample by means of a measuring system such as described below.

According to a variant of the invention, said sample plate, compressive plate, intermediate plate and plate made of rigid, biocompatible and transparent material each independently have a thickness of between 1 mm and a few millimeters. It will be noted that the thickness of the compressive plate may be dependent on that of the elastic membrane.

Preferably, said sample plate, said compressive plate, said compression pad, said elastic membrane and said intermediate plate are all made of polydimethylsiloxane. Such a material has the advantage of being inexpensive. It can be easily molded, it is stable in time and biocompatible. It can be sealed to glass or another layer of PDMS with a simple plasma treatment. It is therefore entirely suitable for producing the multi-layer device according to the present invention. It is also transparent at optical frequencies (240 nm to 1100 nm) and has low auto-fluorescence. As described below, it therefore allows observation by optical means of the deformation of the sample.

For the plate made of rigid, biocompatible and transparent material, this material could for example be glass, polymethyl methacrylate, polystyrene or a cycloolefin copolymer. However, preferably, polystyrene will be used. Such a material is also inexpensive and allows easy handling of the second set of said device. In addition, it allows the easy attachment of a connector, in this case that of a pressure sensor of a measuring system as described below. According to a particularly interesting variant, said device is for single use. According to such a variant, the device can be discarded after use, thus limiting in particular the risk of contamination of the device by the samples.

According to another advantageous variant, said compressive plate, said intermediate plate and said plate having an opening form a monobloc element.

The invention also relates to a system for measuring the rigidity of a biological sample comprising a device for compressing a sample as described above, and further comprising means for applying a pressure on said elastic membrane of said device a pressure sensor measuring the pressure exerted on said elastic membrane, and means for calculating the value of a parameter representative of said rigidity, such as the elastic modulus, of said sample as a function of its deformation and the result of the measurement performed by said pressure sensor.

Such a system makes it possible to apply pressure to a sample positioned in the reception space of said sample plate.

Preferably, said means for applying a pressure on said membrane include a microfluidic pump. Such equipment is quite suitable for easily applying a variable pressure on the sample.

Although the compression device as well as the rigidity measuring system of the invention can be adapted to any measuring bench, the invention also relates to a rig for measuring the rigidity of a biological sample comprising a mounted tray movable in three dimensions designed to accommodate at least one device for compression as described above, a temperature-controlled enclosure within which is provided said movable mounted plate and optical means for viewing the deformation a sample placed in said device.

Lists of figures

The present invention will be better understood with reference to the description of two nonlimiting embodiments which follow a device according to the invention, a system and a measuring bench according to the invention, given with reference to drawings in which:

- Figures 1, 3, 5, 7 and 9 show in perspective the various components of a first embodiment of the compression device according to the present invention;

Figures 2, 4, 6, 8 and 10 show sectional views AA of Figures 1, 3, 5, 7 and 9 respectively; - Figure 11 shows an exploded perspective view of said device before association of its various component parts;

- Figure 12 shows a sectional view of the device after association of its various constituent parts and hosting a sample to be tested before applying a pressure;

- Figure 13 shows a sectional view of the same device receiving the sample when applying a pressure;

FIG. 14 schematically represents a measurement bench including a system for measuring the rigidity of a sample accommodating a compression device according to FIGS. 1 to 13;

- Figure 15 shows a side view of a compression device according to the invention positioned on the plate of the measuring bench shown in Figure 14;

FIG. 16 represents a sectional view of a second embodiment of a device according to the invention;

FIG. 17 represents a perspective view of this second embodiment.

Description of a first embodiment

With reference to FIGS. 1 to 10, the five plates constituting a nonlimiting example embodiment of a compression device according to the present invention are shown separately, each in perspective and in cross-section AA. These five plates are all inscribed in a 1.5 cm square.

The glass plate 1 shown in Figures 1 and 2 is transparent glass, flat and has a thickness of 1mm.

The compressive, intermediate, sample-carrying plates shown in Figures 3 to 10 are all PDMS and were obtained by molding. In the context of the present embodiment, a PDMS obtained by mixing a commercial base and a commercial crosslinking agent in a ratio of 10 to 1 has been used. It may be envisaged in other embodiments to use other materials for making these plates. The upper plate is made of transparent polystyrene about 1mm thick. Other materials may also be contemplated in other embodiments.

The sample plate 2 shown in Figures 3 and 4 has a thickness of 1mm to a few millimeters. It is provided at its center a circular reception area 2a a sample that may also contain a liquid medium necessary to maintain the integrity of the sample. This circular space has a diameter of 1 to 10mm.

