Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
  • C12M47/04—Cell isolation or sorting
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/14—Scaffolds; Matrices
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/16—Particles; Beads; Granular material; Encapsulation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
  • C12M47/20—Heating or cooling

Definitions

• the invention relates to the field of biotechnology and in particular to the structuring of cellular assemblies, for example with a view to reconstructing or modeling living tissues.
• the invention is of particular but in no way limiting interest in the sectors of cell therapy, pharmacological modelling, the food industry, for example for the culture of meat, micro-algae or plants, or even aerospace, in particular for cell culture in microgravity conditions.
• organ-on-a-chip in English
• organoids organoids
• the techniques most used for this purpose include the manipulation of cells within microfluidic devices and the formation of tissues by additive manufacturing.
• Another known technique consists in structuring cells by acoustic levitation: Bouyer et al. A bio-Acoustic Levitational (BAL) Assembly Method for Engineering of Multilayered, 3D Brain-Like Constructs, Using Human Embryonic Stem Cell Derived Neuro-Progenitors, Adv. Mater. 2016, 28, 161-167.
• BAL bio-Acoustic Levitational
• a step of generating a standing acoustic wave in the cavity so as to produce an acoustic radiation force resulting in a displacement of the objects in the cavity said objects comprising: o first objects which present a positive acoustic contrast with respect to the fluid , and o second objects which present a negative acoustic contrast with respect to the fluid.
• the propagation of a standing acoustic wave in the cavity makes it possible to form in the cavity, along the direction of propagation, one or more nodes, that is to say places where the pressure of the fluid is zero, and a or several bellies or anti-nodes, that is to say places where this pressure is maximum.
• the first objects having an acoustic contrast that is to say a density-compressibility factor, positive with respect to the fluid, these will be transported by the acoustic radiation force towards a pressure node.
• the invention thus makes it possible to form in the cavity one or more aggregates of first objects and one or more aggregates of second objects in the form of layers, or sheets, which follow one another along the direction of propagation, this extremely quickly - typically in seconds - and using hardware that is particularly simple to implement and inexpensive.
• Such a layered structure resembles the structure of human organ tissues which typically include layers of cells separated by layers of extracellular matrix.
• the epithelia in particular cardiac or pulmonary or endothelial, may comprise differentiated or non-differentiated layers which rest in a certain number of cases on basal laminae of a protein nature, for example epithelial or muscle cells.
• the blood-brain barrier or the cerebral parenchyma typically comprise layers of interconnected neurons.
• the invention makes it possible to maintain in acoustic levitation all the objects thus structured, under the action of the standing acoustic wave, the generation of which can be maintained for the required duration, for example several hours or days, in order to promote interactions between objects when they are alive, in particular when they are formed by biological cells.
• the invention thus makes it possible to carry out a cell culture in acoustic levitation, by controlling the permeability and consequently the development of the connections and interactions between layers of cells forming the first objects, by a choice of second objects forming one or more layers of chosen porosity. .
• This innovative approach also makes it possible to limit the contact of objects with walls or surfaces, thus preserving their mechanical and functional integrity.
• an “object” designates a living or inert element preferably having a small size compared to the length of the acoustic wave generated in the cavity.
• the objects can typically have a micrometric size, for example between 1 ⁇ m and 100 or several hundreds of ⁇ m.
• said first objects are living elements such as biological cells, for example of the eukaryotic or prokaryotic type.
• Said second objects can themselves be inert elements such as particles comprising a hydrogel, for example based on collagen, gelatin, or even fibrin and extracellular matrix protein.
• the second objects can comprise a compressible elastomer, for example polydimethylsiloxane.
• the invention can however be implemented with objects of different size, that is to say located outside the aforementioned range.
• the objects or some of them may have a size of less than 1 ⁇ m, being for example formed by bacteria or viruses, and/or have a size of several hundreds of ⁇ m.
• the objects in particular the first objects, or some of them, can be multi-cellular elements or artificially formed objects or else objects taken from an organ.
• the fluid in which the objects are suspended is preferably a liquid which, depending on the application envisaged, can comprise water or form a culture medium.
• the invention thus provides a simple solution for reconstituting artificial tissues for research purposes or even in the context of cell therapies.
• the invention moreover provides a particularly precise solution in terms of positioning objects in space and which makes it possible, if necessary, to control the development of intercellular interactions.
• the standing acoustic wave generated in the cavity has a wavelength less than twice a dimension of the cavity in a direction of propagation of the standing acoustic wave.
• this wavelength is less than or equal to this dimension.
• the standing acoustic wave has at least one antinode and at least one or two nodes.
• hydrogel or compressible elastomer to form the second objects makes it possible to form a porous intermediate layer, allowing the development of interactions between the layers of first objects extending from either side of this intermediate layer, when the first objects comprise living cells.
