Patent Application
20230122507
The present invention relates to the characterization, by imaging, of biological tissues, in particular biological microtissues.
In research and in treatment, it is essential to be able to characterize the living biological cells and tissues, in particular during or after a cell culture, in particular in order to monitor the cell proliferation and/or the quality of the cells and of the tissue, and/or in order to follow the differentiation of cells, and/or in order to follow the organization of the tissue, and/or in order to determine the phenotype(s) of the cells forming a tissue, etc.
However, the current imaging techniques, which are in particular those implementing fluorescence microscopy, histology, capacitance measurement, optical density, and standard transmission imaging, do not allow for this.
Fluorescence microscopy is used, coupled with fluorescent probes such as antibodies or endogenic fluorescence, by genetically modifying cells. A plurality of techniques using fluorescence microscopy are generally used, in particular confocal microscopy, light sheet microscopy (or SPIM for “Selective Plane Illumination Microscopy”), multiphotonic microscopy, flow cytometry (“FACS”). These techniques are well-established, but they require fixing (cell death) and/or limiting conditions (marking only of extracellular proteins), or are not compatible with the cell culture with the aim of cellular treatment, such as the addition of non-GMP destructive products or products which cause the genetic modification of cells. They are therefore not suitable for the characterization of living tissues because they are invasive, often destructive, and very slow.
The histology techniques consist in fixing and then marking the tissues. These again involve the destruction of cells and exhibit the same disadvantages as the fluorescence microscopy.
Measuring biomass by means of a capacitance probe proceeds from the principle that the living cells can be considered to be capacitors. This measurement thus takes into account only the accessible outside surface of cells having an integrated membrane. The case of aggregates of cells and microtissues is more complex and will depend on the tightness of connections between cells. In contrast with the previous methods, this is non-invasive, but it makes it possible to have information merely on the only inaccessible volume or on the surface of this volume, which is too limiting and imprecise for the characterization of tissues.
The standard transmission imaging techniques, such as quantitative phase contrast, are rapid and non-invasive. The phase measurement in imaging is a measurement of the local delay of a light beam after interaction with the object studied. The devices used for the phase imaging are based on the phenomenon of optical interference for encoding the phase information into luminous intensity 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 for characterizing fine samples (isolated cells or microtissue slices of thicknesses less than 10 μm), but cannot be used for microtissues or larger tissues. Indeed, the techniques currently used in phase imaging do not allow for quantitative measurement of the phase in a tissue. They remain qualitative, and not quantitative, and can therefore be used only with difficulty for the characterization of thick objects.
Finally, the measurement of the optical density, obtained by phase measurements and allowing access to the mass of the sample, does not allow for characterization of tissues of a size larger than 10 μm.
The object of the invention is to overcome these various problems of the prior art, and to propose a solution for a method for in vitro characterization of living tissues which is complete, rapid and non-invasive and which makes it possible to characterize in particular living biological microtissues, in particular during or at the end of cell culturing, in research or in treatment.
According to the invention, the phase measurement as currently carried out on very fine cells and microtissues necessarily uses a reference beam which results in an absolute measurement which is not suitable because it does not make it possible to measure the mass and to quantify a certain number of parameters.
This is why, in order to achieve the object of the invention, the inventors have developed a method for in vitro characterization of a human, animal or vegetable biological microtissue, the smallest dimension of which is greater than or equal to 20 μm, said method consisting in using a phase measurement technique without a reference beam and which does not make use of fluorescent marking.
Indeed, according to the invention, only the phase measurement techniques without a reference beam, i.e, the direct measurement techniques, or indeed referred to as relative phase measurements, can function for characterizing microtissues, the smallest dimension of which is greater than or equal to 20 μm. Moreover, according to the invention it is important to manage the coherence of the beam used for the lighting. In particular, it is expedient for the coherence in the region of the sample, i.e. that of the speckle generated by the sample (microtissue), to be reduced—preferably the speckle contrast is less than 75% of a maximum unit contrast, yet more preferably 50%, ideally 10%. In practice, this reduction of the speckle is achieved by reducing the spatial coherence and/or the temporal coherence. The spatial coherence of the lighting should preferably be such that the digital illumination opening does not exceed 90%, yet more preferably 50%, ideally 25%, of the digital opening of the imaging system, so as to measure the parameters of the sample in a manner independent of the coherence of the beam.
