Process for characterizing a biological micro-tissue by imaging

Abstract

The invention relates to a method for in vitro characterization of a eukaryotic biological micro-tissue by imaging using a phase measurement technique without a reference beam, and the use of this method in particular for: - controlling the quality of a micro-tissue, - measuring the increase in the biomass of a micro-tissue during its culture and / or its amplification, - monitoring the differentiation and / or the organization of a micro-tissue during its maturation, - determining the phenotype of cells in the micro-tissue, and / or - confirming the absence of undifferentiated cells in the micro-tissue.

Description

Title of the invention: Method for characterizing a biological micro-tissue by imaging

[0001] The present invention relates to the characterization by imaging of biological tissues, in particular biological micro-tissues.

[0002] In research as in therapy, it is essential to be able to characterize living biological cells and tissues, in particular during or after cell culture, in particular to control cell proliferation and / or the quality of the cells and tissue and / or to monitor cell differentiation and / or to monitor the organization of the tissue and / or to determine the phenotype(s) of the cells constituting a tissue, etc.

[0003] However, current imaging techniques, which are in particular those implementing fluorescence microscopy, histology, capacitance measurement, optical density and standard transmission imaging, do not allow this.

[0004] Fluorescence microscopy is used coupled with fluorescent probes such as antibodies or endogenous fluorescence by genetically modifying the cells. Several techniques implementing fluorescence microscopy are generally used, in particular confocal microscopy, light sheet microscopy (or SPIM for "Selective Plane Illumination Microscopy"), multiphoton microscopy, flow cytometry ("facs"). These techniques are very established but they require fixation (cell death) and / or limiting conditions (labeling only of extracellular proteins) or incompatible with cell culture for the purpose of cell therapy, such as the addition of non-GMP products that are destructive or that lead to the genetic modification of the cells. They are therefore not suitable for the characterization of living tissues because they are invasive, often destructive and very slow.

[0005] Histology techniques consist of fixing and then marking tissues. They also result in the destruction of cells and have the same disadvantages as fluorescence microscopy.

[0006] The measurement of biomass by capacitance probe starts from the a priori that living cells can be considered as capacitors. This measurement therefore only takes into account the accessible external surface of cells having an intact membrane. The case of cell aggregates and micro-tissues is more complex and will depend on the tightness of the connections between cells. Unlike the previous methods, this one is non-invasive but it only allows information to be obtained on the non-accessible volume or on the surface of this volume, which is too limiting and imprecise for the characterization of tissues.

[0007] Standard transmission imaging techniques, such as quantitative phase contrast, are fast and non-invasive. Phase measurement in imaging is a measurement of the local delay of a light beam after interaction with the object under study. The devices used for phase imaging are based on the phenomenon of optical interference to encode the phase information into light intensity information. Different techniques in phase imaging for microscopy are described in particular in (Park, Y., Depeursinge, C. & Popescu, G. Quantitative phase imaging in biomedicine. Nature Photon 12, 578-589 (2018) doi:10.1038 / s41566-018-0253-x). Phase imaging is currently used for the characterization of thin samples (isolated cells or micro-tissue sections with thicknesses less than 100 m) but cannot be used for micro-tissues or larger tissues.Indeed, the techniques currently used in phase imaging do not allow a quantitative measurement of the phase in a tissue. They remain qualitative and not quantitative and are therefore difficult to use for the characterization of thick objects.

[0008] Finally, the optical density measurement, obtained by phase measurements and allowing access to the mass of the sample, does not allow the characterization of tissues larger than 100 m.

[0009] The objective of the invention is to overcome these various problems of the prior art, and to propose a solution for a complete, rapid and non-invasive in vitro characterization method for living tissues, which makes it possible to characterize in particular living biological micro-tissues, in particular during or at the end of a cell culture, in research or in therapy. Summary of the invention

[0010] According to the invention, the measurement of the phase as currently carried out on very fine cells and micro-tissues necessarily uses a reference beam, which leads to an unsuitable absolute measurement because it does not allow the mass to be measured and a certain number of parameters to be quantified.

[0011] This is why, to meet the objective of the invention, the inventors have developed a method for in vitro characterization of a human, animal or plant biological micro-tissue whose smallest dimension is greater than or equal to 20 μm, said method consisting of using a phase measurement technique without a reference beam and not requiring the use of fluorescent labeling.

[0012] Indeed, according to the invention, only phase measurement techniques without a reference beam, i.e. indirect measurement techniques or also called relative phase measurements, can work to characterize micro-tissues whose smallest dimension is greater than or equal to 20 μm.

[0013] Advantageously, the use of a phase measurement technique without a reference beam makes it possible to quantitatively characterize living biological micro-tissues at high throughput without destroying or modifying them.

