Apparatus and method for characterizing cells subjected to physical stress

Abstract

The present invention relates to a microfluidic device and method for applying at least one hydrodynamic stress of determined magnitude and duration to suspended cells moving within the device, and characterizing the morphological and physiological state of the cells during and optionally after application of the hydrodynamic stress.

Description

[Technical Field]

[0001] The present invention relates to a microfluidic device and method for applying at least one physical stress of determined magnitude and duration to cells in suspension and for characterizing the morphological and physiological state of the cells subjected to the application of this physical stress.

[0002] The present invention is therefore in the field of microfluidic devices and analytical cell biology. [Background technology]

[0003] Currently known means of describing and characterizing the morphology and physiology of cells are based in particular on the use of cell markers. Morphology itself can be used as a marker of the physiological state of a cell. However, these methods only characterize the physiological state of the cell observed at the time of marking, while requiring appropriate markers specific to that physiological state.

[0004] Furthermore, the efficiency of bioprocesses that use animal cells, especially bioprinting processes, bioproduction of cells for cell therapy, and cell-based methods such as production of therapeutic proteins, viral vaccines, or viral vectors, as well as cell injection or harvesting methods, depends on the quality of the cells used in said bioprocess.

[0005] Cell quality is generally related to physiological characteristics such as cell viability, i.e., the ratio of live cells to cells involved in cell death processes (apoptosis, necrosis, lysis), or cell function (the ability to secrete molecules or, in the case of stem cells, to differentiate into cell subtypes). Therefore, the effectiveness of a bioprocess can be strongly influenced by the changes it brings about in cell quality and, therefore, cell physiology. In bioprocesses, one mechanism that significantly impacts quality, particularly cell viability, is the application of hydrodynamic and mechanical stresses generated by the equipment used, such as bioreactors, syringe and sampler systems, or separation processes. Interactions between the suspending fluid and walls induce stress on cells suspended in the fluid. Direct cell-to-wall interactions result in mechanical shocks. Depending on the nature of the method and the technology used, the intensity and duration of physical stresses acting on cells can vary significantly. For example, cell therapy processes can subject cells to stresses of up to 5,000 Pa, while bioprinting processes can impose stresses for durations of up to 30 ms.

[0006] It is therefore desirable to be able to characterize the physiological state of cells during and / or after the application of physical stress in a simple and reproducible manner, particularly by characterizing their morphology, especially for cells that are likely to be used in bioprocesses.

[0007] It is further desirable to characterize the physiological state of cells when subjected to physical stresses that replicate the physical stress conditions imposed on cells during specific bioprocesses.

[0008] WO 2015 / 024690, entitled "Apparatus and method for determining the mechanical properties of cells," relates to a method and an apparatus for determining the mechanical properties of cells, and discloses a study on the deformation of cells subjected to shear stress. This document discloses an apparatus with a single capillary channel in which suspended cells are subjected to shear stress, and a method for measuring the shape of cells flowing through the apparatus. The method uses binarized images. Measurements are performed in real time.

[0009] EP 3796212 A1, entitled "Device for image-based cell classification, method therefor and use thereof," describes a real-time, label-free cell sorting device that includes a microfluidic network that aligns each cell along its long axis, and a classification unit that includes a neural network that classifies the cells based on their images.

[0010] WO 2019 / 006188, entitled "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 determined mechanical parameters are the elastic modulus E at a flow rate of 100 cells / s, the cell fluidity P, and the transit time T. T , travel time T C , cell size D cell , maximum stretch ε max Using machine learning tools, cells are subjected to calibrated shear stress.

[0011] Therefore, there is a need for devices and methods that not only characterize the mechanical properties of physically stressed cells, but also further characterize their morphology and physiological state.

[0012] Therefore, there is a need for an apparatus and method that can generate at least one physical stress, such as occurs during a bioprocess, and determine and control its nature, intensity, and duration to characterize cells that may be subjected to said stress. "Physical stress" is understood to mean any force acting on cells that can generate cellular stress. Such physical stress can be, in particular, a mechanical shock or a hydrodynamic stress, such as elongation, compression, or shear stress. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] International Publication No. 2015 / 024690 [Patent Document 2] European Patent Application Publication No. 3796212 [Patent Document 3] International Publication No. 2019 / 006188 Summary of the Invention

[0014] The inventors have developed an apparatus for applying at least one physical stress of determined intensity and duration to cells in suspension and for characterizing the cells subjected to said at least one physical stress.

[0015] More particularly, the device according to the invention comprises: - applying at least one hydrodynamic stress of determined magnitude and duration to at least one cell suspended and migrating within said device; and optionally subjecting said cells to at least one mechanical shock. It is structured as follows.

[0016] The device according to the invention is configured to characterize the cells during the application of the stress, and optionally to characterize the cells after the application of the stress.

[0017] The device according to the invention comprises, on the one hand, a microfluidic circuit for applying at least one physical stress and, on the other hand, means for characterizing the cells.

[0018] In the device according to the invention, the at least one physical stress is a stress selected from mechanical stress, in particular a mechanical shock, and / or a hydrodynamic stress, such as a hydrodynamic compressive stress, a hydrodynamic shear stress, and / or a hydrodynamic elongational stress. More particularly, in the device according to the invention, the at least one physical stress is a stress selected from a hydrodynamic stress.

[0019] In the device according to the invention, the means for characterizing the cells are selected from all known technical means for observing cells, in particular imaging or cytometry means. More particularly, in the device according to the invention, the means for characterizing the cells during the application of stress are selected from all known technical means for observing cells, in particular imaging or cytometry means.

[0020] The device according to the invention comprises a microfluidic circuit comprising at least one zone configured to apply at least one physical stress, more particularly hydrodynamic stress, to suspended cells, said zone comprising at least: i) a main flow path configured to circulate a fluid containing said suspended cells; ii) a fluid inlet and a fluid outlet; iii) means for introducing and establishing a flow of said fluid within said flow path.

[0021] Therefore, a first object of the present invention is an apparatus for applying at least one physical stress, more particularly at least one hydrodynamic stress, of determined intensity and duration to at least one suspended cell, and for characterizing said cells during and / or after said application, more particularly during and optionally after said application.

[0022] A second object of the present invention is a method for applying at least one physical stress, more particularly at least one hydrodynamic stress, of determined intensity and duration to at least one suspended cell and characterizing said cells during and / or after said application, more particularly during and optionally after said application.

[0023] Another object of the present invention is a classification model, pre-trained on a training dataset, for characterizing cells and predicting their physiological state during and / or after the application of at least one physical stress according to the device and method of the present invention. Such a classification model saves time and money and improves performance (especially the ability to characterize a large number of cells in a short time) compared to existing commercially available tools.

[0024] Finally, the present invention relates to the use of a device, a method or a classification model according to the present invention for characterizing cells and possibly predicting their physiological state during and / or after the application of at least one physical stress.

[0025] The device and method according to the invention have the advantage of generating at least one physical stress, the nature, intensity and duration of which can be precisely determined and controlled in order to characterize the cells subjected to said stress.

[0026] The device and method according to the invention have the advantage that they allow the application of at least one physical stress, in particular a repetition of the same stress and / or a combination of physical stresses of different natures, in an adjustable sequence, each with a precisely defined intensity and duration. This simple and precise tool allows the reproduction of stresses applied during a bioprocess and the rapid monitoring of the state of at least one cell.

[0027] The devices and methods according to the present invention also have the advantage of being able to predict the physiological state of cells during and / or after they have been subjected to physical stresses, and this prediction can be made up to several days during and after the application of these physical stresses.

[0028] Finally, the device and method according to the invention have the advantage of defining the capacity of a cell to undergo at least one determined physical stress while maintaining a morphology and physiological state compatible with the requirements of the bioprocess. By means of the device and method according to the invention it is possible to define for a given cell a map of its capacity to resist at least one physical stress.

[0029] "Competence" refers to the ability of a cell to withstand at least one physical stress of defined intensity and duration without a change in the physiological state of the cell, which in particular refers to differentiation or a transition from a viable state to a lytic, necrotic, or apoptotic state.

