FR2945818A1 - DEVICE FOR CULTIVATING IN VITRO BIOFILMS AND NON-INVASIVE OBSERVATION SET FOR BIOFILM DEVELOPMENT ON INERT SURFACE OR ON LIVING SURFACE BY CONFOCAL MICROSCOPY - Google Patents

Abstract

The invention provides a device (10) for culturing in vitro biofilms comprising: - a rigid culture chamber (20) of low thermal expansion material, said enclosure comprising one or more channels (21) within which the the biofilms (26) are intended to develop, said channels (21) opening onto one or more faces of said enclosure, - a support (30) of said enclosure (20), - means for dispensing a nutrient medium for supplying said channels (21), characterized in that it further comprises means for controlling a temperature of said enclosure (20). An observation set of a development of biofilms comprising a confocal microscope and a device (10) for culturing in vitro biofilms according to the invention is also proposed.

Description

In natural environments, populations of microorganisms develop, associated with inert or living surfaces, where they form fixed and heterogeneous communities frequently included in an exo-polymeric protective gangue. These complex bacterial structures are called biofilms. The invention relates generally to the non-invasive observation of biofilms. More particularly, the invention relates to devices for non-invasive observation by confocal microscopy of biofilms.

The specific properties of biofilms (morphology, growth, etc.) depend on the living conditions (inert or living surface, availability of nutrients, oxygen, pH, etc.). These microbial community forms are naturally very common in industrial and medical environments where they are often indispensable. However, some biofilms can also be harmful (industrial corrosion, infections, etc.). A better fundamental knowledge of bacterial biofilms is therefore an economic issue and an issue at both the industrial and the public health levels. The new lines of research that are emerging today require deeper insights into the understanding of biofilm development, particularly thanks to non-invasive confocal microscopy. Confocal laser microscopy is now widely used for non-invasive and non-destructive observation of biofilms. It allows you to record images of the biofilm structure, and to follow their growth in real time. The method consists of making successive thin optical sections of the biofilm, generally spaced from each other by about 1 micrometer, the size of a bacterium. A confocal microscope produces a laser light source intended to be directed on a very thin layer of the biofilm (for example 0.4 micrometer). The wavelength of the laser directly excites a fluorophore contained in the bacteria. The confocal microscope comprises a detector capable of recording the emission of photons from the excited fluorophore around a determined spatial position, and of transforming this light signal into a photographic image of the biofilm layer. By successive offsets of the laser (for example 0.6 micrometer), it is possible to acquire a set of images (for example 100 images).

From this set, a 3D reconstruction software allows to virtually reconstruct the biofilm by volumetric projection over a certain thickness (for example 100 micrometers). The method thus makes it possible, by acquiring images with a determined spatial coordinate, to obtain a 3D reconstruction of a part of the biofilm. Other software can deduce from these images quantitative information on the biofilm (biomass, thickness, morphology, surface coverage rate, etc.). By repeating this method over time, it is possible to follow in real time the evolution of biofilms according to determined three-dimensional spatial coordinates. This technology makes it possible to study, on the one hand, the interactions of biofilms with inert or living surfaces that surround them, and on the other hand the structure of the microorganisms constituting the biofilm as a function of their environment. To enable the in vitro development of biofilms, it is necessary to create an enabling environment. To do this, a biofilm observation assembly generally comprises a molded plastic culture chamber consisting of independent channels, sealed and sterile. The channels are thus able to collect biofilms. A nutrient distribution circuit provides an environment conducive to biofilm development without outside intervention. For observation, the channels are hermetically covered with a single glass slide and the enclosure is attached to a confocal microscope. Optionally, the channels may contain living cells (e.g., eukaryotic cells) to observe the development of living surface biofilm. FIG. 1 illustrates a culture chamber 1 according to the state of the art. This culture chamber comprises three channels 2a, 2b and 2c covered by a glass slide 3. FIG. 2 illustrates a diagram of a set of non-invasive observation of the development of biofilms according to the state of the art comprising a culture chamber 1, a distribution system in nutrient medium and a confocal microscope 7. The dispensing system is composed of a reserve in nutrient medium 4, a peristaltic pump 5 ensuring the circulation of the medium in the enclosure of culture 1 through the channels 2a, 2b, 2c and an evacuation 6 of the spent medium.