[0014] This makes it possible in particular to: - measure the increase in the biomass of a micro-tissue during its culture and / or amplification, - determine cell viability, - control the quality of a micro-tissue, - follow the differentiation and / or organization of a micro-tissue during its maturation, - determine the phenotype of cells in the micro-tissue, and / or - confirm the absence of undifferentiated cells in the micro-tissue.

[0015] The invention therefore also relates to the use of the characterization method for these applications in particular. Brief description of the Figures

[0016] [Fig-1]: [Fig.l] is an image of hydrogel capsules containing cells human induced pluripotency, obtained by imaging by phase measurement without reference beam, according to the protocol described in the example.

[0017] [Fig.2]: [Fig.2] is an image of hydrogel capsules containing cells human induced pluripotency, obtained by imaging by measuring the intensity of the phase (absolute measurement), according to the protocol described in the example.

[0018] [Fig.3]: [Fig.3] is a schematic representation of the implementation of a variant of the online process for characterizing biological micro-tissues contained in a bioreactor.

[0019] [Fig.4]: [Fig.4] is a schematic representation of the imaging system of phase measurement without reference beam used for the characterization method according to the invention described in example 1.

[0020] Definitions

[0021] By "local absorption" of the micro-tissue within the meaning of the invention, we mean the attenuation of light due to a loss of local photon by absorption of light and not diffusion.

[0022] By "alginate" within the meaning of the invention linear polysaccharides formed from [3-D-mannuronate and aL-guluronate, salts and derivatives thereof.

[0023] For the purposes of the invention, the term “hydrogel capsule” means a three-dimensional structure formed from a matrix of polymer chains swollen by a liquid, preferably water.

[0024] For the purposes of the invention, the term "human cells" means human cells or immunologically humanized non-human mammalian cells. Even where not specified, the cells, stem cells, progenitor cells and tissues according to the invention are constituted or are obtained from human cells or from immunologically humanized non-human mammalian cells.

[0025] For the purposes of the invention, the term "progenitor cell" means a stem cell already engaged in cellular differentiation (for example into retinal cells) but not yet differentiated. A progenitor cell is a cell that tends to differentiate into a specific type of cell. It is therefore already more specific than a stem cell. Progenitor cells can only divide a limited number of times, being naturally subject to erosion of their telomeres.

[0026] For the purposes of the invention, the term "embryonic stem cell" means a pluripotent stem cell derived from the inner cell mass of the blastocyst. The pluripotency of embryonic stem cells can be assessed by the presence of markers such as the transcription factors OCT4 and NANOG and surface markers such as SSEA3 / 4, Tra-1-60 and Tra-1-81. Embryonic stem cells can be obtained without destroying the embryo from which they originate, for example using the technique described in Chang et al. (Cell Stem Cell, 2008, 2(2)): 113-117). Optionally, embryonic stem cells from humans can be excluded.

[0027] For the purposes of the invention, the term "pluripotent stem cell" or "pluripotent cell" means a cell that has the capacity to form all the tissues present in the entire organism of origin, without being able to form an entire organism as such. These may in particular be induced pluripotent stem cells, embryonic stem cells or MUSE cells (for "Multilineage-differentiating Stress Enduring"). Pluripotent stem cells maintain the length of their telomeres and can retain the capacity to divide without a clear limit on the number of cell cycles, unlike progenitor cells.

[0028] For the purposes of the invention, the term "induced pluripotent stem cell" means a pluripotent stem cell induced to pluripotency by genetic reprogramming of differentiated somatic cells. These cells are in particular positive for pluripotency markers, such as alkaline phosphatase staining and expression of the proteins NANOG, SOX2, OCT4 and SSEA3 / 4. Examples of methods for obtaining induced pluripotent stem cells are described in the articles Yu et al. (Science 2007, 318 (5858): 1917-1920), Takahashi et al (Cell, 207, 131(5): 861-872) and Nakagawa et al (Nat Biotechnol, 2008, 26(1): 101-106).

[0029] By “differentiated” cells within the meaning of the invention we mean cells which exhibit a particular phenotype, as opposed to pluripotent stem cells which are not differentiated.

[0030] By “density” of the micro-tissue within the meaning of the invention, we mean the mass of a unit of volume divided by the mass of the same volume of culture medium.

[0031] By “micro-tissue” or “biological micro-tissue” within the meaning of the invention, we mean a biological tissue or a sample of biological tissue whose largest dimension is less than or equal to 1 cm.

[0032] By "phase", within the meaning of the invention, we mean the delay of the light wavefront, relative phase shift and difference in optical path between the environment of the micro-tissue and the base level of the medium in which it is immersed.

[0033] By “phase measurement technique” within the meaning of the invention, we mean any technique capable of quantitatively measuring the phase of light.

[0034] By "phase measurement technique without reference beam" within the meaning of the invention, we mean techniques capable of tracing the phase component of the light without resorting to the use of an external beam, called "reference", which has not interacted with the micro-tissue.

