- device and method for characterizing cells subjected to a physical stress

Abstract

The present invention relates to a microfluidic device and to a method for applying at least one hydrodynamic stress of defined intensity and duration to cells in suspension, said cell being in motion in said device, and for characterizing the morphology and the physiological state of these cells during, and optionally after, the application of the hydrodynamic stress.

Description

[0001] DESCRIPTION

[0002] Title: DEVICE AND METHOD FOR THE CHARACTERIZATION OF CELLS SUBJECTED TO PHYSICAL STRESS

[0003] Field of invention

[0004] The present invention relates to a microfluidic device and a method for applying at least one physical constraint of a determined intensity and duration to cells in suspension, and for characterizing the morphological and physiological state of the cells subjected to the application of this physical constraint.

[0005] The present invention is therefore in the field of microfluidic devices and analytical cell biology.

[0006] State of the art

[0007] Currently known means of describing and characterizing cell morphology and physiology are based in particular on the use of cell markers. Morphology can itself be used as a marker of the physiological state of a cell. However, these means require, on the one hand, the availability of appropriate and specific markers of a physiological state and, on the other hand, these methods characterize only the physiological state of the cell as observed at the time of labeling.

[0008] Furthermore, the efficiency of bioprocesses using animal cells, such as in particular bioprinting processes, bioproduction of cells for cell therapy, and processes implemented from cells, such as the production of therapeutic proteins, viral vaccines or viral vectors, as well as all processes of injection or collection of cells, depend on the quality of the cells which are used during said bioprocesses.

[0009] Cell quality is generally linked to physiological characteristics such as cell viability, i.e. the proportion of living cells compared to cells engaged in cell death processes (apoptosis, necrosis, lysis) or cell function (capacity to secrete molecules, capacity to differentiate into cell subtypes in the case of stem cells). The efficiency of a bioprocess can therefore be strongly impacted by the alteration it produces on cell quality and therefore on cell physiology. One of the mechanisms that very strongly impacts quality, and in particular cell viability, within a bioprocess is the application of hydrodynamic and mechanical constraints generated by the equipment used, such as a bioreactor, a system using an injection or sampling syringe, or a separation step.The interaction between a suspending fluid and a wall creates a stress on the cells suspended in the fluid. A direct interaction of the cell with the wall produces a mechanical impact on the cell. Depending on the nature of the process and the techniques used, the intensity and duration of a physical stress exerted on a cell vary greatly. For example, a cell therapy process can generate a stress on cells whose intensity can reach 5,000 Pa and a bioprinting process can generate a stress whose duration can reach 30 ms.

[0010] It is therefore desirable to be able to characterize in a simple and reproducible manner the physiological state of cells during and / or after application of a physical constraint, in particular via the characterization of their morphology. In particular, this type of characterization is desirable for cells likely to be used in a bioprocess.

[0011] It is further desirable to characterize the physiological state of cells at the time when they are subjected to physical constraints reproducing the conditions of physical constraint applied to the cells during a particular bioprocess.

[0012] WO 2015 / 024690 "Apparatus and method for determining the mechanical properties of cells" relates to a method and apparatus for determining the mechanical properties of cells, and discloses the study of the deformation of cells subjected to shear stress. This document discloses a device comprising a simple capillary channel in which suspended cells undergo shear stress and a method for measuring the shape of the cells as they circulate in the device. This method uses binarized images. The measurements are made in real time.

[0013] EP 3 796 212 "Device for image-based cell classification, method therefor and use thereof" describes a device for sorting cells in real time and without labeling. This device comprises a microfluidic network leading to the alignment of each of the cells according to its major axis and a classification unit comprising a neural network which sorts the cells according to the images of these.

[0014] WO 2019 / 006188 "Quantitative deformability cytometry: rapid, calibrated measurements of cell mechanical properties" describes a microfluidic device and a quantitative deformability cytometry (q—DC) method for quantitatively assessing the intrinsic mechanical properties of cells. The mechanical parameters determined are: elastic modulus E, cell fluidity P, transit time T T , migration time Te, cell size D ce ii and the maximum stretch £ max, at a rate of 100 cells / s. Machine learning tools are implemented. The cells are subjected to calibrated shear stresses.

[0015] There is therefore a need for a device and a method that can characterize not only the mechanical properties of cells that have undergone physical stress, but also their morphology and physiological state.

[0016] There is therefore a need to have a device and a method capable of generating at least one physical stress such as generated during a bioprocess, the nature, intensity and duration of which are determined and controlled, in order to characterize the cells likely to undergo said stresses. By "physical stress" is meant in particular any force exerted on the cells, capable in particular of generating cellular stress; such a physical stress may in particular be a mechanical impact or a hydrodynamic stress, such as an elongation, compression or shear stress.

[0017] Brief description of the invention

[0018] The inventors have developed a device for applying at least one physical constraint of determined intensity and duration to cells in suspension, and for characterizing the cells having undergone said at least one physical constraint.

[0019] More particularly, a device according to the invention is configured to:

[0020] - the application of at least one hydrodynamic constraint of determined intensity and duration to at least one cell suspended and moving in said device,

[0021] - and for the optional application of at least one mechanical impact to said cell.

[0022] A device according to the invention is configured for the characterization of said cell during the application of said constraint and optionally for the characterization of said cell after the application of said constraint.

[0023] A device according to the invention comprises, on the one hand, a microfluidic circuit for the application of at least one physical constraint and, on the other hand, a means of characterizing the cells.

[0024] In a device according to the invention, said at least one physical constraint is a constraint chosen from mechanical constraints, in particular a mechanical impact, and / or hydrodynamic constraints, such as a hydrodynamic compressive constraint, a hydrodynamic shear constraint and / or a hydrodynamic extension constraint. More particularly, in a device according to the invention, said at least one physical constraint is a constraint chosen from hydrodynamic constraints.

[0025] In a device according to the invention, said means of characterizing the cells is chosen from all known technical means of observing cells, in particular imaging or cytometry means. More particularly, in a device according to the invention, said means of characterizing the cells during the application of the constraint is chosen from all known technical means of observing cells, in particular imaging or cytometry means.

[0026] A device according to the invention comprises a microfluidic circuit comprising at least one segment configured for the application to the cells in suspension of at least one physical constraint, more particularly a hydrodynamic constraint, this segment comprising at least: i) a main channel configured for the circulation of a fluid containing said cell in suspension, ii) a fluid inlet and a fluid outlet, iii) a means for introducing and establishing a flow of said fluid inside said channel.

[0027] The invention therefore has as its first object a device for the application of at least one physical constraint, more particularly at least one hydrodynamic constraint, of determined intensity and duration to at least one cell in suspension, and for the characterization of said cell during and / or after said application, more particularly during and optionally after said application.

[0028] The second subject of the invention is a method for applying at least one physical constraint, more particularly at least one hydrodynamic constraint, of determined intensity and duration to at least one cell in suspension, and for characterizing said cell during and / or after said application, more particularly during and optionally after said application.

[0029] The invention also relates to a classification model, previously trained on a training data set, to characterize a cell and predict its physiological state during and / or after the application of at least one physical constraint, according to a device and a method according to the invention. Such a classification model allows a saving of time, money and performance (in particular allows a large number of cell characterizations during a short period of time) compared to the tools existing on the market.

[0030] The invention finally relates to the use of a device, a method or a classification model according to the invention, to characterize a cell and possibly predict its physiological state during and / or after the application of at least one physical constraint.

[0031] A device and a method according to the invention have the advantage of generating at least one physical constraint, the nature, intensity and duration of which are precisely determined and controlled, in order to characterize the cells having undergone said constraints.

[0032] A device and a method according to the invention have the advantage of allowing the application of a modular sequence of at least one physical constraint, in particular the repetition of the same constraint and / or the combination of physical constraints of a different nature, the intensity and duration of each being precisely defined. It is therefore possible, thanks to this simple and precise tool, to reproduce the constraints applied during a bioprocess and to monitor the state of at least one cell at high speed.

[0033] A device and method according to the invention also have the advantage of making it possible to predict the physiological state of cells during and / or after they have been subjected to physical constraints. This prediction can be carried out during and up to several days after the application of these physical constraints.

[0034] Finally, a device and a method according to the invention have the advantage of defining the capacity of a cell to undergo the application of at least one determined physical constraint while retaining a morphology and a physiological state compatible with the requirements of said bioprocess. It is in fact possible, thanks to a device and a method according to the invention, to define, for given cells, a map of its capacities to resist at least one physical constraint.

[0035] By "capacitance" we mean the capacity of a cell to undergo at least one physical constraint of a determined intensity and duration, without modification of its physiological state. By "modification of its physiological state" we mean in particular a differentiation, or the passage from a viable state to a state of lysis, necrosis, or apoptosis.

[0036] The use of a device and a method according to the invention makes it possible in particular to optimize the bioprocesses, and in particular the associated physical constraints, in order to define the optimal operating conditions and to adapt said constraints to the cells of interest. These conditions, for example, focus on the parameters of the bioprocess (temperature, flow rate, rotation speed), on the suspending fluid (viscosity, osmolarity). It is indeed desirable to optimize the constraints associated with the bioprocesses, in order to adapt said constraints to the data obtained during the characterization and prediction of the physiological state of cells subjected to these physical constraints.