Many criticisms have been issued against such devices that are not entirely satisfactory, and in particular: - The temperature of the culture chamber is imposed by the confocal microscope laser. Thus, for a desired experiment at 37 ° C, it is necessary to frequently move the culture chamber in a chamber at 37 ° C, and it is not possible, after moving in a chamber to the desired temperature of the experience, to place the biofilm precisely at the same observation location, which leads to a bias on the spatial coordinates of image acquisition. In addition, these movements disrupt the development of the biofilm. - The molded plastic culture chambers according to the state of the art are deformed during the experiment, which also induces a bias on the spatial coordinates of image acquisition, in particular along an axis veritcal. Long and heavy manual intervention is required at each image capture, and the system does not allow automated high-speed imaging. - The experiments experience failures due to the presence of bubbles in the nutrient distribution system that tear the biofilm.

An object of the invention is to provide an improved observation set of biofilm development on an inert or living surface, in particular making it possible to overcome at least one of these disadvantages. To this end, the invention proposes a device for culturing in vitro biofilms comprising: a culture chamber made of rigid material chosen for its low thermal expansion properties, said enclosure comprising one or more channels within which the one or more biofilms are intended to develop, the said channel or channels opening on one or more faces of the said enclosure, - a support of the said enclosure, - means for dispensing a nutrient medium for supplying the at least one channel, control means a temperature of said enclosure. According to a possible embodiment of the invention, the support comprises a circulation duct, and the control means are intended to cause a circulation of hot water inside said duct. Advantageously but optionally, the device comprises at least one of the following characteristics: the culture chamber is made of pyrex, the support comprises means for locking the enclosure on the support, the support comprises at least two opposite openings; , an opening being able to allow the supply of the channel (s) of the enclosure by the distribution means in a nutrient medium, another opening being able to allow access to the enclosure of a laser of a confocal microscope the culture chamber has a general rectangular parallelepiped shape, the channel or channels extending in the direction of the length and / or the width of this rectangular parallelepiped, the support is at least partially made of metal the support is at least partially made of steel or aluminum; the distribution system comprises one or more vent filters placed upstream of the channel or channels.

The invention also relates to a set of observation of a development of biofilms comprising a confocal microscope, and a device for culturing in vitro biofilms according to the invention. Advantageously but optionally, the observation assembly comprises at least one of the following characteristics: the assembly comprises a motorized platform which makes it possible to control the relative displacement of the support and the confocal microscope, the set comprises means acquisition of the data from the confocal microscope and data processing means from the confocal microscope so as to build 3D images of the biofilms, the set comprises means for recording the 3D images, the set comprises an interface for enabling a user to enter one or more parameters to the behavior of the confocal microscope, at least one of the parameters is representative of a spatial coordinate of image acquisitions by the confocal microscope, at least one parameters is representative of a duration of imaging by the confocal microscope.

The invention also relates to a computer program product comprising code instructions recorded on a medium usable in a computer, characterized in that it comprises instructions for the implementation of an automatic acquisition of images and a three-dimensional reconstruction within the scope of the invention. The invention has many advantages.

The rigid culture chamber according to the invention makes it possible to cultivate a biofilm or several biofilms in parallel without deforming during the experiment. In addition, it is sufficient to heat the circulating water to the desired temperature to set or control the temperature throughout an experiment without having to move the culture chamber. The biofilm is not disturbed by shocks. In particular, it is possible to cultivate biofilms at 37 ° C so as to create development conditions similar to those of the human body. The growing chamber according to the invention also has a low thermal expansion, which further reduces the bias on the spatial coordinates of image acquisition.

