Methods for characterizing biological microtissues using imaging

Abstract

We propose a solution for a completely rapid and non-invasive method of characterizing living tissues in vitro, enabling the characterization of living biological microtissues, particularly during or at the end of cell culture, in research or therapeutic applications. [Solution] A method for in vitro characterizing eukaryotic microtissues by imaging using a phase measurement technique without reference light, and the use of the above method in particular for: - quality control of microtissues, - measurement of biomass increase of microtissues during culture and / or amplification, - monitoring of differentiation and / or organization of microtissues during maturation, - determination of cellular phenotype of microtissues, and / or - confirmation that there are no undifferentiated cells in the microtissues.

Description

[Technical Field]

[0001] This invention relates to characterizing biological tissues, particularly biological microtissues, by imaging. [Background technology]

[0002] In research and therapy, it is essential to enable the characterization of living cells and biological tissues, particularly during or after cell culture, to control cell proliferation and / or cell and tissue quality, and / or monitor cell differentiation, and / or monitor tissue organization, and / or determine the phenotype of one or more cells that make up a single tissue.

[0003] However, this is not possible with current imaging techniques, particularly those using fluorescence microscopy, histology, capacitance measurement, optical density, and standard transmission imaging.

[0004] Fluorescence microscopy is used in combination with fluorescent probes such as antibodies and endogenous fluorescence by genetically modifying cells. Generally, several techniques using fluorescence microscopy are used, particularly confocal microscopy, light-sheet microscopy (or SPIM: Selective Plane Illumination Microscopy), multiphoton microscopy, and flow cytometry ("facs"). While these techniques are well-established, they require conditions unsuitable for cell culture for cell therapy purposes, such as fixation (cell death) and / or restriction conditions (marking only extracellular proteins), or the addition of destructive non-GMP products or products that induce genetic modification of cells. Therefore, they are often invasive, destructive, and very time-consuming, making them unsuitable for characterizing biological tissues.

[0005] Histological techniques consist of fixing the tissue and then marking it. These techniques also induce cell destruction and have the same drawbacks as fluorescence microscopy.

[0006] Biomass measurement using capacitance probes starts from the a priori reasoning that living cells can be considered as capacitors. Therefore, this measurement only considers the accessible outer surface of cells with intact membranes. The case of cell aggregates and microtissues is more complex and depends on the tightness of the connections between cells. Unlike various previous methods, this is non-invasive, but it is too limited and inaccurate for tissue characterization because it only provides information about inaccessible volumes or the surface of these volumes.

[0007] Standard transmission imaging techniques, such as quantitative phase contrast imaging, are rapid and non-invasive. Phase measurement in imaging involves measuring the local delay of a light beam after it has interacted with the object under investigation. The devices used in phase imaging are based on the phenomenon of light interference, encoding phase information into luminosity information. Various phase imaging techniques 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). Today, phase imaging is used to characterize minute samples (isolated cells or sections of microtissues less than 10 μm thick), but it cannot be used for larger microtissues or tissues. This is because the techniques currently used in phase imaging cannot quantitatively measure the phase within tissue. Because this technique is qualitative rather than quantitative, it is difficult to use for characterizing thicker objects.

[0008] Furthermore, while optical density measurements obtained through phase measurements can determine the mass of a sample, they cannot characterize tissues larger than 10 μm. [Overview of the project] [Problems that the invention aims to solve]

[0009] The object of the present invention is to solve these various problems of the prior art and to propose a solution for a method of characterizing living tissue completely rapidly and non-invasively in vitro, and in particular to enable the characterization of living biological microtissues in research or therapeutic applications, especially during or at the end of cell culture. [Means for solving the problem]

[0010] Current phase measurements performed on extremely fine cells and microtissues inevitably use reference light, but according to the present invention, this leads to inappropriate absolute measurements because it is not possible to measure mass or quantify a specific number of parameters.

[0011] Therefore, in order to achieve the objectives of the present invention, the inventors have developed a method for in vitro characterizing human, animal, or plant microtissues having a minimum dimension of 20 μm or more, the method comprising using a phase measurement technique that does not require a reference light and does not require the use of fluorescent marking.

[0012] In fact, according to the present invention, only phase measurement techniques without reference light, also known as indirect measurement techniques or relative phase measurement techniques, can function for characterizing microstructures with a minimum size of 20 μm or more. Furthermore, according to the present invention, it is important to control the coherence of the beam used for illumination. In particular, coherence at the sample level requires a reduction in speckle generated by the sample (microstructure), preferably with a speckle contrast of less than 75% of the maximum unit contrast, more preferably less than 50%, and ideally less than 10%. In practice, such reduction of speckle is achieved by reducing spatial coherence and / or temporal coherence. The spatial coherence of illumination should preferably be such that the numerical aperture of the illumination does not exceed 90%, more preferably 50%, and ideally 25%, so as to measure sample parameters independently of beam coherence.

[0013] Advantageously, by using phase measurement techniques without reference light, living biological microtissues can be quantitatively characterized in high throughput without destroying or altering them.

[0014] This makes the following possible in particular: - Measuring the increase in microtissue biomass during culture and / or amplification. - Determination of cell viability - Microtissue quality control - Monitoring of differentiation and / or organization of maturing microtissues - Determination of the phenotype of cells in microtissues, and / or - Confirmation that there are no undifferentiated cells in the microtissue.

