System for analyzing cellular microcompartments or cellular tissues

Abstract

The invention relates to an analysis system (2, 20) for cellular microcompartments (MC) or cellular tissues, comprising: a lighting module (3, 30) including a light source (31) and a diaphragm (33); and an objective lens (5); characterized in that the lighting module includes an optical system (7) arranged to conjugate a point located at the center of the diaphragm (33) with an entrance pupil of the objective lens (5), and in that the lighting module is arranged such that the principal emission direction (XLED, X'LED) of the lighting module is offset from the optical axis (X) of the objective lens (5). Figure to be published with the abstract: Fig. 2

Description

Title of the invention: System for analyzing cellular microcompartments or cellular tissues

[0001] The invention relates to the field of cell encapsulation in three-dimensional cell culture compartments. More specifically, the invention relates to a system for analyzing cellular microcompartments or cellular tissues.

[0002] Ex vivo cell culture is a field of growing interest, particularly in the medical and pharmaceutical sectors. Cultured cells can be of any type. They can include differentiated cells with various phenotypes, progenitor cells, and stem cells. Pluripotent stem cells, in particular, are increasingly used. Indeed, in research on genetic diseases, these cells can be used to design cellular models of these diseases. These cells can also be used to test the effects of new drugs, to understand their mechanism of action and safety, or in genetic research, to study regions of the genome involved in cell differentiation.Finally, in the field of cell therapy, pluripotent stem cells can be differentiated into specific cells that can be used to replace damaged or missing cells in the body, such as heart, pancreatic, or liver cells.

[0003] In these various applications, culturing cells in large quantities presents a significant challenge. The research topics mentioned require a substantial quantity of human pluripotent cells. Similarly, the success of cell therapy in humans depends on the availability of industrial quantities of cells, particularly human pluripotent stem cells.

[0004] A significant advance in cell culture techniques is the introduction of three-dimensional culture systems. Three-dimensional cultures are indeed advantageously closer to natural in vivo systems and can be used for numerous applications, particularly in the development of therapies. A particularly suitable technology is that described in application WO2018 / 096277, which consists of three-dimensional cell microcompartments for stem cell culture. This document describes a cell encapsulation device comprising a microfluidic or millifluidic injector, enabling the formation of cell microcompartments in the form of droplets, the outer layer of which is formed by a solution comprising alginate and the core is formed by a solution of cells. These droplets are collected in a calcium bath which stiffens their outer layer to form a shell.

[0005] The microcompartments thus formed allow the cells to be cultured in a liquid medium, while the shell protects the cells from mechanical stresses related to collisions or fusions during culture in liquid suspension.

[0006] In this context, quality control of the microcompartments formed by this type of encapsulation device is of paramount importance. It is indeed necessary to be able to control, non-destructively, whether the microcompartments of a sample are compliant, whether their contents, namely cells or cell aggregates, are viable, degraded, or even non-existent, or whether the cells or cellular microtissues contained within these microcompartments are viable. This need exists for samples from a production batch as well as for samples from research and development applications, in which various parameters are tested, such as solutions, cells, solution concentrations, flow rates, and microcompartment dimensions, in order to identify production conditions suitable for a given application.

[0007] It was thus conceived to employ an imaging system which, from an image of a sample of a production batch, makes it possible to check the number of microcompartments, to control their shape, their size, the number of cells or cellular microtissues they contain, and any other statistical, qualitative or quantitative parameter representative of the quality of the production.

[0008] This system, derived from cell microscopy, combines an illumination module and an optical imaging system, also called an objective, to form an image of an object on an optical sensor. While this type of system provides adequate contrast for cells and microtissues, microcompartments may appear transparent, and their contrast is too low to allow for the clear identification of microcompartments, cells, cellular microtissues, or cell aggregates in the image.

[0009] There is therefore a need for an analysis system that allows for non-destructive control of the quality of cellular microcompartments or cellular tissues, particularly those obtained from a three-dimensional culture process, and which offers improved detection and analysis performance.

[0010] To this end, the invention relates to a system for analyzing cellular microcompartments or cellular tissues, comprising: a. a lighting module comprising a light source and a diaphragm; b. a lens;

[0011] The system according to the invention is characterized in that the lighting module comprises an optical system arranged to conjugate a point located at the center of the diaphragm with an entrance pupil of the lens and in that the lighting module is arranged so that the principal direction of emission of the lighting module is offset from the optical axis of the lens.

[0012] The invention thus proposes to combine a lighting module and a lens, also referred to as an optical imaging system. The lighting module is arranged so that the illumination emitted by its light source is, directly or indirectly, offset, and in particular tilted, rotated, or translated, with respect to the optical axis of the lens. This offset of the emission axis of the lighting module then causes a significant increase in contrast, which makes it possible to clearly identify the cellular microcompartments and their contents in the image acquired by an optical sensor of the analysis system.

[0013] Furthermore, the virtual image of the lens aperture diaphragm, defined by a lens or a diaphragm of the lens, forms an entrance pupil of the lens. This pupil is conjugated with the diaphragm of the illumination module using an optical system, in order to improve the homogeneity of the illumination and contrast, introduced by the offset of the emission axis of the illumination module, over the entire surface of an image acquired by an optical sensor of the analysis system.

[0014] In the context of the present invention, the term "objective" means an optical imaging system capable of forming a magnified image of an object to be analyzed, particularly for use with an optical sensor or an eyepiece. For example, the objective may comprise one or more lenses defining an object plane at which the object to be analyzed is placed and an image plane positioned at the level of an optical sensor. By way of non-limiting example, the objective may have a magnification between 1 and 10, for example, 2. Also by way of non-limiting example, the objective may have a numerical aperture of 0.062.