[0030] In particular, the use of the device and method according to the invention makes it possible to optimize the bioprocess, and in particular the associated physical stresses, in order to define optimal operating conditions and adapt said stresses to the cells of interest. These conditions focus, for example, on the parameters of the bioprocess (temperature, flow rate, rotation speed) and the suspending fluid (viscosity, osmolality). Optimizing the stresses associated with a bioprocess is highly desirable in order to adapt said stresses to data obtained during the characterization and prediction of the physiological state of cells subjected to these physical stresses. DETAILED DESCRIPTION OF THE INVENTION

[0031] According to a first object, the present invention relates to an apparatus for applying at least one physical stress, more particularly at least one hydrodynamic stress, to at least one suspended cell and for characterizing said cell during and / or after said application, said apparatus comprising: - a microfluidic circuit comprising at least one zone, said zone comprising at least: i) a main flow path configured to circulate a fluid containing said suspended cells; ii) a fluid inlet and a fluid outlet; iii) means for introducing and establishing a flow of said fluid within said flow path; and - means for characterizing said cells; The device according to the invention is characterized in that the at least one area - mechanical shock, - hydrodynamic compressive stress, - hydrodynamic shear stress, and - Hydrodynamic extensional stress The method is characterized in that it is configured to apply at least one physical stress selected from the following to the cells.

[0032] In the context of the present invention, the term "so as to" in "so as to apply physical stress" means "configured to apply physical stress." When indicating an interval or range of values, the stated boundaries are considered to be part of said range of values.

[0033] The term "microfluidic circuit" refers to a circuit that allows the flow of small amounts of fluid (10 -18 ~10 -3 This refers to a circuit designed to process

[0034] "Fluid" is understood to mean a deformable medium suitable for circulation and suspension of cells within the device according to the invention.

[0035] Preferably, in the device according to the invention, the fluid is selected from Newtonian fluids, i.e. fluids whose viscosity does not change under shear stress. The fluid has at least one of the following properties: - a Newtonian viscosity of 1 to 1,000 mPa·s, preferably 1 to 100 mPa·s; - a pseudoplasticity index of 0 to 1, preferably 1 mPa·s; - Osmolality is 250-410mOsmol, which is suitable for cell survival under stress-free conditions. - the pH is physiological under ambient air treatment conditions, so that said pH is between 6.5 and 7.8; and - Contains nutrients that can be assimilated by the cells in suspension, such as glucose, glutamine, or nutrient compositions such as culture medium.

[0036] More particularly, in the device and method according to the invention, the fluid for cell suspension is preferably selected from Newtonian fluids, in particular - Physiological buffer solutions (PBS, HBSS, etc., typically used for cell suspensions. These buffer solutions may contain nutritional compounds such as 0.5-6 g / L glucose solution and 2-4 mM glutamine solution.) - Cell culture media such as DMEM, EMEM, α-MEM - Cell separation solutions such as Ficoll and sucrose solutions - Low molar polymer solutions is selected from.

[0037] When applying the at least one physical stress, the cell concentration is preferably less than 100 million cells / mL, preferably 10,000 to 10 million cells / mL, i.e., the cell volume is preferably at most 30% of the total volume of the suspension.

[0038] In the device according to the invention, the reference fluid and the fluid in which the cells are suspended are circulated in a steady flow without pulsation. The device is therefore configured to apply at least one stress to at least one cell moving in suspension within the device. The device is preferably designed to guide the cells along a predetermined flow line into the microfluidic chip.

[0039] In the devices and methods according to the present invention, the intensity of the stress, also referred to in the drawings as "stress level" or "stress," and the residence time under stress, also referred to in the drawings as "stress duration" or "time," are achieved in a controlled manner. The capture and release of suspended cells is also achieved in a controlled manner.

[0040] The pump(s) control the flow rate and flow pattern. The pumps preferably have a flow rate of up to 1.37 x 10 5 This is an ultra-high pressure of kPa.

[0041] The circulation of fluid within the device according to the invention is initiated and maintained by any suitable device known to those skilled in the art, such as a pump.

[0042] In the device according to the invention, the cross section of the channel can be constant or variable, or conical, e.g., nozzle-shaped, with an increasing or decreasing conical cross section at the end of the channel providing additional stress on the cells in terms of controlled capture and release.

[0043] When a fluid containing suspended cells is circulated in a microfluidic circuit, streamlines are formed, allowing the path of the cells to be controlled. The cell to be characterized is positioned on one of the streamlines, allowing for the control of the stress intensity.

[0044] The preferred diameter of the main channel is 10 to 3,000 μm.

[0045] The flow path is 10 -3 ~10 6 kPa, and preferably 1 to 10 4 It is made of a material suitable for circulating a fluid under pressure of 100 kPa and / or a material suitable for circulating a fluid having a viscosity of 1 to 2,000 mPa·s.

[0046] Cell performance varies depending on the origin (clone, species, organ), culture format (number of doublings in culture, medium, environmental conditions of culture, i.e., agitation or not, temperature, pH), and harvesting method (trypsinization, mechanical harvesting). For example, the type of cell culture, particularly 2D or 3D, adherent or suspension culture, and the type of medium used affect cell performance.

[0047] In a particular embodiment of the device according to the invention, the microfluidic circuit consists of a series of channels made of a suitable material selected from PEEK (polyetheretherketone), PVC (polyvinyl chloride), PTFE (polytetrafluoroethylene), FEP (fluorinated ethylene propylene), PDMS (polydimethylsiloxane) or steel, in particular. In another embodiment, the microfluidic circuit consists of a microfluidic chip made of a material selected from PDMS, polyacrylate, SEBS (styrene ethylene butylene styrene), glass, polycarbonate or ceramic.

[0048] In certain embodiments, devices according to the present invention may comprise flow paths arranged in series and / or flow paths arranged in parallel.

[0049] The fluid circulation in the device according to the invention is characterized by at least one of the following parameters: - a flow rate in the channel that takes into account the characterization of the physical stress applied to the cells, of 0 to 5 mL / min, preferably 0 to 1 mL / min, and / or - Flow rates between 5 μm / s and 300 m / s, and / or - When analysis is performed by software, the throughput of analyzed cells can exceed 1,000 cells / min.

[0050] The total duration of time that the cells are present in the flow channel is preferably 1 μs to 10,000 s, preferably 1 μs to 100 s, preferably 1 μs to 1 s, preferably 10 μs to 100 ms.

[0051] "Cells in suspension" is understood to mean any kind of cell, animal or plant, prokaryotic or eukaryotic. Depending on the diameter of the channel and the magnification of the lens used for image analysis, the device according to the invention can be used to characterize, for example, bacterial, algal or fungal cells.

[0052] The present invention particularly relates to an apparatus for applying at least one physical stress and characterizing at least one suspended cell with respect to at least one of the following aspects: - Cell size - Cell shape, especially cell sphericity - Appearance of the adventitia - The appearance of the cytoplasm, especially its granularity - Presence of at least one marker on the cell surface - the protein content of the cells, and - the nucleic acid content of the cell, in particular the amount of DNA, the amount of RNA, the nucleotide sequence of one or more nucleic acids, whether DNA or RNA.

[0053] "Characterization of a cell" is understood to mean the definition of at least one characteristic of said cell. If more than one characteristic of a cell is defined, characterization of said cell includes the definition of a combination of said characteristics.

[0054] Characterization of the cells determines the physiological state of the cells during or after the application of said at least one stress. Physiology studies the role, function, and mechanical, physical, and biochemical organization of cells and their components, particularly organelles. Physiology also studies the interaction of cells with their environment. Determining the physiological state of a cell includes, among other things, determining the state of differentiation and / or nutrition, proliferation, and related functions, such as motility and sensory functions.

[0055] The physiological state is preferably selected from live cells, dead cells, lysed cells, necrotic cells, apoptotic cells, differentiated or non-differentiated cells, diseased or healthy cells.

[0056] The relationship between physiological state and various aspects of characterization is known to those skilled in the art.

[0057] The physiological state of the cells after application of at least one physical stress can further be compared to the physiological state of the cells before application of said physical stress.

[0058] For purposes of the present invention, the "ability" of a cell is defined as the ability of the cell to be subjected to at least one physical stress of defined intensity and duration while maintaining a physiological state suitable for subsequent use.

[0059] According to a particular aspect, the present invention relates in particular to a device configured to apply at least one physical compressive stress to said cells, said device comprising at least one type (A) section comprising or consisting of a first channel joined at the same height with two channels, each of said two channels forming an angle of 30 to 150 degrees with said first channel, said angle also being referred to as the "flow convergence angle".

[0060] "Hydrodynamic compressive stress" is understood to mean the application of a balanced force towards the interior of the cell, also known as "flow focusing". In the device and method according to the invention, the strength of the compressive stress applied to the cell is 10 -3 ~10 3 kPa, preferably 10 -3 ~10 2 kPa, preferably 10 -3 An example of a type A region is shown in Figure 1.