The compressive plate 3 shown in FIGS. 5 and 6 has a thickness of 1.1 mm and has at its center a compression pad 3a connected to the remainder of the plate by an elastic membrane 3b of 50 μιη of thickness defining an annular space 3c . This compression pad 3a has a diameter, at its face in contact with the sample of 2mm. This stud has the same thickness as the rest of the compression plate 3. The annular space 3c has a width of 0.9mm. The size of the circular cross section of the pad 3a is smaller than that of the circular receiving space 2a of the sample plate 2 which measures 2.5mm so as not to generate a frictional force. This compressive plate 3 incorporating the pad 3a and the membrane 3b consists of a single piece.

The intermediate plate 4 shown in Figures 7 and 8 has a thickness of 1.1 mm and has in its center a circular compression chamber 4a passing therethrough. This compression chamber 4a has a diameter of 4mm.

Finally, the upper plate 5 shown in Figures 9 and 10 has in its center a circular opening 5a for receiving means for applying a pressure on said elastic membrane 3b. This opening has a diameter of 1.50 mm.

With reference to FIG. 11, the compression device 10 according to the present invention is obtained by combining:

a first assembly 10a consisting of the glass plate 1 secured by gluing to the sample plate 2; and,

a second assembly 10b constituted by the compressive plate 3 secured to the intermediate plate 4, itself secured to the upper plate 5.

In the present embodiment, the bonding between the plates 1 and 2 on the one hand and 3, 4 and 5 on the other hand was obtained thanks to a known technique of bonding after a plasma treatment of the surfaces

The compression device 10 can be installed on a measuring bench 11 of the type described with reference to FIGS. 1 to 13 and comprising a plate 12 mounted to move in three dimensions. This measuring bench 11 also comprises means 7 for applying a pressure on the elastic membrane 3a of the device 10. In the present embodiment, these means 7 include a micro-fluidic pump for conveying a fluid under pressure in the pressure chamber 4a of the intermediate plate 4 of a compression device 10 as described above positioned on a support 12a of the plate 12 by means of vertical guides 12b as indicated in FIG. measuring bench also has a pressure sensor 8 connected to the upper plate 5 of the device 10 and calculation means 9. Finally, the measuring bench 11 is equipped with optical means comprising a light source 14 and a camera 15 arranged according to an axis parallel to the plate 12 passing through the center of the device 10.

With reference to FIGS. 12, 13 and 15, the measuring bench 11 can be used as follows.

The assembly 10a of the compression device 10 is positioned on the plate 12 and a sample 6 is placed in the receiving space 2a of its sample plate 2. The assembly 10b of the compression device 10 is placed on the set 10a containing the sample. The sample is thus protected from the risks of deterioration before any measurement. The pressure sensor 8 is connected to the upper plate 5 of the compression device 10 by means of micro-fluidic connectors and tips. The light source 14, the camera 15 and the calculation means 9 are activated.

Once the device 10 has been installed on the bench 11, a pressure (symbolized by the arrow in FIG. 13) is applied to the sample 6 by virtue of the microfluidic pump of the means 7 which conveys a fluid under pressure into the chamber of pressure 4a of the intermediate plate 4. Under the effect of the pressure exerted by this fluid, the pad 3a moves vertically more or less depending on the hardness of the sample 6 as shown in Figure 13 for a given pressure. The more or less strong resistance encountered by the pad 3a results in a higher or lower pressure exerted by the sample on the rest of the assembly 10b. Therefore, for the same pressure range applied, a smaller displacement of the pad 3a will be visualized for a more rigid sample 6. This pressure is measured continuously by the pressure sensor 8 which transmits it to the calculation means 9 of the corresponding elastic module of the sample. The deformation of the sample 6 can be followed continuously by the camera 15 whose images can be transmitted on a monitor 16 of the calculation means 9. For example, this deformation could be followed by fluorescence of the sample.

The device and the measuring bench shown in the figures were tested with different calibrated hardness samples made of hydrogel beads having a diameter ranging from 300μιη to 1mm and containing different amounts of polyethylene glycol (PEG) corresponding to different elastic modules, as shown in Table 1 below.

Table 1

Thus, the invention makes it possible to continuously measure a wide range of hardnesses without the need for delicate adaptations (such as, for example, a change of lever arm) of the device or the measuring bench.

Measurements can be made without preliminary adjustment. The compression device is feasible at low cost and easily reproducible. It allows a perfect centering of the sample without the latter having to be the subject of a delicate cut.

Description of a second embodiment

Referring to Figures 16 and 17 showing a second embodiment, the compression plate 3 'having a pad 3'a and a resilient membrane 3'b, the intermediate plate 4' having a pressure chamber 4'a and the plate 5 'provided with an opening 5'a are bonded by gluing after plasma treatment of the surfaces to form a monoblock element (M) in PDMS.

The plate 5 'has a width of 34.2 mm for a length of 39.9 mm and a height of 10 mm. These dimensions allow improved maneuverability. The opening 5 'has a diameter of 2 mm.

This plate 5 'is also provided with two cylindrical bores 51, 52.