[0015] Therefore, the present invention also aims to provide a characterization method for use in particular for these applications. [Brief explanation of the drawing]

[0016] [Figure 1]These are images of hydrogel capsules containing induced pluripotent human cells, obtained by phase measurement imaging without reference light according to the protocol described in the Examples. [Figure 2] These are images of hydrogel capsules containing induced pluripotent human cells, obtained by phase intensity measurement (absolute measurement) imaging without reference light, following the protocol described in the Examples. [Figure 3] This is a schematic diagram illustrating the implementation of a modified embodiment of an online method for characterizing biological microtissues contained in a bioreactor. [Figure 4] This is a schematic diagram showing a phase measurement imaging system without reference light used in the characterization method according to the present invention described in Example 1. [Figure 5a] These are images of microtissues formed by encapsulating artificial human stem cells (Gibco Human Episomal) in alginate capsules after culturing them for 6 days in growth medium (with MTesr1 added) according to the protocol of Example 2, and then photographed according to the method of the present invention. The microtissues consist of stem cells that form a single cyst and are composed of stem cells that meet the criteria of uniformity of cell distribution and roundness, which allow them to be classified into an acceptable category of microtissues. [Figure 5b] These are images of the microtissue formed by encapsulating artificial human stem cells (Gibco Human Episomal) in alginate capsules after culturing for 6 days in growth medium (with MTesr1 added) according to the protocol of Example 2, and then photographed according to the method of the present invention. The microtissue consists of stem cells that form multiple cysts and / or stem cells with heterogeneous cell distribution and roundness that can be classified into the category of unacceptable microtissue. [Figure 5c](Left) An image of a microtissue formed by encapsulating human artificial stem cells (Gibco Human Episomal) into an alginate capsule after culturing for 6 days in a growth medium (added with MTesr1). (Right) An image showing the same object obtained using a multiphoton microscope. Cell nuclei are marked with 10 μg / mL Hoechst 33342 (Thermofisher). Approximately half of the cells are visible. The total number of cells is approximately 500. [Figure 6] The graph shows the results of comparing the mass and area measurements between microtissues of stem cells in alginate capsules at various times (1 - 6 days) after capsule encapsulation, indicating the secondary growth of mass and the diversity of mass and maturity for samples generated simultaneously according to Example 2. [Figure 7] The graph shows the comparison results of mass measurements by area normalization between capsules containing microtissues and empty capsules according to Example 2.

[0017] Definition “Local absorption” of microtissues in the meaning of the present invention means the attenuation of light caused by the loss of local photons due to light absorption rather than diffusion.

[0018] “Alginate” in the meaning of the present invention refers to linear polysaccharides formed from β-D-mannuronic acid and α-L-guluronic acid, their salts and derivatives.

[0019] “Hydrogel capsule” in the meaning of the present invention means a three-dimensional structure formed from a matrix of polymer chains swollen with a liquid, preferably water.

[0020] “Human cells” in the meaning of the present invention means human cells or immunologically humanized non-human mammalian cells. Even if not specified, cells, stem cells, progenitor cells and tissues according to the present invention are composed of human cells or immunologically humanized non-human mammalian cells, or are obtained from them.

[0021] In the context of this invention, "progenitor cells" refers to stem cells that are already involved in cell differentiation (for example, into retinal cells) but have not yet differentiated. Progenitor cells are cells that tend to differentiate into a specific type of cell. Therefore, they are more specific than stem cells. Progenitor cells can only divide a limited number of times and, naturally, are subject to telomere erosion.

[0022] In the context of this invention, "embryonic stem cells" refers to pluripotent stem cells derived from the inner cell mass of a blastocyst. The pluripotency of embryonic stem cells can be evaluated by the presence of markers such as the transcription factor OCT4 and NANOG, and surface markers such as SSEA3 / 4, Tra-1-60, and Tra1-81. 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). Human embryonic stem cells can be excluded as needed.

[0023] In the context of this invention, "pluripotent stem cell" or "pluripotent cell" means a cell that has the ability to form all tissues present in the original organism, but is not capable of forming the entire organism itself. This can particularly involve induced pluripotent stem cells, embryonic stem cells, or MUSE cells ("Multilineage-differentiating Stress Enduring"). Pluripotent stem cells can maintain their telomere length and retain the ability to divide without a clear cell cycle number limitation, unlike progenitor cells.

[0024] In the context of this invention, "induced pluripotent stem cells" refers to pluripotent stem cells that have been induced to be pluripotent by genetic reprogramming of differentiated somatic cells. These cells are particularly positive for pluripotency markers such as alkaline phosphatase staining and the expression of NANOG, SOX2, OCT4, and SSEA3 / 4 proteins. Numerous examples of methods for obtaining induced pluripotent stem cells are described in Yu et al. (Science 2007, 318(5858):1917-1920), Takahasi et al. (Cell, 207, 131(5):861-872), and Nakagawa et al. (Nat Biotechnol, 2008, 26(1):101-106).

[0025] In the context of this invention, "differentiated" cells refer to cells that exhibit a specific phenotype, in contrast to undifferentiated pluripotent stem cells.

[0026] In the sense of this invention, "coherence" of a beam means spatiotemporal coherence, that is, the spatial range and spectral width of the illumination source.

[0027] In the context of this invention, "density" of microtissue means the mass of a unit volume divided by the mass of the same volume of culture medium.

[0028] In the context of this invention, "microtissue" or "biological microtissue" means a sample of biological tissue or biological material tissue with a maximum dimension of 1 cm or less.

[0029] In the sense of the present invention, "phase" means the wavefront delay, relative phase shift, and optical path difference between the environment of the microtissue and the base level of the culture medium in which it is immersed.