[0061] According to another particular aspect, the present invention relates in particular to a device configured to apply at least one hydrodynamic shear stress to said cells, said device comprising at least one type B section comprising or consisting of a channel with a diameter between 10 and 2,000 μm, preferably between 20 and 200 μm.

[0062] "Hydrodynamic shear stress" is understood to mean a mechanical stress applied parallel or tangential to the surface of a material. In the device and method according to the invention, the intensity of the shear stress applied to the cells is between 10 -3 ~10 5 kPa, preferably 10 -3 ~10 4 kPa, preferably 0.1 to 10 3 kPa.

[0063] Hydrodynamic shear stress is applied during cell circulation, particularly in capillary channels with a diameter of preferably 10 to 2,000 μm, more preferably 20 to 200 μm. An example of a type B zone is shown schematically in FIG.

[0064] According to another particular aspect, the present invention relates in particular to a device configured to apply at least one physical extensional stress to said cells, said device comprising at least one C-shaped section comprising or consisting of i) a channel with an increasing or decreasing cross section or ii) a first channel joined to a second channel in which fluid flows in a different, preferably opposite, direction to the first channel.

[0065] "Hydrodynamic extensional stress" is understood to refer to the application of a balanced force or extensional stress to the outside of the cell. In the device and method according to the invention, the intensity of the extensional stress applied to the cell is 10 -3 ~10 3 kPa, preferably 10 -3 ~10 2 kPa, preferably 10 -3 An example of a C-type region is shown in Figure 1.

[0066] According to another particular aspect, the present invention relates in particular to a device configured to apply at least one mechanical shock to said cells, said device comprising at least one D-shaped zone comprising or consisting of a first channel in which the trajectory of the cells strikes an obstacle, in particular a wall of a second channel, etc. An example of a D-shaped zone is shown schematically in Figure 1.

[0067] "Mechanical impact" is understood to mean any type of mechanical impact, such as impact with a wall or inertial collision with a surface. In the device and method according to the invention, the intensity of the mechanical impact applied to the cells is 1-300 m / s, preferably 1-100 m / s, preferably 1-10 m / s. The duration of the impact is preferably less than 1 μs.

[0068] More particularly, the device according to the present invention for applying at least one physical stress and characterizing at least one suspended cell comprises: - at least one Type A area, and / or - at least one type B area, and / or - at least one C-shaped section, and / or - At least one D-shaped area wherein the zones are interdigitated with one another.

[0069] Even more particularly, an apparatus according to the present invention for applying at least one physical stress and characterizing at least one suspended cell comprises: - at least one Type A area, and / or - at least one type B area, and / or - at least one C-shaped area, - and optionally at least one D-shaped section wherein the zones are interdigitated with one another.

[0070] The device according to the invention is particularly designed to apply a sequence comprising one or more repetitions of the same type of physical stress at a determined frequency and intensity, and / or to apply a sequence of multiple physical stresses of different types at a determined frequency and intensity.

[0071] "The application of at least one physical stress of determined intensity and duration" - at least one application of a specific physical stress of determined intensity and duration, and optionally followed by one, two, three, four or more repeated applications of said specific physical stress; and / or - at least one application of a sequence of at least two specific physical stresses of determined intensity and duration, and optionally followed by one, two, three, four or more subsequent applications of at least two physical stresses; and / or - continuous application of any type of physical stress described herein is understood to mean

[0072] According to another particular aspect, the present invention relates to a device configured to apply to said cells at least one sequence comprising or consisting of at least two sequences of physical stresses, in particular of different nature, which sequences can be repeated one, two, three or more times.

[0073] According to another particular aspect, the present invention relates to a device configured to apply to said cells at least one sequence comprising or consisting of at least two successive physical stresses of the same nature, in particular, said device being configured to apply to said cells at least 1, at least 2, at least 3, 4, 5, 6, 7, 8, 9, 10 or even more repeated physical stresses.

[0074] An example of this type of device according to the present invention is shown in Figure 2, which shows a device designed to apply multiple compressive stresses to cells, separated by zones designed to prevent the cells from experiencing stress. These series of physical stresses can be considered equivalent to applying dynamic cell deformations. They are repeated sequences of the same stresses.

[0075] More particularly, the device according to the present invention is adapted to have a total intensity of the at least one physical stress applied to the cells of 10 -3 ~10 5 kPa, preferably 10 -3 ~10 4 kPa, preferably 0.1 to 10 3 kPa, preferably 1 to 10 2 kPa.

[0076] More particularly, the device according to the invention is further characterized in that the total duration of application of said at least one physical stress is between 1 μs and 10,000 s, preferably between 1 μs and 100 s, preferably between 1 μs and 1 s, preferably between 10 μs and 100 ms.

[0077] In the microfluidic device according to the invention, the characterization of the at least one suspended cell is performed during and / or after the application of at least one physical stress to the cells. According to a first embodiment, the characterization of the at least one suspended cell is performed during the application of at least one physical stress to the cells.

[0078] According to another embodiment, the device according to the invention is characterized in that the characterization of the cells is carried out after the application of said at least one stress, said characterization being carried out 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 stress.

[0079] More particularly, the device according to the invention is characterized in that said at least one post-stress characterization of said cells comprises or consists of at least one discrete characterization or at least one characterization carried out over a duration of 1 μs to 10,000 s, preferably 1 μs to 100 s, preferably 1 μs to 1 s, preferably 10 μs to 100 ms.

[0080] More particularly, the device according to the invention is characterized in that the means for characterizing said at least one cell are selected from a cytometer, a microscope, means for analyzing the protein content of the cells, means for analyzing and sequencing the nucleic acids of said cells, and image and / or electrical signal acquisition means combined with signal analysis means, for example the device according to the invention may comprise a microelectrode or a photodiode.

[0081] More particularly, the device according to the invention is characterized in that said signal and / or image analysis means comprise or consist of a central computer unit including software means adapted for signal and / or image analysis.

[0082] Even more particularly, the device according to the invention comprising the signal and / or image analysis means further comprises a first and / or second classification model, the presence of at least a first and / or second classification model having the advantage of speeding up the image analysis process and allowing real-time analysis.

[0083] A "classification model" is understood to mean a previously trained machine learning algorithm, in particular a supervised learning algorithm, as well as a training dataset for training said algorithm and an evaluation dataset. A classification model may consist of a computer program that may be written in any suitable computer language known to those skilled in the art. Said computer program may be executed on a computer to generate technical results. Examples of these technical results are described below.

[0084] The training data set may comprise a training set and a model test set. In this way, the model can be tested on a test basis, and the test set can be used to determine whether the model has learned well. The training set and the test set may be different. Alternatively, the test set may be the same as a portion of the training set.

[0085] Even more particularly, the device according to the invention comprising image analysis means further comprises a first classification model pre-trained on a training dataset and comprising a supervised, unsupervised or semi-supervised machine learning algorithm, said first classification model adapted to predict the physiological state of a given cell based on at least one characteristic of said cell.

[0086] In a specific embodiment of the first classification model, the input data are images with objects. The output data are objects with labels indicating the proportion of objects belonging to a particular class. The training algorithm includes at least 10 epochs, the loss calculation is cross-entropy, and the optimization method is Adam (an improved version of gradient descent). The model uses YOLO transfer learning, and the network is supervised (cell images 1733 / annotations 1733). The model preferably includes 106 convolutional layers. The functions performed by the neurons are convolution, addition, softmax, and upsampling. The neuron / layer connections are made. The training dataset can include a number of data pairs, each of which includes a first data item representing at least one feature of the cell and a second data item representing a physiological state of the cell.

[0087] A training dataset can be previously constructed from data obtained in the laboratory by analyzing the characteristics of cells whose physiological state has been determined.

[0088] In particular, said first classification model can be computer implemented to generate technical results, for example consisting of the classification of cells according to their characteristics.

[0089] Using the first classification model, for example, for a given cell: - the intensity of the physical stress applied to the cell (expressed in Pa) - the duration, or residence time, or "time" of the physical stress applied to the cell (expressed in s) - the physiological state of the cells, i.e. cell viability, necrosis, apoptosis or lysis (expressed in %), or differentiated or undifferentiated state Generate a 3D figure representing the

[0090] The first classification model is considered to have reached a satisfactory level of learning for all profiles in the test set, especially if the classification reaches an F1 score of 70% or higher.