[0037] Detailed description of the invention According to a first object, the invention relates to a device for the application of at least one physical constraint, more particularly at least one hydrodynamic constraint, to at least one cell in suspension and for the characterization of said cell during and / or after said application, the device comprising:

[0038] - a microfluidic circuit comprising at least one segment, said segment comprising at least: i) a main channel configured for the circulation of a fluid containing said cell in suspension, ii) a fluid inlet and a fluid outlet, iii) means for introducing and establishing a flow of said fluid inside said channel, and

[0039] - a means of characterizing said cell; said device according to the invention being characterized in that said at least one segment is configured for the application to said cell of at least one physical constraint chosen from:

[0040] - a mechanical impact,

[0041] - a hydrodynamic compressive stress,

[0042] - a hydrodynamic shear stress and

[0043] - a hydrodynamic extension constraint.

[0044] In the disclosure of the present invention, the term "for" as in particular in the expression "for the application of a physical constraint" means "configured for the application of a physical constraint". When an interval or range of values ​​is indicated, the limits cited are considered as being part of said range of values.

[0045] By "microfluidic circuit" we mean a circuit intended to manipulate small volumes of fluids (10' 18 at 10' 3 liters) in channels with diameters between 5 pm and 3000 pm.

[0046] By “fluid” we mean a deformable medium suitable for circulation in a device according to the invention and for cell suspension.

[0047] Preferably, in a device according to the invention, said fluid is chosen from Newtonian fluids, that is to say, fluids whose viscosity does not vary as a function of shear stress. Said fluid has at least one of the following properties:

[0048] - its Newtonian viscosity between 1 and 1000 mPa.s, preferably between 1 and 100 mPa.s,

[0049] - its pseudoplasticity index is between 0 and 1, and is preferably 1 mPa.s,

[0050] - it is compatible with cell survival in unconstrained conditions, its osmolarity is between 250 and 410 mOsmol,

[0051] - its pH is physiological under handling conditions in ambient air, said pH is therefore between 6.5 and 7.8 and

[0052] - it includes nutrients that can be assimilated by the cells in suspension, such as glucose, glutamine or a nutrient composition such as a culture medium.

[0053] More particularly, in a device and a method according to the invention, the fluid for the cell suspension is preferably chosen from Newtonian fluids, and in particular from:

[0054] - physiological buffers, usually used for suspending cells, such as PBS, HBSS; these buffers are optionally supplemented with nutrient compounds such as a 0.5 to 6 g / l glucose solution and a 2 to 4 mM glutamine solution,

[0055] - cell culture media, such as DMEM, EMEM, alpha-MEM,

[0056] - Cell separation solutions, such as Ficoll and sucrose solution,

[0057] - Low molar mass polymer solutions.

[0058] When applying said at least one physical constraint, the cell concentration is preferably less than 100 million cells per mL, preferably between 0.01 and 10 million cells per mL. In other words, the cell volume preferably represents at most 30% of the total volume of the suspension fluid.

[0059] In a device according to the invention, the circulation of the reference fluid and the fluid in which the cells are suspended is carried out in a stable and pulsation-free flow. Said device is therefore configured for the application of at least one constraint to at least one cell suspended and moving in said device. The device is preferably designed to lead the cells towards a microfluidic chip following a defined current line.

[0060] In a device and method according to the invention, the intensity of the stress, also referred to as "stress level" or "stress" in the figures, and the residence time under stress, also referred to as "duration of application of the stress" or "time" in the figures, take place in a controlled manner. The capture and release of the cells in suspension also take place in a controlled manner.

[0061] The pump(s) control the flow rate and flow pattern. The pumps are preferably ultra-high pressure pumps, up to 1.37xl0 5 kPa.

[0062] The circulation of a fluid within a device according to the invention is initiated and maintained by means of any device suitable for this purpose well known to a person skilled in the art, such as a pump. In a device according to the invention, the section of the channels may be constant or variable, this section may also be conical, for example in the form of nozzles. The presence of a conical section, increasing or decreasing, at the end of a channel imposes an additional constraint on the cells for capture and controlled release.

[0063] When a fluid containing suspended cells is circulated in the microfluidic circuit, current lines are generated and allow the control of the cell trajectory. The cells to be characterized are positioned on one of the current lines allowing the control of the stress intensity.

[0064] The preferred diameter of the main channel is between 10 and 3000 pm.

[0065] The channels are made of a material suitable for the circulation of a fluid subjected to a pressure of between 10' 3 and 10 6 kPa, and preferably between 1 and 10 4 kPa, and / or a material suitable for the circulation of a fluid with a viscosity between 1 and 2000 mPa.s.

[0066] The capacitance of cells depends on their origin (clone, species, organ), their culture method (number of doublings in culture, culture medium, environmental conditions of the culture, i.e. shaking or not, temperature, pH) and their collection method (trypsinization, mechanical harvesting). For example, the culture method of the cells, in particular in 2D or 3D, their culture in adherent or suspension form and the nature of the culture medium used influence the capacitance of the cells.

[0067] In a device according to the invention, according to a particular embodiment, the microfluidic circuit is constituted by a set of channels constituted by a suitable material, notably chosen from the following: PEEK (PolyEtherEtherKetone), PVC (PolyVinyl Chloride), PTFE (PolyTetraFluoroEthene), FEP (Fluorinated ethylene polypropylene), PDMS (PolyDimethylSiloxane) or steel. According to another embodiment, the microfluidic circuit is constituted by a microfluidic chip constituted by a material chosen from: PDMS, polyacrylate, SEBS (StyreneEthyleneButyleneStyrene), glass, polycarbonate or ceramic.

[0068] A device according to the invention may comprise, according to particular embodiments, channels arranged in series and / or channels arranged in parallel.

[0069] The circulation of fluid in a device according to the invention is characterized by at least one of the following parameters:

[0070] - a fluid circulation flow rate, in the channel considered for the characterization of the physical stress applied to the cells, of between 0 and 5 ml / min, preferably between 0 and 1 ml / min and / or - a fluid circulation speed of between 5 pm / s and 300 m / s, and / or

[0071] - a throughput of analyzed cells that can be greater than 1000 cells / minute when the analysis is carried out with software.

[0072] The total duration of presence of the cells in said channel is preferably between 1 ps and 10000 s, preferably between 1 ps and 100 s, preferably between 1 ps and 1 s, preferably between 10 ps and 100 ms.

[0073] By "suspension cell" we mean any type of cell, animal or plant, prokaryotic or eukaryotic. Indeed, depending on the diameter of the channels and the magnification of the objective allowing image analysis, a device according to the invention makes it possible to characterize, for example, bacterial, algal or fungal cells.

[0074] The invention particularly relates to a device for applying at least one physical constraint and characterizing at least one cell in suspension for at least one of the following aspects:

[0075] - the size of the cell,

[0076] - the shape of the cell, in particular the sphericity of the cell

[0077] - the appearance of the external membrane,

[0078] - the appearance of the cytoplasm, in particular its granularity

[0079] - the presence of at least one marker on the surface of the cell,

[0080] - the protein content of the cell and

[0081] - the nucleic acid content of the cell, including the amount of DNA, the amount of RNA, the nucleotide sequence of one or more nucleic acids, whether DNA or RNA.

[0082] By "cell characterization" is meant the definition of at least one characteristic of said cell. In the case where more than one cellular characteristic is defined, the characterization of said cell includes the definition of the combination of said characteristics.

[0083] Cell characterization leads to the determination of the physiological state of the cells during or after the application of said at least one constraint. Physiology studies the role, functioning and mechanical, physical and biochemical organization of cells and their components, in particular cell organelles. Physiology also studies the interactions between a cell and its environment. Determining the physiological state of cells includes in particular the state of differentiation and / or the determination of nutrition; proliferation and relationship functions, such as mobility and sensory functions. This physiological state is preferably chosen from the following: living cell, dead cell, lysed cell, necrotic cell, apoptotic cell, differentiated cell or undifferentiated cell, pathological cell or healthy cell.

[0084] The relationship between the physiological state and the various aspects of characterization are known to those skilled in the art.

[0085] The physiological state of the cells after the application of at least one physical stress can further be compared to the physiological state of the cells before the application of said physical stress.

[0086] For the purposes of the present invention, the capacity of cells to undergo at least one physical constraint of determined intensity and duration while maintaining a physiological state compatible with subsequent use is defined as the “capacitance” of the cells.

[0087] According to a particular aspect, the invention particularly relates to a device configured for the application to said cell of at least one physical compressive stress, said device comprising at least one segment of type (A) comprising, or consisting of, a first channel joined, at the same level, by two channels, each making an angle of between 30 and 150 degrees with said first channel, this angle is also called "flow focusing angle", or flow focusing angle.