[0035] By "tissue" or "biological tissue" within the meaning of the invention, we mean the common meaning of tissue in biology, that is to say the intermediate level of organization between the cell and the organ. A tissue is a set of similar cells of the same origin (most often from a common cell lineage, although they can find their origin by association of distinct cell lineages), grouped in clusters, networks or bundles (fibers). A tissue forms a functional whole, that is to say its cells contribute to the same function. Biological tissues regenerate regularly and are assembled together to form organs. A tissue can comprise differentiated cells and stem cells. Typically, pluripotent stem cells form an epithelial-type tissue, called epiblastic (citation: Self-organization of the human embryo in the absence of maternal tissues, Shahbazi et al., Nat Cell Biol. 2016, doi: 10.1038 / ncb3347)..

[0036] By “texture” of a micro-tissue within the meaning of the invention, we mean the local roughness of the image and its local frequency content. Detailed description

[0037] The subject of the invention is therefore a method for in vitro characterization of a eukaryotic biological micro-tissue, in particular a human, animal or plant micro-tissue, the smallest dimension of which is greater than or equal to 20 μm, even more preferably greater than or equal to 30 μm.

[0038] The biological micro-tissue is preferably a micro-tissue whose largest dimension is less than or equal to 10 mm, even more preferably less than or equal to 1mm and in particular less than or equal to 500pm.

[0039] The biological micro-tissue may be a micro-tissue comprising eukaryotic cells, in particular human cells, or animal (non-human) cells, in particular amniote cells and in particular mammalian cells, or plant cells.

[0040] The biological micro-tissue when it is a human or animal micro-tissue can for example be chosen from epithelial, connective, muscular or nervous micro-tissues. According to one embodiment, the micro-tissue may in particular comprise: - differentiated cardiac cells or retinal cells or neural cells or liver cells or chondrocytes or keratinocytes or lymphoid cells or hematopoietic stem cells or mesenchymal stem cells, and / or: - progenitor stem cells - endothelial cells.

[0041] According to another embodiment, the micro-tissue may comprise or consist of pluripotent cells in the form of an epiblast.

[0042] The biological micro-tissue when it is a human or animal micro-tissue, for example, can be chosen from the different phases of embryonic or fetal development, in particular in the early phases of development in the context of in vitro fertilization for reproductive purposes (in humans or animals) or research (in humans or animals) or animal production.

[0043] The biological micro-tissue when it is a plant micro-tissue can for example be chosen from meristems, parenchymas, conductive tissues, support tissues, covering or protective tissues, secretory tissues and nourishing tissues.

[0044] The micro-tissue may be surrounded at least partially by an extracellular matrix. The cellular matrix layer may consist of cellular matrix secreted by cells of the micro-tissue and / or by added extracellular matrix. The extracellular matrix layer may form a gel. It preferably comprises a mixture of proteins and extracellular compounds necessary for the culture of the cells constituting the micro-tissue. Preferably, the extracellular matrix comprises structural proteins, such as collagen, laminins, entactin, vitronectin, as well as growth factors, such as TGF-beta and / or EGF. The extracellular matrix layer may consist of or comprise Matrigel® and / or Geltrex® and / or a hydrogel-type matrix of plant origin such as modified alginates or of synthetic origin or of a copolymer of poly(N-isopropylacrylamide) and poly(ethylene glycol) (PNIPAAm-PEG) type Mebiol®.

[0045] According to a variant, the micro-tissue may be a micro-tissue encapsulated in a micro-compartment or a capsule comprising an external hydrogel layer, such as, for example, the micro-compartments described in patent application WO2018 / 096277. This is referred to as a hydrogel capsule. Preferably, the hydrogel used is biocompatible, i.e. it is not toxic to the cells. The hydrogel capsule must allow the diffusion of oxygen and nutrients to feed the cells contained in the micro-compartment and allow their survival. The external hydrogel layer may be an external layer comprising alginate. It may consist exclusively of alginate. The alginate may in particular be a sodium alginate, composed of 80% α-L-guluronate and 20% β-D-mannuronate, with an average molecular mass of 100 to 400 kDa and a total concentration of between 0.5 and 5% by mass.The hydrogel capsule helps protect cells from the external environment and limit uncontrolled cell proliferation.

[0046] The micro-tissue can be in any three-dimensional form, that is to say it can have the shape of any object in space. It can be in the form, for example, of a hollow or solid ovoid, a hollow or solid cylinder, a hollow or solid tuboid or tube, a hollow or solid spheroid or sphere, or monolayers partially folded on themselves (2.5D). It is the outer layer of the micro-tissue or the extracellular matrix layer when present, which gives the micro-tissue its size and shape. An example of a solid micro-tissue is the cardiac spheroid used in bioproduction (https: / / doi.Org / 10.1016 / j.bbamcr.2015.ll.036)

[0047] According to one embodiment of the invention, the micro-tissue may be a human or animal biological micro-tissue intended to be grafted into humans or animals.