[0015] In one embodiment of the invention, the optical system of the lighting module may have a focal length, and the center of the diaphragm of the lighting module may be arranged upstream of the optical system of the lighting module, at a distance equal to a multiple, in particular two, of this focal length, while the lens may be arranged downstream of the optical system of the lighting module, so that its entrance pupil is located at a distance equal to a multiple, in particular two, of this focal length. The conjugation is in this case referred to as 2f-2f. The optical system of the lighting module may, in particular, be a converging lens.

[0016] In the context of the present invention, the cell microcompartments may be obtained using a cell encapsulation system, the system comprising at least: a. two containers, one of the containers being intended to hold a cell solution and the other of the containers being intended to hold a solution capable of gelling, b. a milli-fluidic or micro-fluidic encapsulation device connected to the containers and arranged to form cellular microcompartments, the outer layer of which is the gelling solution and the core the cell solution, c. a collection tank containing a stiffening solution and arranged to collect the cellular microcompartments formed by the encapsulation device.

[0017] The present invention is thus of interest for various applications, and in particular in the quality control of cellular microcompartments or cellular tissues obtained from: a. research and development applications, requiring less than one milliliter of solution volume in the container, and in which different parameters are tested, such as solutions, cells, solution concentrations, flow rates, microcompartment dimensions, in order to identify production conditions suitable for a given application; b. small-scale production applications, requiring between one millilitre of volume and one litre of solution in the container, allowing a small volume of microcompartments to be obtained in order to test cell culture in a bioreactor, to conduct research on genetic diseases or to test the effects of new drugs; c. large-scale production applications, requiring at least one liter of solution, or even several tens of liters, in the container, in order to produce complete batches of microcompartments for cell therapies.

[0018] In the context of the present invention, and by way of non-limiting example, the term "microfluidic device" means any device having one or more inlets and one or more outlets connected by a plurality of channels with a cross-section on the order of a hundred micrometers and capable of directing the flow of one or more fluids from the inlet(s) to the outlet(s). The term "millifluidic device" also means any device having one or more inlets and one or more outlets connected by a plurality of channels with a cross-section on the order of a millimeter and capable of directing the flow of one or more fluids from the inlet(s) to the outlet(s).

[0019] If desired, the encapsulation system may be provided for to include a third container connected to an input of the encapsulation device by one or more distributors and intended to include, or including, an intermediate solution, such as an intermediate solution not containing a divalent cation such as Ca2+ Mg2+ to avoid too early crosslinking of the hydrogel in the collection tank, preferably an isotonic solution not containing a divalent cation such as Ca2+ Mg2+ such as for example a sorbitol solution.

[0020] According to one embodiment, the cell solution may contain culture medium and / or an extracellular matrix and / or an extracellular matrix substitute and / or an aqueous solution. According to another embodiment, the intermediate solution may contain an extracellular matrix and / or an extracellular matrix substitute. Where applicable, the encapsulation device will be arranged to form, within the collection tank, cell microcompartments, the outer layer of which is the gel-ready solution, an intermediate layer forming a cell matrix or extracellular matrix substitute, and the core the cell solution. This cell matrix allows the cells in the cell solution to grow and multiply. For example, the extracellular matrix substitute may comprise a mixture of proteins and extracellular compounds necessary for cell culture, and more specifically for pluripotent cells.Preferably, the extracellular matrix or extracellular matrix substitute may comprise structural proteins, such as laminins containing the α1, α4, or α5 subunits, the α31 or α32 subunits, and the γ1 or γ3 subunits, entactin, vitronectin, laminins, collagen, and growth factors, such as TGF-β and / or EGF. The extracellular matrix may be an aqueous solution and / or a hydrogel, preferably a hydrogel, different from the hydrogel forming the outer layer, such as, for example, a hydrogel comprising or composed of alginate, fibrin, laminin, fibronectin, entactin, hyaluronic acid, and / or collagen. It may also be an extracellular matrix or an extracellular matrix substitute such as Matrigel®. For example, the cell solution comprises a plurality of cells. The cells can be of all cell types.Preferably, the cells are chosen from human, animal, and plant eukaryotic cells, and even more preferably from pluripotent stem cells, progenitor cells, cells undergoing differentiation, and differentiated cells. Where appropriate, these pluripotent stem cells may be induced pluripotent stem cells (iPSCs), MUSE cells (Multilineage-differentiating Stress Enduring) found in the skin and bone marrow of adult mammals, or embryonic stem cells (ESCs). In a particular embodiment, and for legal or ethical reasons, the term "stem cells" excludes human embryonic stem cells or cells derived from the destruction of human embryos.

[0021] In one embodiment, the gelling solution comprises or is constituted by a hydrogel, such that the outer layer is a three-dimensional structure formed from a matrix of polymer chains swollen by a liquid, preferably water. For example, the gelling solution comprises or is constituted by alginate and, preferably, consists of alginate. In the context of the invention, "alginate" means linear polysaccharides formed from 3-D-mannuronate (M) and α-L-guluronate (G), salts and derivatives thereof. Advantageously, the alginate is a sodium alginate, composed of more than 60%, or even more than 80%, of G and less than 40%, or even less than 20%, of M, with an average molecular mass of 100 to 400 kDa and a total concentration of between 0.5% and 5% by mass.

[0022] In the microcompartments obtained by means of the encapsulation system, the cells present in the internal part can be isolated and / or in the form of at least one layer and / or in the form of at least one three-dimensional aggregate and / or in the form of at least one three-dimensional cellular microtissue, optionally with at least one lumen.

