Patent Application
20220250057
The present invention relates to a microfluidic chip for manipulating objects such as micro- or nanoparticles with a size comprised between 0.1 μm and 300 μm. More particularly, it relates to the levitation of cells by applying acoustic waves. The following refers mainly to cells, but the present invention applies to any particle sensitive at least to acoustic waves.
In a general way, the principle of acoustic levitation and the formation of particle (or cell) aggregates in acoustic levitation is recapped in
More precisely, under the effect of the acoustic radiation force, particles (or cells) in suspension are forced to migrate towards the acoustic pressure nodes. Once in the different acoustic levitation planes, the radial component of the acoustic radiation force forces the particles to collect together and form an aggregate at each level. The frequency of the ultrasonic wave is defined so as to obtain several pressure nodes distributed over the height h of the channel.
In current acoustofluidic applications, the acoustic radiation force is used to separate the species as a function of their physical and mechanical properties, namely their density and their compressibility, as well as their size, since the acoustic force is directly dependent on these parameters, as defined in the following equation:
F
ac=4π·r3·kac·E0·Φ·sin(2kac·z)
Where r is the radius of the particle, Φ is the acoustic contrast factor, kac is the acoustic wavenumber and E0 is the acoustic energy. This defines the sensitivity of the particles (or cells) to the acoustic radiation force. The denser and larger the particle is, the greater the acoustic force that it is subjected to is and the faster the particle migrates towards the pressure node. This force is usually used to separate flowing particles or cells as a function of these characteristics.
Document WO 2019/002551 A1 is known, which describes a method for separating flowing particles by applying acoustic waves in order to levitate them. This document also describes the principle of optical exclusion. The present invention refers to this document WO 2019/002551 A1 for the methods of levitating the particles and for the application of the principle of optical exclusion.
The object of the present invention is a novel use of the principle of acoustic levitation with optical manipulation.
Another object of the invention is a device making an optimized manipulation of aggregates created by acoustic levitation possible.
Yet another object of the invention is a novel method for creating the aggregates.
At least one of the above-named objectives is achieved with a microfluidic chip comprising:
The resonant cavity comprises walls for containing the cells. In other words, the cells can penetrate an area in the resonant cavity where they are no longer affected by the flow in the passage channel.
They can therefore be subjected to the action of the acoustic and optical waves.
They can also remain immobile, relative to the movement of the flow in the passage channel. Immobile means that they are subjected to small movements of the medium, but not to the linear displacement movement of the flow.
The rate of the flow can be adjusted so that it does not involve the cells forming in particular one or more aggregates. The acoustic force is then great enough to oppose the flow.
With the microfluidic chip according to the invention, it is possible to use the acoustic waves and the optical beams simultaneously so as to hold the cells in levitation over one or more layers by acoustic waves while manipulating them by optical beams according to the technique of optical exclusion.
The microfluidic chip according to the invention constitutes an optoacoustic bioreactor in which the cells can be cultured in acoustic levitation and manipulated by means of specific illumination so as to structure the aggregates in successive layers of cells. By structuring is meant a spatial distribution of the layers of cells which are different from one another.
In the resonant cavity, the cells are bathed in a liquid which does not disrupt the acoustic waves or the optical beams used and is suitable for cell culture.
The resonant cavity is advantageously intended for a culture of cells in acoustic levitation. It is clear to a person skilled in the art that any micro- or nanoparticle that is sensitive to acoustic waves and possibly to certain optical beams, endogenously or exogenously, can be used in the invention.
Using both optical and acoustic properties, therefore, it is possible to spatially structure cell aggregates in acoustic levitation. This operation can be repeated several times, by successive injections of different types of cells, which are marked (fluorescent biological markers) or not, and which react to different optical wavelengths.
The acoustic radiation force due to the acoustic waves makes it possible to create particle aggregates which can be held in acoustic levitation for as long as necessary. This force is dimensioned in order to hold this aggregate in acoustic levitation in the resonant cavity, which constitutes an acoustic trap.
According to an advantageous characteristic of the invention, the microfluidic chip can moreover comprise at least one second optical emitter, the two emitters being arranged so as to emit through two opposite walls of the resonant cavity respectively. Preferably, the two optical emitters emit at different wavelengths. Such an arrangement makes it possible to act, by means of the technique of optical exclusion, on different types of cells simultaneously or sequentially. This also makes it possible to act on aggregates symmetrically because there is an attenuation of the intensity of the beams passing through a thickness of fluid and encountering aggregates along its axis of propagation. Owing to this setup, the top aggregate is acted on in the same way as the bottom aggregate.