Advantageously, the use of a phase measuring technique without a reference beam makes it possible to quantitatively characterize living biological microtissues at high speed, without destroying them or modifying them.
This makes it possible in particular to:
The invention thus also relates to the use of the characterization method, for these applications in particular.
“Local absorption” of the microtissue within the meaning of the invention means the attenuation of the light due to a local photon loss by absorption of the light and not diffusion.
Within the meaning of the invention, “alginate” means linear polysaccharides formed from β-D-mannuronate and α-L-guluronate, and salts and derivatives thereof.
Within the meaning of the invention, “hydrogel capsule” means a three-dimensional structure formed from a matrix of polymer chains swollen by a liquid, preferably water.
Within the meaning of the invention, “human cells” means human cells or cells of non-human mammals which are immunologically humanized. Even if this is not specified, the cells, the stem cells, the progenitor cells and the tissues according to the invention are made up of or are obtained from human cells or from cells of non-human mammals which are immunologically humanized.
Within the meaning of the invention, “progenitor cell” means a stem cell already engaged in cellular differentiation (for example in cells of the retina), but not yet differentiated. A progenitor cell is a cell which tends to be differentiated into a specific type of cell. It is therefore already more specific than a stem cell. The progenitor cells can divide only a limited number of times, being naturally subject to erosion of their telomeres.
Within the meaning of the invention, “embryonic stem cell” means a pluripotent stem cell derived from the internal cellular mass of the blastocyst. The pluripotency of embryonic stem cells can be evaluated 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. The embryonic stem cells can be obtained without destroying the embryo from which they originate, for example by means of the technique described in Chang et al. (Cell Stem Cell, 2008, 2(2)): 113-117). Optionally, the embryonic stem cells of humans may be excluded.
Within the meaning of the invention, “pluripotent stem cell” or “pluripotent cell” means a cell which is capable of forming all the tissues present in the whole original organism, without, however, being able to form an entire organism per se. They may in particular be induced pluripotent stem cells, embryonic stem cells, or MUSE cells (for “Multilineage-differentiating Stress Enduring”). The pluripotent stem cells retain the length of their telomeres and can preserve the ability to divide without a clear limit of the number of cellular cycles, unlike progenitor cells.
Within the meaning of the invention, “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 the pluripotency markers, such as the alkaline phosphatase coloration and the expression of the proteins NANOG, SOX2, OCT4 and SSEA3/4. Examples of methods making it possible to obtain 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).
Within the meaning of the invention, “differentiated” cells means cells which have a particular phenotype, in contrast with pluripotent stem cells which are not differentiated.
Within the meaning of the invention, “coherence” of the beam means the spatia-temporal coherence, i.e. the spatial extent and the spectral width of the illumination source.
Within the meaning of the invention, “density” of the microtissue means the mass of one unit volume divided by the mass of the same volume of culture medium.
Within the meaning of the invention, “microtissue” or “biological microtissue” means a biological tissue or a sample of biological tissue, the largest dimension of which is less than or equal to 1 cm.
Within the meaning of the invention, “phase” means the delay of the luminous wavefront, relative phase shift, and optical path difference between the environment of the microtissue and the base level of the medium into which it is plunged.
Within the meaning of the invention, “phase measurement technique” means any technique capable of quantitatively measuring the phase of the light.
Within the meaning of the invention, “phase measurement technique without a reference beam” means the techniques capable of going back to the phase component of the light, without having to resort to the use of an external beam, referred to as “reference,” which has not interacted with the microtissue.
Within the meaning of the invention, “tissue” or “biological tissue” means the common sense of tissue in biology, i.e. the intermediate organization level between the cell and the organ. A tissue is a set of similar cells of the same origin (most often originating from a common cellular lineage, although they may originate by association of distinct cell lineages), grouped in a cluster, network or beam (fiber). A tissue forms a functional unit, i.e. the cells thereof contribute to the same function. The biological tissues regenerate regularly and are assembled together to form organs. A tissue may comprise differentiated cells and stem cells. Typically, the pluripotent stem cells form a tissue of the epithelia type, qualified as an epiblast (citation: Self-organization of the human embryo in the absence of maternal tissues, Shahbazi et al., Nat Cell Biol. 2016, doi: 10.1038/ncb3347).
Within the meaning of the invention, “texture” of a microtissue means the local roughness of the image and its local frequency content.