[0023] According to one embodiment, at least one cellular microcompartment obtained by means of the system comprises at least one cell layer and at least one lumen. When the microcompartment comprises at least one lumen, at least one cell layer, the intermediate solution layer of the inner part, and the outer layer are preferentially arranged successively around said lumen, this is referred to as a cyst-shaped conformation. Thus, according to one embodiment, at least one cellular microcompartment obtained by means of the system comprises at least one cyst, the hollow center, or lumen, of which is preferentially aqueous. In the context of the invention, a "cyst" is understood to be a three-dimensional, spherical, monolayer arrangement of cells or epithelial cells surrounding a central lumen. This cyst-shaped conformation reduces the pressures experienced by the cells.This configuration also reduces cell mortality and increases the culture amplification factor. Consequently, this reduces the number of passages and dissociations required, and the culture time needed to reach the final cell count.

[0024] The cellular microcompartments obtained by means of the encapsulation system preferably comprise one or more cysts, and / or one or more tissues and / or micro-tissues and / or cell aggregates with or without lumen(s).

[0025] Advantageously, the encapsulation system is arranged so that each cellular microcompartment obtained by means of this system is closed. In one embodiment, the encapsulation system is arranged so that each cellular microcompartment obtained by means of this system has a shape spherical or teardrop-shaped. Preferably, the diameter of such a microcompartment is between 10 pm and 1 mm, more preferably between 50 pm and 700 pm, even more preferably greater than 200 pm, preferably less than 600 pm.

[0026] In another embodiment, the encapsulation system can be arranged so that each cellular microcompartment obtained by means of this system has an elongated shape, in particular an ovoid or tubular shape.

[0027] In one embodiment, the encapsulation system comprises at least one organ capable of electrically charging at least one of the solutions with an electrical potential and the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), of which an external flow is the solution capable of gelling and an internal flow is the cell solution, and a nozzle connected to the body to receive said concentric flow and forming the outlet of the encapsulation device, the encapsulation device being arranged to form, at the outlet of the nozzle, a concentric jet from the concentric flow such that this jet is fractionated into cellular microcompartments.

[0028] In this embodiment, the encapsulation device may be a microfluidic or millifluidic device capable of generating a concentric jet containing the cell solution at its center, optionally surrounded by the intermediate solution, itself optionally surrounded by the gelling solution. The increase in hydrodynamic instabilities within the jet forces it to fragment into droplets; this effect is known as Plateau-Rayleigh instability. These droplets, once immersed in the stiffening solution, form the cell microcompartments. Electrically charging at least one of the solutions passing through the encapsulation device improves the fragmentation of the jet into droplets. This technique is notably called "electro-jetting."It should be noted that the relative sizes of the outer layer and the core of the microcompartments can be adjusted by modifying the flow ratios of the two solutions at the distributors.

[0029] In the case of electro-jetting, an electric field-generating element, such as a metallic ring positioned downstream of the encapsulation device's outlet, may be added so that the jet or the cellular microcompartments pass through this ring. If necessary, the electric field-generating element may be connected to an electrical potential, for example, to ground. This electric field helps, in particular, to promote the dispersion of the cellular microcompartments.

[0030] In another embodiment, the encapsulation system comprises at least one element capable of electrically charging at least one of the solutions with an electrical potential, and the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the distributor(s), of which one The external flow is the solution suitable for gelling, and the internal flow is the cell solution. A nozzle connected to the body receives this concentric flow and forms the outlet of the encapsulation device. The encapsulation device is designed to form the cell microcompartments directly from the concentric flow at the nozzle outlet. The encapsulation device is thus of the "electro-dripping" type, forming the microcompartments one after another directly from the nozzle, without a jet.

[0031] Regardless of the embodiment envisaged, the outlet of the encapsulation device may be positioned above the collection tank, so that the microcompartments fall by gravity into this collection tank. Advantageously, the collection tank and the encapsulation device are arranged at a distance from each other such that the cellular microcompartments formed by the encapsulation device pass through a gaseous volume, in particular air, defined by a closed and sterile enclosure before being collected by the collection tank.

[0032] In yet another embodiment, the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), of which an external flow is the solution suitable for gelling and an internal flow is the cell solution, and a nozzle connected to the body to receive said concentric flow and forming the outlet of the encapsulation device, the encapsulation device and the collection tank being arranged so that the nozzle of the encapsulation device is in contact with the stiffening solution contained in the collection tank and / or so that the nozzle of the encapsulation device is immersed in the stiffening solution contained in the collection tank, the encapsulation device being arranged to form, directly in the stiffening solution, said cell microcompartments from the concentric flow.

[0033] In the context of the present invention, cellular tissues may be obtained from cells encapsulated in one or more three-dimensional closed microcompartments, in particular obtained from an encapsulation system as described above. Where appropriate, the microcompartment(s) may be cultured in a culture medium in a closed chamber, in particular a bioreactor.

[0034] In one embodiment of the invention, the lighting module is arranged so that the main emission direction of the lighting module is inclined with respect to the optical axis of the lens.

[0035] Advantageously, the illumination module can be tilted in its entirety relative to the optical axis of the lens. In this example, the diaphragm extends in a plane inclined to the optical axis at an angle strictly between 0° and 90°. This embodiment simplifies the overall size of the optical assembly of the analysis system and the mounting of the illumination module.

[0036] Alternatively, only the light source may be inclined so that its emission axis is inclined relative to the optical axis of the lens. The diaphragm thus extends in a plane oriented at 90° to the optical axis of the lens.

[0037] Preferably, the lighting module is arranged so that the optical axis of the lens passes through said point located at the center of the diaphragm. The diaphragm of the lighting module, the optical system of the lighting module, and the lens are thus centered.

[0038] In another embodiment of the invention, the lighting module is arranged so that the main emission direction of the lighting module is translated relative to the optical axis of the lens.