[0030] In the context of this invention, "phase measurement technology" means any technology capable of quantitatively measuring the phase of light.

[0031] In the context of this invention, "phase measurement technique without reference light" means a technique that can determine the phase component of light without relying on the use of an external beam called "reference light" that does not interact with microtissue.

[0032] In the context of this invention, "tissue" or "biological tissue" refers to the common biological understanding of tissue, i.e., an intermediate level of organization between cells and organs. A tissue is a collection of similar cells of the same origin, grouped into clusters, networks, or bundles (fibers) (although their origin can sometimes be traced back to different cell lineages, they usually originate from a common cell lineage). Tissues form functional collections, that is, collections of cells that contribute to the same function. Biological tissues regenerate periodically and come together to form various organs. Tissues can include differentiated cells and stem cells. Generally, pluripotent stem cells form epithelial tissue called epiblasts (Reference: Self-organization of the human embryo in the absence of maternal tissues, Shahbazi et al., Nat Cell Biol. 2016, doi:10.1038 / ncb3347).

[0033] In the context of this invention, "texture" of a microstructure means the local roughness of an image and its local frequency components. [Modes for carrying out the invention]

[0034] Therefore, the present invention aims to provide a method for in vitro characterizing eukaryotic microtissues, particularly those of humans, animals, or plants, having a minimum dimension of 20 μm or more, more preferably 30 μm or more. This method involves characterizing the entire microtissue, not just a portion of it.

[0035] The biological microtissue is preferably a microtissue with a maximum dimension of 10 mm or less, more preferably 1 mm or less, particularly 500 μm or less, and particularly 200 μm or less.

[0036] Biological microtissues can include microtissues containing eukaryotic cells, particularly human cells, or animal cells (non-human), particularly amniotic cells and particularly mammalian cells, or plant cells.

[0037] In the case of human or animal microtissues, the biological microtissues can be selected from, for example, epithelial, connective, muscular, or nerve microtissues.

[0038] According to one embodiment, the microtissue is, in particular, - Differentiated cardiac cells or retinal cells or nerve cells or hepatocytes or chondrocytes or keratinocytes or lymphoid cells or hematopoietic stem cells or mesenchymal stem cells, and / or: -Progenitor stem cells -endothelial cells It can include...

[0039] According to another embodiment, the microtissue may include or be composed of pluripotent cells in the form of epiblasts.

[0040] The biological microtissue, if it is a human or animal microtissue, is selected from various stages of embryonic or fetal development, particularly in the early stages of development, in in vitro fertilization for reproduction (human or animal) or research (human or animal) or animal production.

[0041] In the case of plant microtissues, for example, biological microtissues can be selected from among meristematic tissue, parenchyma, conduction tissue, supporting tissue, covering or protective tissue, secretory tissue, and vegetative tissue.

[0042] Microtissues may be at least partially surrounded by an extracellular matrix. The cell matrix layer may consist of cell matrix secreted by the cells of the microtissue and / or added extracellular matrix. The extracellular matrix layer can form a gel. This matrix layer preferably contains a mixture of proteins and extracellular compounds necessary for culturing the cells constituting the microtissue. Preferably, the extracellular matrix contains structural proteins such as collagen, laminin, entactin, and vitronectin, as well as growth factors such as TGF-beta and / or EGF. The extracellular matrix layer may consist of or contain a hydrogel-type matrix having plant-derived or synthetic-derived poly(N-isopropylacrylamide) copolymers and poly(ethylene glycol) copolymers (PNIPAAm-PEG) such as Matrigel® and / or Geltrex® and / or modified alginate.

[0043] According to modified embodiments, the microtissue can be encapsulated in a microcompartment, such as the microcompartment described in International Publication No. 2018 / 096277, or encapsulated in a capsule containing a hydrogel outer layer. A hydrogel capsule will be described. Preferably, the hydrogel used is biocompatible, i.e., non-toxic to cells. The hydrogel capsule needs to diffuse oxygen and nutrients to supply the cells contained in the microcompartment, enabling the cells to survive. The hydrogel outer layer may be an alginate-containing outer layer. This outer layer may consist solely of alginate. The alginate can be sodium alginate, in particular, composed of 80% α-L-guluronic acid and 20% β-D-mannuronic acid, with an average molecular weight of 100-400 kDa and a total concentration of 0.5-5% by mass. The hydrogel capsule can particularly protect cells from the external environment and limit uncontrolled cell proliferation.

[0044] Microtissues can take on any three-dimensional shape, i.e., the shape of any object in space. This can be, for example, a hollow or solid oval, hollow or solid cylindrical, hollow or solid tubular or tube, a hollow or solid ellipsoid or sphere, or a single layer (2.5D) partially folded in itself. The dimensions and shape of a microtissue are determined by its outer layer or, if present, the extracellular matrix layer. One example of a solid microtissue is a cardiac spheroid used in bioproduction. (https: / / doi.org / 10.1016 / i.bbamcr.2015.11.036)

[0045] According to one embodiment of the present invention, the microtissue may be a human or animal biological microtissue intended to be transplanted into a human or animal.

[0046] When using this method, microtissues can be prepared on frozen or unfrozen living microtissues.

[0047] The method according to the present invention includes characterizing microtissues by imaging using a phase measurement technique without reference light.