The plates 3 'and 4' both have a width of 15 mm and a length of 15 mm. Their cumulative thickness is 6 mm. The elastic membrane 3'b may have a thickness ranging from 50 μιη to 200 μιη. The pad 3'a may have a diameter ranging from 4.5 to 9.5 mm in contact with the sample. The intermediate plate 4 'has a thickness of 3 mm and has at its center a circular pressure chamber 4'a therethrough. This pressure chamber 4 'has a diameter of 13 mm.

This second embodiment also comprises a PDMS sample plate 2 'whose reception space 2' of a sample is constituted by the part central of its upper surface. The plate 2 'has a width of 34.2 mm for a length of 39.9 mm and a height of 10 mm.

This sample plate 2 'is also provided with two cylindrical bores 21, 22 designed to face the bores 51, 52 when the one-piece element (M) is positioned on the sample plate 2', and having the same diameter than these. Slides (not shown) can be placed in these holes (51,21; 52,22) to guide the monoblock element M on the sample plate 2. Thus, any lateral sliding between the plate 2 'and the element monoblock (M) is prevented. The sample being placed in the center of the sample plate, these guides make it possible to always align the stud with the sample. A thumbwheel or other adjustment mechanism (not shown) may be used to adjust the distance between the plate 2 'and the one-piece member (M).

A plate of rigid material (not shown) is plasma bonded to the lower surface of the sample plate 2 '.

Claims

1. Device (10) for compressing a sample, in particular a biological sample, of micrometric or millimetric dimensions, characterized in that it comprises a plate (1) of rigid, biocompatible and transparent material to which a sample plate is secured. (2) delimiting a receiving space (2a) of said sample, a compressive plate (3) receiving a compression pad (3a) centered with respect to said receiving space (2a) of said sample and an elastic membrane (3b) integral with the said compressive plate (3) and said compression pad (3a) adapted to organize the mobility of said compression pad (3a) in a vertical movement under the effect of a pressure, an intermediate plate (4) secured to said compressive plate (3) and delimiting a pressure chamber (4a), and a plate (5) provided with an opening (5a) for receiving means for applying a pressure on said elastic membrane (3b).

2. Device according to claim 1 characterized in that it comprises:

said plate of rigid material, transparent and biocompatible (1) on the upper part of which is secured

said sample plate (2) delimiting said receiving space (2a) of said sample,

said compressive plate (3) placed on an upper face of said sample-carrying plate (2) and accommodating

said compression pad (3a) centered with respect to said receiving space (2a) of said sample and

said elastic membrane (3b) integral with said compressive plate (3) and said compression pad (3a) adapted to organize the mobility of said compression pad (3a) in a vertical movement under the effect of a pressure,

said intermediate plate (4) secured to an upper portion of said compressive plate (3) and delimiting said pressure chamber (4a), and

said plate (5) provided with an opening (5a) for receiving means for applying a pressure on said elastic membrane (3a) fixed to an upper face of said intermediate plate (4).

3. Device according to claim 1 or 2 characterized in that said compression pad (3a), said elastic membrane (3b) and said compressive plate (3) are integrated in a single element.

4. Device according to claim 1 to 3 characterized in that said elastic membrane (3b) has a thickness between 5 μιη and 200 μιη.

5. Device according to any one of claims 1 to 4 characterized in that said sample plate (2), compressive plate (3) intermediate plate (4) and plate of rigid material, biocompatible and transparent (5) independently present each a thickness between 1 mm and a few mm.

6. Device according to any one of claims 1 to 5 characterized in that said sample plate (2), said compressive plate (3), said compression pad

(3a), said elastic membrane (3b) and said intermediate plate (4) are all made of polydimethylsiloxane.

7. Device according to any one of claims 1 to 6 characterized in that said plate of rigid material, biocompatible and transparent (5) is polystyrene.

8. Device according to any one of claims 1 to 7 characterized in that it is disposable.

9. Device according to any one of claims 1 to 8 characterized in that said compressive plate, said intermediate plate and said plate having an opening form a monobloc element.

10. System for measuring the rigidity of a biological sample comprising a device (10) for compressing said sample according to any one of claims 1 to 9, means (7) for applying a pressure on said elastic membrane (3a). ) of said device, a pressure sensor (8) measuring the pressure exerted on said elastic membrane (3b), means (9) for calculating the value of a parameter representative of said rigidity, such as the Young's modulus, of said sample as a function of its deformation and of the result of the measurement made by said sensor pressure (8).

11. Measuring system according to claim 10 characterized in that said means (7) for applying a pressure on said membrane include a micro-fluidic pump.

Measurement bench (11) for the rigidity of a biological sample comprising a movable mounted plate (12) in three dimensions adapted to accommodate at least one device (10) for compressing said sample according to any of the claims. 1 to 9, a chamber (13) regulated in temperature inside which is provided said movable mounted plate (12) and optical means (14, 15) for visualizing the deformation of a sample placed in said device ( 10).