[0088] By "hydrodynamic compressive stress" is meant the application of balanced forces towards the interior of the cells, this stress is also referred to as "Flow focusing". In a device and a method according to the invention, the intensity of the compressive stress applied to the cells is between 10' 3 and 10 3 kPa, preferably between 10' 3 and 10 2 kPa, preferably between 10' 3and 10 kPa. Figure 1 schematically represents an example of a type A segment.

[0089] According to another particular aspect, the invention particularly relates to a device configured for the application to said cell of at least one hydrodynamic shear stress, said device comprising at least one type B segment comprising, or consisting of, a channel with a diameter of between 10 and 2000 pm, preferably between 20 and 200 pm.

[0090] By "hydrodynamic shear stress" is meant a mechanical stress applied parallel or tangential to the face of a material. In a device and a method according to the invention, the intensity of the shear stress applied to the cells is between 10' 3 and 10 5 kPa, preferably between 10' 3 and 10 4 kPa, preferably between 0.1 and 10 3kPa. A hydrodynamic shear stress is applied in particular during the circulation of cells in a capillary channel, the diameter of which is preferably between 10 and 2000 pm, preferably between 20 and 200 pm. Figure 1 schematically represents an example of a type B segment.

[0091] According to another particular aspect, the invention particularly relates to a device configured for the application to said cell of at least one physical extension constraint, said device comprising at least one type C segment comprising, or consisting of, i) a channel of increasing or decreasing section or ii) a first channel joined by a second in which the circulation of the fluid takes place in a different direction, preferably opposite, to that of the first channel.

[0092] By "hydrodynamic extensional stress" is meant the application of balanced forces towards the outside of the cells, or an elongational stress. In a device and a method according to the invention, the intensity of the extensional stress applied to the cells is between 10' 3 and 10 3 kPa, preferably between 10' 3 and 10 2 kPa, preferably between 10' 3 and 10 kPa. Figure 1 schematically represents an example of a type C segment.

[0093] According to another particular aspect, the invention particularly relates to a device configured for the application to said cell of at least one mechanical impact, said device comprising at least one D-type segment comprising, or consisting of, a first channel within which the line of path of the cells encounters an obstacle, such as in particular the wall of a second channel. Figure 1 schematically represents an example of a D-type segment.

[0094] By "mechanical impact" is meant any type of mechanical shock, such as for example a shock upon encountering a wall or an inertial collision on the surface. In a device and a method according to the invention, the intensity of the mechanical impact applied to the cells is between 1 and 300 m / s, preferably between 1 and 100 m / s, preferably between 1 and 10 m / s. The duration of the impact time is preferably less than 1 ps.

[0095] More particularly, a device according to the invention for the application of at least one physical constraint and the characterization of at least one cell in suspension, comprises a microfluidic circuit comprising or consisting of:

[0096] - at least one type A segment, and / or

[0097] - at least one type B segment, and / or

[0098] - at least one segment of type C, and / or - at least one segment of type D, said segments being combined with each other.

[0099] Even more particularly, a device according to the invention for the application of at least one physical constraint and the characterization of at least one cell in suspension, comprises a microfluidic circuit comprising or consisting of:

[0100] - at least one type A segment, and / or

[0101] - at least one type B segment, and / or

[0102] - at least one type C segment,

[0103] - and optionally at least one type D segment, said segments being combined with each other.

[0104] A device according to the invention is in particular designed for the application of a sequence comprising one or more iterations of a physical constraint of the same nature, at a determined frequency and intensity, and / or for the application of a sequence of several physical constraints of different nature, at a determined frequency and intensity.

[0105] By "application of at least one physical constraint of determined intensity and duration" we mean:

[0106] - at least one application of a particular physical constraint of determined intensity and duration, optionally followed by the application of one, two, three, four or more repetitions of the application of said particular physical constraint, and / or

[0107] - at least one application of a sequence of at least two particular physical constraints of determined intensity and duration, optionally followed by the application of one, two, three, four or more sequences of at least two physical constraints, and / or

[0108] - the application of any type of physical constraint sequence as described in this application.

[0109] According to another particular aspect, the invention particularly relates to a device configured for the application to said cell of at least one sequence comprising, or consisting of, at least two successive physical constraints of a different nature. The sequence can be repeated one, two, three or more times.

[0110] According to another particular aspect, the invention particularly relates to a device configured for the application to said cell of at least one sequence comprising, or consisting of, at least two successive physical constraints of the same nature. According to this particular aspect, the device is configured for the application to said cell of a physical constraint repeated at least 1 time, at least twice, at least 3, 4, 5, 6, 7, 8, 9, 10 times, or even more.

[0111] An example of this embodiment of a device according to the invention is shown diagrammatically in Figure 2, which represents a device designed for the application to the cells of a large number of compressive stresses, separated by segments designed so that the cells do not undergo stress. These sequences of physical stresses can be considered as equivalent to the application of dynamic deformation of the cells. They are repetitions of the same stress sequence.

[0112] More particularly, a device according to the invention is characterized in that the total intensity of said at least one physical constraint applied to the cell is between 10' 3 kPa and 10 5 kPa, preferably between 10' 3 kPa and 10 4 kPa, preferably between 0.1 kPa and 10 3 kPa, preferably between 1 kPa and 10 2 kPa.

[0113] More particularly, moreover, a device according to the invention is characterized in that the total duration of the application of said at least one physical constraint is between 1 ps and 10000 s, preferably 1 ps and 100 s, preferably between 1 ps and 1 s, preferably 10 ps and 100 ms.

[0114] In a microfluidic device according to the invention, the characterization of said at least one cell in suspension is carried out during the application to said cell of at least one physical constraint and / or after the application to said cell of at least one physical constraint. According to a first embodiment, the characterization of said at least one cell in suspension is carried out during the application to said cell of at least one physical constraint.

[0115] According to another embodiment, a device according to the invention is characterized in that the characterization of said cell takes place after the application of said at least one constraint, this characterization is carried out during a period of between 0 and 120 days, preferably between 0 and 30 days, preferably between 0 and 1 day after the application of said at least one physical constraint.

[0116] More particularly, a device according to the invention is characterized in that the characterization of said cell after the application of said at least one constraint comprises, or consists of, at least one punctual characterization or at least one characterization carried out for a duration of between 1 ps and 10000 s, preferably 1 ps and 100 s, preferably between 1 ps and 1 s, preferably 10 ps and 100 ms.

[0117] More particularly, a device according to the invention is characterized in that the means for characterizing said at least one cell is chosen from: a cytometer, a microscope, a means for analyzing the protein content of the cell, a means for analyzing and sequencing the nucleic acids of said cell, and a means for capturing an image and / or an electrical signal, combined with a means for analyzing the signal. For example, a device according to the invention may comprise a microelectrode or a photodiode.

[0118] More particularly, a device according to the invention is characterized in that said signal and / or image analysis means comprises, or is constituted by, a central computer unit comprising software means adapted for signal and / or image analysis.

[0119] Even more particularly, a device according to the invention comprising a signal and / or image analysis means further comprises a first and / or a second classification model. The presence of at least a first and / or a second classification model has the advantage of accelerating the image analysis process and allowing real-time analysis.

[0120] The term "classification model" means a previously trained machine learning algorithm, in particular during supervised learning, as well as a training dataset, allowing the training of said algorithm, and an evaluation dataset. A classification model may consist of a computer program, said computer program being able to be written in any suitable computer language, known to a person skilled in the art. Said computer program is capable of being implemented on a computer to generate a technical result. Examples of these technical results are described below.

[0121] The training dataset may include a training set and a test set of the model. The model may thus be tested on the test set and the test set may be used to determine whether the model has been trained satisfactorily or not. The training set and the test set may be different. Alternatively, the test set may correspond to a part of the training set.

[0122] Even more particularly, a device according to the invention comprising an image analysis means further comprises a first classification model, previously trained with a training data set, and comprising a supervised, unsupervised or semi-supervised machine learning algorithm. Said first classification model is suitable for predicting the physiological state of a given cell from at least one characteristic of said cell. In the case of the first classification model, according to a particular embodiment, the input data are images with objects. The output data are objects with a label: a percentage of belonging to a specific class. The training algorithm comprises at least 10 epochs, the loss calculation is cross-entropy and the optimizer is Adam (enhanced gradient descent).The model is transfer learning with YOLO, the nature of the network is a supervised model (1733 cell images / 1733 annotations). The model preferably includes 106 convolutional layers. The functions performed in a neuron are: convolution, addition, softmax, "up sampling". Neuron / layer connections are made. The training dataset can include a multitude of data pairs, each of the data pairs comprising a first data representing at least one characteristic of said cell and a second data representing a physiological state of said cell.

[0123] The training data set can be previously constituted from data obtained in the laboratory by analysis of characteristics of cells whose physiological state has been determined.

[0124] Said first classification model can in particular be implemented on a computer to generate a technical result consisting, for example, of a classification of a cell according to its characteristics.