[0048] Preferably, it is important to control the coherence of the beam used for illumination. In particular, coherence at the microtissue level requires low speckle generated by the sample (microtissue), preferably with a speckle contrast of less than 75% of the maximum unit contrast, more preferably less than 50%, and ideally less than 10%. In practice, such speckle reduction is achieved by reducing spatial coherence and / or temporal coherence. The spatial coherence of the illumination device should preferably be such that the numerical aperture of the illumination does not exceed 90%, more preferably 75%, and ideally 50%, of the numerical aperture of the imaging system, so as to measure the parameters of the sample (microtissue) independently of the beam coherence.

[0049] In one particularly suitable embodiment, this method is carried out with a spatially semi-coherent beam, namely: - The spectral width of the light source, also called the illumination wavelength, which corresponds to the temporal coherence of the illumination, is at least 5 nm and at most 600 nm in the visible region, ideally about 100 nm, for example 100 nm; also - The illumination numerical aperture (corresponding to illumination spatial coherence) is at least 5% and at most 90% of the imaging system's numerical aperture, ideally around 50%, for example 50%.

[0050] This feature ensures equivalent quantitative measurements of phase, particularly regardless of the sample (microtissue) or illumination parameters, by measuring the parameters of the microtissue as a whole.

[0051] Preferably, a phase measurement technique without a reference light is selected from the following: -Wavefront analysis - Dynamic modulation of phase or luminosity in the pupil of an illumination or imaging system. - Photon multiplexing imaging with changes in the focal plane

[0052] When the phase measurement technique without reference light is wavefront analysis, it is preferably performed using wavefront gradient imaging, particularly wavefront gradient imaging techniques selected from the following: - 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) - Modified (or unmodified) 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 segmentation (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)).

[0053] Preferably, the reference-free phase measurement technique used in the method according to the present invention is the modified Hartmann method, as it offers the best compromise in terms of stability, sensitivity, and miniaturization when characterizing microtissues.

[0054] When the phase measurement technique without reference light involves dynamic modulation of the phase or luminosity in the pupil of an illumination or imaging system, the technique is preferably carried out using the following: - Tychography (Zheng, G., Horstmeyer, R. & Yang, C. "Wide-field, high-resolution Fourier ptychographic microscopy" Nat. Photonics 7,739 (2013)) or - Selective phase modulation of specific frequencies within the pupil (Wang, Z. et al. "Spatial light interference microscopy" (SLIM). Opt. Express 19, 1016-1026 (2011)).

[0055] Preferably, the phase measurement technique without reference light used in the method according to the present invention is a ptychography technique, because the quantification of phase in this technique is more direct than selective phase modulation, which gives only less quantitative images.

[0056] When the phase measurement technique without reference light is photomultiplexing with a change in the focal plane, it is preferably performed using the following: - 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 tomographic imaging for partially coherent light microscopy. Opt. Express 25, 15699-15712 (2017) or Barty, A., Nugent, KA, Paganin, D. & Roberts, A. "Quantitative optical phase microscopy". Opt. Lett. 23, 817-819 (1998)).

[0057] Preferably, the phase measurement technique without reference light used in the method according to the present invention is a high-speed simultaneous technique, although it is more complex than sequential multiplane imaging.

[0058] Whatever the phase measurement technique without reference light may be, this method preferably involves measuring the phase of light passing through a microstructure and, optionally, measuring its intensity.

[0059] Preferably, the method according to the present invention is a method for in vitro characterizing a eukaryotic microtissue by imaging using a phase measurement technique without reference light, the method comprising at least an investigation of the organization of cells within the microtissue, preferably at least an investigation of the topology of the microtissue, and / or an investigation of the relative arrangement of cells within the microtissue.

[0060] According to one preferred embodiment, this method involves the following measurements: - Measurement of microtissue density from phase measurement, and - If necessary, measure the local absorption of the microtissue from the phase and intensity measurements of light passing through the microtissue. Includes. The density of microtissue can be measured from phase measurements as follows: 1) Separate the area of ​​the image containing microtissue (called the useful region) from the rest of the image (background, generally called the culture medium). 2) Subtract the background phase value from the effective region phase. 3) After this subtraction, the phase is converted to the optical path difference (in meters) as needed and summed over the entire effective area. 4) Multiply this value by the area of ​​the basic pixels of the image and return it to the target plane: the value will be obtained in cubic meters. 5) This value can be adjusted for each tissue using a specific refractive increment (Barer, "Interference microscopy and mass determination," Nature, 1952) (average 0.18 μm). 3 Divide by ( / pg): In this way, you obtain a measurement of the so-called dry mass (total mass - mass of culture medium) integrated into the entire sample (microtissue).

[0061] Comparative examples and explanations of this technology are available (Zangle, T. et Teitell, MA, "Live-cell mass profiling: an emerging approach in guantitative biophysics," Nature Methods, 2014).

[0062] Density is expressed in units of mass (grams).

[0063] Local absorption in microtissues can be measured by measuring the phase and intensity of light transmitted through the microtissue, as follows: By simultaneously measuring the phase φ and intensity I, the electromagnetic field E = Ie iφ We obtain this. By extracting the real and imaginary parts (via the Fourier transform) in the digital Fourier space of the electromagnetic field, we can decompose this complex value. Returning (via the inverse Fourier transform) to the direct space of the real components of the Fourier space, we can determine the components of local absorption. The absorption measurement is given by photons / cm 2 It is represented as follows.

