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EP-4133251-B1 - METHOD FOR DETERMINING THE VIABILITY OF CELLS

EP4133251B1EP 4133251 B1EP4133251 B1EP 4133251B1EP-4133251-B1

Inventors

  • ALLIER, Cédric
  • HERVÉ, Lionel
  • ESTEBAN, GEOFFREY
  • PISANESCHI, Martin
  • HILL, Melissa

Dates

Publication Date
20260506
Application Date
20200408

Claims (14)

  1. A method for determining a state of a cell, the cell being placed in a sample, in contact with a culture medium, the method comprising: a) illuminating (100) the sample with a light source (11) and acquiring (110) an image of the sample with an image sensor (16), the image sensor lying in a detection plane; b) from the acquired image, locating a position of the cell in a plane parallel to the detection plane; the method further comprising : c) from the acquired image, estimating (130) a refractive index of the cell or a relative refractive index of the cell, the relative refractive index corresponding to the refractive index of the cell relative to the refractive index of the culture medium. the refractive index or the relative refractive index being a complex quantity; d) from the estimation of the refractive index or of the relative refractive index, determining (140) an index of interest of the cell, said index of interest comprising a real part of the refractive index or of the relative refractive index; e) from the index of interest, classifying (150) a state of the cell among predetermined states, the predetermined states comprising at least one apoptosis state and one living state.
  2. The method according to claim 1, wherein: - in d), the index of interest is determined depending on a real part and on an imaginary part of the refractive index or of the relative refractive index.
  3. The method according to Claim 2, wherein the index of interest is determined from a difference or from a weighted difference between the real part and the imaginary part of the refractive index or of the relative refractive index.
  4. The method according to any one of the preceding claims, wherein e) comprises : - taking into account a threshold ; - comparing the index of interest with the threshold.
  5. The method according to any one of the preceding claims, wherein - e) comprises taking into account a reference range, the reference range comprising indices of interest corresponding to living cells ; - the apoptosis state corresponds to an index of interest outside of the reference range.
  6. The method according to claim 5, wherein : - the predetermined states comprise at least one dead state ; - the dead state and the apoptosis state correspond to indices of interest lying respectively on one side and on another side of the reference range.
  7. The method according to any one of the preceding claims, wherein: - b) to e) are carried out for a plurality of cells, each cell of the plurality of cells being considered as living cells, so as to obtain a distribution of the index of interest of said cells ; - in e), the reference range is defined from said distribution.
  8. The method according to Claim 7, wherein e) comprises : - taking into account a threshold; - comparing the index of interest with the threshold ; and wherein in e), the threshold is defined from said distribution.
  9. The method according to Claim 7, wherein e) comprises determining a reference value from said distribution, the state of each cell being determined depending on a deviation between the reference value and the index of interest of each cell.
  10. The method according to any one of the preceding claims, wherein: - step a) is carried out at various times, so as to obtain an image of the sample at each time; - steps b) to e) are carried out successively using the images obtained at each time; - in each e), the predetermined states comprise at least one dead cellular state; - the method comprising a step f) of computing a viability at each time, the viability depending on a number of cells considered to be living, dead and in apoptosis, respectively.
  11. The method according to any one of the preceding claims, wherein: - a) is carried out at various times, so as to obtain an image of the sample each time; - steps b) to e) are carried out successively using the images obtained at each time; - the method further comprises emitting a warning when a number of cells considered to be in the apoptosis state exceeds a predetermined value.
  12. The method according to any one of the preceding claims, wherein : - in a), an exposure light wave propagates towards the image sensor along a propagation axis; - step c) comprises : c-i. on the basis of the acquired image, applying a propagation operator, for a plurality of reconstruction distances from the detection plane, so as to estimate, at each reconstruction distance, a complex amplitude of the exposure light wave ; c-ii. on the basis of the complex amplitude estimated, at various reconstruction distances, obtaining a profile representing a variation of the complex amplitude of the exposure light wave along an axis parallel to the propagation axis and passing through the position of the cell ; c-iii. associating each cell with a set of parameters, at least one parameter of the set of parameters depending on the refractive index of the cell or on a relative refractive index of the cell ; c-iv. modelling a cell, taking into account a value of each parameter of the set of parameters, and modelling an exposure light wave, propagating toward the image sensor, under the effect of an illumination, with the light source, of the modelled cell ; C-v. on the basis of the modelled exposure light wave, forming a modelled profile representing a variation in the complex amplitude of the modelled exposure light wave, along an axis parallel to the propagation axis ; c-vi. comparing the profile obtained in c-ii) with the modelled profile resulting from c-v), so as to determine the value of said at least one parameter of the cell. c-vii. deriving the refractive index of the cell or the relative refractive index of the cell from the parameters associated to the cell.
  13. The method according to claim 12, wherein steps c-iii) to c-vi) are implemented iteratively, such that, in each iteration, the profile modelled in c-v) gets gradually closer to the profile obtained in c-ii).
  14. The method according to anyone of claim 1 to 11, wherein : - in step a), an exposure light wave propagates towards the image sensor ; - the sample lies in a sample plane ; - the sample is described by sets of parameters, each set of parameters being respectively defined at a plurality of radial positions, in the sample plane, each set of parameters comprising at least an optical parameter of the sample, at least one optical parameter being an optical path difference induced by the sample at each radial position ; - step c) comprises : c-i. taking into account sets of parameters, describing the sample, in the sample plane ; c-ii. on the basis of the sets of parameters, forming a complex image of the sample in the sample plane; c-iii. applying a propagation operator to the complex image formed in c-ii), in order to compute an image of the sample in the detection plane; c-iv. comparing the image acquired in a) and the image computed in c-iii), in order to compute a validity indicator ; c-v. updating the sets of parameters, so as to make the validity indicator tend toward a preset value ; c-vi. reiterating c-ii) to c-v) taking into account the sets of parameters updated in c-v), until the validity indicator is considered as reaching the preset value ; c-vii. estimating a radial position of a cell ; c-viii. estimating the refractive index or the relative refractive index of the cell from the sets of parameters defined at said at least one radial position of the cell.