The invention thus makes it possible to acquire images in fixed spatial coordinates and to follow the evolution of the biofilms at points determined at the beginning of an experiment, and which do not vary. The observation assembly according to the invention makes it possible to observe the development of biofilms on a maintained or living inert surface, which are pre-implanted human or animal cells such as bacterial biofilms. The observation assembly according to the invention makes it possible to obtain three-dimensional reconstructions of one or more biofilms automatically, without the need for manual intervention after the initiation of the experiment.

Other features, objects and advantages of the present invention will appear on reading the detailed description which follows, with reference to the appended drawings, given by way of non-limiting examples and in which: FIG. Biofilm culture according to the state of the art; FIG. 2 is a simplified representation of an observation set of the biofilm development according to the state of the art; FIG. 3 illustrates a device for In vitro culture of biofilms according to a possible embodiment of the invention, Figure 4 shows a top view of a portion of the device shown in Figure 3, Figure 5 illustrates biofilm culture enclosures in a possible embodiment. FIG. 6 illustrates a culture chamber support according to one possible embodiment of the invention, and FIG. 7 illustrates a set of observations of a development of biofilms according to one embodiment. possible of the invention.

With reference to FIG. 3, a device 10 for culturing in vitro biofilms according to one possible embodiment of the invention comprises a rigid culture chamber 20 comprising one or more channels 21, a support 30 and a distribution system in a nutrient medium. The dispensing system comprises a reserve in nutrient medium 4, a peristaltic pump 5, a used medium collection tank 8 and connecting tubes 11, 12, 13. The peristaltic pump 5 ensures the circulation of the nutrient medium. The support has an inlet 311 and an outlet 312 connected to a thermoregulation circuit 40 to allow control of the temperature of the culture chamber by circulating water inside the support.

Thus, the temperature of the chamber 20 is kept constant during an experiment and it does not need to move regularly in a dedicated room. The support will be described below in detail with reference to FIG. 6. Advantageously, the thermoregulation system 40 comprises tubes 41 and 42, a circulation pump 43 and a heated water bath 44. Advantageously, the dispensing system in nutrient medium comprises a vent filter 9 associated with each channel and placed upstream of this channel in the direction of circulation of the nutrient medium, so as to eliminate air and prevent bubbles from entering the channels and tear the biofilm. Preferably, but not exclusively, the vent filter (s) 9 are arranged on the support 30 so as to eliminate the bubbles closest to the channel (s) (21). Advantageously, the evacuation of the spent medium can be done in various other ways. Advantageously, the connecting tubes 11, 12, 13 are of flexible material. FIG. 4 shows a view from above of the support of the device 10 illustrated in FIG. 3. Advantageously, the culture chamber 20 comprises a given number (for example 6) of distinct channels 21. Thus, a given number of biofilms can be grown independently under the same experimental conditions.

FIGS. 5a to 5d show culture enclosures 20 according to possible embodiments of the invention. Figures 5a and 5d illustrate a culture chamber 20 having 6 channels 21 according to a possible embodiment of the invention. A face 22 and a face 23 of the culture enclosure 20 have been distinguished. FIG. 5a illustrates the culture enclosure 20 outside the scope of use. Advantageously, the culture chamber 20 comprises two plugs 25 associated with each channel 21 so as to plug the openings and to keep the culture chamber 20 sterile before use. Preferably, the channels 21 are parallel. Figure 5d illustrates a side sectional view of the culture chamber 20 as part of a possible use according to the invention. The culture chamber 20 is disposed on the support 30. A biofilm 26 is disposed inside a channel 21. Advantageously, the face 23 is directed upwards, so as to be able to feed the channel 21 in a nutrient medium vertically and from above through the opening 22. FIG. 5b shows, according to one possible embodiment of the invention, a culture chamber comprising three channels 21. Preferably, the channels 21 are parallel. FIG. 5c shows, according to possible embodiments of the invention, single-channel culture enclosures 20 of various shapes: T-shaped channel 211, cross-shaped channel 212, closed-loop channel 213. Optionally, the closed circuit channel 213 has a substantially rectangular shape. Preferably, but not exclusively, the channels 21 are, with reference to FIGS. 5a, 5b and 5c, the channels 21 extend in the direction of the length and / or the width of the rectangular parallelepiped. The culture chamber according to the invention is made of a rigid material, so as not to be deformed during an experiment to allow acquisitions of images according to the fixed spatial coordinates over time. Advantageously, the culture chamber is of low thermal expansion material. In the context of the invention, the term "low thermal expansion material" is understood to mean a material whose coefficient of thermal expansion between 20 ° C. and 300 ° C. is less than 50 ° 10 -7 / ° C. Advantageously, the culture chamber is made of material with high thermal inertia.