According to an embodiment of the invention, the acoustic wave generator can comprise an upper element and a lower element sandwiching at least part of the resonant cavity on two opposite walls; the upper element, which is fixed or removable, being an upper transducer or an acoustic wave reflector; and the lower element being a lower transducer, the upper and lower transducers being capable of emitting acoustic waves. Furthermore, the upper element and/or the lower element are transparent to the light beams provided for illuminating the cells.
In other words, the generator comprises a lower transducer combined with a reflector capable of reflecting the acoustic waves from the lower transducer so as to create the acoustic radiation force in the resonant cavity. However, the reflector can be replaced with an upper transducer generating the same acoustic waves as the lower transducer in a synchronous manner so as to also create the acoustic radiation force in the resonant cavity.
Preferably, at least one of the two transducers is transparent to the optical wavelength or else is annular in order to allow the optical illumination to pass into its centre.
The removable nature of the upper element may make it possible to evacuate or recover the cells forming aggregates.
According to an embodiment of the invention, the resonant cavity is closed at its lower end by the lower transducer. The lower transducer thus constitutes the lower wall of the resonant cavity. In such an embodiment, the lower transducer is in direct contact with the culture medium. This configuration makes it possible to improve the energy efficiency of the resonant cavity.
According to an advantageous characteristic of the invention, the lower transducer can be arranged inside the resonant cavity.
A removable lower transducer capable of sliding inside the resonant cavity is thus provided. Preferably, the dimensions of the transducer make it possible to ensure that the resonant cavity is sealed. In this case, the usable volume forming the culture medium is smaller than the total volume of the resonant cavity. It is thus possible to define different volumes of the resonant cavity. It is thus a cavity with a variable volume and therefore a variable number of levitation planes.
Advantageously, the resonant cavity can have at least one stop for blocking the head of the lower transducer once it has been inserted in the resonant cavity.
According to an embodiment of the invention, the resonant cavity can be closed at its lower end by a fixed or removable film which is transparent to the acoustic waves originating from the lower transducer arranged outside the resonant cavity.
This film can be made from polydimethylsiloxane (PDMS) material or of cyclic olefin copolymer (COC) or can be an adhesive PCR (Polymerase Chain Reaction) film which is easily detachable and makes a very good transfer of the ultrasonic waves possible. A removable film makes the passage or recovery of cell aggregates at the end of culturing possible, for example.
Preferably, the resonant cavity is a cylinder the side walls of which are constituted by the block. The upper base can be formed by the reflector or the upper transducer. The lower base can be formed by a film or directly by the lower transducer.
According to an embodiment, the passage channel can lead into the resonant cavity at the upper end of a side wall of the cylinder. Such an arrangement makes it possible to supply cells to the cavity through the upper end of the side wall of the cylinder.
According to the invention, the resonant cavity can have a stop arranged so that the head of the lower transducer can be inserted to reduce the height of the usable volume in the resonant cavity until it is equal to the height of the passage channel.
Ideally, the usable volume makes it possible to produce one or more monolayer or multi-layer cell aggregates.
According to an advantageous embodiment of the invention, the passage channel can be made on the upper surface of the block and a bonded or removable strip can cover all of the surface of the block, including the upper end of the resonant cavity; this strip being transparent to the optical beams provided to illuminate the cells of the resonant cavity from the outside. In addition, when a transducer is arranged opposite it, this strip reflects the acoustic waves from the transducer on the internal side of the resonant cavity.
Preferably, the passage channel and the cylinder can be arranged perpendicular to one another. The block can then moreover comprise two microchannels passing through the block from one side to the other, parallel to the cylinder and connected respectively to the two free ends of the passage channel; the first microchannel being intended for the arrival of cells in the passage channel and the second microchannel being intended for evacuating cells from the passage channel.
By way of non-limitative example, the reflector is a strip made from glass, from polymethyl methacrylate (PMMA), from quartz, from silicon, from polydimethylsiloxane (PDMS) or from cyclic polyolefin copolymer (COC). Such a strip is designed to ensure a good transmission on the one hand and a good reflection on the other. Preferably, the reflector can be designed starting from a material identical to that of the block and having an internal surface treated to reflect acoustic waves.
The chip according to the invention can comprise several microchannels made in the thickness of the block and leading into the resonant cavity for cells to enter and/or exit. Preferably, these microchannels are aligned respectively with the pressure nodes provided in the resonant cavity because of the acoustic radiation force. These microchannels can be used, for example, to inject cells of different natures from those injected through the passage channel, to inject nutrients, and also to evacuate cells.