The invention thus relates to a method for in vitro characterization of a eukaryote biological microtissue, in particular a human, animal or vegetable microtissue, the smallest dimension of which is greater than or equal to 20 μm, even more preferably greater than or equal to 30 μm. The method consists in characterizing the biological microtissue in its entirety, and not only a portion of the microtissue.
The biological microtissue is preferably a microtissue, the largest dimension of which is less than or equal to 10 mm, even more preferably less than or equal to 1 mm, and in particular less than or equal to 500 μm, in particular less than or equal to 200 μm.
The biological microtissue may be a microtissue comprising eucaryote cells, in particular human cells, or animal (non-human) cells, in particular cells of amniotes and in particular cells of mammals, or vegetable cells.
When it is a human or animal microtissue, the biological microtissue may for example be selected from the epithelial, connective, muscular or nerve microtissues,
According to an embodiment, the microtissue may in particular comprise:
According to another embodiment, the microtissue may comprise or be made up of pluripotent cells in the form of an epiblast.
When it is a human or animal microtissue, the biological microtissue may, for example, be selected from the different phases of embryonic or fetal development, in particular the early phases of development within the context of in vitro fertilization for purposes of reproduction (in humans or animals) or research (in humans or animals), or animal production.
When it is a vegetable microtissue, the biological microtissue may, for example, be selected from the meristems, the parenchyma, the conducting tissues, the supporting tissues, the covering or protective tissues, the secretion tissues, and the nutritive tissues.
The microtissue may be surrounded, at least in part, by an extracellular matrix. The cellular matrix layer may be formed by the cellular matrix excreted by the cells of the microtissue and/or by the added extracellular matrix. The extracellular matrix layer may form a gel. It preferably comprises a mixture of proteins and extracellular compounds required for the growth of cells forming the microtissue. 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 matrix of the hydrogel type of vegetable origin, such as modified alginates, or of synthetic origin, or of copolymer of poly(N-isopropylacrylamide) and polyethylene glycol) (PNIPAAm-PEG) of the Mebiol® type.
According to a variant, the microtissue may be a microtissue that is encapsulated in a microcompartment or a capsule comprising an external hydrogel layer, such as the microcompartments described in the patent application WO2018/096277. Reference is made to a hydrogel capsule. Preferably, the hydrogel used is biocompatible, i.e. it is not toxic for the cells. The hydrogel capsule must allow the diffusion of oxygen and of nutrients for feeding the cells contained in the microcompartment and to allow their survival. The external hydrogel layer may be an external layer comprising alginate. It may be formed exclusively of alginate. The alginate may in particular be a sodium alginate, made up of 80% α-L-guluronate and 20% β-D-mannuronate, having an average molecular mass of 100 to 400 kDa and a total concentration of between 0.5% and 5% by mass. The hydrogel capsule in particular makes it possible to protect the cells from the external medium and to limit the uncontrolled proliferation of cells.
The microtissue may be provided in any three-dimensional shape, i.e. it may be in the shape of any object in space. It may be provided for example in the shape of a hollow or solid egg, a hollow or solid cylinder, a tuboid or a hollow or solid tube, a spheroid or a hollow or solid sphere, or of monolayers partially folded back on themselves (2.5 D). It is the external layer of the microtissue or the layer of extracellular matrix, when this is present, which confers its size and its shape to the microtissue. An example of a solid microtissue is the cardiac spheroid used in bioproduction (https://doi.org/10.1016/j.bbamcr.2015.11.036).
According to an embodiment of the invention, the microtissue may be a human or animal biological microtissue intended to be grafted in humans or animals.
During implementation of the method, the microtissue may be realized on a living frozen or non-frozen microtissue.
The method according to the invention comprises the characterization of the microtissue by imaging, by a phase measurement technique without a reference beam.
Preferably, it is important to manage the coherence of the beam used for the lighting. In particular, it is expedient for the coherence in the region of the microtissue, i.e. that of the speckle generated by the sample (microtissue), to be reduced—preferably the speckle contrast is less than 75% of the unit contrast, yet more preferably 50%, ideally 10%. In practice, this reduction of the speckle is achieved by reducing the spatial coherence and/or the temporal coherence. The spatial coherence of the lighting should preferably be such that the digital illumination opening does not exceed 90%, yet more preferably 75%, ideally 50%, of the digital opening of the imaging system, so as to measure the parameters of the sample (microtissue) in a manner independent of the coherence of the beam.