[0048] The micro-tissue during the implementation of the method can be produced on a frozen or non-frozen living micro-tissue.

[0049] The method according to the invention comprises the characterization of the micro-tissue in imaging by a phase measurement technique without reference beam.

[0050] Preferably, the phase measurement technique without reference beam is chosen from: - wavefront analysis - dynamic modulation of the phase or light intensity in the pupil of the illumination or imaging system - multiple light intensity imaging with modification of the focus plane.

[0051] When the phase measurement technique without reference beam is wavefront analysis, it is preferably carried out using wavefront gradient imaging, and in particular a wavefront gradient imaging technique. wave chosen from: - the Shack-Hartmann method (Gong, H. et al. Optical path difference microscopy with a Shack Hartmann wavefront sensor. Opt. Lett. (2017) oi:10.1364 / OL.42.002122), - the modified (or not) Hartmann method (Bon, P., Maucort, G., Wattellier, B. & Monneret, S. Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells. Opt. Express 17, 13080-13094 (2009)), - pupil partitioning (Parthasarathy, AB, Chu, KK, Ford, TN & Mertz, J. Quantitative phase imaging using a partitioned detection aperture. Opt. Lett. 37, 4062-4064 (2012)), - speckle field imaging (Berto, P., Rigneault, H. & Guillon, M. Wavefront sensing with a thin diffuser. Opt. Lett. 42, 5117-5120 (2017)).

[0052] Preferably, the phase measurement technique without reference beam used in the method according to the invention is the modified Hartmann method because it is the technique making it possible to obtain the best compromise in terms of stability, sensitivity and compactness for the characterization of micro-tissues.

[0053] When the phase measurement technique without reference beam is the dynamic modulation of the phase or the light intensity in the pupil of the illumination or imaging system, it is preferably carried out using: - ptychography (Zheng, G., Horstmeyer, R. & Yang, C. Wide-field, high-resolution Fourier ptychographic microscopy. Nat. Photonics 7, 739 (2013)), or - selective phase modulation of certain frequencies in the pupil (Wang, Z. et al. Spatial light interference microscopy (SLIM). Opt. Express 19, 1016-1026 (2011)).

[0054] Preferably, the phase measurement technique without reference beam used in the method according to the invention is the ptychography technique because the quantification of the phase is more direct than selective phase modulation which only gives relatively quantitative images.

[0055] When the phase measurement technique without reference beam is multiple light intensity imaging with modification of the focusing plane, it is preferably carried out using: - simultaneous multiplane imaging (Descloux, A. et al. Combined multi-plane phase retrieval and super-resolution optical fluctuation imaging for 4D cell microscopy. Nat. Photonics 12, 165-172 (2018)) or sequential multiplane imaging (Soto, JM, Rodrigo, JA & Alieva, T. Label-free quantitative 3D tomography imaging for partially coherent light microscopy. Opt. Express 25, 15699-15712 (2017) or Barty, A., Nugent, KA, Paganin, D. & Roberts, A. Quantitative opticalphase microscopy. Opt. Lett. 23, 817-819 (1998)).

[0056] Preferably, the phase measurement technique without reference beam used in the method according to the invention is the simultaneous technique, because it is fast, even if it presents a greater complexity than sequential multiplane imaging.

[0057] Whatever the technique for measuring the phase without a reference beam, the method preferably comprises measuring the phase and possibly the light intensity of the light having passed through the micro-tissue.

[0058] According to a preferred embodiment, the method comprises measuring: - the density of the micro-tissue, from the phase measurement, and - possibly from the local absorption of the micro-tissue, from the measurement of the phase and the measurement of the luminous intensity of the light having passed through the micro-tissue.

[0059] The density of the micro-tissue can be measured from the phase measurement as follows: 1) the area of ​​the image containing the micro-tissue (called the useful area) is separated from the rest (called the background, generally the culture medium); 2) the value of the background phase is subtracted from the phase of the useful area; 3) after this subtraction, the phase is converted if necessary into optical path difference (expressed in metric units) and summed over the entire useful area; 4) this value is multiplied by the surface area of ​​an elementary pixel of the image, brought back into the object plane: we obtain a value in metric units cubed; 5) this quantity is divided by the specific refraction increment (Barer, Interference microscopy and mass determination, Nature, 1952) which is 0.18 pmVpg on average and which can be adjusted for each tissue: we thus obtain a measurement of the so-called dry mass (total mass - mass of the culture medium) integrated over the entire sample. A comparison and description of this technique is available (Zangle, T. and Teitell, MA, Live-cell mass profiling: an emerging approach in quantitative biophysics. Nature Methods, 2014) Density is expressed in mass units (g).