[0125] The said first classification model makes it possible to generate a three-dimensional diagram, representing, for example, for a given cell:

[0126] - the intensity of the physical stress applied to the cells, expressed in Pa,

[0127] - the duration of application of the physical constraint applied to the cells, or residence time, or “time”, expressed in s,

[0128] - the physiological state of the cells, i.e. cell viability, necrosis, apoptosis or lysis, expressed as a percentage, or the differentiated or undifferentiated character.

[0129] The first classification model is considered to have reached a satisfactory level of learning on all the profiles in the test set if the classification reaches a minimum Fl score of 70%.

[0130] Even more particularly, when a device according to the invention comprises an image analysis means, said image analysis means further comprises a second classification model, previously trained with a training data set, and comprising a supervised, unsupervised or semi-supervised machine learning algorithm, said second classification model being suitable for detecting and monitoring the deformation of a given cell, in response to at least one physical constraint.

[0131] Said first classification model can in particular be implemented on a computer to generate a technical result consisting, for example, of monitoring the morphological evolution of a cell according to different applications of physical constraints.

[0132] Preferably, said second classification model uses at least one neural network whose functions are as follows: i) Locate and isolate the cell from said image ii) Validate the presence of a single cell iii) Improve the quality of the image then process the image by applying a mask to the cell and determining its outline, iv) position and measure the major and minor axis of the ellipse describing the outline of the cell. The deformation being defined as the ratio between the major axis and the minor axis.

[0133] According to a particular embodiment, the second classification model comprises: input data 440 images of cells cut and adjusted in gray level, output data: binary segmentation mask. The training data comprises 420 training images. For the training algorithm at least 10 epochs are necessary, the loss calculation is cross entropy and the optimizer is Adam (enhanced gradient descent). The nature of the network is U-Net. The number of layers is: 5 contraction layers, 5 expansion layers, i.e. 10 in total. The functions performed in a neuron are: 2D convolution between image and filter, i.e. compress the image, extract the characteristic vector which contains the object of interest and decompress the image. The neuron / layer connections are characterized in that the layers are composed of two convolutions both followed by activation functions (ReLU).

[0134] An example of locating and isolating a cell is shown in Figure 3. An example of positioning and measuring the major and minor axes of the ellipse is shown in Figure 4.

[0135] It is considered that said second classification model has reached a satisfactory level of learning on all the profiles of the test set if the classification reaches in particular a minimum Fl score of 65%, preferably at least 80%.

[0136] According to a second subject, the invention relates to a method for applying at least one physical constraint of determined intensity and duration to at least one cell in suspension, and for characterizing said cell after said application, the method comprising the following steps: a) depositing and circulating a fluid containing said at least one cell in suspension in a microfluidic circuit comprising at least one segment, said segment comprising at least: i) a main channel configured for circulating a fluid containing said cell, ii) a fluid inlet and a fluid outlet, iii) means for introducing and establishing a flow of said fluid inside said channel, and b) characterizing said cell after applying said at least one constraint,a method according to the invention is characterized in that said at least one segment is configured for the application to said cell of at least one physical constraint chosen from:,

[0137] - a mechanical impact,

[0138] - a hydrodynamic compressive stress,

[0139] - a hydrodynamic shear stress and

[0140] - a hydrodynamic extension constraint.

[0141] A method according to the invention particularly relates to the application of a physical constraint and the characterization of at least one cell in suspension, said characterization relates to at least one of the following aspects: the size, the shape, the appearance of the external membrane, the appearance of the cytoplasm, the presence of at least one marker on the surface of the cell, the protein content of the cell and the nucleic acid content of the cell.

[0142] More particularly, the subject of the invention is a method according to the invention comprising the application of at least one physical constraint to at least one cell in suspension, said physical constraint being characterized in that the cell is subjected to:

[0143] - at least one mechanical impact of intensity between 1 and 300 m / s, preferably between 1 and 100 m / s, preferably between 1 and 10 m / s and / or for a duration of less than 1 ps and / or

[0144] - at least one shear stress of intensity between 10' 3 kPa and 10 5 kPa, preferably between 10' 3 kPa and 10 4 kPa, preferably between 0.1 kPa and 10 3 kPa, preferably between 1 kPa and 10 2 kPa and / or for a duration between 1 ps and 10,000 s, preferably between 1 ps and 100 s, preferably between 10 ps and 1 s, and / or

[0145] - at least one compressive stress of intensity between 10'3 kPa and 10 5 kPa, preferably between 10' 3 kPa and 10 4 kPa, preferably between 0.1 kPa and 10 3 kPa, preferably between 1 kPa and 10 2 kPa and / or for a duration between 1 ps and 10 s, preferably between 1 ps and 1 s, preferably between 10 ps and 10 ms and / or

[0146] - at least one extension constraint of intensity between 10' 3 kPa and 10 5 kPa, preferably between 10' 3 kPa and 10 4 kPa, preferably between 0.1 kPa and 10 3 kPa, preferably between 1 kPa and 10 2 kPa and / or for a duration between 1 ps and 10 s, preferably between 1 ps and 1 s, preferably between 10 ps and 10 ms.

[0147] More particularly, in a method according to the invention, the parameters i) fluid flow rate and ii) fluid viscosity are chosen to reproduce stress intensities and residence time reproducing the stresses that are experienced by the cells during a particular bioprocess. As the fluid circulates through the device, a streamline is generated. As the fluid circulates through the device, the cell follows the streamline developed by the fluid movement and its interaction with the channel geometry. By calculating or measuring the hydrodynamic stress on this line and the cell movement speed, the stress and residence time for the cell can be deduced.

[0148] Preferably, in a method according to the invention, the fluid flow rate is between 10' 3 and 10 ml / min, preferably between 10' 3and 1 ml / min. Preferably, in a method according to the invention, the residence time of the cells is between 1 ps and 10,000 s, preferably between 1 ps and 1 s, preferably between 10 ps and 100 ms.

[0149] More particularly, the subject of the invention is a method according to the invention comprising the application of at least one physical constraint to at least one cell in suspension, then the characterization of said cell, the method further comprising a step of predicting the physiological state of a cell by a first classification model previously trained, from the characteristics determined during step b).

[0150] More particularly, the invention also relates to a method according to the invention comprising the application of at least one physical constraint to at least one cell in suspension, then the characterization of said cell, the method further comprising a step of predicting the physiological state of a cell by a first classification model previously trained, from the characteristics determined during step b), said first classification model comprising: a machine learning algorithm, a supervised, semi-supervised or unsupervised learning neural network, previously trained with a training data set.

[0151] More particularly, the invention also relates to a method according to the invention further comprising a step of monitoring the deformation of a cell at different times during its stay in said microfluidic channel, by a second classification model previously trained, from the characteristics determined during step b). According to a third aspect, the invention also relates to a classification model, previously trained on a training data set to predict, in a method according to the invention, a physiological state of a cell after the application of at least one physical constraint.

[0152] According to a fourth aspect, the invention relates to the use of a device or method according to the invention, or of a classification model according to the invention for the characterization of cells of the following type: prokaryotic cell, eukaryotic cell, animal cell, plant cell, human cell, stem cell, epithelial cell, fibroblast, blood cell, genetically modified cell or synthetic cell mimic.

[0153] More particularly according to this fourth aspect, the invention relates to the use of a device or a method according to the invention, or of a classification model according to the invention for determining the “capacitance” of a cell.

[0154] Even more particularly according to this fourth aspect, the invention relates to the use of a device or a method according to the invention, or of a classification model according to the invention for the definition of at least one parameter of a bioprocess.

[0155] Even more particularly, the subject of the invention is the use of a device or a method according to the invention, or of a classification model according to the invention for the definition of at least one parameter of a bioprocess chosen from: bioprinting, cell therapy and bioproduction.

[0156] The present invention will be better understood by reading the following examples, which are given to illustrate the invention and not to limit its scope. In particular, it will be possible to imagine variants of the invention comprising only a selection of the characteristics described below, isolated from the other characteristics described, if this selection of characteristics is sufficient to confer a technical advantage or to differentiate the invention from the prior art.

[0157] Figures

[0158] Figure 1 is a schematic representation of examples of segments A, B, C, and D.

[0159] Figure 2 is a schematic representation of an example of a device in which several types of physical stresses are applied to the cells, here a sequence of shear and elongation stresses. The lower graph represents an enlargement of the upper graph. The cell is subjected to repeated stress. This type of stress can also be defined as dynamic deformation, or oscillatory deformation.

[0160] Figure 3 shows the steps in isolating a cell.

[0161] Figure 4 shows the measurement of the minor axis and major axis of the ellipse.

[0162] Figure 5, in Example 2, represents the viability state of human mesenchymal stem cells AD-MSC after application of shear stresses of varying intensity and duration. The percentage of cells in a particular physiological state is given as a function of the duration of application (time) expressed in seconds and the intensity (stress) expressed in Pa, of the shear stress. Figure 5 thus represents the percentage of viable cells (panel A), lysed cells (panel B), necrotic cells (panel C) and cells undergoing apoptosis (panel D).

