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EP-4127669-B1 - METHOD FOR CHARACTERISING A BIOLOGICAL MICRO TISSUE USING IMAGING

EP4127669B1EP 4127669 B1EP4127669 B1EP 4127669B1EP-4127669-B1

Inventors

  • ALESSANDRI, Kévin, Pascal, Stéphane
  • BON, PIERRE

Dates

Publication Date
20260513
Application Date
20210326

Claims (20)

  1. Method for in vitro characterization of a frozen or unfrozen living eukaryotic biological microtissue of which the smallest dimension is greater than or equal to 20 micrometers and of which the largest dimension is less than or equal to 1 cm, by imaging using a reference-beam-free phase measurement technique, the spectral range of the illumination being a minimum of 5 nm in the visible range and a maximum of 600 nm, and the spatial coherence of the lighting being such that the illumination numerical aperture is a minimum of 5% and a maximum of 90% of the numerical aperture of the imaging system, said method comprising at least the study of the organization of the cells in the microtissue.
  2. Method for in vitro characterization of a microtissue according to the preceding claim, characterized in that the study of the organization of the cells in the microtissue comprises the study of the topology of the microtissue and/or the relative positioning of the cells in the microtissue.
  3. Method for in vitro characterization of a microtissue according to either of the preceding claims, characterized in that the spatial and/or temporal coherence of the lighting is selected in such a way that the contrast of the speckle generated by the microtissue is less than 75% of the maximum unit contrast.
  4. Method for in vitro characterization of a microtissue according to claim 1, the microtissue being a human, animal or plant microtissue.
  5. Method for in vitro characterization of a microtissue according to any of the preceding claims, characterized in that the largest dimension is less than or equal to 1 mm.
  6. Method for in vitro characterization of a microtissue according to any of the preceding claims, characterized in that the reference-beam-free phase measurement technique is selected from: - wavefront analysis - dynamic modulation of the phase or luminous intensity in the pupil of the illumination or imaging system - multiple luminous-intensity imaging with modification of the plane of focus.
  7. Method for in vitro characterization of a microtissue according to any of the preceding claims, characterized in that the reference-beam-free phase measurement technique is wavefront analysis and in that it is performed using wavefront gradient imaging.
  8. Method for in vitro characterization of a microtissue according to the preceding claim, characterized in that wavefront gradient imaging is selected from the Shack-Hartmann, modified or unmodified Hartmann, pupil partitioning and speckle field imaging methods.
  9. Method for in vitro characterization of a microtissue according to any of claims 1 to 5, characterized in that the reference-beam-free phase measurement technique is the dynamic modulation of the phase or luminous intensity in the pupil of the illumination or imaging system and in that it is performed using the ptychography technique or the selective phase modulation of certain frequencies in the pupil.
  10. Method for in vitro characterization of a microtissue according to any of claims 1 to 5, characterized in that the reference-beam-free phase measurement technique is multiple luminous-intensity imaging with modification of the plane of focus and in that it is performed using simultaneous or sequential multi-planar imaging.
  11. Method for in vitro characterization of a microtissue according to any of the preceding claims, characterized in that it comprises measurement of the phase and luminous intensity of the light having passed through the microtissue.
  12. Method for in vitro characterization of a microtissue according to any of the preceding claims, characterized in that it is performed online on the contents of a bioreactor.
  13. Method for in vitro characterization of a microtissue according to any of claims 1 to 12, characterized in that it is performed: i) in flow cells, or ii) by spot sampling outside the bioreactor, or iii) to sort microtissues online or offline.
  14. Method for in vitro characterization of a microtissue according to any of the preceding claims, characterized in that it comprises measuring: - the density of the microtissue, based on the phase measurement, and - optionally, the local absorption of the microtissue, based on the phase measurement and measurement of the luminous intensity of the light having passed through the microtissue.
  15. Method for in vitro characterization of a microtissue according to any of the preceding claims, characterized in that it comprises measuring at least one of the following parameters: - dimensions of the microtissue, - dimensions of at least one of the microtissue cells, - number of cells in the microtissue, - global and local mass of the microtissue, - global and local density of the microtissue, - mass distribution in the microtissue, - topology of the microtissue - relative positioning of the cells in the microtissue, - viability of the microtissue cells, - texture of the microtissue.
  16. Method for in vitro characterization of a microtissue according to any of the preceding claims, characterized in that the microtissue is encapsulated in a microcompartment comprising an outer hydrogel layer.
  17. Method for in vitro characterization of a microtissue according to any of the preceding claims, characterized in that the microtissue is in the form of an ovoid, tuboid, spheroid or sphere, or of partially folded monolayers (2,5D).
  18. Method for in vitro characterization of a microtissue according to any of the preceding claims, characterized in that the microtissue is at least partially surrounded by an extracellular matrix.
  19. Method for in vitro characterization of a microtissue according to any of the preceding claims, characterized in that the microtissue is a human or animal biological microtissue intended for grafting in humans or animals.
  20. Method for in vitro characterization of a microtissue according to any of the preceding claims, characterized in that the microtissue is a human or animal biological microtissue selected from epithelial, connective, muscular or nervous microtissues.