[0091] Even more particularly, when the device according to the invention comprises image analysis means, said image analysis means further comprises a second classification model pre-trained on a training data set and comprising a supervised, unsupervised or semi-supervised automatic learning algorithm, said second classification model adapted to detect and monitor deformation of a given cell in response to at least one physical stress.

[0092] In particular, said first classification model can be implemented in a computer to generate technical results, for example in monitoring morphological changes in cells due to various applications of physical stress.

[0093] Preferably, the second classification model uses at least one neural network having the following functions: i) locating and isolating cells from said image; ii) Verify the presence of a single cell. iii) After improving the image quality, the image is processed by masking the cells and determining their contours. iv) The major and minor axes of an ellipse describing the cell outline are placed and measured, and the deformation is defined as the ratio of the major and minor axes.

[0094] According to a specific embodiment, the second classification model includes 440 grayscale-adjusted sliced ​​cell images as input data and segmentation binary masks as output data. The training data includes 420 training images. The training algorithm requires at least 10 epochs, the loss calculation is cross-entropy, and the optimization method is Adam (an improved version of gradient descent). The network type is U-Net. There are 10 layers in total, including five contraction layers and five dilation layers. The functions performed by the neurons are two-dimensional convolution between the image and the filter, i.e., image compression, extraction of a feature vector containing the object of interest, and image decompression. The neuron / layer connection is characterized in that the layer consists of two convolutions, both followed by an activation function (ReLU).

[0095] An example of cell localization and isolation is shown in Figure 3. An example of how the major and minor axes of an ellipse can be positioned and measured is shown in Figure 4.

[0096] The second classification model is considered to have reached a satisfactory level of learning for all profiles in the test set if the classification reaches an F1 score of 65% or higher, preferably at least 80%.

[0097] According to a second object, the present invention relates to a method for applying at least one physical stress of determined intensity and duration to at least one suspended cell and for characterizing said cell after said application, said method comprising the steps of: a) injecting and circulating a fluid containing said at least one suspended cell in a microfluidic circuit comprising at least one zone, said zone comprising at least: i) a main flow path configured to circulate the fluid containing said cells; ii) a fluid inlet and a fluid outlet; iii) means for introducing and establishing the flow of said fluid in said flow path; and b) characterizing the cells after application of the at least one stress; The method according to the invention comprises the step of: - mechanical shock, - hydrodynamic compressive stress, - hydrodynamic shear stress, and - Hydrodynamic extensional stress The method is characterized in that it is configured to apply at least one physical stress selected from the following to the cells.

[0098] The method according to the invention particularly relates to the application of physical stress and characterization of at least one of the suspended cells, said characterization relating to at least one of the following aspects: size, shape, outer membrane appearance, cytoplasmic appearance, the presence of at least one marker on the cell surface, cellular protein content, and cellular nucleic acid content.

[0099] More particularly, the present invention relates to a method according to the present invention, comprising applying at least one physical stress to at least one suspended cell, said physical stress causing said cell to: at least one mechanical impact with an intensity of 1 to 300 m / s, preferably 1 to 100 m / s, preferably 1 to 10 m / s, and / or a duration of less than 1 μs; and / or - 10 -3 ~10 5 kPa, preferably 10 -3 ~10 4 kPa, preferably 0.1 to 10 3 kPa, preferably 1 to 10 2 kPa and / or at least one shear stress with a duration of 1 μs to 10,000 s, preferably 1 μs to 100 s, preferably 10 μs to 1 s, and / or - 10 -3 ~10 5 kPa, preferably 10 -3 ~10 4 kPa, preferably 0.1 to 10 3 kPa, preferably 1 to 10 2 kPa and / or at least one compressive stress with a duration of 1 μs to 10 s, preferably 1 μs to 1 s, preferably 10 μs to 10 ms, and / or - 10 -3 ~10 5 kPa, preferably 10 -3 ~10 4 kPa, preferably 0.1 to 10 3 kPa, preferably 1 to 10 2 kPa and / or at least one elongation stress with a duration of 1 μs to 10 s, preferably 1 μs to 1 s, preferably 10 μs to 10 ms. It is characterized by receiving.

[0100] More specifically, in the method according to the present invention, the parameters i) fluid flow rate and ii) fluid viscosity are selected to reproduce stress intensities and residence times that replicate the stresses experienced by cells during a particular bioprocess. As the fluid flows through the device, streamlines are generated. As the fluid flows through the device, cells follow the streamlines formed by the movement of the fluid and its interaction with the channel geometry. By calculating or measuring the hydrodynamic stresses and cell migration speeds on these streamlines, the stresses and residence times of the cells can be estimated.

[0101] Preferably, in the method according to the invention, the fluid flow rate is 10 -3 ~10 mL / min, preferably 10 -3 Preferably, in the method according to the present invention, the residence time of the cells is 1 μs to 10,000 s, preferably 1 μs to 1 s, preferably 10 μs to 100 ms.

[0102] More particularly, the present invention relates to a method according to the present invention comprising characterizing at least one suspended cell after applying at least one physical stress to said cell, said method further comprising predicting the physiological state of the cell by a pre-trained first classification model based on the features determined in step b).

[0103] More particularly, the present invention also relates to a method according to the present invention comprising characterizing at least one suspended cell after applying at least one physical stress to said cell, the method further comprising predicting a physiological state of the cell by a pre-trained first classification model based on the features determined in step b), wherein said first classification model comprises a machine learning algorithm, a supervised, semi-supervised or unsupervised learning neural network pre-trained on a training dataset.

[0104] More particularly, the present invention also relates to a method according to the present invention, further comprising the step of monitoring the deformation of the cell at different times during its residence in the microfluidic channel by a second pre-trained classification model based on the features determined in step b).

[0105] According to a third aspect, the present invention also relates to a classification model, pre-trained on a training dataset, for predicting the physiological state of cells after the application of at least one physical stress in a method according to the invention.

[0106] According to a fourth aspect, the present invention relates to the use of a device or a method according to the invention, or a classification model according to the invention, for characterizing cell types such as prokaryotic cells, eukaryotic cells, animal cells, plant cells, human cells, stem cells, epithelial cells, fibroblasts, blood cells, genetically modified cells, or synthetic cell mimics.

[0107] More particularly, according to a fourth aspect, the present invention relates to the use of a device or a method according to the invention, or a classification model according to the invention, for determining the performance of a cell.

[0108] Even more particularly, according to a fourth aspect, the present invention relates to the use of a device or a method according to the invention, or a classification model according to the invention, for defining at least one parameter of a bioprocess.

[0109] Even more particularly, the present invention relates to the use of a device or method according to the present invention, or a classification model according to the present invention, for defining at least one parameter of a bioprocess selected from bioprinting, cell therapy, and bioproduction.

[0110] The present invention will be better understood upon reading the following examples, which are presented to illustrate the invention and not to limit its scope: In particular, where a selection of features is sufficient to confer a technical advantage or to differentiate the invention over the prior art, it is possible to imagine a variant of the invention that includes only this selection of features disclosed below, in isolation from the other features disclosed. [Brief explanation of the drawings]