According to a particularly suitable embodiment, the method is implemented using a spatially semi-coherent beam, i.e.:
This feature makes it possible in particular to guarantee a quantitative phase measurement that is comparable, whatever the sample (microtissue) and the illumination, while measuring the parameters of the microtissue in its entirety.
Preferably, the phase measurement technique without a reference beam is selected from:
When the phase measurement technique without a reference beam is wavefront analysis, it is preferably performed by means of imaging of gradients of the wavefront, and in particular a wavefront gradient imaging technique selected from:
Preferably, the phase measurement technique without a reference beam, used in the method according to the invention, is the modified Hartmann method, because this is the technique which makes it possible to achieve the best compromise in terms of stability, sensitivity and compactness, for the characterization of microtissues.
When the phase measurement technique without a reference beam is the dynamic modulation of the phase or of the luminous intensity in the pupil of the illumination or imaging system, it is preferably performed by means of:
Preferably, the phase measurement technique without a reference beam, used in the method according to the invention, is the ptychography technique, because the phase quantification there is more direct than the selective phase modulation, which provides images that are only slightly quantitative.
When the phase measurement technique without a reference beam is the multiple imaging of luminous intensity with modification of the focusing plane, it is preferably performed by means of:
Preferably, the phase measurement technique without a reference beam, used in the method according to the invention, is the simultaneous technique, because it is rapid, even if it is more complex than the sequential multiplane imaging.
Whatever the phase measurement technique without a reference beam, the method preferably comprises the measurement of the phase and optionally of the luminous intensity of the light that has passed through the microtissue.
Preferably, the method according to the invention is a method for in vitro characterization of a eukaryote biological microtissue by imaging by means of a phase measurement technique without a reference beam, said method comprising at least the study of the organization of cells in the microtissue, preferably at least the topology of the microtissue and/or the relative positioning of cells in the microtissue.
According to a preferred embodiment, the method comprises measuring:
The density of the microtissue can be measured from the phase measurement as follows:
1) the zone of the image containing the microtissue (referred to as the useful zone) is separated from the remainder (referred to as the base, generally the culture medium);
2) the phase value of the base is subtracted from the phase of the useful zone;
3) following this subtraction, the phase is converted, if required, into the optical path difference (expressed in metric units) and added to the entirety of the useful zone;
4) said value is multiplied by the surface of an elementary pixel of the image, brought into the object plane—a value in cubic metric units is obtained;
5) said magnitude is divided by the specific refraction increment (Barer, Interference microscopy and mass determination, Nature, 1952) which is 0.18 μm3/pg on average and which can be adjusted for each tissue—thus, a measurement of the “dry” mass is obtained (total mass—mass of culture medium), integrated over the entire sample (microtissue).
A comparison and description of this technique is available (Zangle, T. and Teitell, M. A., Live-cell mass profiling: an emerging approach in quantitative biophysics, Nature Methods, 2014).
The density is expressed in mass units (g).
The local absorption of the microtissue can be measured from the phase measurement and the luminous intensity measurement of the light that has passed through the microtissue, in the following manner. By means of a combined measurement of the phase φ and of the intensity I, the electromagnetic field is obtained E=Ieiφ. Said complex magnitude can be broken down 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, from the Fourier space, it is possible to go back to the component of the local absorption.
The measurement of the absorption is expressed in photon/cm2.
Preferably, the method according to the invention comprises the measurement of at least one of the following parameters:
The dimensions of the microtissue can be measured from the phase measurement as follows. The dimensions in the plane of the image are extracted by automatic trimming (edge detection algorithm of the Otsu type for example, or manual trimming), and the dimensions obtained by an adjustment of the trimming by an ellipsis (in the case of an egg-shaped microtissue).
The dimensions are expressed in micrometers.
The dimensions of one or more cell(s) of the microtissue can be measured from the phase measurement as follows. When the optical resolution is better than the size of a cell, manual or automatic trimming within the microtissue is performed (edge or watershed detection algorithm). The dimensions are then obtained by an adjustment of each automatic trimming by an ellipsis.
The dimensions of a cell are expressed in micrometers.
The overall mass of the microtissue can be measured from the phase measurement as follows. The sum of the phase information (within the meaning of the optical path, expressed in μm) over the zone containing the microtissue (obtained by automatic or manual trimming) is then multiplied by the surface of a phase pixel returned into the space of the object (expressed in μm2), then multiplied by the specific increment (generally 0.18 pg/μm3) in order to obtain the overall mass measurement.