[0163] Figure 6, in Example 3, represents the viability state of fibroblast cells after application of a shear stress. Fibroblasts are characterized after the application of shear stresses of varying intensity and duration. The percentage of cells in a particular physiological state is given as a function of the duration of application (time), expressed in seconds, and the intensity (stress), expressed in Pa, of the shear stress. Figure 6 thus represents the percentage of viable cells (frame A), lysed cells (frame B), necrotic cells (frame C) or undergoing apoptosis (frame D).

[0164] Figure 7, in Example 4, represents the proportion of undifferentiated AD-MSC stem cells (white circle) and differentiated cells (black circle) after application of shear stress, as a function of the duration of application (time), expressed in seconds, and the intensity (stress), expressed in Pa of the shear stress.

[0165] Figure 8, in Example 5, represents the viability state of HEK293T cells after application of an elongation stress. HEK293T cells are characterized after the application of elongation stresses of varying intensity and duration. The percentage of cells in a particular physiological state is given as a function of the duration of application (time), expressed in seconds, and the intensity (stress), expressed in Pa, of the elongation stress. Figure 8 thus represents the percentage of cells in apoptosis (panel A), viable (panel B), necrotic (panel C) or lysed (panel D).

[0166] Figure 9, in Example 6, represents the viability status of fibroblast cells after application of shear stress, where viability is measured by cytometry (histogram bars) or by trypan blue staining (black circles). Figure 10, in Example 7, represents the viability status of HEK293T cells after application of shear stresses of varying intensity and duration. The percentage of viable cells, measured by trypan blue staining, is given as a function of the duration of application (time) expressed in seconds, and the intensity (stress), expressed in Pa, of the shear stress. The percentage of viable cells is expressed after a small number of passages (HEK293T P07, white triangles) or after a higher number of passages (HEK293T P18, black circles).

[0167] Figure 11, in Example 8, represents the viability status of fibroblast cells after application of shear stresses of varying intensity and duration, according to two series of measurements carried out on the same cell sample (P06), represented respectively by white triangles and black circles. P06 denotes the 6 ème generation of cell culture passage.

[0168] Figure 12, in example 9, represents in three dimensions and on a logarithmic scale the percentage of cell viability as a function of the intensity of a hydrodynamic constraint and the residence time.

[0169] Figure 13, in Example 9, represents in two dimensions the percentage of viability of fibroblasts on the y-axis, as a function of the intensity of a hydrodynamic shear stress, on the x-axis, and of the residence time, represented according to the dot pattern.

[0170] Figure 14, in Example 9, represents in two dimensions the percentage of cell viability as a function of the intensity of a hydrodynamic constraint and the residence time.

[0171] Figure 15, in Example 10, is a histogram representing cell viability as a function of shear stress intensity, in kPa. For each stress intensity, the viability value was measured after several experiments.

[0172] Figure 16, in Example 10, is a graphical representation of the standard deviation (light bar), standard error (black bar), and coefficient of variation (gray bar) of the various measurements, as a function of shear stress intensity, in kPa.

[0173] Figure 17, in Example 10, is a histogram plotting percent cell viability versus shear stress intensity, in kPa.

[0174] Figure 18, in example 11, represents: i) on the left, the percentage of cell viability of different types of cells as a function of the intensity of the hydrodynamic stress applied, in kPa, ii) on the right, for each of the cells studied, the percentage of cell viability as a function of the hydrodynamic stress that is applied.

[0175] Figure 19, in Example 12, represents the percentage of cell viability of cells as a function of the intensity of the applied shear stress, in kPa. The symbols P07 (circle), P08 (square), P09 (triangle) and P10 (star) represent the percentage of cell viability of cells defined according to the number of passages of the cell in culture.

[0176] Figure 20, in Example 12, represents the percentage of cell viability of cells as a function of the intensity of the applied shear stress, in kPa. P09, P10 and Pli represent the percentage of cell viability of cells defined according to the number of passages of the cell in culture.

[0177] Figure 21, in example 13, represents the succession of the same hydrodynamic stress.

[0178] Figure 22, in Example 13, represents the percentage of cell viability of cells as a function of the intensity of the applied hydrodynamic stress, in kPa.

[0179] Figure 23, in Example 13, represents the percentage of cell viability of cells as a function of the number of cycles of application of the stress, for a stress of 0.2 kPa.

[0180] Figure 24, in Example 14, depicts in images the steps of locating and isolating a cell from an original image.

[0181] Figure 25, in example 15, represents in image the steps of detecting the axes of a cell from an original image.

[0182] Figure 26, in example 15, represents the deformability of a cell as a function of its initial diameter, under the effect of a shear stress.

[0183] Figure 27, in example 15, represents the deformability of a cell as a function of its initial diameter, under the effect of a mechanical impact stress.

[0184] Figure 28, in example 16, schematically represents an “autoencoder” type neural network with an example of transformation of an initial image (input) into a final image (output) after processing by this network.

[0185] Figure 29, in example 16, represents the differences in image processing by the supervised network (U-net) of an initial image preprocessed or not by an unsupervised network (autoencoder), line 1 represents the results without application of autoencoder, line 2 represents the results with application of a “variational autoencoder”, line 3 represents the results with application of a “denoising autoencoder”.

[0186] Figures 30 A and 30 B, in example 17, represent the contour results after applying the different neural networks and the associated metrics, then the difference in calculating the deformability compared to a contour made manually (Control at 0).

[0187] Figure 31, in Example 18, represents a classification result of three cells with, from left to right, a necrotic cell, an apoptotic cell and a living cell.

[0188] EXAMPLES

[0189] Example 1: Materials and methods for the characterization of cells after the application of physical stress

[0190] AD-MSCs are cultured in the culture medium marketed under the name MSC-Growth, with a seeding concentration of around 2500 cells per cm 2 The medium was changed every two days. To achieve 70% confluence, the cells were incubated for seven days in a T175 flask.

[0191] The fibroblast cells are cultured in the culture medium marketed under the name DMEM GLUTAMAX - Gibco, with a seeding concentration of around 5500 cells per cm 2 To achieve 80% confluence, the cells were incubated for seven days in a T175 flask.

[0192] HEK293T cells are cultured in the culture medium marketed under the name DMEM GLUTAMAX - Gibco with a seeding concentration of around 12,000 cells per cm 2 To achieve 80% confluence, the cells were incubated for four days in a T175 flask. During the incubation period, the temperature was maintained at 37°C and the CO2 concentration was around 5%.

[0193] To detach the cells from the flask surface, first the culture medium was removed. The cells attached to the flask were rinsed with 15 ml of PBS. Then, 5 ml of 0.5% Trypsin-EDTA was added to the flask. The trypsin application time was two minutes at 37°C, then 10 ml of culture medium containing fetal calf serum was added to the flask to stop the reaction. The suspension was placed in a tube and centrifuged at 1200 rpm / 210 g for 5 minutes. The liquid was removed and the cells were suspended in a solution of PBS and Ficoll at a concentration of one million per ml.

[0194] The microfluidic device for shear stress consists of a PDMS microfluidic channel with a diameter of 50 micrometers and a length of 10 cm. The cells were suspended in a fluid (a solution of PBS and Ficoll) with a viscosity of 1.91 mPa.s.

[0195] The microfluidic device for elongational stress consists of a main microfluidic channel with a diameter of 162 micrometers and a length of 1 cm. The channel cross-section first decreases from 162 to 30 micrometers and then increases from 30 to 162 micrometers. This change in cross-section occurs over a distance of 360 micrometers. The viscosity of the suspending fluid (a solution of PBS and Ficoll with a volume concentration of 60% Ficoll) is 1.91 mPa.s.

[0196] The cell suspension and cell-free fluid are injected into the device using syringe pumps and 3 mm diameter PEEK tubing.

[0197] Cells are injected into the device at a flow rate of 25 to 800 microliters per minute. Measurements and cell harvesting are performed after hydrodynamic stability within the system has been achieved.

[0198] After passing through the stress zone, the cells were harvested. The harvested cell suspension was centrifuged to remove the suspending fluid (Ficoll solution and PBS) and replace it with the labeling buffer.

[0199] For the viability test, cells are labeled with annexin V, a marker for apoptotic cells, or propidium iodide, a marker for necrotic cells. For this, a population of 100,000 cells corresponding to each injection rate was suspended in 100 microliters of labeling buffer. Then 2 microliters of annexin V marker and 2 microliters of propidium iodide were added to the buffer. The cell suspension with viability markers was incubated for 15 minutes in a dark place. Then, the cells were centrifuged and rinsed with the labeling buffer.

[0200] For the stemness and differentiation status testing of AD-MSC stem cells, the cells were labeled using the BD Human Mesenchymal Stem Cell Analysis Kit (BDB562245). The kit contains the positive hMSC markers: CD90, CD105, CD73 and CD 44 and the negative hMSC markers: CD34, CD11b, CD19, CD45 and HLA-DR. For the labeling procedure, 100,000 cells were suspended in 100 microliters of BD Stain Buffer (FBS), then 5 microliters of each positive marker and 20 microliters of each negative marker were added to the buffer. The cell suspension with the markers was incubated for 15 minutes in a dark place. Then, the cells were centrifuged and rinsed with the labeling buffer.