[0111] [Figure 1] 1 is a schematic diagram of an example of areas A, B, C, and D. [Figure 2] This is a schematic diagram of an example of a device in which multiple types of physical stress are applied to cells, in this case a sequence of shear and extensional stresses. The bottom image is an enlarged version of the top image. The cells are subjected to repeated stresses. This type of stress can also be defined as dynamic or oscillatory deformation. [Figure 3] FIG. 1 shows the steps involved in cell separation. [Figure 4] FIG. 1 illustrates the measurement of the minor and major axes of an ellipse. [Figure 5] 1A and 1B are graphs showing the viability of AD-MSC human mesenchymal stem cells (HMSCs) after shear stress of different intensities and durations in Example 2. The percentage of cells in a specific physiological state is shown as a function of the duration (time, expressed in seconds) and intensity (stress, expressed in Pa) of the shear stress. Graph A shows the percentage of live cells (Graph B), lysed cells (Graph C), necrotic cells (Graph D), and apoptotic cells (Graph D), respectively. [Figure 6] Graphs showing the viability of fibroblasts after shear stress in Example 3. Fibroblasts were characterized after shear stress of different intensities and durations. The percentage of cells in a specific physiological state is shown as a function of shear stress duration (time, expressed in seconds) and intensity (stress, expressed in Pa). Graphs A show the percentage of viable cells (Graph A), lysed cells (Graph B), necrotic cells (Graph C), and apoptotic cells (Graph D), respectively. [Figure 7] 1 is a graph showing the ratio of undifferentiated AD-MSC stem cells (white circles) to differentiated cells (black circles) after application of shear stress as a function of the duration (time, expressed in s (seconds)) and strength (stress, expressed in Pa) of shear stress in Example 4. [Figure 8]Graphs showing the viability of HEK293T cells after application of tensile stress in Example 5. HEK293T cells were characterized after application of tensile stress of different intensities and durations. The percentage of cells in a specific physiological state is shown as a function of the duration (time, expressed in seconds) and intensity (stress, expressed in Pa) of the applied tensile stress. Accordingly, the percentage of apoptotic cells (Graph A), viable cells (Graph B), necrotic cells (Graph C), or lysed cells (Graph D) is shown, respectively. [Figure 9] 1 is a graph showing the viability of fibroblasts after shear stress in Example 6, where viability is measured by cytometry (histogram bars) or trypan blue staining (black circles). [Figure 10] 1 is a graph showing the viability of HEK293T cells after shear stress of different intensities and durations in Example 7. The percentage of viable cells, measured by trypan blue staining, is shown as a function of shear stress duration (time, expressed in seconds) and intensity (stress, expressed in Pa). The percentage of viable cells is shown for low passage (HEK293T P07, open triangles) or high passage (HEK293T P18, filled circles). [Figure 11] 1 is a graph showing the viability of fibroblasts after application of shear stress of different intensities and durations, based on two measurements performed on the same cell sample (P06), represented by white triangles and black circles, respectively, in Example 8. P06 indicates the sixth cell culture passage. [Figure 12] 10 is a graph showing cell viability as a function of hydrodynamic stress intensity and residence time in three dimensions on a logarithmic scale in Example 9. [Figure 13] 1 is a two-dimensional graph showing fibroblast viability (y-axis) as a function of hydrodynamic shear stress intensity (x-axis) and residence time (represented by dots) in Example 9. [Figure 14] 10 is a graph showing cell viability in two dimensions as a function of hydrodynamic stress intensity and residence time in Example 9. [Figure 15]1 is a histogram showing cell viability as a function of shear stress intensity (kPa) in Example 10. For each stress intensity, viability values ​​were measured after several experiments. [Figure 16] FIG. 1 shows the standard deviation (light bars), standard error (black bars), and coefficient of variation (gray bars) of various measurements as a function of shear stress intensity (kPa) in Example 10. [Figure 17] 1 is a histogram showing cell viability as a function of shear stress intensity (kPa) in Example 10. [Figure 18] In Example 11, i) the left side shows the viability of various cell types as a function of the applied hydrodynamic stress intensity (kPa), and ii) the right side shows the cell viability as a function of the applied hydrodynamic stress intensity for each of the cells examined. [Figure 19] 1 is a graph showing cell viability as a function of applied shear stress intensity (kPa) in Example 12. Symbols P07 (circle), P08 (square), P09 (triangle), and P10 (star) indicate cell viability defined according to cell passage number in culture. [Figure 20] 1 is a graph showing cell viability as a function of the intensity (kPa) of applied shear stress in Example 12. P09, P10, and P11 show cell viability defined according to the cell passage number in culture. [Figure 21] FIG. 13 shows the same hydrodynamic stress sequence in Example 13. [Figure 22] 10 is a graph showing cell viability as a function of the intensity (kPa) of applied hydrodynamic stress in Example 13. [Figure 23] 12 is a graph showing cell viability as a function of the number of stress cycles for a stress of 0.2 kPa in Example 13. [Figure 24] FIG. 13 shows images of the process of identifying and separating the positions of cells from the original image in Example 14. [Figure 25] FIG. 16 shows images of the step of detecting cell axes from an original image in Example 15. [Figure 26] FIG. 15 is a diagram showing the deformability of cells under shear stress based on the initial diameter of the cells in Example 15. [Figure 27] FIG. 15 shows the deformability of cells based on their initial diameter under the influence of mechanical impact stress in Example 15. [Figure 28] FIG. 20 is a diagram illustrating an example of an “autoencoder” neural network and the transformation from an initial image (input) to a final image (output) after processing by this network in Example 16. [Figure 29] In Example 16, these images show the difference in image processing by a supervised network (U-net) of an initial image with or without preprocessing by an unsupervised network (autoencoder). The first column shows the results when an autoencoder is not applied, the second column shows the results when a "variational autoencoder" is applied, and the third column shows the results when a "noise-removing autoencoder" is applied. [Figure 30A] FIG. 16 shows contour results and associated metrics after applying various neural networks in Example 17. [Figure 30B] 10 is a graph showing the difference between manually extracted contours (control taken as 0) and calculated deformability values ​​in Example 17. [Figure 31] FIG. 10 shows the results of classifying three types of cells, from left to right: necrotic cells, apoptotic cells, and live cells, in Example 18. [Example]

[0112] Example 1: Materials and methods for characterizing cells after physical stress AD-MSC cells were cultured in MSC growth medium at approximately 2,500 cells / cm. 2 The cells were cultured at a seeding density of 1000 x g / ml. The medium was changed every 2 days. The cells were incubated in a T175 flask for 7 days to reach 70% confluence.

[0113] Fibroblasts were cultured in Gibco's DMEM GLUTAMAX medium at approximately 5,500 cells / cm. 2 The cells were cultured at a seeding density of 1000 μg / ml. The cells were incubated in a T175 flask for 7 days to reach 80% confluence.

[0114] HEK293T cells were grown in Gibco's DMEM GLUTAMAX medium at approximately 12,000 cells / cm. 2 The cells were cultured at a seeding density of 1000 μg / ml. The cells were incubated in a T175 flask for 4 days to reach 80% confluence. The temperature was maintained at 37°C and the CO2 concentration was approximately 5% during incubation.

[0115] To detach the cells from the flask surface, the culture medium was first removed. The cells attached to the flask were rinsed with 15 mL of PBS. Next, 5 mL of trypsin-EDTA (0.5%) was added to the flask. After the trypsin was allowed to act for 2 minutes at 37°C, 10 mL of culture medium containing fetal bovine 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 1 million cells / mL.

[0116] The microfluidic shear stress device consisted of a PDMS microfluidic channel with a diameter of 50 μm and a length of 10 cm. Cells were suspended in a fluid with a viscosity of 1.91 mPa·s (a solution of PBS and Ficoll).

[0117] The microfluidic device for extensional stress consists of a main microfluidic channel with a diameter of 162 μm and a length of 1 cm. The channel cross section first decreases from 162 μm to 30 μm, then increases from 30 μm to 162 μm. This change in cross section occurs over a distance of 360 μm. The viscosity of the suspending fluid (a solution of PBS and Ficoll with a volume concentration of 60% Ficoll) is 1.91 mPa·s.

[0118] The cell suspension and cell-free solution are pumped into the device using a syringe pump and 3 mm diameter PEEK tubing.

[0119] Cells are injected into the device at a flow rate of 25-800 μL / min. Measurements and cell recovery are performed after hydrodynamic stability is achieved within the device.

[0120] The cells are collected after passing through the stress region, and the collected cell suspension is centrifuged to remove the suspending fluid (PBS and Ficoll solution) and replace it with labeling buffer.

[0121] For viability testing, cells were stained with annexin V, a marker for apoptotic cells, or propidium iodide, a marker for necrotic cells. For this, 100,000 cells corresponding to each injection rate were suspended in 100 μL of labeling buffer. Then, 2 μL of annexin V marker and 2 μL of propidium iodide were added to the buffer. The cell suspension with the viability marker was incubated in the dark for 15 minutes. The cells were then centrifuged and rinsed with labeling buffer.

[0122] To test the stemness and differentiation status of AD-MSCs, cells were labeled using the BD Human Mesenchymal Stem Cell Analysis Kit (BDB562245), which includes hMSC-positive markers CD90, CD105, CD73, and CD44, as well as hMSC-negative markers CD34, CD11b, CD19, CD45, and HLA-DR.

[0123] To stain, 100,000 cells were suspended in 100 μL of BD Stain Buffer (FBS), and then 5 μL of each positive marker and 20 μL of each negative marker were added to the buffer. The cell suspension with markers was incubated in the dark for 15 minutes. The cells were then centrifuged and rinsed with labeling buffer.

[0124] Cells are examined by cytometry (FACS Canto II) and characterized according to their state (viable, lysed, necrotic, apoptotic or stemness). Each measurement point corresponds to the analysis of at least 50,000 cells.