The overall mass is expressed in micrograms.
The local mass of the microtissue can be measured from the phase measurement as follows. The same method as for the above point is applied, but adding the phase only over a selected portion of the microtissue.
The local mass is expressed in micrograms.
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 image phase (referred to as thickness) is obtained: a) by a 3D reconstruction of the object in different imaging planes, b) or by a hypothesis on the shape of the object (generally egg-shaped), c) or by a hypothesis on the average optical refraction index of the microtissue and of the medium which makes it possible, by dividing the phase (within the meaning of the optical path difference) by the difference in the refractive index, to trace back to the thickness of the microtissue. The three dimensions are combined in order to obtain the volume of the sample (microtissue). By dividing the mass by the volume, the volume mass is returned.
The overall density is expressed in g/cm3.
The local density of the microtissue can be measured from the phase measurement as follows. The same protocol as for the measurement of the overall density is used, but limiting the measured zone to a portion of the microtissue. The local density is expressed in g/cm3.
The distribution of mass in the microtissue can be measured from the phase measurement as follows. Local mass measurements are carried out on sub-portions of the microtissue covering all or some of the microtissue. A statistical analysis of its masses (standard deviation, mean/median type) is performed.
The mass distribution is expressed in grams.
The organization of cells in the microtissue is to be taken within the histological meaning of the term, as understood by a person skilled in the art, and denotes the topology of the tissue and the relative positioning of the cells and the elements of the extracellular matrix. The viability of cells of the microtissue can be measured from the phase measurement as follows: local mass measurements are carried out on the sub-portions of the microtissue covering all or some of the microtissue. A statistical analysis of its masses (standard deviation, mean/median type) is performed.
The mass distribution is then correlated to a conventional histological analysis in order to generate an analyzed and annotated training dataset. Algorithm-based and/or directed machine learning (neural network type for example) can then be performed on this set of data, in order to automate the method.
The organization of cells in the microtissue is thus qualified by an expert system, human or otherwise, based on the histological classification.
The phenomena of cell death cause a change in the density and the size of the cells which can be detected in phase. The viability of cells of the microtissue can be measured from the phase measurement as follows: local mass measurements are carried out on the sub-portions of the microtissue covering all or some of the microtissue. A statistical analysis of its masses (standard deviation, mean/median type) is performed. The mass distribution is then correlated to conventional viability measurements such as ethidium bromide (dead cells) and calcein (living cells) identifying the percentage of living cells in order to generate an analyzed and annotated training dataset. Algorithm-based and/or directed machine learning (neural network type for example) can then be performed on this set of data, in order to automate the method.
The viability of the cells in the microtissue is thus expressed in percentage of living cells of the total cells.
The texture of the microtissue can be measured from the phase measurement as follows. The measurement of spatial variation statistics of the phase, including the standard deviation and frequency distribution of structures of the image within the region of interest, makes it possible to determine texture parameters.
The texture is expressed in phase units and in (phase unit)/μm.
According to an embodiment, the method according to the invention can be performed in vitro on microtissues which have been previously taken from a human, an animal, or a vegetable. The method may make it possible, for example, to characterize the quantity of an islet of Langerhans originating from the cadaver (in particular its viability) prior to a transplant in a diabetic patient, or indeed to characterize a pre-implantation embryo.
According to another embodiment, the method according to the invention can be performed in vitro on microtissues comprising pluripotent or progenitor stem cells intended to be differentiated, or on microtissues comprising cells undergoing differentiation, or on microtissues comprising differentiated cells obtained by cell culture from pluripotent or progenitor stem cells. The method according to the invention can be performed in vitro on microtissues made up of pluripotent or progenitor stem cells intended to be differentiated, or on microtissues made up of cells undergoing differentiation, or on microtissues made up of differentiated cells obtained by cell culture from pluripotent or progenitor stem cells.
According to a variant, the method according to the invention is implemented online, on the contents of a bioreactor. An example of such a variant applied to the cell culture in capsules or microcompartments is shown in
This advantageously makes it possible to verify the quality of the microtissues, in particular online, during differentiation or maturation. The method according to the invention, in particular in the context of implementation during differentiation or maturation of cells forming a microtissue in a bioreactor, can thus be performed:
The method according to the invention has several advantages compared with the methods currently used. In particular, it can be implemented without destroying or modifying the microtissues studied, it is rapid to implement, requires simple equipment, and makes it possible to measure numerous physical parameters for characterizing the microtissues which was not possible using the methods of the prior art.