[0060] The local absorption of the micro-tissue can be measured from the measurement of the phase and the measurement of the light intensity of the light having passed through the micro-tissue as follows. By a joint measurement of the phase and the intensity 1, we obtain the electromagnetic field £ _ This complex quantity can be decomposed by extracting the real and imaginary part in the digital Fourier space of the electromagnetic field (via a Fourier transform). By returning to the direct space (via an inverse Fourier transform) of the real component of the Fourier space we can go back to the component of the local absorption. The measurement of absorption is expressed in photon / cm2.

[0061] Preferably, the method according to the invention comprises the measurement of at least one of the following parameters: - dimensions of the micro-tissue - dimensions of at least one of the cells of the micro-tissue - number of cells in the micro-tissue - overall and local mass of the micro-tissue - global and local density of micro-tissue - mass distribution in the micro-tissue - organization of cells in the micro-tissue: topology of the micro-tissue and relative positioning of cells in the micro-tissue - viability of microtissue cells - texture.

[0062] The dimensions of the micro-tissue can be measured from the phase measurement as follows. The dimensions in the image plane are extracted by automatic clipping (e.g., Otsu-type edge detection algorithm, or manual clipping) and the dimensions are obtained by adjusting the clipping by an ellipse (in the case of an ovoid micro-tissue). Dimensions are expressed in micrometers.

[0063] The dimensions of one or more cells of the micro-tissue can be measured from the phase measurement as follows. When the optical resolution is better than the size of a cell, a manual or automatic clipping is carried out within the micro-tissue (edge ​​detection algorithm or watershed line). The dimensions are then obtained by adjusting each automatic clipping by an ellipse. The dimensions of a cell are expressed in micrometers.

[0064] The overall mass of the micro-tissue can be measured from the phase measurement as follows. The sum of the phase information (in the sense of the optical path, expressed in pm) on the area containing the micro-tissue (obtained by automatic or manual clipping) is then multiplied by the surface of a phase pixel brought back into the object space (expressed in pm2) then multiplied by the specific increment (generally 0.18pg / pm3) to obtain the overall mass measurement. The overall mass is expressed in micrograms.

[0065] The local mass of the micro-tissue can be measured from the phase measurement as follows. The same method as for the previous point is applied but by summing the phase only over a selected part of the micro-tissue. The local mass is expressed in micrograms.

[0066] The overall density of the microtissue can be measured from the phase measurement as follows. The mass is measured from the phase. The transverse dimensions in the image plane are obtained with the phase image. The dimension in the orthogonal plane of the phase image (called thickness) is obtained: a) either by a 3D reconstruction of the object in different imaging planes, b) or from an assumption on the shape of the object (generally ovoid), c) or from an assumption on the average optical refractive index of the micro-tissue and the medium which allows, by dividing the phase (in the sense of the optical path difference) by the difference in refractive index, to go back to the thickness of the micro-tissue. The three dimensions are combined to obtain the volume of the sample. By dividing the mass by the volume, we go back to the density. The overall density is expressed in g / cm3

[0067] The local density of the micro-tissue can be measured from the phase measurement as follows. The same protocol as for the measurement of the global density is used but restricting the measured area to a sub-part of the micro-tissue. The local density is expressed in g / cm3.

[0068] The mass distribution in the microtissue can be measured from the phase measurement as follows. Local mass measurements are performed on sub-parts of the microtissue covering all or part of the microtissue. A statistical analysis of its masses (such as standard deviation / standard deviation, mean / median) is performed. The mass distribution is expressed in grams.

[0069] The organization of the cells in the micro-tissue is to be taken in the histological sense of the term as appreciated by a person skilled in the art and qualifies the topology of the tissue and the relative positioning of the cells and the extracellular matrix elements. The viability of the cells of the micro-tissue can be measured from the phase measurement as follows: local mass measurements are carried out on sub-parts of the micro-tissue covering all or part of the micro-tissue. A statistical analysis of its masses (type standard deviation / standard deviation, mean / median) is carried out. The mass distribution is then correlated with a conventional histological analysis to generate a training dataset that is analyzed and annotated. Algorithmic and / or directed machine learning (such as a neural network) can then be performed on this dataset to automate the process. The organization of cells in the micro-tissue is therefore qualified by an expert system, human or not, based on histological classification.

[0070] Cell death phenomena cause a change in density and size of cells detectable in Phase. The viability of the cells of the micro-tissue can be measured from the measurement of the phase as follows: local mass measurements are carried out on sub-parts of the micro-tissue covering all or part of the micro-tissue. A statistical analysis of its masses (type standard deviation / standard deviation, mean / median) is carried out. The mass distribution is then correlated with Common viability measures such as ethidium bromide (dead cells) and calcein (live cells) identifying the percentage of live cells to generate a training dataset that is analyzed and annotated. Algorithmic and / or directed machine learning (such as neural networks) can then be performed on this dataset to automate the process. The viability of cells in the micro-tissue is therefore expressed as a percentage of living cells out of the total number of cells. The texture of microtissue can be measured from the phase measurement as follows. Measurement of the spatial variation statistics of the phase including the standard deviation and frequency distribution of the image structures within the region of interest allows the determination of texture parameters.