[0064] Preferably, the method according to the present invention includes measuring at least one of the following parameters: - Dimensions of microstructures - Dimensions of at least one cell in the microtissue - Number of cells in microtissues -Total mass and local mass of microtissue - Overall density and local density of microtissues - Mass distribution within microtissue -Organization of cells within the microtissue: topology of the microtissue and / or relative arrangement of cells within the microtissue. - Cell viability of microtissues - Texture

[0065] The dimensions of microtissues can be measured from phase measurements as follows: Dimensions within 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 ellipses (in the case of oval-shaped microtissues).

[0066] Dimensions are expressed in micrometers.

[0067] The dimensions of one or more cells in a microtissue can be measured from phase measurements as follows: If the optical resolution is better than the cell size, manual or automated clipping is performed within the microtissue (edge ​​detection algorithm or watershed). The dimensions are then obtained by adjusting each automated clipping with an ellipse.

[0068] Cell dimensions are expressed in micrometers.

[0069] The total mass of the microtissue can be measured from the phase as follows:

[0070] Next, the sum of the phase information of the region containing the microstructure (obtained by automatic or manual clipping) (in terms of the optical path, expressed in μm) is added to the target space (μm). 2 The area of ​​the phase pixel (represented by ) is multiplied, and then a specific increment (generally 0.18 pg / μm3) is multiplied to obtain the measured total mass.

[0071] The total mass is expressed in micrograms.

[0072] The local mass of microtissue can be measured from phase measurements as follows:

[0073] The same method as above is applied, but the phases are summed only in selected portions of the microtissue.

[0074] Local mass is expressed in micrograms.

[0075] The overall density of the microtissue can be measured from phase measurements as follows: Mass is measured from the phase. The lateral dimensions of the image plane are obtained from the phase image. The dimensions of the orthogonal planes of the phase image (called thickness) are obtained not from a) 3D reconstruction of the object across various imaging planes, but from b) assumptions about the shape of the object (usually oval) or c) assumptions about the average optical refractive index of the microtissue and culture medium. This allows us to determine the thickness of the microtissue by dividing the phase by the difference in refractive index (in the sense of optical path difference). Combining the three dimensions gives the volume of the sample (microtissue). Dividing the mass by the volume gives the density.

[0076] The total density is g / cm³ 3 It is represented as follows.

[0077] The local density of microtissue can be measured from phase measurements as follows:

[0078] The same protocol as for measuring overall density is used, but the measurement area is limited to the lower part of the microtissue. Local density is measured in g / cm³. 3 It is represented as follows.

[0079] The mass distribution within the microtissue can be measured from phase measurements as follows: Local mass measurements are performed on the sub-parts of the microtissue that cover all or part of it. Statistical analysis of the mass (standard deviation (deviation standard) / standard deviation (ecart-type), mean deviation / median deviation type) is then performed.

[0080] The mass distribution is expressed in grams.

[0081] The organization of cells within a microtissue should be interpreted in the histological sense of the term, as understood by those skilled in the art, and represents the topology of the tissue as well as the relative arrangement of cells and extracellular matrix elements. The cell viability of a microtissue can be measured from the phase measurements as follows: Local mass measurements are performed on the sub-part of the microtissue that covers all or part of it. A statistical analysis of its mass (standard deviation (deviation standard) / standard deviation (ecart-type), mean deviation / median deviation type) is performed.

[0082] In this case, the mass distribution is correlated with conventional histological analysis to generate an analyzed and annotated training dataset. Then, to automate the process, algorithms and / or directed automated learning (e.g., neural network type) can be run on this dataset.

[0083] Therefore, the organization of cells within a microtissue, whether human or not, is certified by an expert system based on histological classification.

[0084] Cell death phenomena cause changes in cell density and size that are detectable in phases. Cell viability in microtissues can be measured from phase measurements as follows: Local mass measurements are performed on sub-parts of the microtissue covering all or part of it. Statistical analysis of this mass (deviation standard / ecart-type, mean / median type) is performed. In this case, the percentage of living cells is identified by correlating the mass distribution with common viability measures such as ethidium bromide (dead cells) and calcein (live cells), and an analysis and annotated training dataset is generated. Then, to automate the process, algorithms and / or directed automated learning (e.g., neural network type) can be run on this dataset.

[0085] Therefore, the cell viability within the microtissue is expressed as the percentage of living cells out of the total number of cells.

[0086] The texture of microstructures can be measured from phase measurements as follows: Texture parameters can be determined by measuring statistics of spatial variations in phase, including the standard deviation and frequency distribution of image structures within the region of interest.

[0087] Texture is expressed in phase units, represented as (phase units) / μm.

[0088] According to one embodiment, the method according to the present invention can be performed in vitro on microtissues previously collected from humans, animals, or plants. This method makes it possible, for example, to characterize the quality (particularly their viability) of cadaver-derived islets of Langerhans before transplantation into diabetic patients, or to characterize pre-implantation embryos.

[0089] According to another embodiment, the method according to the present invention can be performed in vitro on a microtissue comprising pluripotent stem cells or progenitor cells intended to differentiate, or on a microtissue comprising cells in the process of differentiation, or on a microtissue comprising differentiated cells obtained by cell culture from pluripotent stem cells or progenitor cells. The method according to the present invention can be performed in vitro on a microtissue composed of pluripotent stem cells intended to differentiate, or on a microtissue composed of cells in the process of differentiation, or on a microtissue composed of differentiated cells obtained by cell culture from pluripotent stem cells or progenitor cells.