The method can thus be used for numerous applications. In particular, the invention relates to the use of the method, for:
According to a particular embodiment, the microtissue may 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, highly reproducible, and predictive of the success of the implantation of the embryo in the mother. In particular, in order to describe this structure on an embryo intended to be reimplanted, only solutions of imaging without marking are conceivable. In order to improve the yield of implantations and reduce the risk of failure, or, in contrast, of multiple embryos, clinics are developing ever more precise monitoring of the fertilized embryo in pre-implantation, in particular using video monitoring of the development. The method according to the invention makes it possible to more efficiently eliminate the embryos having an abnormal structure, by adding a relevant information source and without marking, and thus non-destructively.
According to another embodiment, the microtissue is a microtissue produced for the purpose of bioproduction of medicine or food.
The invention is now illustrated by way of an embodiment of the method according to the invention, compared with an example of a characterization method of the prior art.
In this example, the method relates to the analysis of a human microtissue contained in a microcompartment, as described in example 1 of the application WO2018/096277 (example 1: protocol for obtaining cellular microcompartments from human cells induced to pluripotency).
A microcompartment has been analyzed according to an imaging process by measuring the intensity, as described in (Bon P. et al, Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells, 2009 Optical Society of America). The intensity has been obtained by demodulation of low frequencies via processing in the Fourier space of an interferogram obtained using the protocol set out in
A microcompartment was analyzed according to a method according to the invention. The operating protocol is described in the following: a halogen light is used for transmission illumination of the sample, a microscope objective (×20, digital opening 0.5) mounted on an inverted microscope is used for forming the image of the sample on a self-referenced interferometer (detector sensitive inter alia to the phase). The imaging technique used is the imaging of gradients of the wavefront, and in particular the modified Hartmann method. The protocol is also shown schematically in
The results obtained are set out in
It is noted that the image obtained by means of the method according to the invention makes it possible to measure the local density of the sample and to decorrelate it from the absorption by comparison with the image obtained by the technique of the prior art. In particular, the method according to the invention has made it possible to measure:
In this example, the method relates to the analysis of a plurality of human microtissues contained in microcompartments, as described in example 1 of the application WO2018/096277 (example 1: protocol for obtaining cellular microcompartments from human cells induced to pluripotency). Pluripotent human stem cells (Gibco Human Episomal PSC) were encapsulated in an extracellular matrix surrounded by a porous wall of alginate and cultivated for 6 days in a growth medium in a growth medium (supplemented MTesr1).
The microtissues were analyzed according to the method according to the invention. The operating protocol is described in the following: a halogen light is used for transmission illumination of the sample, a microscope objective (×20 NA, digital opening 0.45) mounted on an inverted microscope is used for forming the image of the sample on a self-referenced interferometer (detector sensitive inter alia to the phase). The imaging technique used is quantitative phase imaging by interferometry. The protocol is also shown schematically in
The results obtained are set out in
In
In
It is noted that the method according to the invention makes it possible, by means of statistical analysis of roundness and homogeneity of texture, to characterize the acceptable microtissues and the non-acceptable microtissues, even if dense.
It is also noted that the marking of the nuclei of cells of the microtissues makes it possible to correlate the data of the texture and the roundness with the number of cells.
In this example, the method relates to the analysis of a human microtissue contained in a microcompartment, as described in example 1 of the application WO2018/096277 (example 1: protocol for obtaining cellular microcompartments from human cells induced to pluripotency) compared with the same microcompartment without microtissue.
The microcompartments with and without microtissues were analyzed according to the method according to the invention. The operating protocol is described in the following: a halogen light is used for transmission illumination of the sample, a microscope objective (×20 NA, digital opening 0.45) mounted on an inverted microscope is used for forming the image of the sample on a self-referenced interferometer (detector sensitive inter alia to the phase). The imaging technique used is quantitative phase imaging by interferometry. The digital illumination opening is 0.13 (spatial coherence of the illumination), the illumination wavelength is 550±100 nm (spectral range of the illumination, or temporal coherence of the illumination).
The results obtained are set out in
It can be seen in
| Number | Date | Country | Kind |
|---|---|---|---|
| FR2003036 | Mar 2020 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/057977 | 3/26/2021 | WO |