Description

Technical field The technical field of the invention is the analysis of images of cells in order to determine a state of said cells. It is in particular a question of determining a living or dead cellular state, including the occurrence of apoptosis. Prior art Cells are cultured in bioreactors in many pharmaceutical or medical fields, in order to produce molecules for therapeutic purposes. Which may, for example, be proteins, vaccines or antibodies. However, cells are fragile. The composition of the biological medium, and hydrodynamic stresses (stirring of the medium) to which the cells are subjected may limit the viability of the cells. When the viability of the cells decreases, i.e. when the number of dead cells increase, the yield of the production decreases. It is therefore important to detect, or even to limit as much as possible, the death of cells cultivated in bioreactors. There are two types of cellular death, which may occur in vivo but also in vitro: necrosis and apoptosis. Necrosis occurs accidentally, and rapidly, following a malfunction of a cell, in particular, following a perturbation of the culture medium. Apoptosis is a programmed cellular death by self-destruction, the process of which may take several hours. In a bioreactor, apoptosis is also considered to be influenced by the culture physicochemical conditions, for example a lack of oxygen, a lack of nutrients, or the accumulation of toxic metabolites. The hydrodynamic conditions in the bioreactor may also lead to apoptosis, notably when the culture medium is stirred too vigorously. It will be understood that the optimization of culture conditions is essential to improving the yield of production in a bioreactor. Currently, on the industrial scale, the main optical devices for estimating cellular viability are based on the use of viability markers, based on color (marking with trypan blue) or fluorescence (marking with propidium iodide). An optical method not using marking has been described in US10481076. US8260063 describes an apparatus for observing cells by fluorescence microscopy. US2010/093015 describes an apparatus for analysing cells by Raman spectroscopy. The publication Curl Claire et al « Refractive Index Measurement in Viable Cells Using Quantitative Phase-Amplitude Microscopy and Confocal Microscopy » describes the use of phase microscopy for the observation of cells. DESCRIPTION OF THE INVENTION An object of the invention is a method for determining a state of a cell as defined in claim 1. In one embodiment, in d), the index of interest comprises a real part of the refractive index or of the relative refractive index. In one embodiment, in d), the index of interest is determined depending on a real part and/or on an imaginary part of the refractive index or of the relative refractive index. The index of interest may be determined from a difference or from a weighted difference between the real part and the imaginary part of the refractive index or of the relative refractive index By from the acquired image, what is meant is using the acquired image so as to locate the position of the cell. This may comprise reconstructing a complex image of an exposure light wave, propagating between the sample and the image sensor. In one embodiment, step e) comprises : taking into account a threshold ;comparing the index of interest with the threshold. In one embodiment, step e) comprises taking into account a reference range, the reference range comprising index of interest corresponding to living cells. The apoptosis state corresponds to an index of interest outside of the reference range. The predetermined states may comprise at least one dead state. The dead state and the apoptosis state may correspond to an index of interest lying respectively on one side and on another side of the reference range. In one embodiment, steps b) to d) are carried out for a plurality of cells, each cell of the plurality of cells being considered as a living cell, so as to obtain a distribution of the index of interest of said cells ;in step e), the reference range is defined from the distribution. The method may comprise determining the threshold based on the distribution of the index of interest. The method may comprise determining a reference value from the distribution. The state of each cell may be determined depending on a deviation between the reference value and the index of interest of said cell. In one embodiment, a) is carried out at various times, so as to obtain an image of the sample each time;b) to e) are carried out successively using the images obtained at each time;in each step e), the predetermined states comprise at least one dead cellular state;the method comprises a step f) of computing a viability at each time, the viability depending on a number of cells considered to be living, dead and in apoptosis, respectively. In one embodiment, step a) is carried out at various times, so as to obtain an image of the sample each time;steps b)