• the invention not only makes it possible to produce a cell culture in acoustic levitation but also, alternatively or additionally, to initiate or continue such a process by maintaining the objects in position using a matrix.
• the method may comprise, after positioning the objects under the action of the acoustic radiation force, a step of introducing a substance into the cavity so as to form a matrix capable of supporting the first objects.
• This substance is preferably a biocompatible active substance promoting the phase change of the medium constituted by the fluid.
• This substance may comprise a hydrogel prepolymer or other element capable of forming a matrix in the form of a gel.
• This substance may include a catalyst and/or a photoinitiator.
• a matrix in the form of a gel makes it possible to adequately maintain the objects in position in space while being elastically deformable. Furthermore, it is preferred that the matrix be porous, whether it is in the form of a gel or in another form.
• the porosity of the matrix makes it permeable and perfusable, so as to allow the development of cellular connections.
• said substance comprises a photopolymerizable material
• the method comprising, after introduction of the substance into the cavity, a step of light stimulation of the substance so as to polymerize it.
• the invention thus makes it possible to sculpt a support matrix for the structured set of objects, in particular the first objects.
• the method may comprise, after positioning the objects under the action of the acoustic radiation force, a step of incubating the objects.
• the cavity and its contents can be placed in an incubator for this purpose.
• Incubation promotes the differentiation, self-organization and maturation of cell layers.
• the method comprises, after positioning the objects under the action of the acoustic radiation force, a step of heating the second objects so as to merge them.
• the heating step can be carried out using a laser sheet.
• Such a heating step is preferably carried out before forming this matrix.
• the method comprises, after positioning the objects under the action of the acoustic radiation force, a step of encapsulating the first objects.
• this encapsulation step comprises an introduction into the cavity of third objects having a positive acoustic contrast with respect to the fluid.
• the third objects can thus be transported by the force of acoustic radiation towards a pressure node to form around the first objects which are there a protective shell.
• the third objects can comprise hydrogel balls or another material making it possible to form a porous protective shell.
• the encapsulation step is preferably implemented, but not necessarily, when the use of a support matrix is not resorted to.
• the invention can also be implemented for cell therapy purposes, for example by in vivo injection of a culture or proto-culture produced using the principles described in this document.
• Fig. 1 is a schematic view of a device comprising a cavity and a transducer able to generate a standing acoustic wave in the cavity, the cavity containing a fluid with objects in suspension which are distributed relatively homogeneously in the cavity before undergoing the acoustic wave effects;
• Fig. 2 is a schematic view of the device of FIG. 1, in which the objects have been moved by an acoustic radiation force produced by the acoustic wave so as to be respectively aligned on a node or an antinode of this wave;
• Fig. 3 is a schematic view illustrating a phenomenon of diffusion between layers of cells
• Fig. 4 is a schematic view illustrating a phenomenon of development of cell extensions
• Fig. 5 is a schematic view illustrating a cell migration phenomenon
• Fig. 6 is a schematic view of the device of FIG. 2, the objects being maintained in the configuration of FIG. 2 using a gel matrix.
• This device comprises on the one hand a receptacle which forms a cavity 1 capable of containing a fluid and/or various substances in the form, for example, of a liquid or else of a gel.
• the cavity 1 extends along a direction Al, which in this example corresponds to a vertical direction.
• Cavity 1 has along direction Al a dimension B1 which corresponds in this example to a height of cavity 1.
• the cavity 1 here has a generally cylindrical shape.
• the cavity 1 can have another geometry, for example a rectangular section.
• the device of FIGS. 1 and 2 comprises an acoustic wave generation system.
• this system comprises a piezoelectric transducer 2 arranged at the level of a first end of the cavity 1 in the direction Al, in this case vertically below the cavity 1, as well as an acoustic reflector 3 which delimits a second end of the cavity 1 in the direction Al, in this case being arranged vertically above the cavity 1.
• This system is configured to be able to generate in the cavity 1 and to propagate in the fluid that it contains a standing acoustic wave 4, according to a direction of propagation which corresponds to the direction Al.
• the standing wave 4 thus generated can have a frequency identical to the resonance frequency of the cavity 1, which consequently forms a resonator.
• this standing wave 4 can have a different frequency from the resonant frequency of cavity 1.
• the system is configured to be able to generate, in particular, a wave 4 having a wavelength A less than or equal to twice the height B1 of the cavity 1, in order to form along the direction Al at least a pressure node and at least one pressure belly.
• the transducer 2 is a broadband transducer equipped with an ultrasonic source.
• Such a transducer 2 makes it possible to modify the position of the node(s) of wave 4 along the direction Al and/or the distance between different nodes of wave 4, by varying the frequency of this wave 4.