[0090] According to a modified embodiment, the method according to the present invention is performed online on the contents of a bioreactor. An example of such a modified embodiment applied to cell culture in capsules or microcompartments is shown in Figure 3. In this example, capsules 12, each containing microtissue, are suspended in culture medium 14 within a bioreactor 10. An output means 16 positioned on the bioreactor allows the capsules 12 to flow into their culture medium 14 and pass through a phase measurement imaging system 18 without reference light. At the output of this system 18, capsules containing microtissue that meet quality standards defined by the bioreactor user, so-called normal capsules 12-1 in the culture medium 14, are reintroduced into the bioreactor 10 via an input means 20, while undesirable capsules 12-2 that do not meet quality standards defined by the bioreactor user are recovered and removed via a removal means 22. The output means 16 may be, for example, a tube and a peristaltic pump, and the inlet means 20 may be, for example, a tube. The discharge means 22 may be, for example, a piezoelectric valve system. System 18 can be any imaging system suitable for phase measurement without reference light, such as one of the systems described in this application.

[0091] This advantageously allows for online checking of microtissue quality, particularly during differentiation or maturation. Therefore, the method according to the present invention can be carried out as follows, particularly when performed during the differentiation or maturation of cells forming microtissue within a bioreactor: i) In a flow cell. That is, the contents of a bioreactor-type culture enclosure are continuously recirculated in a sterile fluid system for the purpose of analyzing and / or sorting the contents of the culture enclosure, or ii) In a single sampling made outside the bioreactor to analyze a portion of the bioreactor at a specific point in time. Generally, this is done during sampling for the purpose of analyzing and / or reseeding a second bioreactor in the case of a "seed train" and / or volume rise, and / or for the purpose of dividing the contents of the bioreactor into multiple enclosures or multiple quality control conditions. iii) Select the microtissues offline when emptying the bioreactor for the purpose of purification or continuation of a series of production and / or differentiation and / or conditioning.

[0092] The method according to the present invention has many advantages over currently used methods. In particular, it can be implemented without destroying or altering the microtissue under investigation, is quick to implement, requires simple equipment, and can measure many physical parameters that characterize the microtissue, which was not possible with conventional methods.

[0093] Therefore, this method can be used for many purposes. In particular, the present invention aims to use the above method for the following: - Microtissue quality control: In fact, by implementing the method according to the present invention, it is possible to measure characteristics such as the size, density, cell number or texture of the microtissue, thereby checking the quality of the microtissue, and / or - Measuring the increase in biomass of microtissues during culture and / or amplification: In fact, by the method according to the present invention, the total or local mass of microtissues can be measured, thereby enabling monitoring of the increase in the number of cells in microtissues during culture, differentiation and / or amplification, and / or - Monitoring of the differentiation and / or evolution of the topology of maturing microtissues, particularly the spatial relative positions of the cells that constitute them: In fact, by the method according to the present invention, it is possible to measure the mass distribution within the microtissue and / or the organization of cells within the microtissue and / or the cell viability of the microtissue during the differentiation and / or maturation of cells in the microtissue, thereby providing information on the differentiation and / or maturation of cells, and / or - Determination of the phenotype of cells in microtissue. In fact, by the method according to the present invention, the mass of each cell and the texture of the microtissue can be measured, thereby providing information about the phenotype of the cells, and / or - Confirmation that there are no undifferentiated cells in the microtissue: In fact, by measuring the mass and / or density of cells, the texture of the microtissue and / or the organization of cells within the microtissue, information can be obtained about the differentiation of cells in the microtissue, and, as a result, information about the absence of cell differentiation in some cases.

[0094] According to one particular embodiment, the microtissue can be an embryo. Therefore, the method according to the present invention can be used for screening embryos obtained by in vitro fertilization. The histological structure of a healthy embryo is representative, highly reproducible, and predicts the success of embryo implantation in the mother. In particular, to describe the structure of such embryos that require re-implantation, only unmarked imaging solutions can be considered. To improve implantation yield and reduce the risk of failure, or conversely, the risk of multiple embryos, hospitals are developing increasingly accurate monitoring of fertilized embryos before implantation, especially video monitoring of development. The method according to the present invention can more effectively exclude embryos with abnormal structures by adding relevant information sources without marking, and therefore non-destructive sources.

[0095] According to another embodiment, the microtissue is a microtissue prepared for the purpose of the biological production of pharmaceuticals or the biological production of food.

[0096] Below, one embodiment of the method according to the present invention will be described in comparison with an example of a prior art characterization method.

[0097] Examples Example 1 In this embodiment, the method relates to the analysis of human microtissue contained in a single microcompartment, as described in Example 1 of International Publication No. 2018 / 096277 (Example 1: Protocol for obtaining cellular microcompartments from pluripotent human cells).

[0098] Microcompartments were analyzed using an imaging method based on intensity measurement, as described in the following literature (Bon P et al., "Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells," 2009 Optical Society of America). Intensity was obtained by demodulating low frequencies through Fourier space processing of the interferogram obtained using the protocol shown in Figure 4. The results are shown in Figure 2.

[0099] Microsections were analyzed according to the method according to the present invention. The operating protocol is as follows: the sample is transmitted and illuminated using halogen light, and an image of the sample is formed using a self-reference interferometer (particularly a phase-sensing detector) with a microscope objective lens (×20, numerical aperture 0.5) mounted on an inverted microscope. The imaging technique used is wavefront gradient imaging, specifically the modified Hartmann method. The protocol is similarly shown in Figure 4. Figure 4 shows the illumination system, sample, microscope, and phase-sensing detector. Enlarged views are shown to illustrate the modified Hartmann setup used to obtain Figures 1 and 2.