In the context of the invention, the term "high thermal inertia material" is understood to mean a material having the following properties: a density of between 2000 and 2500 kg / m 3; a mass heat capacity of 25 ° C. above 740 J.kg. K-1 - a thermal conductivity between 20 ° C and 150 ° C between 0.0110 and 0.0130 ° C - 1 ° C - 1 ° C. Preferably, but not exclusively, the culture chamber is Pyrex, a material which has good properties in the context of the invention: - low thermal expansion, - high thermal inertia, - good hydrolytic resistance, - good resistance to compression (3600 bars at 20 ° C.) and pulling (142 bars at 20 ° C.), good transparency, resistance to heat without deformation up to 300 ° C. at atmospheric pressure.

The culture chamber 20 and the channels 21 are then easily reproducible, for example by rolling or extrusion. The heat resistance makes it possible to recycle and autoclave the culture chamber between two experiments. Preferably, but not exclusively, the culture chamber has a height between 1 and 12 millimeters. Figure 6 illustrates a growing chamber support 30 according to a possible embodiment of the invention in exploded form. The support 30 comprises a conduit 31, an inlet 311 and an outlet 312. Thus, the support allows a circulation of water in the conduit 31. The water is for example heated in a balloon so as to maintain the culture chamber at a desired temperature of the experiment, thus allowing control of the temperature. Advantageously, the support comprises a locking system of the culture chamber 20 comprising here, preferentially but not exclusively, jaws 351, 352 and a retaining ring 33. Thus, the biofilm is not disturbed by parasitic movements during its development. Preferably, but not exclusively, the jaws 351, 352 are screw jaws.

Advantageously, the retaining ring 33 is fixed to the support 30 by screws 331. Thus, the clearance between the holding ring 33 and the support 30 is adjustable and adaptable to several culture enclosures 20. Advantageously, the support 30 provides at least two opposite openings 342 and 343. Thus one can feed the channels 21 of the culture chamber 20 in nutrient medium by the top of the support 30 through the opening 342. On the other hand, the opening 342 can observe the development of biofilms in the channels 21 from below the support 30. In particular, it makes it possible to target the biofilms with a laser of a confocal microscope.

Advantageously, the support 30 is made of heavy material so as to ensure good stability of the culture chamber during the experiment. Preferably, but not exclusively, the support 30 is partially made of metal such as steel and / or aluminum. The support 30 of heavy material combined with the locking system ensures the immobility of the culture chamber 20 during an experiment. Thus, it is possible to acquire images with fixed spatial coordinates on the biofilm, and to follow the evolution of the biofilm at certain points. Preferably, the support 30 comprises an interlocking device to be adapted to any existing confocal microscope.