Description

The present invention relates to the characterization by imaging of biological tissues, in particular of biological micro-tissues. In both research and therapy, it is essential to be able to characterize living biological cells and tissues, particularly during or after cell culture, notably to control cell proliferation and/or cell and tissue quality and/or to monitor cell differentiation and/or to monitor tissue organization and/or to determine the phenotype(s) of the cells constituting a tissue, etc. However, current imaging techniques, particularly those employing fluorescence microscopy, histology, capacitance measurement, optical density and standard transmission imaging, do not allow this. Fluorescence microscopy is used in conjunction with fluorescent probes such as antibodies or endogenous fluorescence to genetically modify cells. Several techniques employing fluorescence microscopy are commonly used, including confocal microscopy, selective plane illumination microscopy (SPIM), multiphoton microscopy, and flow cytometry (facs). These techniques are well-established, but they require fixation (resulting in cell death) and/or limit conditions (labeling only extracellular proteins) or are incompatible with cell culture for cell therapy purposes, such as the addition of non-GMP products that are destructive or cause genetic modification of cells. Therefore, they are not suitable for characterizing living tissues because they are invasive, often destructive, and very slow. Histological techniques involve fixing and then labeling tissues. These techniques also result in cell destruction and present the same drawbacks as fluorescence microscopy. Biomass measurement using capacitance probes is based on the assumption that living cells can be considered capacitors. This measurement therefore only takes into account the accessible outer surface of cells with an intact membrane. The case of cell aggregates and microtissues is more complex and depends on the tightness of the connections between cells. Unlike previous methods, this one is non-invasive, but it does not allow for to have information only on the inaccessible volume or on the surface of that volume, which is too limiting and imprecise for tissue characterization. Standard transmission imaging techniques, such as quantitative phase contrast, are fast and non-invasive. Phase measurement in imaging is a measurement of the local delay of a light beam after interaction with the object under study. Devices used for phase imaging are based on the phenomenon of optical interference to encode phase information into light 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 Phase imaging is currently used to characterize thin samples (isolated cells or microtissue sections less than 10 µm thick) but is not suitable for larger microtissues or tissues. Indeed, current phase imaging techniques do not allow for quantitative measurement of phase within a tissue. They remain qualitative rather than quantitative and are therefore difficult to use for characterizing thick tissues. The document Robles et al "Epi-mode tomographic quantitative phase imaging in thick scattering samples", PROGRESS IN BIOMEDICAL OPTICS AND IMAGING, SPIE - INTERNATIONAL SOCIETY FOR OPTICAL ENGINEERING BELLINGHAM, WA, US, vol 11251, February 20, 2020 This presents a quantitative phase imaging technique for thick samples. Although it allows for depth imaging, this method is not optimized for the rapid and non-invasive characterization of live micro-tissues in culture. The article from Hu Junbao et al. “Higher Order Transport of Intensity Equation Methods”, IEEE PHOTONICS JOURNAL, IEEE, USA, vol 11, no. 3, June 2019 , describes advanced intensity transport equation methods for phase imaging. However, these techniques require the acquisition of multiple images, limiting their use for the rapid characterization of live tissues in culture. The document Bon Pierre et al., “Self-interference 3D super-resolution microscopy for deep tissue investigations”, NATURE METHODS, NATURE PUB. GROUP, NEW YORK, vol. 15, n°6, April 30, 2018 , presents a 3D super-resolution microscopy technique for depth imaging. Despite its high resolution, this method is complex and poorly suited to the rapid and non-invasive characterization of cultured biological microtissues. The article Bon Pierre et al. “Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells”, OPTICS EXPRESS, OSA PUBLISHING, US, vol. 17, n°15, July 20, 2009 , describes an interferometry technique for quantitative phase imaging. This approach, although applicable to living cells, is limited to thin samples and is not suitable for thick cultured microtissues. The demand US 2018/113064 A1 describes a method for determining