[0100] The results obtained are shown in Figure 1.

[0101] The images obtained by the method according to the present invention can measure the local density of the sample compared to the images obtained by the prior art, and it is confirmed that this local density and absorption can be made uncorrelated. In particular, the following measurements have become possible by the method according to the present invention: - The total dimensions of each microtissue. These are 91 μm (maximum) and 59 μm (minimum), and are calculated by measuring the diameter of each microtissue on image D in pixels. The magnification g of the entire imaging system y and knowing the physical size Tpix of one pixel, the dimensions of the microtissue are D × Tpix / g y and will be.

[0102] - The diameter of the lumen region at the center of the microtissue by analysis of the dimensions of the region having a uniform grain size and a low phase shift (i.e., a darker image) at the center of the microtissue. It is 38 μm for the largest microtissue and 25 μm for the smallest. - The dry mass of the object (the integral of the density of the object). It is 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), 39 μm for the largest microtissue and 16 μg for the smallest. Briefly, each microtissue is automatically clipped by determining the edges, the background phase value is evaluated by polynomial fitting, subtracted from the image, all the phase information of each microtissue is summed, and this is converted to mass by the following formula: m = 0.18 pg / μm 3 ×∬ microtissuPhase DS cell count. The largest microtissue has 608 ± 176 cells, and the smallest has 244 ± 64 cells. This value is obtained through two complementary approaches. The cell count can be estimated by knowing the average cell size (5 μm) and the volume of the area where cells reside within the microtissue (microtissue volume - lumen volume) (421 cells and 180 cells, respectively). Another calculation of the cell count can be derived by knowing the average mass of stem cells (50 pg) and the total mass of the microtissue (784 cells and 312 cells, respectively).

[0103] Example 2 In this example, the method relates to the analysis of multiple human microtissues contained in a microcompartment, as described in Example 1 of International Publication No. 2018 / 096277 (Example 1: Protocol for obtaining cellular microcompartments from pluripotent human cells). Human pluripotent stem cells (Gibco Human Episomal IPSCs) were encapsulated in an extracellular matrix surrounded by a porous alginate wall and cultured in growth medium (with MTesr1 added) for 6 days.

[0104] Microtissue was analyzed according to the method of the present invention. The operating protocol was as follows: the sample was transmitted and illuminated using halogen light, and an image of the sample was formed using a self-reference interferometer (particularly a phase-sensing detector) with a microscope objective lens (×20NA, numerical aperture 0.45) mounted on an inverted microscope. The imaging technique used was quantitative phase imaging by interferometry. The protocol was similarly shown in Figure 4. The numerical aperture of illumination was 0.13 (spatial coherence of illumination), and the wavelength of illumination was 550 ± 100 nm (spectral range of illumination or temporal coherence of illumination).

[0105] The results obtained are shown in Figures 5a, 5b, and 5c. These results allow us to characterize the texture and roundness of microsections having acceptable microstructure (Figure 5a) and unacceptable microstructure (Figure 5b).

[0106] In Figure 5a, the microtissue consists of stem cells that form a single cyst and are composed of stem cells that meet the criteria of uniformity and roundness of cell distribution, allowing them to be classified into an acceptable microtissue category.

[0107] In Figure 5b, the microtissue consists of stem cells that form multiple cysts and / or stem cells with heterogeneous cell distribution and roundness that may be classified as unacceptable microtissue.

[0108] The method according to the present invention allows for the characterization of acceptable and unacceptable microstructures of the same density through statistical analysis of roundness and texture uniformity.

[0109] Figure 5c shows one of the microtissues obtained according to the method of Example 2 on the left, and the same microtissue obtained using multiphoton microscopy on the right (cell nuclei are marked with 10 μg / mL Hoechst 33342 (Thermo Fisher)). Compared to the present invention, approximately half of the cells are visible with multiphoton microscopy. The total number of cells is approximately 500.

[0110] Furthermore, it has been confirmed that marking the cell nuclei of microtissues allows for the correlation between texture and roundness data and cell count.

[0111] Example 3 In this embodiment, the method relates to the analysis of human microtissue contained in a single microcompartment compared with the same microcompartment without microtissue, as described in Example 1 of International Publication No. 2018 / 096277 (Example 1: Protocol for obtaining cellular microcompartments from pluripotent human cells).

[0112] Microsectional areas were analyzed according to the method of the present invention, with and without the presence of microtissue. The operating protocol was as follows: the sample was transmitted and illuminated using halogen light, and an image of the sample was formed using a self-reference interferometer (particularly a phase-sensing detector) with a microscope objective lens (×20NA, numerical aperture 0.45) mounted on an inverted microscope. The imaging technique used was quantitative phase imaging by interferometry. The numerical aperture of illumination was 0.13 (spatial coherence of illumination), and the wavelength of illumination was 550 ± 100 nm (spectral range of illumination or temporal coherence of illumination).

[0113] The results obtained are shown in Figures 6 and 7.

[0114] Figure 6 clearly shows the secondary evolution and variance of cell growth at specific dates, which determine population growth and select microtissues during maturation.

[0115] Figure 7 confirms that phase imaging makes it possible to characterize the density of cells present in microtissues.

Claims

1. A method for in vitro characterizing a microtissue of a frozen or unfrozen living eukaryotic microtissue having a minimum dimension of 20 μm or more and a maximum dimension of 1 cm or less, by imaging using a phase measurement technique without reference light, wherein the spectral range of the illumination is at least 5 nm and at most 600 nm in the visible region, and the spatial coherence of the illumination is such that the numerical aperture of the illumination is at least 5% and at most 90% of the numerical aperture of the imaging system, and the method includes at least an investigation of the organization of cells within the microtissue.