[0201] The cells are studied by cytometry (FACS Canto II) and characterized according to their state: viable cell, lysis, necrosis, apoptosis or stemness. Each measurement point corresponds to an analysis of at least 50,000 cells.

[0202] From the results obtained, a mathematical model for predicting the physiological state of cells as a function of the intensity and duration of the stress is established. The model is a mathematical expression that makes a relationship between the parameters, for example a polynomial expression, a power law, a sum of sines or other 2D or 3D mathematical expressions. For example, we can use a power law in the form y = ax k+ c which establishes a relationship between x and y. In this model a is a constant of proportionality, k is the exponent and c is the error term. Using this method, we model the physiological state of the cells before and / or after passing through the stress zone as a function of the stress intensity and the residence time under stress. For this, the model is established as follows: Physiological state = A x Stress 6 + C; Residence time = D / Stress. The parameters A, B, C and D differ for each cell type, stress type and passage number.

[0203] Example 2: Characterization of the viability state of AD-MSCs stem cells after the application of shear stress

[0204] AD-MDCs stem cells were cultured and then subjected to shear stress of varying intensity and duration. After application of these stresses, the viability status of the cells was characterized as a function of the intensity of the shear stress and the residence time of the cells under stress. The characterization of the physiological state (viable, lysis, necrosis or apoptosis) of the cells was carried out as described in Example 1.

[0205] The mathematical model defined in this case is as follows: State = A x Constraint B + C ; Time = D / Constraint.

[0206] The values ​​A, B, C and D as defined for each of the possible physiological states are as follows:

[0207] Table 1

[0208] The results obtained are presented in Figure 5. These results show that human mesenchymal stem cells AD-MSC are sensitive to the intensity of the shear stress and the residence time under the stress. We observe that AD-MSC cells have a mechanical capacitance to the stress of approximately 1000 Pa with a residence time of approximately 0.25 s. Beyond these values, AD-MSC stem cells no longer withstand the stress and undergo mortality. The mortality pathway by lysis or necrosis is the most present, however the level of apoptosis is negligible in this range of stress and residence time.

[0209] Example 3: Physiological state of fibroblasts after application of shear stress

[0210] Fibroblast cells were cultured and then cell samples were subjected to shear stresses of varying intensity and duration. After application of these stresses, the viability or lysis, necrosis or apoptosis of the cells was determined as described in Example 1.

[0211] The mathematical model defined in this case is as follows: State = A x Constraint B + C ; Time = D / Constraint

[0212] The values ​​A, B, C and D as defined for each of the possible physiological states are as follows:

[0213] Table 2

[0214] The results obtained are presented in Figure 6. These results show that fibroblast cells are highly sensitive to shear stress and residence time under stress. We can see that fibroblast cells up to 900 Pa and for 0.04 s, are able to withstand shear stress without being damaged, i.e. without showing any signs of lysis, apoptosis or necrosis. These values ​​are the mechanical capacitance of fibroblast cells facing Tl shear stress. On the other hand, beyond this mechanical capacitance, the physiological response, i.e. the loss of viability, of fibroblasts is considerable. Indeed, fibroblasts do not resist the stress and the viability level of 85% at 900 Pa suddenly drops to 30% at 1000 Pa and reaches a level of less than 5% viability at 2000 Pa.Fibroblast cell mortality is caused rather by the lysis pathway and other physiological states are negligible.

[0215] Example 4: Differentiation state of human mesenchymal stem cells AD-MSC after application of shear stress

[0216] AD-MSC stem cells were cultured as described in Example 1. As a prerequisite, AD-MSC stem cells were characterized by cytometry to confirm their stemness before applying a shear stress of varying intensity and duration. After passing through the stress zone, the cells were harvested and resuspended in DMEM (+) culture medium. The differentiation state was characterized after their re-culture. Seeding was performed at 80% confluence. After three weeks of incubation, the cells were harvested, labeled using the “BD Human Mesenchymal Stem Cell Analysis Kit (BDB562245)” and analyzed by flow cytometry (FACS Canto II). Each measurement point corresponds to a minimum of 30,000 cells. The characterization was done by flow cytometry which allows here to identify the state of the cells after application of the constraint in a binary way: differentiated or undifferentiated.The procedure of cytometer characterization was carried out as indicated in Example 1.

[0217] The mathematical model defined in this case is as follows: Undifferentiated state: Time = D / Stress where D = 30.6 in the range of 0.147 s < time < 0.39 s and 78 < stress < 2082 Pa

[0218] The results obtained are presented in Figure 7. These results show the absence of differentiated cells. This indicates that human mesenchymal stem cells AD-MSC maintain their stemness after undergoing shear stress in the range represented in the figure. The physiological state of AD-MSC cells, here the maintenance of stemness, was therefore not affected by the stress and residence time in the range 0-2100 Pa and 0.015-0.4 s.

[0219] Example 5: Viability status of HEK293T cells after application of an elongation stress

[0220] HEK293T cells were cultured and then cell samples were subjected to elongation stresses of varying intensity and duration. After application of these stresses, the viability or lysis, necrosis or apoptosis of the cells was determined as described in Example 1.

[0221] The results obtained are presented in Figure 8. These results show that HEK293T cells have a high survival rate against elongational stress in the stress range between 10 and 110 Pa and the residence time between 0.1 ms and 0.8 ms. The states of lysis, necrosis and apoptosis are negligible compared to the viable state.

[0222] Example 6: Viability status of fibroblast cells after application of shear stress, as measured by two protocols

[0223] Fibroblast cells were cultured and then cell samples were subjected to shear stresses of varying intensity and duration. After application of these stresses, the viability of the cells was determined by two different protocols: flow cytometry analysis and trypan blue counting. Viability is illustrated as a function of shear stress intensity. The residence time under stress corresponds to the following equation: time (s) = 30.6 / shear stress intensity (Pa)

[0224] The cells were harvested after passing through the stress zone. They were then divided into two batches for analysis by flow cytometry and Trypan Blue. For flow cytometry analysis, the batch was labeled with Annexin V and propidium iodide. Subsequently, the labeled cells were analyzed by flow cytometry. Each measurement point corresponds to a minimum of 50,000 cells. For Trypan Blue analysis, the cell batch was mixed with Trypan Blue. Three counts were performed on the entire surface of the Malassez slide. The error bar corresponds to these three counts. The results are presented in Figure 6.

[0225] Example 7: Viability status of HEK293T cells after application of shear stress

[0226] HEK293T cells were cultured and then cell samples were subjected to shear stress of varying intensity and duration, as described in Example 1. HEK293T cells were harvested after passage through the stress zone and then mixed with Trypan Blue. Three counts were performed across the entire surface of the Malassez slide. The value corresponds to the average of these three counts.

[0227] The results obtained are presented in Figure 10. These results show that the number of cell passages in the cell culture clearly affects the mechanical capacitance of HEK293T cells. These cells with a P07 passage (7 passages) perfectly resist the stress with a 100% viability rate for stresses below 200 Pa. But beyond this mechanical capacitance, the same HEK293T cells passage P07 experience a drop in viability. However, the same cell line but with a P18 passage (18 passages) loses this mechanical capacitance and does not resist the slightest stress.

[0228] Example 8: Demonstration of the reproducibility of measurements

[0229] The characterization of the physiological state of fibroblast cells after application of a shear stress was carried out in duplicate. Both measurements were made on the same cell sample (P06). The reproducibility of the experiment has an average error of 3.45% of viability. The analyses carried out by the system are therefore reproducible. The results obtained are presented in Figure 11.

[0230] Example 9: Mechanical capacity of cells as a function of stress intensity and residence time under stress

[0231] Residence time is defined as the period during which cells are exposed to hydrodynamic stress. In a microfluidic device and method according to the invention, the stress is maintained at a constant intensity, while the residence time depends on the flow rate and viscosities in which the cells are suspended. This approach allows the two parameters to be decoupled and their effect to be studied independently. Fibroblasts were suspended in fluids with a viscosity of 1, 5, 10, 15 or 20 mPa.s and injected through the microfluidic capillary tube with a flow rate between 117 and 942 pl / min. This configuration covered the shear stress intensity in a range between 0.16 and 25.59 kPa and a residence time between 12.5 and 100.7 ms.

[0232] Figure 12 and Figure 13 show a mapping of fibroblast cell viability as a function of stress intensity and residence time. These two figures represent the same data with a 3D and 2D view and linear and logarithmic scales. The white dots are the experimental data and the surface is the fit of the mathematical model. It is observed that within the indicated ranges, for a given residence time, increasing the stress intensity decreases cell viability. However, for a constant stress intensity, cell viability is independent of residence time. Figure 14 represents fibroblast viability only as a function of stress intensity, while the gray shades identify the residence time.Thanks to the three representations of the same data, it is possible to conclude that the viability and mechanical capacity of fibroblasts do not depend on the residence time in the range of 12.5 to 100.7 ms. These results indicate that, in this case, the viability of fibroblasts can only be characterized as a function of the intensity of the stress.