Advantageously, the height of the resonant cavity can be a function of the number of pressure nodes to be created and the wavelength of the acoustic waves generated by the generator. The resonant cavity is designed to contain a superimposition of several cell layers, while the height of the passage channel is preferably adapted to the size of a cell.
The resonant cavity is taller than the passage channel, for example several times the diameter of a cell, in particular at least 10 times the diameter. It is possible to try to put the first pressure node at the level of the passage channel, therefore a channel height of the order of λ/4.
Preferably, the passage channel can have a rectilinear shape, such that the section of the passage channel between an inlet end and the resonant cavity is colinear with the section of the passage channel between the resonant cavity and the other, outlet, end of the passage channel.
According to an embodiment of the invention, the block can be made from polydimethylsiloxane (PDMS) or from cyclic olefin copolymer (COC).
In particular, the block is designed starting from a gas-permeable material in order to facilitate the gas exchanges between the medium, the contents of the resonant cavity, and the outside if necessary. The channels are etched or moulded into the block and can be installed in an incubator so as to ensure the optimum culture conditions in terms of gas and temperature.
In particular, the resonant cavity can be dimensioned with a height greater than the diameter of the passage channel leading into this resonant cavity. Thus, the passage channel, which is a conduit with a small diameter, typically suitable for conveying the cells only in a line, is clearly distinguished from the resonant cavity, which has a larger dimension capable of containing cell aggregates in one plane and in one volume.
By way of non-limitative example, the resonant cavity can have a diameter between 1 and 50 mm, a height of the resonant cavity comprised between 5 and 15 mm and a height of the passage channel equal to 450 μm.
The microfluidic chip according to the invention can moreover comprise at least one additional microchannel made in the block in the same plane as the passage channel.
This is a simplified arrangement which makes it possible to supply cells to and evacuate cells from the resonant cavity, which cells can form aggregates in the same plane as the cells originating from the passage channel, which is, in fact, a main microchannel.
This additional microchannel can also be used to inject other cells, biomarkers, or else to wash the culture medium or recover the production of the cells during their culturing.
According to another aspect of the invention, a method for manipulating cells in acoustic levitation in a microfluidic chip as defined above is proposed. This method comprises the following steps:
According to the invention, the selective opto-acousto-fluidic exclusion principle is used, which describes the fact that one particle or cell reacts to certain optical wavelengths while other particles or cells do not react at all.
According to an advantageous embodiment of the invention, the injected cells have different natures, and the steps of generating acoustic waves and illuminating can be carried out as follows:
The present invention thus makes it possible to produce radially structured monolayer cell aggregates. The aggregate can thus be composed of successive rings of different cells, and thus make the co-culture of cells possible, steps which are indispensable for the formation of organoids.
By cell aggregate is meant a layer of cells satisfying all of the following characteristics: at least two cells included in said layer, in particular at least 10%, better 25%, preferably 50% of the cells included in said layer, are in contact, and said layer has, over at least part of its length, a succession of cells during the displacement in at least one of its transverse dimensions.
According to an advantageous embodiment of the invention, a three-dimensional structure formed of several layers of aggregates is produced by carrying out the following steps:
The invention makes it possible to create several monolayers (cell sheets) one above another, which brings a considerable time saving for the cell culture since it is no longer necessary to trypsinize a sheet of cells, to detach it from a possible solid substrate in order then to deposit it on another. This process is more particularly suitable for MSC-type (Mesenchymal Stromal Cells) stem cell cultures, where the cells are cultured to manufacture cell sheets which are then superimposed, but it is relevant to any type of cell culture intended to form cell sheets.
With the invention, it is possible to envisage the manufacture, in acoustic levitation, of spheroids, organoids or tumoroids which could be held and cultured in acoustic levitation.
The invention makes it possible to carry out a cell culturing while holding the aggregate or the aggregates obtained immobile in acoustic levitation for the duration of the culturing.
Such a system has thus made it possible to culture MSCs in acoustic levitation for 18 days. At the end of this culturing, the cells were living and could proliferate normally once deposited on a substrate. The system has thus been validated for culturing over short periods (a few hours) and over long periods (a few weeks). This is made possible owing to the flow of nutrients and the porosity of the materials used, which makes the gas exchanges in an incubator possible. The acoustic bioreactor was placed in an incubator in order to make the culturing of cells possible. The compactness of the present system makes it possible to place several chips in an incubator. It is thus possible to carry out the culturing of a number of sheets or spheroids in acoustic levitation in parallel (a few tens per well, and a few wells in the incubator).