[0125] Based on the results obtained, a mathematical model is developed to predict the physiological state of the cells from the stress intensity and duration. A model is a mathematical expression that relates parameters, such as a polynomial, power law, sum of sines, or other two- or three-dimensional equation. For example, y=ax, which establishes the relationship between x and y. k A power law of the form a + c can be used. In this model, a is a proportionality constant, k is an exponent, and c is an error term. This model is used to model the physiological state of cells before and / or after passing through a stress zone, based on the stress intensity and stress residence time. For this purpose, the model is established as follows: Physiological state = A x stress B + C; residence time = D / stress. Parameters A, B, C, and D vary with cell type, type of stress, and passage number.

[0126] Example 2: Characterization of AD-MSC Stem Cell Viability Following Shear Stress AD-MSC stem cells were cultured and subjected to shear stress of various intensities and durations. After these stresses were applied, the cell viability was characterized based on the shear stress intensity and the cell residence time under the stress. The physiological state of the cells (survival, lysis, necrosis, or apoptosis) was characterized as described in Example 1.

[0127] The mathematical model defined in this case is as follows: State = A x stress B + C; time = D / stress.

[0128] The defined values ​​A, B, C, and D for each of the possible physiological states are as follows:

[0129] [Table 1]

[0130] The results are shown in Figure 5. These results indicate that human AD-MSCs are sensitive to the intensity of shear stress and the residence time under stress. Our observations suggest that AD-MSCs have the mechanical capacity to withstand a stress of approximately 1,000 Pa with a residence time of approximately 0.25 s. Above this value, AD-MSCs cannot tolerate the stress and die. While lysis or necrosis are the most common pathways of death, the rate of apoptosis is negligible within this range of stress and residence time.

[0131] Example 3: Physiological state of fibroblasts after shear stress Fibroblasts were cultured and cell samples were subjected to shear stresses of various intensities and durations, after which cell viability, or lysis, necrosis, or apoptosis, was determined as described in Example 1.

[0132] The mathematical model defined in this case is as follows: State = A x stress B + C; time = D / stress.

[0133] The defined values ​​A, B, C, and D for each of the possible physiological states are as follows:

[0134] [Table 2]

[0135] The results are shown in Figure 6. These results indicate that fibroblasts are extremely sensitive to shear stress and the duration of exposure to stress. Our observations suggest that fibroblasts can withstand shear stress up to 900 Pa for 0.04 s without sustaining damage, i.e., without exhibiting lysis, apoptosis, or necrosis. These values ​​represent the mechanical capacity of fibroblasts to withstand shear stress. However, exceeding this mechanical capacity significantly reduces the physiological response of fibroblasts, resulting in a significant loss of viability. Indeed, fibroblasts are unable to tolerate the stress, with a viability of 85% at 900 Pa suddenly dropping to 30% at 1,000 Pa and below 5% at 2,000 Pa. Fibroblast death is primarily due to lytic pathways, with other physiological pathways being minimal.

[0136] Example 4: Differentiation state of AD-MSC human mesenchymal stem cells after shear stress application AD-MSC stem cells were cultured as described in Example 1. As a prerequisite, the AD-MSC stem cells were characterized by cytometry to confirm their stemness before the application of shear stress of different intensities and durations. After passing through the stress zone, the cells were collected and resuspended in DMEM(+) medium. The differentiation state was characterized by re-culturing the cells. Seeding was performed at 80% confluence. After 3 weeks of incubation, the cells were collected, labeled with the BD Human Mesenchymal Stem Cell Analysis Kit (BDB562245), and analyzed by flow cytometry (FACS Canto II). Each measurement point represented at least 30,000 cells. Characterization was performed using flow cytometry to distinguish the state of the cells after stress application as either differentiated or undifferentiated. The cytometric characterization procedure was performed as described in Example 1.

[0137] The mathematical model defined in this case is as follows: Undifferentiated state: Time = D / Stress, where D = 30.6, in the range 0.147 s < Time < 0.39 s and 78 < Stress < 2082 Pa.

[0138] The results are shown in Figure 7. These results demonstrate the absence of differentiated cells. This indicates that AD-MSC human mesenchymal stem cells maintained their stemness even after exposure to shear stress within the range shown in the figure. Therefore, the physiological state of AD-MSC cells, in this case, the maintenance of stemness, was not affected by stresses ranging from 0 to 2100 Pa and residence times ranging from 0.015 to 0.4 s.

[0139] Example 5: Survival status of HEK293T cells after application of extension stress HEK293T cells were cultured and then subjected to stretching stresses of varying magnitude and duration. After these stresses, cell viability, lysis, necrosis, or apoptosis were determined as described in Example 1.

[0140] The results are shown in Figure 8. These results indicate that HEK293T cells have a high survival rate against extension stress in the stress range of 10 to 110 Pa and dwell time of 0.1 to 0.8 ms. Lysis, necrosis, and apoptosis are negligible compared to the survival state.

[0141] Example 6: Fibroblast viability after shear stress measured by two methods After culturing fibroblasts, cell samples were subjected to shear stress of varying intensity and duration. After these stresses, cell viability was determined using two methods: cytometry analysis and trypan blue counting. Viability was determined based on the shear stress intensity. The residence time under stress was calculated as follows: Time (s) = 30.6 / Shear stress intensity (Pa)

[0142] The cells were collected after passing through the stress zone. They were then divided into two batches for cytometric analysis and trypan blue analysis. For cytometric analysis, the batches were stained with Annexin V and propidium iodide. The labeled cells were then analyzed using a cytometer. Each measurement point represented at least 50,000 cells. For trypan blue analysis, the cell batches were mixed with trypan blue. Triplicate counts were performed across the entire surface of the Malassez counting vessel. The error bars correspond to the triplicate counts. The results are shown in Figure 6.

[0143] Example 7: Survival of HEK293T cells after shear stress After culturing HEK293T cells, cell samples were subjected to shear stress of varying intensity and duration as described in Example 1. The HEK293T cells were collected after passing through the stress zone and then mixed with trypan blue. Three counts were performed across the entire surface of the Malassez counting vessel. Values ​​represent the average of these three counts.

[0144] The results are shown in Figure 10. These results indicate that the number of cell passages in cell culture clearly affects the mechanical capacity of HEK293T cells. Cells at P07 (7th passage) show 100% viability and can fully withstand stress below 200 Pa. However, when this mechanical capacity is exceeded, HEK293T cells at the same passage P07 show a decrease in viability. However, even with the same cell line, at P18 (18th passage), this mechanical capacity is lost and they are unable to withstand even slight stress.

[0145] Example 8: Demonstration of measurement reproducibility Characterization of the physiological state of fibroblasts after shear stress was performed twice. Both measurements were performed on the same cell sample (P06). The mean error in viability was 3.45% during experimental reproducibility. Therefore, the analysis performed with this system is reproducible. The results obtained are shown in the accompanying Figure 11.

[0146] Example 9: Cellular mechanical capacity based on stress intensity and stress dwell time Residence time is defined as the period during which cells are subjected to hydrodynamic stress. In the microfluidic device and method of the present invention, the stress is maintained at a constant intensity, while the residence time depends on the flow rate and viscosity of the cell suspension. This approach allows us to decouple the two parameters and study their effects separately. Fibroblasts were suspended in fluids with viscosities of 1, 5, 10, 15, or 20 mPa·s and injected through a microfluidic capillary at flow rates ranging from 117 to 942 μL / min. This configuration corresponded to shear stress intensities ranging from 0.16 to 25.59 kPa and residence times ranging from 12.5 to 100.7 ms.

[0147] Figures 12 and 13 show mappings of fibroblast cell viability as a function of stress intensity and dwell time. These two figures show the same data in 3D and 2D views, and on linear and logarithmic scales. The white dots represent experimental data, and the surfaces represent mathematical model fits. Within the range shown, for a given dwell time, increasing stress intensity decreases cell viability. However, at a constant stress intensity, cell viability is independent of dwell time. Figure 14 shows only fibroblast viability as a function of stress intensity, with dwell time identified by different shades of gray. From these three plots of the same data, we can conclude that fibroblast viability and mechanical capacity are independent of dwell time in the range of 12.5–100.7 ms.

[0148] These results indicate that in this case, fibroblast viability can only be characterized based on stress intensity.