[0039] Advantageously, the lighting module may include an optical deflection element arranged to deflect the light beam emitted by the light source in a direction translated relative to the optical axis of the lens.

[0040] In one embodiment, the light source may be arranged so that the light beam emitted by the source is oriented in an emission direction substantially orthogonal to the optical axis of the objective lens, the optical deflection element being arranged to deflect this light beam in a principal emission direction of the illumination module substantially parallel to the optical axis of the objective lens but offset by a given distance from this optical axis. This distance may, for example, be 1.25 mm. These features thus make it possible to reduce the axial size of the microscope by offsetting the light source and the diaphragm, while simultaneously enhancing the contrast of the analysis system.

[0041] Preferably, the optical deflection element may be a plane mirror placed between the diaphragm of the lighting module and the optical system and oriented at 90° with respect to the optical axis of the lens.

[0042] Advantageously, the optical deflection element can be mounted on an adjustable device to modify the distance between the optical axis of the lens and the principal emission direction of the illumination module, such as a block mounted on a rail and movable in a direction orthogonal to the optical axis of the lens, for example, by means of a screw. These features allow a user to first achieve ideal focus by aligning the principal emission direction of the illumination module and the optical axis of the lens, and then moving the optical deflection element to enhance contrast.

[0043] The characteristics described above relating to the optical deflection element may also be considered for tilting the main emission direction of the lighting module rather than translating it.

[0044] Alternatively, it may be provided that all or part of the lighting module, and in particular the light source, can be translated to move the main emission direction of the lighting module.

[0045] In a non-limiting embodiment of the invention, the focal length of the optical system of the lighting module is approximately 50 mm, and the optical system of the lighting module is located 100 mm from the center point of the diaphragm. The lens may be positioned 90 mm from the optical system of the lighting module, and its entrance pupil may be arranged so that it is located 10 mm downstream of its front face. The conjugate between the diaphragm and the entrance pupil is thus a 2f-2f conjugate. Preferably, the aperture of the diaphragm is approximately 4 mm. This configuration is optimal with regard to the contrast of the image formed by the lens on an optical sensor of the analysis system.

[0046] In one embodiment of the invention, the lighting module includes a diffuser arranged downstream of the diaphragm. The diffuser further improves the homogeneity of the lighting and the contrast of the image formed by the lens on an optical sensor of the analysis system.

[0047] For example, the diffuser may be made of frosted glass. Alternatively, the diffuser may be made of a light-colored material, for example, white polypropylene.

[0048] Advantageously, the light source comprises a light-emitting diode.

[0049] In one embodiment of the invention, the analysis system comprises an optical sensor arranged downstream of the lens so as to acquire at least one image of an object interposed between the lens and the illumination module. This optical sensor may be a CCD (Charge-Coupled Device) type sensor, a CMOS (Complementary Metal-Oxide-Semiconductor) type sensor, a PMT (photomultiplier) type sensor, or an APD (avalanche photodiode) type sensor. The optical sensor may be a pixel sensor, with pixels having, for example, a dimension of 2 µm, so as to capture elementary image portions of 1 µm in the object plane.

[0050] Advantageously, the analysis system comprises a processing unit arranged to implement at least one algorithm for detecting cellular microcompartments and / or cellular tissues and / or cells, and / or cell aggregates contained within these microcompartments in said image of the object acquired by the optical sensor. Thanks to the contrast enhancement introduced by the invention, the processing unit can thus, by means of image processing algorithms and / or machine learning algorithms, reliably segment the encapsulated microcompartments, cells, or cellular tissues within the image of the object. within these microcompartments, see distinguishing single cells, groups, clusters and aggregates of cells, and cysts.

[0051] In the context of the present invention, the term "processing unit" means one or more electronic, computer, and / or software components designed to perform calculation, comparison, and image storage operations in a computer memory. The processing unit may be equipped with one or more microcontrollers and / or processors arranged to execute instructions from one or more computer programs in order to implement one or more image processing algorithms. Alternatively, the processing unit may be integrated into a machine comprising the lighting module, the lens, and the optical sensor, or alternatively, the processing unit may be located remotely from this machine while being connected to it by wired or wireless means.

[0052] For example, the processing unit may be arranged to implement one or more lighting correction algorithms in the image of the object acquired by the optical sensor. This or these lighting correction algorithms may, in particular, include a sequence of a background smoothing algorithm, for example of the Lo regularization type, a background segmentation algorithm, for example of the adaptive segmentation or thresholding type, and an algorithm for correcting each pixel of the image by means of a correction coefficient derived from the segmentation algorithm.

[0053] Alternatively or cumulatively, the processing unit may, for example, be arranged to implement one or more cell pre-detection algorithms in the image of the object acquired by the optical sensor. This or these pre-detection algorithms may, in particular, include a sequence of one or more cell edge detection algorithms, for example by image thresholding, or image transformation, or of only one channel of the image, and a weighted cell segmentation algorithm in the image using a mask of the detected edges, for example of the partitioning or K-means clustering type.

[0054] Alternatively or cumulatively, the processing unit may, for example, be configured to implement one or more algorithms for detecting and segmenting microcompartments, whether empty or filled, in the image of the object acquired by the optical sensor. This or these detection and segmentation algorithm(s) may include one or more algorithms for segmenting the contours of the microcompartments, in particular of the Watershed type, of the morphological closure and opening type, and / or of the machine learning type, previously trained to segment the contours of microcompartments in an image. It may, in particular, be considered to use a convolutional neural network, or CNN, and in particular of the Stardist type, is particularly suitable for detecting cellular structures in an image.