Other characteristics and advantages of the invention will become apparent on reading the detailed description of implementations and embodiments which are in no way limitative, in the light of the attached figures, in which:
The embodiments which will be described hereinafter are in no way limitative; variants of the invention can in particular be implemented comprising only a selection of characteristics described hereinafter in isolation from the other characteristics described, if this selection of characteristics is sufficient to confer a technical advantage or to differentiate the invention with respect to the state of the prior art. This selection comprises at least one, preferably functional, characteristic without structural details, or with only a part of the structural details if this part alone is sufficient to confer a technical advantage or to differentiate the invention with respect to the state of the prior art.
In particular, all the variants and all the embodiments described are provided to be combined together in all combinations where there is no objection to this from a technical point of view.
In the figures, elements common to several figures keep the same reference.
Although the invention is not limited thereto, a microfluidic chip will now be described which is suitable for culturing cell aggregates in acoustic levitation.
A set of components of an example of a microfluidic chip according to the invention is represented in
For supply to the resonant cavity 6, a passage channel 7, 8 is etched on the upper surface of the block 5 such that this passage channel and the inside of the cavity are accessible. The passage channel has a first part 7 intended for the entry of the cells from an inlet microchannel 9 towards the resonant cavity. It also has a second part 8 intended for the evacuation of cells from the resonant cavity 6 towards an outlet microchannel 10. The two, inlet and outlet, microchannels are made in the thickness of the block, like the resonant cavity, and lead onto the lower surface of the block 6, the back of this block being more accessible to different devices for the supply to and management of the microfluidic chip according to the invention.
Preferably, this block is designed starting from a biocompatible material capable of ensuring gas exchanges, if necessary, between the resonant cavity and the outside (incubator). It is designed in order that the microfluidic chip according to the invention can ensure a flow of nutrients and a flow of culture medium, if necessary, within the resonant cavity. Of course, it makes it possible to inject the cells and evacuate them and makes it possible to create the aggregates with large dimensions within the resonant cavity.
The microfluidic chip is installed in an incubator so as to ensure the optimum culture conditions (gas and temperature).
A glass strip intended to cover, in particular by plasma bonding, the upper surface of the block 5 can also be seen. It can be a strip produced together with the block 5. This glass strip 11 has, at least on its internal wall facing the resonant cavity, an internal surface capable of reflecting acoustic waves from a transducer 12 provided opposite, on the side of the lower surface of the block 6. Advantageously, the glass strip is transparent to the optical beams from an optical emitter 13 arranged above the strip 11.
The strip 11 can be designed starting from one or a combination of the following materials: glass, PMMA, quartz, silicon, COC, PDMS, so as to ensure a good transmission on the one hand and a good reflection on the other.
The transducer 12 is composed of a stainless steel cylinder containing a piezoelectric element the operating frequency of which can be chosen as a function of the height of the resonant cavity. This frequency can be chosen between 0.1 MHz to 10 MHz for resonance cavities the thickness (height) of which can vary from a few mm to a few tens of μm.
Other inlets/outlets (not represented) of the microchannel type 9, 10 and passage channel type 7, 8 can be produced for supplying the resonant cavity with identical or different cells, biomarkers, or else for washing the culture medium or recovering the production of the cells during their culturing.
An embodiment is illustrated in
The flow 17 of cells and the creation of aggregate 18 from cells trapped in the resonant cavity 6 under the action of the acoustic waves emitted by the transducer 12 are illustrated in
In an embodiment such as can be seen in
An embodiment is illustrated in
In addition to the cell culture, the manufacture of spatially structured organoids or spheroids with different layers of cells is advantageously provided.
The microfluidic chip according to the invention makes it possible in particular to inject particles or cells into the resonant cavity, and therefore to produce cell aggregates in the cavity. This makes it possible to produce cell cultures over long periods of time by providing the culture medium needed to the aggregated cells in acoustic levitation.
The invention also makes it possible to create composite and structured layers of cells, which can be useful from a tissue engineering perspective. In order to do this, the emitter 13 is used to illuminate the cells at specific wavelengths and to carry out the technique of optical exclusion.
A mixture of two types of particles or cells can be injected, these will form an aggregate which mixes the two species under the action of acoustic waves.
If it is desired to organize the aggregate spatially, in particular to structure the aggregate in successive layers, which are annular and concentric, it is possible to use the optical exclusion principle.