[0233] In a method according to the invention, the stress intensity and the residence time are completely decoupled. One of these parameters can be kept constant and the other parameter can be varied. Thus, we are able to have a detailed overview of the effect of residence time and shear stress intensity as two independent parameters.

[0234] Example 10: Device reproducibility and precision

[0235] In order to achieve reproducibility of experiments and results, the inventors measured the mechanical capacity of MDCK (Madin-Darby Canine Kidney) cells 6 times. To rule out biological variability that will affect the evaluation of errors related to machine performance and procedure, a population of cells was divided into 6 batches. The measurement of mechanical capacity was performed on each batch of cells. The cells were exposed to shear stresses with an intensity of 0.175, 0.469, 2.716 and 6.397 kPa, and then their viability was measured by cytometer. The standard deviation, standard error and coefficient of variation were calculated with the following formulas:

[0236] The results show that the standard error for the 6 tests is less than 1% in the worst case. This example perfectly demonstrates the precision and performance of the machine and the procedure that leads to reliable and very precise measurements. This precision allows for in-depth analysis with significant reliability as shown in Figures 15 to 17.

[0237] Example 11: Mechanical capacity of different cell lines

[0238] With a device according to the invention, it is possible to measure the mechanical capacity against hydrodynamic constraints of different cells: primary line, immortalized cell line, stem cells, etc. The mechanical capacity of the following cell lines was analyzed: Adipose tissue-derived mesenchymal stem cell (AD-MSC), Wharton's jelly-derived mesenchymal stem cells (WJ-MSC), Bone marrow-derived mesenchymal stem cell (BM-MSC), human dermal fibroblasts, Human Umbilical Vein Endothelial Cells (HUVEC), Cancer-associated fibroblasts (CAF), Human embryonic kidney 293T cells (HEK293T) and Madin-Darby canine kidney (MDCK).

[0239] Cells were suspended in two fluids with a viscosity of 1 and 5 mPa.s and injected through a 50 μm diameter microfluidic channel with a flow rate ranging from 85 to 942 μl / min and subjected to a shear stress of 0.11 to 6.4 kPa. The collected cells were labeled with propidium iodide and analyzed by cytometry to quantify the necrotic cell population induced by shear stress. To confirm that cells are only damaged by shear stress upon injection into the microfluidic channel and not by the static syringe pressure, fibroblast cells were injected without passing through the microfluidic channel and analyzed by cytometry. We confirmed that cell viability was unchanged compared to the control cell population viability of 97%.Furthermore, in the case of adherent cells, special care was taken during the experiment to limit their death due to suspension conditions. Furthermore, the time between cell harvest and exposure to shear stress was only 5 min, and identical for all cell lines.

[0240] Figure 18 represents the viability of cell lines after the application of shear stress. The points are the experimental data and the lines are the fitting curves. Such a cell viability evolution profile can be described as having two phases. First, cell viability shows a plateau up to a certain shear stress limit, i.e., the ability of cells to withstand shear stress. Then, after the shear stress that corresponds to the mechanical capacity of the cell, cell viability deteriorates and undergoes a sharp decrease that can reach less than 5% of viable cells. Before the shear stress related to the mechanical capacity of each cell, the viability varies by less than 5% compared to the unsheared control cell population.It is also noteworthy that while all cell types studied show similar trends, each cell line has a specific sensitivity to shear stress as mechanical capacity, AD-MSC: 0.64 kPa, WJ-MSC: 0.35 kPa, BM-MSC: 0.48 kPa, fibroblasts: 0.64 kPa, HUVEC: 0.32 kPa and HEK293T: 0.43 KPa.

[0241] Example 12: Evolution of mechanical capacity with cell aging and incubation time

[0242] To explore the link between cellular aging and its mechanical capacity, fibroblasts were subcultured (passaged) once a week and subjected to shear stress. The experiments were continued for four passages, from P07 to P10. Passaging is the process of subculturing animal cells, with "P07" meaning the 7 èmegeneration of cell passage in culture. Subculture practices were strictly identical between different passages. The viability of fibroblast cells after being subjected to several shear stress intensities was measured for each passage number by flow cytometry. The cells were suspended in fluids with viscosities of 1 and 5 mPa.s, then exposed to shear stress between 0.159 and 6.14 kPa. The flow rate was in the range of 117 to 942 µl / min for both suspension fluids.

[0243] Figure 19 shows that sequential subculture practices do not affect the mechanical capacity of fibroblasts in the face of shear stress. Indeed, they exhibit similar mechanical capacity regardless of their passage number. Up to the shear stress corresponding to the mechanical capacity of the cell 0.577 kPa, the viability of fibroblasts is not affected (96%), while the control viability is 97%. In the range of 0.639 kPa to 4.78 kPa, the viability degrades sharply from 92% to 5% and reaches a plateau below 5%.

[0244] A similar study was applied to HEK293T cells. Unlike fibroblasts, HEK293T cells were subcultured twice weekly from passage number P09 to P11. Therefore, the cell growth time alternates between 3 and 4 days between successive subcultures.

[0245] Cells were suspended in fluids with viscosities of 1 and 5 mPa.s and then exposed to shear stress in the range of 0.11–6.4 kPa. The flow rate was in the range of 85–942 µl / min for both suspension fluids. Cell viability was analyzed by flow cytometry.

[0246] As shown in Figure 20, the mechanical capacity of HEK293T cells is affected by the variation of the incubation period for successive cell subcultures. The incubation period before the application of stress and before the measurement is 3 or 4 days. HEK293T with a 3-day incubation period demonstrates a mechanical capacity of 0.516 kPa, while HEK293T cells with a longer incubation period suffered a weakening of the mechanical capacity which is 0.319 kPa.

[0247] Example 13: Repeating a series of constraints

[0248] To explore the effect of repeated identical stresses on the mechanical capacity of the cell, AD-MSCs were suspended in a fluid with a viscosity of 1 mPa.s and exposed to repeated hydrodynamic stresses. This type of stress is defined as a succession of the same stress in a cyclic manner. Figure 21 shows this succession. The cells undergo a shear stress, then they enter a shear-free zone and are sheared. This cycle was performed for a batch of cells, with a number of cycles equal to 77, 154, 231, 308, 385 and 462, with a stress of 0.2 kPa. This stress is lower than the mechanical capacity of the cells, 0.6 kPa, as shown in Figure 22. The cells, after being exposed to the different numbers of cycles, were analyzed by a flow cytometer to determine cell viability.Figure 23 represents cyclic stress below the mechanical capacity of AD-MSC cell, it does not affect cell viability.

[0249] Example 14: Cell Location and Isolation

[0250] Cell localization and isolation are performed using an algorithm from the original images taken by the fast camera. They consist firstly in the creation of a mask to reduce the size of the original image by superposition. Secondly, image processing functions such as erosion and dilation are applied to reduce the background noise of the images and reveal the location of the cell in a more contrasted way. Then, isolation is performed by defining the region of interest (ROI) (Figure 24).

[0251] Example 15: Positioning and measuring the minor and major axes

[0252] The positioning and measurement of the minor and major axes are performed using a supervised learning model. The first step consists of applying the supervised model (here U-Net) to the resulting cell photo in Figure 24. This allows the identification of the cell size and shape. Second, an edge extraction image processing function is performed to position the cell outline on the image from Figure 24. Then, the minor and major axes are measured, the major axis being the largest diameter and the minor axis being the smallest diameter. These results can be displayed in a graph representing the deformability level as a function of cell size. Here, our results demonstrate the accuracy of our image processing deformability measurements for shear and crash tests.

[0253] Example 16: Improving image quality using an unsupervised model

[0254] The images resulting from the cell localization and isolation steps are not always of good quality to allow the measurement of the major and minor axes. This is because these images are noisy enough to prevent the supervised model from segmenting the cell. In order to improve this image quality, the application of an unsupervised model is necessary. Here, it is shown that the application of an “autoencoder” type neural network allows the reconstruction of the image resulting from the ROI extraction of better quality (Figure 28). Figure 29 demonstrates the relevance of this approach. In Figure 29, line 1 represents the results without the application of “autoencoder”, line 2 represents the results with the application of a “variational autoencoder”, line 3 represents the results with the application of a “denoising autoencoder”).

[0255] Example 17: Improving cell detection and deformability measurements by coupling an unsupervised and supervised model

[0256] The coupling of an unsupervised and supervised model can be used to improve cell detection within an image as well as its deformability measurement. In this example, we demonstrate this approach by pairing autoencoder neural networks and U-Net on images from shear measurement (Figure 30A).

[0257] The numerical values ​​are as follows (Table 3):

[0258] Table 3

[0259] We can observe that the application of the supervised network (U-Net) alone does not allow to detect and follow all the kinematics of the cell while the coupling with an unsupervised model (autocoder VAE or DAE) improves the detection and measurement (higher Precision, Recall and F& score) (Figure 30B).

[0260] Also, the improvement of the measurement is described by subtracting the deformability measurement compared to a standard manual approach. That is, each image was processed by the user without image processing or application of a learning model.

[0261] Example 18: Prediction of the physiological state of a cell by a classification model using supervised learning.