[0071] Texture is expressed in phase unit and (phase unit) / pm.

[0072] According to one embodiment, the method according to the invention can be carried out in vitro on micro-tissues that have been previously taken from a human being, an animal or a plant. The process can allow exeloke to characterize the quality of an islet of Langerhans from a cadaver (in particular its viability) before transplantation in a diabetic patient, or even to characterize a pre-implantation embryo.

[0073] According to another embodiment, the method according to the invention can be carried out in vitro on micro-tissues comprising pluripotent or progenitor stem cells intended to be differentiated or on micro-tissues comprising cells in the process of differentiation or on micro-tissues comprising differentiated cells obtained by cell culture from pluripotent or progenitor stem cells. The method according to the invention can be carried out in vitro on micro-tissues consisting of pluripotent layer cells intended to be differentiated or on micro-tissues consisting of cells in the process of differentiation or on micro-tissues consisting of differentiated cells obtained by cell culture from pluripotent or progenitor stem cells.

[0074] According to a variant, the method according to the invention is implemented online on the content of a bioreactor. An example of such a variant applied to cell culture in capsules or microcompartments is shown in [Fig.3]. In this example, capsules 12 each containing a microtissue are suspended in a culture medium 14 in a bioreactor 10. Output means 16 provided on the bioreactor allow the capsules 12 to be removed from their culture medium 14 and passed into a non-reference beam phase measurement imaging system 18. At the output of this system 18, the capsules containing microtissues meeting the quality criteria defined by the bioreactor user, said normal capsules 12-1 in their culture medium 14 are reintegrated via input means 20 into the bioreactor 10 and the undesirable capsules 12-2 do not not meeting the quality criteria defined by the user of the bioreactor, are recovered via elimination means 22 to be eliminated. The outlet means 16 may be for example a tube and a peristaltic pump. The inlet means 20 may be for example a tube. The elimination means 22 may be for example a piezoelectric valve system. The system 18 may be any imaging system suitable for measuring the phase without a reference beam such as one of those described in the present application.

[0075] This advantageously makes it possible to check the quality of the micro-tissues, in particular online during differentiation or maturation. The method according to the invention, in particular in the context of an implementation during differentiation or maturation of the cells forming a micro-tissue in a bioreactor, can thus be carried out: i) in flow cells, i.e. by continuous recirculation of the contents of a bioreactor-type culture chamber within a sterile fluidic system for the purposes of analysis and / or sorting of the contents of said culture chamber, or ii) in one-off sampling outside the bioreactor to analyze at a particular point in time a part of said bioreactor, typically during sampling for analysis purposes and / or re-seeding of a second bioreactor as part of a "seed train" and / or as part of an increase in volume and / or fragmentation of the contents of the bioreactor into several enclosures or quality control conditions, or iii) to sort the micro-tissues offline during emptying of the bioreactor for the purposes of purification or continuation of a production sequence and / or differentiation and / or conditioning.

[0076] The method according to the invention has many advantages over the methods currently used. In particular, it can be implemented without destroying or modifying the micro-tissues studied, it is quick to implement, requires simple equipment, and makes it possible to measure many physical parameters to characterize the micro-tissues, which was not possible with the methods of the prior art.

[0077] The method can thus be used for numerous applications. In particular, the invention relates to the use of the method for: - control the quality of a micro-tissue: in fact, the implementation of the method according to the invention makes it possible to measure characteristics of the micro-tissue such as its size, its density, the number of cells or its texture which make it possible to verify the quality of a micro-tissue, and / or - measure the increase in the biomass of a micro-tissue during its culture and / or its amplification: in fact, the method according to the invention makes it possible to measure the overall or local mass of a micro-tissue and thus allows the monitoring of the increase in the number of cells in a micro-tissue during its culture, its differentiation and / or its amplification, and / or - follow the differentiation and / or the evolution of the topology of a micro-tissue during its maturation, and in particular the relative position in space of the cells composing it: in fact, the method according to the invention makes it possible to measure the distribution of mass in the micro-tissue and / or the organization of the cells in the micro-tissue and / or the viability of the cells of the micro-tissue, during differentiation and / or maturation of the cells of the micro-tissue, which gives information on the differentiation and / or maturation of said cells, and / or - determine the phenotype of cells of the micro-tissue, in fact, the method according to the invention makes it possible to measure the mass of each cell and the texture of the micro-tissue, which gives information on the phenotype of said cells, and / or - confirm the absence of undifferentiated cells in the micro-tissue: in fact, the measurement of cell mass and / or cell density, the texture of the micro-tissue and / or the organization of cells in the micro-tissue provides information on the differentiation of the cells in the micro-tissue and consequently the possible absence of differentiation of said cells.