2. The method for in vitro characterizing a microtissue according to claim 1, characterized in that the investigation of the organization of cells within the microtissue includes an investigation of the topology of the microtissue and / or an investigation of the relative arrangement of cells within the microtissue.

3. The method according to any one of claims 1 to 2, characterized in that the contrast of speckles generated by microtissue is less than 75% of the maximum unit contrast.

4. The method for in vitro characterizing a microtissue according to claim 1, wherein the microtissue is a human, animal, or plant microtissue.

5. An in vitro characterization method for microtissue according to any one of claims 1 to 4, characterized in that the maximum dimension is 10 mm or less.

6. Phase measurement techniques without reference light, - Wavefront analysis - Dynamic modulation of phase or luminosity in the pupil of an illumination or imaging system - Photochromic multiplexing imaging with changes in the focal plane A method for in vitro characterizing microtissue according to any one of claims 1 to 5, characterized in that the selection is made from among the following.

7. A method for characterizing a microstructure according to any one of claims 1 to 6, characterized in that the phase measurement technique without reference light is wavefront analysis, and this technique is performed using wavefront gradient imaging.

8. The method for characterizing microtissue according to claim 7, characterized in that the wavefront gradient imaging is selected from the Shack-Hartmann method, modified or unmodified Hartmann method, pupillary segmentation, and speckle field imaging.

9. A method for characterizing a microtissue according to any one of claims 1 to 5, characterized in that the phase measurement technique without reference light is dynamic modulation of phase or luminosity in the pupil of an illumination or imaging system, and this technique is performed using a technique of ptychography or selective phase modulation of a specific frequency in the pupil.

10. A method for characterizing a microtissue according to any one of claims 1 to 5, characterized in that the phase measurement technique without reference light is photometric multiplexing with a change in the focal plane, and this technique is performed using simultaneous or sequential multiplane imaging.

11. A method for characterizing a microstructure according to any one of claims 1 to 10, characterized by including the measurement of the phase and luminosity of light that has passed through the microstructure.

12. A method for characterizing microtissues according to any one of claims 1 to 11, characterized in that it is performed online on the contents of a bioreactor.

13. i) In a flow cell, or ii) By a single sample taken outside the bioreactor, or iii) To sort microtissues online or offline A method for characterizing microtissue according to any one of claims 1 to 12, characterized in that it is carried out.

14. The following measurements: -Density of microtissue from phase measurements, and - If necessary, local absorption of microtissues from measurements of the phase and intensity of light passing through the microtissue. A method for characterizing microtissue according to any one of claims 1 to 13, characterized by including the following:

15. At least one of the following parameters: - Dimensions of microtissues - Dimensions of at least one cell in the microtissue - Number of cells in the microtissue - Total mass and local mass of microtissue - Overall density and local density of microtissues - Mass distribution within microtissue - Topology of microtissues - Relative arrangement of cells within microtissue - Cell viability of microtissues - Texture of microstructures A method for characterizing microtissue according to any one of claims 1 to 14, characterized by including the measurement of [a specific value].

16. A method for characterizing a microtissue according to any one of claims 1 to 15, characterized in that the microtissue is encapsulated within a microcompartment including the outer layer of a hydrogel.

17. A method for characterizing microtissue according to any one of claims 1 to 16, characterized in that the microtissue takes the form of an oval, tubular, ellipsoidal, or spherical shape, or a single layer (2.5D) that is partially folded by itself.

18. A method for characterizing a microtissue according to any one of claims 1 to 17, characterized in that the microtissue is at least partially surrounded by an extracellular matrix.

19. A method for characterizing a microtissue according to any one of claims 1 to 18, characterized in that the microtissue is a living microtissue of a human or animal intended to be transplanted into a human or animal.

20. A method for characterizing a microtissue according to any one of claims 1 to 19, characterized in that the microtissue is a living microtissue of a human or animal, selected from among epithelial, connective, muscle, or nerve microtissues.

21. A method for characterizing a microtissue according to any one of claims 1 to 20, characterized in that the microtissue includes pluripotent cells that take the form of differentiated cardiac cells or retinal cells or nerve cells or hepatocytes or chondrocytes or keratinocytes or lymphoid cells or hematopoietic stem cells or mesenchymal stem cells or epiblasts.

22. A method for in vitro characterizing a microtissue according to any one of claims 1 to 21, characterized in that the microtissue is a microtissue prepared for the purpose of biological production of a drug or food.

23. A method for characterizing a microtissue according to any one of claims 1 to 19, characterized in that the microtissue is a plant-derived biological microtissue selected from among meristematic tissue, parenchyma, conduction tissue, supportive tissue, covering or protective tissue, secretory tissue, and vegetative tissue.

24. - Quality control of microtissues, and / or - Measurement of the increase in biomass of microtissues during culture and / or amplification, and / or - Monitoring of differentiation and / or organization of maturing microtissues, and / or - Determination of the phenotype of microtissue cells, and / or - Confirmation that there are no undifferentiated cells in the microtissue, and / or - Determining the cell viability of microtissues Use of the method according to any one of claims 1 to 23 for

25. A use of the method according to any one of claims 1 to 19 for screening embryos obtained by in vitro fertilization, wherein the embryo is a microtissue.

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