[0149] In the method according to the present invention, stress intensity and dwell time are completely decoupled. It is possible to keep one of these parameters constant while varying the other. In this way, the effects of dwell time and shear stress intensity as two independent parameters can be understood in detail.

[0150] Example 10: Instrument repeatability and precision To ensure reproducibility of experiments and results, we measured the mechanical capacity of Madin-Darby canine kidney (MDCK) cells six times. To eliminate biological variations that could affect the evaluation of machine and procedural performance errors, the cell population was divided into six batches. Mechanical capacity was measured for each cell batch. Cells were subjected to shear stresses of 0.175, 0.469, 2.716, and 6.397 kPa, and then viability was measured using a cytometer. The standard deviation, standard error, and coefficient of variation were calculated using the following formulas:

[0151]

number

[0152] The results show that the standard error over six trials is as low as 1% in the worst case. This example perfectly demonstrates the precision and performance of the machine and procedure, which results in highly accurate and reliable measurements. This precision allows for reliable and sophisticated analysis, as shown in Figures 15-17.

[0153] Example 11: Mechanical capabilities of different cell lines The device according to the present invention can measure the mechanical capacity of various cells, including primary cell lines, immortalized cell lines, and stem cells, against hydrodynamic stress. The mechanical capacity of the following cell lines was analyzed: adipose tissue-derived mesenchymal stem cells (AD-MSCs), Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), human dermal fibroblasts, human umbilical vein endothelial cells (HUVECs), cancer-associated fibroblasts (CAFs), human embryonic kidney 293T cells (HEK293T), and Madin-Darby canine kidney (MDCK).

[0154] Cells were suspended in two fluids with viscosities of 1 mPa·s and 5 mPa·s and injected into a 50 μm diameter microfluidic channel at flow rates of 85–942 μL / min, subjecting them to shear stresses ranging from 0.11 to 6.4 kPa. The recovered cells were stained with propidium iodide and analyzed by cytometry to quantify the shear stress-induced necrotic cell population. To confirm that cells were damaged solely by the shear stress of injection into the microfluidic channel, rather than by static syringe pressure, fibroblasts were injected without passing through the microfluidic channel and analyzed by cytometry. Cell viability was confirmed to be unchanged compared to the 97% viability of the control cell population. Furthermore, special care was taken during the experiment to minimize cell death due to suspension in adherent cells. The time between cell collection and shear stress exposure was exactly 5 min, consistent across all cell lines.

[0155] Figure 18 shows the viability of cell lines after shear stress application. The dots represent experimental data, and the lines are fitted curves. This change in cell viability can be described as having two phases. First, cell viability plateaus up to a certain shear stress limit, indicating the cells' ability to withstand shear stress. Subsequently, above a shear stress corresponding to the cells' mechanical capacity, cell viability deteriorates, dropping to less than 5% viable cells. Before the shear stress corresponding to each cell's mechanical capacity is applied, the viability is less than 5% different from that of the control cell population without shear stress application. While all cell types examined show similar trends, it is also noteworthy that each cell line exhibits specific sensitivity to shear stress as a measure of mechanical capacity (AD-MSCs: 0.64 kPa, WJ-MSCs: 0.35 kPa, BM-MSCs: 0.48 kPa, fibroblasts: 0.64 kPa, HUVECs: 0.32 kPa, and HEK293T: 0.43 kPa).

[0156] Example 12: Changes in mechanical capacity with cell aging and incubation time To investigate the relationship between cell aging and mechanical capacity, fibroblasts were subcultured (passaged) once a week and subjected to shear stress. The experiment continued for four passages, from P07 to P10. Passaging is a method of subcultivating animal cells. "P07" refers to the seventh cell passage in culture. The subculturing method was strictly consistent across different passages. The viability of fibroblasts after exposure to several shear stresses was measured at each passage by flow cytometry. Cells were suspended in fluids with viscosities of 1 mPa·s and 5 mPa·s and then subjected to shear stresses ranging from 0.159 to 6.14 kPa. The flow rates for both suspension fluids were 117 to 942 μL / min.

[0157] Figure 19 shows that the serial subculture procedure does not affect the mechanical capacity of fibroblasts against shear stress. In fact, cells have similar mechanical capacity regardless of the number of passages. Up to a shear stress of 0.577 kPa, which corresponds to the mechanical capacity of the cells, fibroblast viability is unaffected (96%), and the control viability was 97%. Between 0.639 kPa and 4.78 kPa, viability deteriorates sharply from 92% to 5%, reaching a plateau below 5%.

[0158] A similar study was performed on HEK293T cells. Unlike fibroblasts, HEK293T cells were subcultured twice weekly from passage P09 to P11. As a result, cell growth periods alternated between 3 and 4 days with each successive subculture.

[0159] Cells were suspended in fluids with viscosities of 1 mPa·s and 5 mPa·s and then subjected to shear stresses ranging from 0.11 to 6.4 kPa. The flow rates for both suspension fluids were 85 to 942 μL / min. Cell viability was analyzed by cytometry.

[0160] As shown in Figure 20, the mechanical capacity of HEK293T cells is affected by varying the incubation period for successive cell subcultures. The incubation period before stress application and measurement was 3 or 4 days. The mechanical capacity of HEK293T cells incubated for 3 days was 0.516 kPa, while the mechanical capacity of HEK293T cells incubated for longer periods was 0.319 kPa, a decrease.

[0161] Example 13: Repeated series of stresses To examine the effect of repeated application of the same stress on the mechanical capacity of cells, AD-MSC cells were suspended in a fluid with a viscosity of 1 mPa·s and repeatedly subjected to hydrodynamic stress. This type of stress is defined as successive cycles of the same stress. Figure 21 illustrates this sequence. Cells are subjected to shear stress, enter a shear-free region, and then are subjected to shear stress again. This cycle was performed on a batch of cells at a stress of 0.2 kPa for cycles of 77, 154, 231, 308, 385, and 462. This stress is less than the mechanical capacity of the cells, 0.6 kPa, as shown in Figure 22. After various cycles of stress, the cells were analyzed using a flow cytometer to measure cell viability. Figure 23 shows cyclic stresses below the mechanical capacity of AD-MSC cells that do not affect cell viability.

[0162] Example 14: Cell Localization and Isolation Cell localization and isolation are performed using algorithms based on raw images captured by a high-speed camera. First, a mask is created that reduces the size of the raw image by superimposition. Next, image processing functions such as erosion and dilation are applied to reduce background noise in the image and increase contrast to reveal the cell locations. Regions of interest (ROIs) are then defined and isolation is performed (Figure 24).

[0163] Example 15: Arrangement and measurement of minor and major axes The location and measurement of the short and long axes are performed using a supervised learning model. In the first step, a supervised model (in this case, U-Net) is applied to the resulting cell photograph, shown in Figure 24. This identifies the cell size and shape. Next, an "edge extraction"-type image processing function is applied to locate the cell outline on the image extracted from Figure 24. The short and long axes are then measured, with the long axis being the maximum diameter and the short axis being the minimum diameter. These results can be displayed as a graph showing the degree of deformation based on cell size. Here, the results demonstrate the accuracy of image processing-based deformability measurements in shear and impact tests.

[0164] Example 16: Image quality improvement using unsupervised models The images obtained during the cell localization and separation process are not always of suitable quality for measuring the long and short axes. This is because these images are noisy enough to prevent cell segmentation using supervised models. To improve this image quality, the application of unsupervised models is required. Here, we show that applying an "autoencoder" neural network can improve the quality of the image reconstruction obtained from ROI extraction (Figure 28). Figure 29 demonstrates the validity of this method. The first column of Figure 29 shows the results without applying an autoencoder, the second column shows the results with a variational autoencoder, and the third column shows the results with a denoising autoencoder.

[0165] Example 17: Combining unsupervised and supervised models to improve cell detection and deformability measurements A combination of unsupervised and supervised models can be used to improve the detection of cells in images and the measurement of their deformability. In this example, we demonstrate this approach by combining an autoencoder and a U-Net neural network with an image obtained from a shear measurement (Figure 30A).

[0166] The figures are as follows (Table 3).

[0167] [Table 3]

[0168] When applied alone, a supervised network (U-Net) cannot detect and track all cell movements, but when combined with an unsupervised model (VAE or DAE autoencoder), it can be observed that detection and measurement are improved (higher precision, recall, and F1 score) (Figure 30B).

[0169] Therefore, the improvement in the measurements is accounted for by subtracting the deformability measurements from the standard manual approach, i.e., each image was processed by the user without applying any image processing or learning models.