• the device which has just been described, or any similar device, is implemented in order to position objects of small size, typically of micrometric size, within the cavity 1 according to a spatial organization determined by one or more parameters of wave 4, in particular its frequency.
• the cavity 1 is filled with a fluid 5 and objects 6 and 7 suspended in this fluid 5.
• the objects 6 are biological cells
• the fluid 5 forms a culture medium for these cells 6
• the objects 7 are polydimethylsiloxane beads.
• each of the objects 6 and 7 has a size between 1 ⁇ m and 100 ⁇ m and the height B1 of the cavity 1 is several centimeters.
• each of the objects 6 has a density p 01 greater than the density p of the fluid 5.
• each of the objects 7 has a density p 01 less than the density p of the fluid 5.
• the objects 6 are also chosen so that the speed c 01 of propagation of an acoustic wave in these objects 6 is greater than the speed c of propagation of this acoustic wave in the fluid 5.
• the objects 7 are chosen so that the speed c o2 of propagation of the acoustic wave in these objects 7 is lower than the speed c of propagation of this acoustic wave in the fluid 5.
• the transducer 2 After placing the fluid 5 in the cavity 1 and the objects 6 and 7 suspended in the fluid 5 in the manner illustrated in Figure 1, the transducer 2 is actuated so as to generate a standing acoustic wave 4 in the cavity 1.
• This wave 4 makes it possible to produce an acoustic radiation force which is exerted on the objects 6 and 7.
• This acoustic radiation force FRA can in particular be described according to the following model, known per se, by K. Yosioka and Y. Kawasima:
• v 0 is the speed of wave 4
• k the wave number
• F y a density-compressibility factor
• z the position of the object 6 or 7 considered along the direction Al, c i.e. along the direction of propagation of wave 4.
• the density-compressibility factor F y can be defined as follows: where p ox is the density p 01 or p o2 of the object 6, or respectively 7, considered, and c ox is the propagation speed c 01 or c o2 of wave 4 within object 6, or respectively 7, considered.
• the objects 6 Given the respective density and the respective speed of propagation of the acoustic wave of the objects 6 and 7 with respect to the fluid 5, the objects 6 have a positive density-compressibility factor, or acoustic contrast, while that the objects 7 have a negative density-compressibility factor, or acoustic contrast.
• the wave 4 has a wavelength A equal to the height B1 of the cavity 1, respectively forming along the direction Al a first node at a coordinate Cl, an antinode at a C2 coordinate and a second node at a C3 coordinate.
• the invention thus makes it possible to spatially organize the objects 6 and 7 in the form of spaced layers along the direction Al and to keep them thus positioned in acoustic levitation, under the action of the wave 4.
• the objects 7 form an intermediate layer, located at mid-height of the cavity 1, while the objects 6 form two layers extending on either side of the intermediate layer.
• the objects 7 being polydimethylsiloxane beads, their aggregation or grouping in the form of a layer makes it possible to form a porous barrier which allows the development of interactions between the layers of cells 6, this without contact with the walls of the cavity 1.
• the invention thus makes it possible to produce a cell culture in acoustic levitation.
• the invention also makes it possible to control the interactions between layers of cells 6 since it is in particular possible to choose different materials, geometries and sizes for the objects 7, these parameters having a direct impact on the porosity of the barrier which they constitute under the action of the acoustic radiation force.
• it is thus possible to trigger or authorize the diffusion of solutes or cell secretion 10 (FIG. 3), the development of cell extensions 11 of the neuronal axon type (FIG. 4), or even the migration of cells 6 (Figure 5).
• the objects 7 comprise hydrogel particles which, after positioning under the action of the acoustic radiation force as described below, submerged by local heating, for example using a laser sheet.
• the invention also makes it possible to continue the cell culture, or to initiate it after positioning the objects 6 and 7 in the manner described above, by producing in the cavity 1 a support matrix.
• a substance based on hydrogel prepolymer can be introduced into the cavity 1.
• Such a substance makes it possible to form a porous matrix 20 in the form of a gel, making it possible to support the layers of objects 6 and 7 (FIG. 6).
• this substance also comprises a photopolymerizable material which is subjected, after introduction into the cavity 1, to a light stimulation resulting in the polymerization of the matrix.
• the acoustic wave 4 can then be interrupted so that the cell culture occurs within such a matrix, for example by placing the container in an incubator.
• the invention makes it possible to reconstruct and stimulate complex architectures comprising different layers of cells separated by a variety of objects making it possible to control the interactions between the cellular layers, using a method and a device that are particularly simple to implement.

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• 2021
  • 2021-09-28 FR FR2110209A patent/FR3127501A1/fr active Pending
• 2022
  • 2022-09-27 JP JP2024515323A patent/JP2024534340A/ja active Pending
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