The optical exclusion principle makes it possible to eject particles or cells in acoustic levitation under the effect of an optical illumination at a given wavelength suitable for the cell or particle which it is desired to exclude. This effect is dependent on the optical absorption properties of the particles/cells. Cells marked with a fluorescent marker also react to an illumination at an absorption wavelength of the fluorescent marker, see in particular
The invention makes it possible to form a 2D aggregate structured in the plane by successive bands at the periphery of the aggregate. A radially structured aggregate 25 as illustrated in
In order to do this, a mixture of two cells C1 and C2 is injected, of which one absorbs a given optical wavelength λopt1 and the other does not. In this case, an aggregate can be structured easily. In fact, if the aggregation area is illuminated at the wavelength λopt1, this will prevent C1 from aggregating under the effect of the acoustic force. The C2 species will therefore form a first aggregate. It is then sufficient to stop the illumination at the wavelength λopt1 in order that the C1 species forms aggregates around the first aggregate.
Using both optical and acoustic properties, therefore, it is possible to spatially structure cell aggregates in acoustic levitation. This operation can be repeated several times, by means of successive injections of different types of cells, which are marked or not, and which react to different optical wavelengths.
In
In order to do this, the use of a piezoelectric transducer (PZT) 19 which is transparent, as can be seen in
A (non-packaged) annular transducer (PZT) can also be used. In this case, it is possible to illuminate through the ring and therefore to couple two optical sources simultaneously.
The double illumination can also be carried out with the embodiment from
Another advantage of the culturing in levitation is that it is possible to create several pressure nodes in a cavity and thus to form cell aggregates in levitation one above another. As shown in
It is possible to use the optical exclusion effect on several cell aggregates in acoustic levitation and therefore to structure objects in the volume of the resonant cavity.
The aggregation area is therefore centred on the axis of the transducer. It is then possible to structure several superimposed aggregates, 26 as represented in
The creation of several monolayers one above another represents a considerable time saving for the cell culture.
The present invention therefore proposes new means for cell culturing suitable for replacing the traditional techniques of cell culturing on solid substrates. In fact, in the case of a traditional cell culturing, the cells, such as stem cells for example, will multiply on the solid substrate, but also move in order to come back into contact with the other cells. The culturing is regarded as terminated when the cells arrive “at confluency”, i.e. are in contact with one another and thus form a “monolayer” layer of cells (a single layer of cells).
The present invention relates to the design of an optoacoustic bioreactor in which the cells can be cultured in acoustic levitation and the cell aggregates can be manipulated and structured by specific illumination so as to form structured aggregates.
The inventors have shown that the opto-acousto-fluidic effect can be quantified by an ejection velocity Vej of the illuminated objects. This involves showing that the objects in levitation according to the invention leave the illuminated area at a velocity which is in particular a function of the wavelength of the illumination signal. These objects are micro- or nanoparticles with sizes comprised between 0.1 μm and 300 μm and sensitive to the wavelengths used.
Generally, the ejection velocity can be measured for different species of particles, as a function of the optical wavelength, the intensity of the illumination, the magnification of the objective lenses of the microscope. These parameters make it possible to control the power of the illumination. In
In
It is observed that the size of the samples influences the ejection velocity. It is also observed that the red-coloured particles have a much higher ejection velocity than the fluorescent particles.
The curve from
The table below shows opto-acousto-fluidic responses of various cells.
The acoustic frequency is 1.91 MHz with an amplitude of 9 V. The flow rate is 0.15 ml/h.
The formation of an aggregate of white particles with very few trapped red particles is observed. The figure on the left corresponds to a time of 5 min, whereas the figure on the right shows the aggregate formed after 20 min. The white particles are thus gradually concentrated, with optical exclusion of the red particles.
The photos in
The cells comprise red blood cells (/100) and MDA cancer cells (/100). The illumination is obtained by a signal at 460 nm, at 80% of the maximum power for a magnification of ×10. The acoustic frequency is 1.59 MHz with an amplitude of 10 V. The flow rate is 0.15 ml/h.
In the photo on the right, the layers are almost all transformed into crowns.
The aggregates are formed of 15-μm particles of red polystyrene. The illumination is obtained by a signal at 460 nm, at 60% of the maximum power for a magnification of ×10. The acoustic frequency is 1.91 MHz with an amplitude of 9 V. Illumination sequence: 460 off-on, i.e. first the aggregates are formed in white light (460 OFF), followed by illumination at 460 nm (ON) to form the crown of particles in acoustic levitation.
A layered annular structure can be seen in
| Number | Date | Country | Kind |
|---|---|---|---|
| 1906031 | Jun 2019 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/065732 | 6/5/2020 | WO |