[0055] Alternatively or cumulatively, the processing unit may, for example, be configured to implement one or more algorithms for detecting and segmenting cells or cellular tissues in microcompartments segmented within the image of the object acquired by the optical sensor. This or these detection and segmentation algorithm(s) may, in particular, include one or more algorithms for segmenting cell contours in regions of interest defined by the contours of the microcompartments, for example, of the adaptive segmentation or thresholding type, and / or one or more algorithms for combining cells segmented by one or more pre-detection algorithms and microcompartments segmented by segmentation algorithms.

[0056] Alternatively or cumulatively, the processing unit may, for example, be configured to implement one or more algorithms for detecting and segmenting groups, clusters, and aggregates of cells within the segmented microcompartments in the image of the object acquired by the optical sensor. This or these detection and segmentation algorithm(s) may include one or more partitioning or clustering algorithms, particularly of the K-means type, and optionally one or more classification algorithms for the groups resulting from the partitioning algorithm(s) to identify groups of cells and aggregates of cells, for example, by thresholding the sizes of said groups.

[0057] Alternatively or cumulatively, it may for example provide that the processing unit is arranged to implement one or more classification algorithms for cellular microcompartments and / or cellular tissues and / or cells detected and segmented in said image of the object acquired by the optical sensor.This or these classification algorithms may include one or more machine learning algorithms, in particular of the convolutional neural network type and in particular of the ResNet type, trained beforehand to classify cellular microcompartments and / or cellular tissues and / or segmented cells in an image among a set of predetermined classes, in particular among the following classes: empty microcompartment, microcompartment containing one or more isolated cells, microcompartment containing one or more cellular aggregates, namely clusters of unorganized cells, microcompartment containing one or more cellular microtissues, namely clusters of cells organized for example spherically, smooth, microcompartment containing one or more rough cellular microtissues, open microcompartment, isolated cell, . small aggregate of cells, large aggregate of cells not organized into microtissue, microtissues.

[0058] It may be provided that the processing unit is arranged to implement, automatically or by prior selection by a user, a sequential combination of several of the correction, pre-detection, segmentation and detection algorithms listed above.

[0059] Advantageously, the processing unit is arranged to generate, from the image of the object acquired by the optical sensor and from the cellular microcompartments and / or cellular tissues and / or cells and / or groups, clusters, and aggregates of cells detected and segmented in this image by the algorithm(s), a new image in which these cellular microcompartments and / or cellular tissues and / or cells and / or groups, clusters, and aggregates of cells are identified. The processing unit may, for example, enhance or colorize contours or an interior surface. Preferably, the analysis system includes a screen, and the processing unit is arranged to display the new image on said screen.

[0060] In one embodiment of the invention, the processing unit is arranged to determine, from the result of said detection algorithm, at least one value of a geometric and / or qualitative and / or quantitative parameter of the cellular microcompartments and / or cellular tissues and / or cells contained in said object.

[0061] For example, the processing unit may be arranged to implement at least one algorithm for classifying the shape of the cellular microcompartments and / or cellular tissues and / or cells detected and segmented in the image, for example by calculating a geometric parameter such as circularity or by comparing the shape to predetermined geometric shapes, such as a circle, an ellipsoid or a drop.

[0062] It may be provided that the processing unit to determine a qualitative parameter of each cellular microcompartment and / or cellular tissue and / or cell detected and segmented in the image, for example by calculating a distance from the contour of said cellular microcompartment and / or cellular tissue and / or cell to a contour of the shape with which this cellular microcompartment and / or cellular tissue and / or cell has been classified.

[0063] The processing unit may be provided for in order to determine a quantitative parameter of the cellular microcompartments and / or cellular tissues and / or cells detected and segmented in the image, for example by counting these cellular microcompartments and / or cellular tissues and / or cells in said image, counting the cells contained in each microcompartment in said image or by calculating the dimensions of these cellular microcompartments and / or cellular tissues and / or cells or the average number of cells contained in the microcompartments, including such as the length of their minor axis, their major axis, the area of ​​their surface, their equivalent diameter.

[0064] The invention also relates to a method for analyzing cellular microcompartments or cellular tissues produced using an encapsulation system as described and implemented by an analysis system according to the invention.

[0065] The present invention is now described by means of purely illustrative and in no way limiting examples of the scope of the invention, and from the accompanying drawings, in which the various figures represent:

[0066] [Fig-1] represents, schematically and partially, a view of a system encapsulation of cells in microcompartments;

[0067] [Fig.2] represents, schematically and partially, a cross-sectional view of an analysis system according to one embodiment of the invention;

[0068] [Fig.3] represents, schematically and partially, an image of cellular microcompartments from the system of [Fig.1], obtained using the analysis system of [Fig.2];

[0069] [Fig.4] schematically and partially represents a new image obtained after processing the image of [Fig.3] by the processing unit of [Fig.2]; and

[0070] [Fig.5] represents, schematically and partially, a cross-sectional view of an analysis system according to another embodiment of the invention.

[0071] In the following description, identical elements, by structure or by function, appearing on different figures retain, unless otherwise specified, the same references.

[0072] A cell encapsulation system for the production of cell microcompartments intended to be analyzed by an analysis system according to an embodiment of the invention is shown in [Fig.1].

[0073] The system comprises two containers 11 and 12. A first container 11 contains a solution comprising a plurality of human pluripotent stem cells. A second container 12 contains a gelling solution, comprising, for example, a hydrogel such as alginate. The system may also include a third container containing an intermediate solution, for example, an isotonic solution such as sorbitol.

[0074] The system also includes a milli-fluidic or micro-fluidic encapsulation device 13 arranged to form, from the solutions in containers 11 and 12, cellular microcompartments whose outer layer is the alginate solution and whose core is the cell solution. The encapsulation device 13 has several inlets, each connected to one of the containers 11, 12 via a distributor 14.