[0078] According to a particular embodiment, the micro-tissue can be an embryo. Thus, the method according to the invention can be used for screening embryos obtained by in vitro fertilization. The histological structure of a healthy embryo is typical, very reproducible and predictive of the success of implantation of the embryo in the mother. In particular, to describe this structure on an embryo to be reimplanted, only imaging solutions without labeling are possible. To improve the yield of implantations and reduce the risk of failure or, on the contrary, of multiple embryos, clinics are developing increasingly precise monitoring of the fertilized embryo in preimplantation, in particular with video monitoring of development. The method according to the invention makes it possible to exclude embryos with an abnormal structure more effectively by adding a relevant and label-free, therefore non-destructive, source of information.

[0079] According to another embodiment, the micro-tissue is a micro-tissue produced for the purposes of drug bioproduction or food bioproduction.

[0080] The invention is now illustrated by an example of implementation of the method according to the invention compared to an example of a characterization method of the prior art. Examples

[0081] In this example, the method relates to the analysis of a human micro-tissue contained in a microcompartment, as described in example 1 of application WO2018 / 096277 (example 1: protocol for obtaining cellular microcompartments from human cells induced to pluripotency).

[0082] A microcompartment was analyzed using an intensity measurement imaging method, as described in (Bon P. et al, Quadriwave lateral shearing inter-ferometry for quantitative phase microscopy of living cells, 2009 Optical Society of America). The intensity was obtained by demodulation of low frequencies via processing in Fourier space of an interferogram obtained with the protocol shown schematically in [Fig.4]. The results obtained are presented in [Fig.2].

[0083] A microcompartment was analyzed according to a method according to the invention. The operating protocol is described as follows: halogen light is used to illuminate the sample in transmission, a microscope objective (x20, numerical aperture 0.5) mounted on an inverted microscope is used to form the image of the sample on a self-referenced interferometer (detector sensitive among other things to the phase). The imaging technique used is wavefront gradient imaging and in particular the modified Hartmann method. The protocol is also shown schematically in [Fig.4]. [Fig.4] shows the illumination system, the sample, the microscope and the phase-sensitive detector. A zoom is shown to describe the modified Hartmann setup used to obtain Figures 1 and 2. The results obtained are presented in [Fig.l].

[0084] It is noted that the image obtained with the method according to the invention makes it possible to measure the local density of the sample and to decorrelate it from the absorption in comparison with the image obtained by the technique of the prior art. In particular, the method according to the invention made it possible to measure: -the total dimensions of each micro-tissue which are 91pm (for the largest) and 59pm (for the smallest) and calculated by measuring the diameter in pixels of each micro-tissue on the image D. Knowing the total magnification of the imaging system gy and the physical size of a pixel measured Tpix, the dimension of a micro-tissue is then D x Tpix / gy. - The diameter of the light zone in the center of each micro-tissue by an analysis of the dimensions of the zone presenting a homogeneous granularity and a weaker phase shift (i.e. darker image) in the center of the micro-tissue, measured at 38pm for the largest micro-tissue and 25pm for the smallest. - the dry mass of the object (the integral over the object of the density) which is 39pg for the largest micro-tissue and 16pg for the smallest, calculated as described in the publication (Aknoun S. et al., Living cell dry mass measurement using quantitative phase imaging with quadriwave lateral shearing interferometry: an accuracy and sensitivity discussion, J / of Biomedical Optics, 2015). Briefly, this involves automatically cropping each micro-tissue by determining the edges, evaluating the phase value of the background by polynomial fit, subtracting it from the image, summing all the phase information of each micro-tissue and converting this into mass by the following equation: m = 0. Ï8pg / pm3 x JJ phase • ds microtissue - the number of cells which is 608+176 cells for the largest micro-tissue and 244+64 cells for the smallest micro-tissue. This value is obtained by two complementary approaches. Knowing the average size of a cell (5pm) and the volume of the area where there are cells in the micro-tissue (volume of the micro-tissue - volume of the lumen) we deduce a value of the number of cells (respectively 421 and 180 cells). Knowing the average mass of a stem cell (50pg) and the total mass of the micro-tissue we deduce another evaluation of the number of cells (respectively 784 and 312 cells).

Claims

Claims

1. Method for in vitro characterization of a eukaryotic biological micro-tissue whose smallest dimension is greater than or equal to 20 pm, by imaging using a phase measurement technique without a reference beam, said method comprising at least the study of the organization of the cells in the micro-tissue.

2. Method for in vitro characterization of a micro-tissue according to claim 1, the micro-tissue being a human, animal or plant micro-tissue.

3. Method for in vitro characterization of a micro-tissue according to one of the preceding claims, characterized in that the largest dimension is less than or equal to 10 mm.