With reference to FIG. 7, an observation set 50 of a biofilm development according to the invention comprises a device for culturing in vitro biofilms 10 according to the invention and a confocal microscope 7. Advantageously, the set of observation 50 comprises a motorized platform 51 connected to the confocal microscope 7 and the support 30 of the device 10 is fixed on the motorized platform 51. The motorized platform makes it possible to move the support 30 with respect to the confocal microscope 7, and thus to move relative to the confocal microscope 7 or the biofilms 26 contained in the channel (s) 21 of the culture chamber 20 disposed on the support 30. Advantageously, the observation assembly 50 comprises means 52 for acquiring the images of the confocal microscope 7 connected directly or indirectly to the confocal microscope 7. Advantageously, the observation assembly 50 comprises recording means 54 connected directly or indirectly to the [0008] Advantageously, the observation set 50 includes an interface 55 which allows a user to define parameters of the experiment. This interface is connected directly or indirectly to the confocal microscope. Preferably, but not exclusively, the interface 55 comprises a display device, for example a screen, and an interaction device such as a keyboard or a mouse. Preferably, but not exclusively, the user can define spatial coordinates of the image acquisition, a duration of the experiment, the number of layers of biofilms at each image taking, the thickness of these layers or the interval between two layers. Advantageously, at least two of the devices 53, 54 and the interface 55 are grouped in a computer. Advantageously, the taking of images, the 3D construction and the recording are automated. Thus, no user intervention in taking pictures is necessary after the beginning of the experiment. Three-dimensional image acquisition and reconstruction are performed automatically, according to user-determined spatial coordinates prior to the initiation of the experiment. According to the invention, automatic three-dimensional image acquisition and reconstruction is performed by a computer program product comprising code instructions recorded on a computer usable medium. The computer program product includes instructions for performing sorting of three-dimensional reconstructions in computer files based on a reference of the experiment. The reference includes, for example, a date and an experience number. Preferably, the computer program product comprises instructions for implementing a repositioning of the microscope for successive images taken according to fixed spatial coordinates, for one or more biofilms in parallel.

The computer program product according to the invention was the subject of a deposit with bailiff dated March 30, 2009.

Claims (6)

REVENDICATIONS1. Device (10) for culturing in vitro biofilms, characterized in that it comprises: - a culture chamber (20) made of rigid material with low thermal expansion, said chamber comprising one or more channels (21) inside which the or the biofilms (26) are intended to develop, said channels (21) opening on one or more faces of said enclosure, - a support (30) of said enclosure (20), - means for dispensing a nutrient medium for supplying said channels (21), and - means for controlling a temperature of said enclosure (20). 2. Device according to the preceding claim, wherein the culture chamber (20) is Pyrex Cl 3. Device according to the preceding claim, wherein said support (30) comprises a circulation duct (31), said control means being adapted to provide controlled temperature circulation of water within said duct. 4. Device according to any one of the preceding claims, wherein the support (30) comprises locking means of the enclosure (20) on said support (30). 25 5. Device according to any one of the preceding claims, wherein the support (30) comprises at least two openings (342, 343) opposite, an opening (342) for allowing the supply or channels (21) of the enclosure (20) by the distribution means in nutrient medium, another opening (343) to allow access to the enclosure of a laser confocal microscope. 30 6. Device according to any one of the preceding claims, wherein the culture chamber (20) has a generally rectangular parallelepiped shape, the channel or channels (21) extending in the direction of the length and / or the width of this rectangular parallelepiped. 35. Device according to any one of the preceding claims, wherein the support (30) is at least partially made of metal. 8. Device according to claim 7, wherein the support (30) is at least partially made of steel or aluminum. 9. Device according to any one of the preceding claims, wherein the dispensing system comprises one or more vent filters (9) placed upstream of the channel or channels (21). 10. Support (30) for culture chamber as defined in any one of the preceding claims. 11. Observation assembly (50) of a biofilm development comprising a confocal microscope (7), characterized in that it comprises a device (10) according to any one of claims 1 to 9. 12. Together observation device according to the preceding claim, comprising a motorized platform (51) which makes it possible to control the relative displacement of the support (30) and the confocal microscope (7). 13. Observation assembly according to one of claims 11 and 12, comprising acquisition means (52) data from the confocal microscope (7) and processing means (53) data from the confocal microscope (7). ) so as to build 3D images of the biofilms. 14. Observation assembly according to the preceding claim, comprising means for recording (54) 3D images. An observation assembly according to one of claims 11 to 14, comprising an interface (55) for allowing a user to enter one or more parameters relating to the behavior of the confocal microscope (7). 16. An observation assembly according to the preceding claim, wherein at least one of the parameters is representative of an image acquisition spatial coordinate by the confocal microscope (7). An observation assembly according to one of claims 15 and 16, wherein least one of the parameters is representative of an imaging time by the confocal microscope (7).