[0075] It should be noted that the dispenser 14 for distributing the alginate solution is equipped, in the example described, with a device capable of electrically charging the alginate solution with an electrical potential. Alternatively, the alginate solution may be charged directly in its container 12 via an electrode immersed in the solution.

[0076] The encapsulation device 13 is a microfluidic device comprising a body, including the inlets, and a nozzle connected to a single outlet of the body and forming a single outlet of the device 13. The body and the nozzle may be made of glass or another material suitable for the pharmaceutical industry. The body and the nozzle may form a single piece or, conversely, be made separately and then assembled to form the encapsulation device 13.

[0077] The body allows the formation of a concentric flow from the solutions supplied by the containers 11, 12 via the distributors 14, with an external flow formed by the alginate solution and an internal flow formed by the cell solution. The nozzle thus receives the concentric flow. Given the flow rates of the solutions and the electrostatic force generated by the electrical charges carried by the alginate solution, the encapsulation device 13 thus generates, at the nozzle outlet, a concentric jet from the concentric flow. This concentric jet is fragmented, under the effect of Plateau-Rayleigh instability, into cellular microcompartments, the outer layer of which is the alginate solution and the core the cell solution.

[0078] The encapsulation device 13 is thus of the "electro-jetting" type. It should be noted that the relative sizes of the outer layer and the core of the microcompartments can be adjusted by modifying the flow ratios of the two solutions using the distributors 14, while the overall size of the microcompartments can be controlled by adjusting the overall flow rate of the solutions and the electrical potential of the alginate solution.

[0079] As an alternative, the flow rates of the solutions and the electrical potential may be sized so that the encapsulation device 13 is of the "electro-dripping" type, and thus forms the microcompartments one after the other directly from the nozzle.

[0080] The system further comprises a collection tank 15 arranged below the encapsulation device 13 and containing a first solution for collecting the cellular microcompartments formed by this encapsulation device 13 and falling by gravity into the collection tank 15. The collection tank 15 and the encapsulation device 13 are arranged at a distance from each other such that the cellular microcompartments formed by the encapsulation device pass through a gaseous volume, in particular air, defined by a closed and sterile enclosure before being collected by the collection tank 15.

[0081] Alternatively, the encapsulation device 13 may be arranged so that a nozzle of the encapsulation device is immersed in the stiffening solution contained in the collection tank 15, the encapsulation device 13 being arranged to form, directly in the stiffening solution, said cellular microcompartments from the concentric flow.

[0082] The stiffening solution comprises a surfactant and a calcium salt enabling the crosslinking of an alginate solution, and thus enabling the stiffening of the outer layer of each cell microcompartment upon its immersion in the collection tank.

[0083] Each cellular microcompartment obtained by means of this first stage 1 is thus closed and, in the example described, has a spherical or elongated teardrop shape.

[0084] In one embodiment, the container comprising the cell solution and / or the container comprising the intermediate solution includes at least one extracellular matrix and / or an extracellular matrix substitute. In this case, the encapsulation device may form cellular microcompartments, the outer layer of which is a hydrogel derived from the alginate solution, the intermediate layer is a cellular matrix and / or an extracellular matrix substitute derived from the cell solution and / or the intermediate solution, and at least one layer and / or base and / or aggregate and / or cyst of cells derived from the cell solution. In this example, the electrically charged solution with said electrical potential may be the alginate solution and / or the sorbitol intermediate solution.

[0085] It should be noted that the collection tank 15 includes a withdrawal outlet 151 for the stiffening solution and microcompartments immersed in this solution.

[0086] It may be provided that the lower part of the collection tank 15 is arranged so that the stiffening solution it contains is electrically connected to ground.

[0087] In the described example, containers 11 and 12, dispensers 14, encapsulation device 13, and collection tank 15 form an encapsulation stage 1 for generating the cellular microcompartments of the encapsulation system. The cellular microcompartments can be harvested, via the withdrawal outlet 151, in a harvesting circuit and then cultured in a bioreactor, for example, so that the encapsulated cells form microtissues in these microcompartments.

[0088] [Fig.2] A system of analysis 2 is shown allowing the analysis of cellular microcompartments from the system of [Fig.1] to be carried out indifferently before or after their culture in a bioreactor.

[0089] The analysis system 2 comprises a wide-field visible light microscopy block, including an illumination module 3, a stage 4 for supporting an object to be analyzed, the stage being hollow or transparent, an objective 5 and an optical sensor 6.

[0090] The lighting module 3 comprising a light source 31 capable of emitting visible light. In the example described, this light source 31 comprises a light-emitting diode. The light beam emitted by the light source 31 is thus oriented globally along an emission axis X, pn.

[0091] The lighting module 3 includes a diffuser 32, above the light source 31. The diffuser 32 is for example made of frosted glass.

[0092] The lighting module 3 also includes a diaphragm 33, above the diffuser 32. This diaphragm 33 allows the amount of light coming from the lighting module 3 to be limited.

[0093] The objective 5 comprises an optical imaging system including, in the example described, a stack of several lenses. These lenses may be meniscus lenses, hemispherical lenses, doublets, or triplets. This imaging system thus presents an object plane located at the level of the stage and therefore of the object to be analyzed, and an image plane positioned at the level of the optical sensor 6. The objective 5 can thus form a magnified image of the object to be analyzed on the optical sensor 6.

[0094] The virtual image of the aperture diaphragm of the lens 5, defined by the lenses and / or a diaphragm of the lens, defines an entrance pupil of the lens. Furthermore, the focal points of the lens 5 together define an optical axis X of the lens 5.

[0095] In order to improve the homogeneity of the lighting and contrast of the image formed on the optical sensor 6, the lighting module 3 includes an optical system 7, interposed between the diaphragm 33 and the plate 4.