4. Method for in vitro characterization of a micro-tissue according to one of the preceding claims, characterized in that the technique for measuring the phase without reference beam is chosen from: - wavefront analysis - dynamic modulation of the phase or of the light intensity in the pupil of the illumination or imaging system - multiple imaging of light intensity with modification of the focusing plane.

5. Method for characterizing a micro-tissue according to one of the preceding claims, characterized in that the technique for measuring the phase without a reference beam is wavefront analysis and in that it is carried out using imaging of the gradients of the wavefront.

6. Method for characterizing a micro-tissue according to the preceding claim, characterized in that the imaging of the wavefront gradients is chosen from the Shack-Hartmann method, modified Hartmann or not, pupil partitioning and speckle field imaging.

7. Method for characterizing a micro-tissue according to one of claims 1 to 4, characterized in that the technique for measuring the phase without reference beam is the dynamic modulation of the phase or the light intensity in the pupil of the illumination or imaging system and in that it is carried out using the ptychography technique or the selective phase modulation of certain frequencies in the pupil.

8. Method for characterizing a micro-tissue according to one of claims 1 to 4, characterized in that the phase measurement technique without reference beam is multiple light intensity imaging with changing the focus plane and in that it is performed using simultaneous or sequential multiplane imaging.

9. Method for characterizing a micro-tissue according to one of the preceding claims, characterized in that it comprises measuring the phase and the light intensity of the light having passed through the micro-tissue.

10. Method for characterizing a micro-tissue according to one of the preceding claims, characterized in that it comprises the measurement: - of the density of the micro-tissue, from the measurement of the phase, and - possibly of the local absorption of the micro-tissue, from the measurement of the phase and the measurement of the light intensity of the light having passed through the micro-tissue.

11. Method for characterizing a micro-tissue according to one of the preceding claims, characterized in that it comprises the measurement of at least one of the following parameters: - dimensions of the micro-tissue, - dimensions of at least one of the cells of the micro-tissue, - number of cells in the micro-tissue, - overall and local mass of the micro-tissue, - overall and local density of the micro-tissue, - mass distribution in the micro-tissue, - topology of the micro-tissue - relative positioning of the cells in the micro-tissue, - viability of the cells of the micro-tissue, - texture of the micro-tissue.

12. Method for characterizing a micro-tissue according to one of the preceding claims, characterized in that the micro-tissue is encapsulated in a micro-compartment comprising an external hydrogel layer.

13. Method for characterizing a micro-tissue according to one of the preceding claims, characterized in that the micro-tissue is in the form of an ovoid, a tuboid, a spheroid or a sphere, or of monolayers partially folded on themselves (2.5D).

14. Method for characterizing a micro-tissue according to one of the preceding claims, characterized in that the micro-tissue is at least partially surrounded by an extracellular matrix.

15. Method for characterizing a micro-tissue according to one of the preceding claims, characterized in that the micro-tissue is a human or animal biological micro-tissue intended to be grafted in humans or the animal.

16. Method for characterizing a micro-tissue according to one of the preceding claims, characterized in that the micro-tissue is a human or animal biological micro-tissue chosen from epithelial, connective, muscular or nervous micro-tissues.

17. Method for characterizing a micro-tissue according to one of the preceding claims, characterized in that the micro-tissue comprises differentiated cardiac cells or retinal cells or neural cells or liver cells or chondrocytes or keratinocytes or lymphoid cells, or hematopoietic stem cells or mesenchymal stem cells or pluripotent cells in the form of an epiblast.

18. Method for in vitro characterization of a micro-tissue according to one of the preceding claims, characterized in that the micro-tissue is a micro-tissue produced for the purposes of bioproduction of medicine or food.

19. Method for characterizing a micro-tissue according to one of claims 1 to 14, characterized in that the micro-tissue is a plant biological micro-tissue chosen from meristems, parenchymas, conductive tissues, support tissues, covering or protective tissues, secretory tissues and nourishing tissues.

20. Method for characterizing a micro-tissue according to one of the preceding claims, characterized in that it is carried out online on the contents of a bioreactor.

21. Method for characterizing a micro-tissue according to one of claims 1 to 20, characterized in that it is carried out: i) in flow cells, or ii) in one-off sampling outside the bioreactor, or iii) to sort the micro-tissues online or offline.

22. Use of a method according to one of the preceding claims, for: - controlling the quality of a micro-tissue, and / or - measuring the increase in the biomass of a micro-tissue during its culture and / or its amplification, and / or - monitoring the differentiation and / or the organization of a micro-tissue during its maturation, and / or - determining the phenotype of cells of the micro-tissue, and / or - confirming the absence of undifferentiated cells in the micro-tissue, and / or -determine the viability of micro-tissue cells.

23. Use of a method according to one of claims 1 to 21, for screening embryos obtained by in vitro fertilization, the embryo being the micro-tissue.

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