[0170] Example 18: Predicting the physiological state of cells using classification models with supervised learning Supervised learning models can be used to classify the physiological state of cells through image analysis. This example shows three cell classifications annotated as necrotic (A), apoptotic (B), and viable (C).

Claims

1. 1. An apparatus for applying at least one hydrodynamic stress of determined magnitude and duration to at least one cell suspended and moving within the apparatus, and characterizing the cell during and, optionally, after application of the stress, comprising: a microfluidic circuit comprising at least one zone, said zone comprising at least: i) a flow path configured to circulate a fluid containing said suspended cells; ii) a fluid inlet and a fluid outlet; iii) means for introducing and establishing a flow of said fluid within said flow path; and - means for characterizing said cells during the application of said stress, The device, characterized in that the at least one zone is configured to apply at least one hydrodynamic stress selected from hydrodynamic compressive stress, hydrodynamic shear stress, and hydrodynamic extensional stress to the cells, and optionally to apply at least one mechanical shock to the cells.

2. 10. The apparatus of claim 1, wherein the characterization of the cells comprises at least one criterion selected from size, shape, outer membrane appearance, cytoplasmic appearance, the presence of at least one cell surface marker, cellular protein content, and cellular nucleic acid content.

3. 3. The device according to claim 1, wherein the at least one zone A configured to apply a compressive stress to the cells comprises or consists of a first channel joined at the same height as two channels, each of the two channels forming an angle of 30 to 150 degrees with the first channel.

4. 4. The device according to any one of claims 1 to 3, characterized in that the at least one zone B configured for applying a shear stress to the cells comprises or consists of a channel with a diameter of 10 to 2,000 μm, preferably 20 to 200 μm.

5. 5. The device according to claim 1, wherein the at least one zone C configured to apply an extensional stress to the cells comprises or consists of: i) a channel with an increasing or decreasing cross section; or ii) a first channel coupled to a second channel in which fluid circulates in a different, preferably opposite, direction to the first channel.

6. 6. The device according to claim 1, wherein the at least one zone D configured for applying a mechanical shock to the cells comprises or consists of a first channel, inside which the flow line of the cells encounters an obstacle, such as in particular a wall of a second channel.

7. 7. Device according to any one of claims 1 to 6, characterized in that the circuit comprises at least one area A and / or at least one area B and / or at least one area C and optionally at least one area D, combined with one another.

8. 8. The device according to claim 1, wherein the circuit is configured to apply to the cells at least one sequence comprising or consisting of at least two sequences of physical stresses of different nature.

9. 8. The device according to any one of claims 1 to 7, characterized in that the circuit is configured to apply to the cells at least one sequence comprising or consisting of at least two successive hydrodynamic stresses of the same nature and, optionally, at least two successive mechanical stresses of the same nature.

10. The intensity of at least one hydrodynamic stress applied to the cells is 10 －３ ~10 ５ kPa, preferably 10 －３ ~10 ４ kPa, preferably 0.1 to 10 ３ kPa, preferably 1 to 10 ２ 10. Apparatus according to any one of claims 1 to 9, characterized in that the pressure is between 100 kPa and 10,000 kPa, and / or the total duration of application of the at least one hydrodynamic stress is between 1 μs and 100 s, preferably between 1 μs and 100 s, preferably between 1 μs and 1 s, preferably between 10 μs and 100 ms.

11. 11. The device according to any one of claims 1 to 10, characterized in that the characterization of the cells after the application of the at least one stress is performed between 0 and 120 days, preferably between 0 and 30 days, preferably between 0 and 1 day after the application of the at least one physical stress.

12. 12. Apparatus according to any one of claims 1 to 11, characterized in that the characterization of the cells during the application of the at least one stress comprises or consists of at least one discrete characterization or at least one characterization performed for a duration of 1 μs to 10,000 s, preferably 1 μs to 100 s, preferably 1 μs to 1 s, preferably 10 μs to 100 ms.

13. 13. The device according to any one of claims 1 to 12, characterized in that the means for characterizing the at least one cell are selected from a cytometer, a microscope, means for analyzing the protein content of a cell, means for analyzing and sequencing the nucleic acids of the cell, and image capture means combined with image analysis means.

14. 14. Apparatus according to claim 13, characterized in that the image analysis means comprise or consist of a central computer unit containing software means adapted for image analysis.

15. 15. The apparatus according to claim 13 or 14, characterized in that the image analysis means further comprises a first classification model pre-trained on a training dataset and comprising a supervised, unsupervised or semi-supervised machine learning algorithm, the first classification model being adapted to predict the physiological state of a given cell from the cellular features.

16. 16. The apparatus according to any one of claims 13 to 15, characterized in that the image analysis means further comprise a second classification model pre-trained on a training dataset and comprising a supervised, unsupervised or semi-supervised automatic learning algorithm, wherein the second classification model is adapted to detect and monitor deformation of a given predetermined cell in response to at least one physical stress.

17. 1. A method for applying at least one physical stress of determined intensity and duration to at least one suspended cell, and characterizing said cell during and optionally after said application, comprising the steps of: a) injecting and circulating a fluid containing said at least one suspended cell in a microfluidic circuit comprising at least one zone, said zone comprising at least: i) a main flow path configured to circulate the fluid containing said cells; ii) a fluid inlet and a fluid outlet; iii) means for introducing and establishing the flow of said fluid in said flow path; and b) characterizing said cells during and optionally after the application of said at least one stress, The method, characterized in that the at least one zone is configured to apply at least one hydrodynamic stress selected from hydrodynamic compressive stress, and / or hydrodynamic shear stress, and / or hydrodynamic extensional stress, and optionally at least one mechanical shock to the cells.

18. 18. The method of claim 17, wherein the characterization of the cells comprises at least one criterion selected from size, shape, outer membrane appearance, cytoplasmic appearance, and the presence of at least one surface marker.

19. The cells -10 －３ ~10 ５ kPa, preferably 10 －３ ~10 ４ kPa, preferably 0.1 to 10 ３ kPa and / or at least one shear stress with a duration of 1 μs to 10,000 s, preferably 1 μs to 100 s, preferably 10 μs to 1 s, preferably 10 μs to 10 ms, and / or -10 －３ ~10 ３ kPa, preferably 10 －３ ~10 ２ kPa, preferably 10 －３ At least one compressive stress with an intensity of up to 10 kPa and / or a duration of 1 μs to 10 s, preferably 1 μs to 1 s, preferably 10 μs to 10 ms, and / or -10 －３ ~10 ３ kPa, preferably 10 －３ ~10 ２ kPa, preferably 10 －３ At least one elongation stress with an intensity of up to 10 kPa and / or a duration of 1 μs to 10 s, preferably 1 μs to 1 s, preferably 10 μs to 10 ms and optionally at least one mechanical impact with an intensity of 1 to 300 m / s and / or a duration of less than 1 μs.

19. The method of claim 17 or 18, wherein the

20. 20. The method according to any one of claims 17 to 19, further comprising predicting the physiological state of the cells by means of a first pre-trained classification model based on the features determined in step b).

21. 21. The method of any one of claims 17 to 20, further comprising predicting the physiological state of the cell by a pre-trained first classification model, wherein the first classification model comprises a machine learning algorithm, a supervised learning neural network, or a multi-class probabilistic classification algorithm pre-trained on a training dataset.

22. 22. The method according to any one of claims 17 to 21, further comprising monitoring the deformability of the cells at different times during their residence in the microfluidic channel by a second pre-trained classification model based on the features determined in step b).

23. 23. The method according to any one of claims 17 to 22, wherein the classification model, pre-trained on a training dataset, predicts the physiological state of the cells during and / or after the application of at least one hydrodynamic stress.

24. Use of the device of any one of claims 1 to 16, or the method of any one of claims 17 to 22, or the classification model of claim 23, for characterizing cells of the following types: prokaryotic cells, eukaryotic cells, animal cells, plant cells, human cells, stem cells, epithelial cells, fibroblasts, blood cells, genetically modified cells, or synthetic cell mimics.

25. Use of the device according to any one of claims 1 to 16, the method according to any one of claims 17 to 22 or the classification model according to claim 23 for determining the performance of a cell.

26. 26. Use of an apparatus according to any one of claims 1 to 16, a method according to any one of claims 17 to 22, or a classification model according to claim 23 for defining at least one parameter of a bioprocess.

27. 27. The use according to claim 26, characterized in that the bioprocess is selected from bioprinting, cell therapy, and biomanufacturing.

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