[0096] This optical system 7 operates a conjugation between the diaphragm 33, namely a point located at the center of the diaphragm 33, and the entrance pupil of the lens 5.

[0097] In the example described, the optical system 7 thus comprises a converging lens centered with the optical axis X and presenting an object plane Fl and a focal plane F2, positioned on either side of the converging lens at a focal distance f.

[0098] The diaphragm 33 is thus placed upstream of the optical system 7, so that the center of the diaphragm 33 is positioned at a distance 2f from this optical system 7, while the lens 5 is placed downstream of the optical system 7, so that its entrance pupil is also positioned at a distance 2f from this optical system 7. In other words, the light rays emanating from this center of the diaphragm 33 are deflected by the optical system 7 to converge towards a position P at which the entrance pupil of the objective 5 is located.

[0099] The conjugation performed by the optical system 7 is thus called 2f-2f conjugation. Although this conjugation is optimal, other conjugations may be considered without departing from the scope of the present invention.

[0100] By way of non-limiting example, the microscopy block shown in [Fig.2] has the following dimensions: a. Focal length f of optical system 7: 50 mm b. distance 2f between optical system 7 and the center of diaphragm 33: 100 mm; c. focal length of lens 5: 30 mm; d. distance between the plate 4 and the optical system 7: 60 mm; e. distance between objective 5 and the entrance pupil: 10 mm; f. diaphragm opening: 4 mm; g. magnification of the objective lens 5:2; h. numerical aperture of lens 5: 0.062.

[0101] In the example of [Fig.2], the lighting module 3 is arranged so that the emission axis X,m of the light source 31 is inclined with respect to the optical axis X of the lens 5.

[0102] More specifically, the entire lighting module 3 is rotated about an axis Y, orthogonal to the optical axis X. The lighting module 3 remains oriented however so that the optical axis X passes through the center of the diaphragm 33, this diaphragm 33, the lens 5 and the optical system 7 being thus centered.

[0103] This shift in the XLED emission axis then causes a notable strengthening of the contrast which makes it possible to clearly identify the cellular microcompartments and their contents in the image acquired by the optical sensor 6.

[0104] Alternatively, it may be possible to consider pivoting around another axis Z orthogonal to the optical axis X, or to offset the lighting module 3 from the optical axis X in one and / or the other of the directions Y and Z.

[0105] As an alternative, it may be provided that only the light source 31 is inclined so that its emission axis X, pp is inclined with respect to the optical axis of the lens.

[0106] In the example of [Fig.2], the optical sensor 6 may be a CCD (Charge-Coupled Device) type sensor, a CMOS (Complementary Metal-Oxide-Semiconductor) type sensor, a PMT or photomultiplier type sensor, or an APD or avalanche photodiode type sensor.

[0107] In view of the description of the microscopy block which has been described, the optical sensor 6 thus acquires an image of the object to be analyzed with homogeneous illumination and homogeneous and enhanced contrast.

[0108] An example of an image I acquired by the microscopy block shown in [Fig. 2] is shown in [Fig. 3]. This image I thus shows a plurality of cellular microcompartments MC formed by the system of [Fig. 1] and comprising isolated C cells or aggregates A of C cells.

[0109] With further reference to [Fig.2], the analysis system comprises a processing unit 8 and a screen 9.

[0110] The processing unit 8 arranged to implement at least one algorithm for detecting cellular microcompartments and / or cellular tissues and / or cells, cellular tissues and / or cell aggregates contained in these microcompartments in said image I represented in [Fig.3].

[0111] Thanks to the contrast enhancement introduced by the combination of the optical system 7 and the offset of the emission axis X,m with respect to the optical axis X, the processing unit can thus, by means of image processing algorithms and / or machine learning algorithms, reliably segment in the image I the microcompartments MC, the C cells or the cellular tissues encapsulated in these microcompartments, see distinguish the individual cells, the groups, clusters and aggregates of A cells, and the cysts.

[0112] In the example described, the processing unit 8 and the screen 9 are integrated into a machine comprising the microscopy unit. Alternatively, the processing unit 8 may be located remotely from the microscopy unit but connected to it by wired or wireless means to receive the images acquired by the optical sensor 6.

[0113] In the example described, the processing unit 8 is thus arranged to apply to the image I of [Fig.3] a sequence of algorithms for lighting correction, pre-detection of C cells, detection and segmentation of MC microcompartments, detection and segmentation of C cells in segmented MC microcompartments and detection and segmentation of aggregates A of C cells in MC microcompartments.

[0114] It may be foreseen that the processing unit 8 applies to the image I a different sequence of image processing algorithms than that described above.

[0115] Following this sequence of algorithms, the microcompartments MC, as well as the cells C and the aggregates A of cells C, are detected and segmented within the image I. The processing unit can then generate an AI-augmented image from the image I, in particular to highlight the contours of the microcompartments MC, the cells and the aggregates, and to label each microcompartment, for example with an identifying number. This AI-enhanced image can then be displayed on screen 9 to help an operator analyze this image and therefore the sample that has been observed.

[0116] An example of an augmented image IA obtained from image I is shown in [Fig.4].

[0117] Furthermore, since the MC microcompartments as well as the C cells and the A aggregates of C cells are detected and segmented, the processing unit 8 can perform on the one hand a count of the microcompartments and, for each MC compartment, a count of the C cells or the A aggregates contained in that MC compartment.

[0118] The processing unit 8 can further classify each microcompartment MC, according to its shape or content, into one of the following classifications: empty microcompartment, microcompartment containing one or more isolated cells, microcompartment containing one or more cell aggregates, namely clusters of unorganized cells, microcompartment containing one or more cellular microtissues, namely clusters of cells organized for example spherically, smooth, microcompartment containing one or more rough cellular microtissues, open microcompartment.