[0262] A supervised learning model can be used to classify the physiological state of the cell by image analysis. Here the example shows a classification result of 3 cells annotated as necrotic (A), apoptotic (B) and alive (C).

Claims

CLAIMS [Claim 0001] Device for applying at least one hydrodynamic stress of determined intensity and duration to at least one cell suspended and moving in said device, and for characterizing said cell, during the application of said stress and optionally for characterizing said cell after the application of said stress, the device comprising: - a microfluidic circuit comprising at least one segment, said segment comprising at least: i) a main channel configured for the circulation of a fluid containing said cell in suspension, ii) a fluid inlet and a fluid outlet, iii) means for introducing and establishing a flow of said fluid inside said channel, and - a means for characterizing said cell during the application of said stress, characterized in that said at least one segment is configured for the application to said cell of at least one hydrodynamic stress chosen from: a hydrodynamic compression stress, a hydrodynamic shear stress and a hydrodynamic extension stress, and for the optional application of at least one mechanical impact to said cell. [Claim 0002] Device according to the preceding claim, in which the characterization of said cell has as its object at least one criterion chosen from: the size, the shape, the appearance of the external membrane, the appearance of the cytoplasm, the presence of at least one marker on the surface of the cell, the protein content of the cell and the nucleic acid content of the cell. [Claim 0003] Device according to one of the preceding claims, characterized in that said at least one segment A, configured for the application to said cell of a compressive stress, comprises, or is constituted by, a first channel joined, at the same level, by two channels, each making an angle of between 30 and 150 degrees with said first channel. [Claim 0004] Device according to one of the preceding claims, characterized in that said at least one segment B, configured for the application to said cell of a shear stress, comprises, or is constituted by, a channel with a diameter of between 10 and 2000 pm, preferably between 20 and 200 pm. [Claim 0005] Device according to one of the preceding claims, characterized in that said at least one segment C, configured for the application to said cell of an extension constraint, comprises, or is constituted by, i) a channel of increasing or decreasing section or ii) a first channel joined by a second channel in which the circulation of the fluid takes place in a different direction, preferably opposite, to that of the first channel. [Claim 0006] Device according to one of the preceding claims, characterized in that said at least one segment D, configured for the application to said cell of a mechanical impact, comprises, or is constituted by, a first channel within which the line of path of the cells encounters an obstacle, such as in particular the wall of a second channel. [Claim 0007] Device according to any one of the preceding claims, characterized in that said circuit comprises: at least one segment A and / or at least one segment B and / or at least one segment C, and optionally at least one segment D, combined with each other. [Claim 0008] Device according to any one of the preceding claims, characterized in that said circuit is configured for the application to said cell of at least one sequence comprising, or consisting of, at least two successive physical constraints of a different nature. [Claim 0009] Device according to any one of claims 1 to 7, characterized in that said circuit is configured for the application to said cell of at least one sequence comprising, or consisting of, at least two successive hydrodynamic constraints of the same nature, and optionally at least two successive mechanical constraints of the same nature. [Claim 0010] Device according to any one of the preceding claims, characterized in that the intensity of at least one hydrodynamic stress applied to the cell is between 10' 3 kPa and 10 5 kPa, preferably between 10' 3 kPa and 10 4 kPa, preferably between 0.1 and 10 3 kPa, preferably between 1 and 10 2 kPa and / or in that the total duration of the application of said at least one hydrodynamic constraint is between 1 ps and 10000 s, preferably between 1 ps and 100 s, preferably between 1 ps and 1 s, preferably 10 ps and 100 ms. [Claim 0011] Device according to any one of the preceding claims, characterized in that the characterization of said cell after the application of said at least one constraint is carried out for a period of between 0 and 120 days, preferably between 0 and 30 days, preferably between 0 and 1 day after the application of said at least one physical constraint. [Claim 0012] Device according to any one of the preceding claims, characterized in that the characterization of said cell during the application of said at least one constraint comprises, or consists of, at least one punctual characterization or at least one characterization carried out for a duration of between 1 ps and 10000 s, preferably 1 ps and 100 s, preferably between 1 ps and 1 s, preferably 10 ps and 100 ms. [Claim 0013] Device according to any one of the preceding claims, characterized in that said means for characterizing said at least one cell is chosen from: a cytometer, a microscope, a means for analyzing the protein content of the cell, a means for analyzing and sequencing the nucleic acids of said cell, and an image capture means combined with an image analysis means. [Claim 0014] Device according to the preceding claim, characterized in that said image analysis means comprises or is constituted by: a central computer unit comprising software means adapted for image analysis. [Claim 0015] Device according to one of claims 13 or 14, characterized in that said image analysis means further comprises a first classification model, previously trained with a training data set, and comprising a supervised, unsupervised or semi-supervised machine learning algorithm, said first classification model being suitable for predicting the physiological state of a given cell from characteristics of said cell. [Claim 0016] Device according to one of claims 13 to 15, characterized in that said image analysis means further comprises a second classification model, previously trained with a training data set, and comprising a supervised, unsupervised or semi-supervised machine learning algorithm, said second classification model being suitable for detecting and monitoring the deformation of a given cell in response to at least one physical constraint. [Claim 0017] Method for applying at least one physical stress of determined intensity and duration to at least one cell in suspension, and for characterizing said cell during said application, and optionally after said application, the method comprising the following steps: a) depositing and circulating a fluid containing said at least one cell in suspension in a microfluidic circuit comprising at least one segment, said segment comprising at least: i) a main channel configured for the circulation of a fluid containing said cell, ii) a fluid inlet and a fluid outlet, iii) means for introducing and establishing a flow of said fluid inside said channel, and b) characterizing said cell during the application of said at least one stress; and optionally after the application of said at least one stress, characterized in that said at least one segment is configured for the application to said cell of at least one hydrodynamic stress chosen from: a hydrodynamic compressive stress and / or a hydrodynamic shear stress and / or a hydrodynamic extension stress, and optionally at least one mechanical impact. [Claim 0018] Method according to the preceding claim, in which the characterization of said cell has as its object at least one criterion chosen from: the size, the shape, the appearance of the external membrane, the appearance of the cytoplasm and the presence of at least one surface marker. [Claim 0019] Method according to one of claims 17 or 18, in which the cell is subjected to: - at least one shear stress of intensity between 10' 3 kPa and 10 5 kPa, preferably between 10' 3 kPa and 10 4 kPa, preferably between 0.1 kPa and 10 3 kPa and / or for a duration between 1 ps and 10,000 s, preferably between 1 ps and 100 s, preferably between 10 ps and 1 s, preferably between 10 ps and 10 ms, and / or - at least one compressive stress of intensity between 10' 3 kPa and 10 3 kPa, preferably between 10' 3 kPa and 10 2 kPa, preferably between 10' 3 kPa and 10 kPa and / or for a duration between 1 ps and 10 s, preferably between 1 ps and 1 s, preferably between 10 ps and 10 ms and / or - at least one extension constraint of intensity between 10' 3 kPa and 10 3 kPa, preferably between 10' 3 kPa and 10 2 kPa, preferably between 10' 3and 10 kPa and / or for a duration of between 1 ps and 10 s, preferably between 1 ps and 1 s, preferably between 10 ps and 10 ms, - and optionally at least one mechanical impact of intensity between 1 and 300 m / s and / or for a duration of less than 1 ps. [Claim 0020] Method according to one of claims 17 to 19, characterized in that it further comprises a step of predicting the physiological state of a cell by a first previously trained classification model, from the characteristics determined during step b). [Claim 0021] Method according to one of claims 17 to 20, characterized in that it further comprises a step of predicting the physiological state of a cell by a first previously trained classification model, characterized in that said first classification model comprises: a machine learning algorithm, a supervised learning neural network or a multi-class probabilistic classification, previously trained with a training dataset. [Claim 0022] Method according to one of claims 17 to 21, characterized in that it further comprises a step of monitoring the deformability of a cell at different times during its stay in said microfluidic channel, by a second classification model previously trained, from the characteristics determined during step b). [Claim 0023] Classification model, previously trained on a training data set to predict, in a method according to any one of claims 17 to 22, a physiological state of a cell during and / or after the application of at least one hydrodynamic constraint. [Claim 0024] Use of a device according to one of claims 1 to 16, or of a method according to one of claims 17 to 22 or of a classification model according to claim 23 for the characterization of cells of the following type: prokaryotic cell, eukaryotic cell, animal cell, plant cell, human cell, stem cell, epithelial cell, fibroblast, blood cell, genetically modified cell or synthetic cell mimic. [Claim 0025] Use of a device according to one of claims 1 to 16, of a method according to one of claims 17 to 22 or of a classification model according to claim 23, for determining the capacitance of a cell. [Claim 0026] Use of a device according to one of claims 1 to 16, of a method according to one of claims 17 to 22 or of a classification model according to claim 23 for the definition of at least one parameter of a bioprocess. [Claim 0027] Use according to claim 26, characterized in that said bioprocess is chosen from: bioprinting, cell therapy and bioproduction.

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