[0119] The processing unit 8 can also, from the contour of each compartment MC, adjust the dimensions of a given geometric shape, such as an ellipse or a drop, so that said shape approaches this contour optimally, and then estimate the distance between the contour and the shape in order to obtain an indicator relating to the quality of the microcompartment.

[0120] It may be foreseen that the processing unit 8 determines other indicators allowing control of the quality of the production process implemented by the system of [Fig.1], such as a ratio of the number of empty microcompartments to the total number of microcompartments, an average number of cells contained in the microcompartments, average dimensions of the microcompartments, the ratio of the number of microcompartments with an abnormal shape to the total number of microcompartments, the ratio of the number of microcompartments belonging to one or each of the classifications to the total number of microcompartments.

[0121] All the data determined by the processing unit 8 can be stored in a computer file.

[0122] An analysis system 20 according to another embodiment of the invention is shown in [Fig.5].

[0123] Similar to the embodiment of [Fig. 2], the analysis system 20 comprises a wide-field visible light microscopy unit, including an illumination module 30, a stage 4 for supporting an object to be analyzed, the stage being hollow or transparent, an objective lens 5, an optical sensor 6, an optical system 7, interposed between the diaphragm 33 and the plate 4 and performing a conjugation between the diaphragm 33 and lens entrance pupil 5, a processing unit 8 and a screen 9.

[0124] On the other hand, unlike the embodiment of [Fig.2], the light source 31 is arranged so that its emission axis X, Er> is oriented in an emission direction substantially orthogonal to the optical axis X of the lens 5. In the example described, this XLED axis is parallel to the Z direction, but it may be provided that this XLED axis is parallel to the Y direction or to a combination of the Y and Z directions.

[0125] The lighting module 30 also includes a flat mirror 34 placed between the diaphragm 33 and the optical system 7. This flat mirror 34 is oriented at 90° with respect to the optical axis X of the lens so as to reflect the light beam emitted by the light source 31 along the XLED axis in a principal emission direction X\Er> substantially parallel to the optical axis X of the lens 5.

[0126] In addition, this plane mirror 34 is mounted on an adjustable device, not shown, to be moved along the Y and / or Z direction and to modify the distance dl between the optical axis X of the lens and the principal emission direction X'i.Er>.

[0127] It is therefore possible to achieve an ideal focus beforehand by aligning the main emission direction X'LED and the optical axis X of the lens, 5 and then moving the flat mirror 34 and therefore the X'LED axis to modify the distance dl and thus enhance the contrast.

[0128] The preceding description clearly explains how the invention achieves its stated objectives, namely, to provide an analytical system for non-destructively monitoring the quality of cellular microcompartments or cellular tissues, particularly those obtained from a three-dimensional culture process, and which offers improved detection and analysis performance. It is understood that these objectives are achieved by means of an imaging system employing a microscopy unit combining an optical system for conjugating an entrance diaphragm and pupil, and an offset of the emission axis of an illumination module relative to the optical axis of the objective lens. This combination provides homogeneous and enhanced contrast.

[0129] In any event, the invention cannot be limited to the embodiments specifically described in this document, and extends in particular to all equivalent means and to any technically operative combination of these means.

Claims

Demands

1. Analysis system (2, 20) for cellular microcompartments (MC) or cellular tissues, comprising: a. an illumination module (3, 30) including a light source (31) and a diaphragm (33); b. an objective (5); characterized in that the illumination module includes an optical system (7) arranged to conjugate a point located at the center of the diaphragm (33) with an entrance pupil of the objective (5) and in that the illumination module is arranged so that the principal emission direction (X, pn, X'LED) of the illumination module is offset with respect to the optical axis (X) of the objective (5).

2. Analysis system (2) according to the preceding claim, characterized in that the lighting module (3) is arranged so that the principal emission direction (X, pp) of the lighting module (3) is inclined with respect to the optical axis (X) of the objective (5).

3. Analysis system (20) according to claim 1, characterized in that the lighting module (3) is arranged so that the main emission direction (X'LED) of the lighting module is translated with respect to the optical axis (X) of the lens (5).

4. Analysis system (2, 20) according to any one of the preceding claims, characterized in that the focal length (f) of the optical system (7) of the lighting module (3) is substantially 50 mm and in that the optical system of the lighting module is placed 100 mm from the point at the center of the diaphragm (33).

5. Analysis system (2, 20) according to any one of the preceding claims, characterized in that the lighting module (3) comprises a diffuser (32) arranged downstream of the diaphragm (33).

6. Analysis system (2, 20) according to the preceding claim, characterized in that the diffuser (32) is made of frosted glass.

7. Analysis system (2, 20) according to any one of the preceding claims, characterized in that the light source (31) comprises a light-emitting diode.

8. An analysis system (2, 20) according to any one of the preceding claims, characterized in that it comprises an optical sensor (6) arranged downstream of the lens (5) so as to acquire at least an image (I) of an object interposed between the lens (5) and the optical system (7) of the lighting module (3).

9. Analysis system (2, 20) according to the preceding claim, characterized in that it comprises a processing unit (8) arranged to implement at least one algorithm for detecting cellular microcompartments (MC) and / or cellular tissues and / or cells (C), cellular tissues and / or aggregates (A) of cells contained in these microcompartments in said image (I) of the object acquired by the optical sensor (6).

10. Analysis system (2, 20) according to the preceding claim, characterized in that the processing unit (8) is arranged to determine, from the result of said detection algorithm, at least one value of a geometric and / or qualitative and / or quantitative parameter of the cellular microcompartments (MC) and / or cellular tissues and / or cells (C) contained in said object.

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