Cell Counting

Abstract

The invention presents a non-contact device for counting the concentration of particles suspended in a liquid fluid, comprising a container associated with a stirring means for the homogeneous suspension of the particles, which includes: an image acquisition means having a field adapted to form an image containing a plurality of particles after sedimentation, focused on the inner surface of the bottom of said container, and a light source for illuminating the inner surface of the bottom of the container; a computer controlling the acquisition of a time-stamped sequence of at least two images (Ii, ti) in the focal plane of sedimentation of said suspended particles; the counting, in each of said images (Ii, ti), of the number of particles Pi; the determination of a set of values ​​(Pi, ti); the determination of the concentration CP of particles, at time t of the last image (Ii, ti) acquired by applying a function F to said set of values ​​(Pi,ti), the function F being a function of the hydrodynamic drag force. Figure of the abstract: 1,

Description

Title of the invention: Cell counting Field of invention

[0001] The present invention relates to a method for counting cells online and in situ in a biological culture medium, in particular in the field of personalized cellular immunotherapy and more particularly the collection and automated processing of cell subsets, in particular CART-T (Chimeric Antigenic Receptor - T) cells in peripheral blood with the aim of locally reinjecting these cells to repair tissues. The invention relates more particularly to automatic cell counting methods for monitoring the proliferation of T lymphocytes in culture, in a closed system and in real time. The objective is to be able to reach a target concentration of T lymphocytes (approximately 1 billion cells in 600 ml, i.e. a concentration of 1.66 M / ml) at which the following steps of the innovative therapy based on Car-t cells can be initiated.

[0002] Cell counting or counting corresponds to the determination of the number of cells contained in a volume and is carried out in the state of the art on a sub-sample or sub-part of the culture and very largely involves at least one sampling step, in particular for the following reasons: • to facilitate counting and save time: it seems very tedious and time-consuming to count the millions / billions of cells in culture, • counting is often intrusive for cells and the idea is therefore to be able to apply a treatment that is possibly deleterious or modifies cellular homeostasis for a sub-sample of the culture without impacting the rest of the cells in culture.

[0003] Cell counting represents for biologists a daily manipulation carried out in the context of cell cultures. It is essential that it be precise for the well-being of the cells in culture but also for any experiment involving cells in order to be able to adapt the concentrations of reagents or to be able to obtain images that can be analyzed in imaging for example.

[0004] Several automated counters with imagers that contain a micro-camera and image processing software have been developed in recent years. These counters nevertheless involve a step of taking a part of the sample and the use of markers aimed at improving the contrast between living cells and noise to facilitate their automatic detection. For example, we can cite Trypan blue which, used in excess, colors the membranes of living cells. improving contrast (and completely coloring dead cells blue) or the use of fluorescent protein and membrane probes capable of attaching to living eukaryotic cells.

[0005] Concerning the imaging of cells, there are two main imaging technologies: the classic microscope coupled with a camera or holographic reconstruction which consists of the projection of the diffraction of the cells onto a CMOS sensor without a lens by a point light source. Holographic reconstruction has the advantage of imaging a large surface but the light source must be as direct as possible which is difficult to achieve for cultures (very high height of the medium, roughness of the walls of the pocket, high cell concentration, presence of air bubbles and aggregates).

[0006] Regarding image analysis for automatic cell counting, there are two main methods: • The image analysis technique using filters, thresholding and segmentation of varying complexity. There is no generic algorithm; each application requires a suitable algorithm. This method is not very effective for the classification of poorly differentiated elements, i.e. the identification of cells of the same size but with slightly different morphology, • The supervised learning method from thousands of manually labeled cell images. Learning requires a very high computing power (96 GB of GPU) but is only done once. A hyperparameter model is then created to analyze images quickly and classify the detected elements. The computing power (CPU or GPU) is then lower and depending on the number of classes to be identified, the image can be processed live. State of the art

[0007] FR3034103 is known in the prior art describing a device for measuring the concentration of microorganisms in a liquid sample, comprising a light source, a sample support, an image sensor, an optical device located between said sample support and said image sensor, a microprocessor and a counting program. The optical device consists of a single transparent lens. This document also describes a method for monitoring the development of microorganisms in a liquid, characterized in that it comprises the following steps: • taking a sample of said liquid, • introducing said sample into a device mentioned above, • detecting an image of said sample by said image sensor • determination of the concentration of microorganisms contained in said sample by counting microorganisms on said image, by means of said program.

[0008] Patent EP2025744 describes a method for counting cells online and in situ in a biological culture medium, comprising the following steps: • a plurality of measurements of the capacitance of said medium or a plurality of measurements of the conductance of said medium, at distinct frequencies varying within a predetermined measurement frequency range, • an extraction of information on variation in permittivity due to [3-dispersion in said medium, from said capacitance measurements, and • processing of said permittivity variation information to deliver cell counting information in said medium.

[0009] Patent application WO2018220198 describes a device and a method for detecting and imaging biocontaminant elements, comprising a matrix of detector pixels having an active face on which light is incident, a light-transparent support which covers the active face and lighting means which produce the incident light on the active face, display means. The invention is characterized in that the incident light is the light emitted by the biocontaminant elements and in that the device further comprises at least one filter, a polarizer and an analyzer, said polarizer and said analyzer being positioned in a crossed configuration to overcome laser granularities in the images. Disadvantages of the prior art

[0010] The solutions of the prior art require taking a sample of the solution. The step of taking a sample and pipetting the cell culture makes it possible in particular to minimize a technical obstacle well described in the literature which corresponds to the presence of counting artifacts (air bubbles, etc.) likely to deteriorate the precision of the counting method by pipetting steps. In our case, cell culture and cell counting in a bag and without taking a sample required direct agitation of the bag, which on the contrary facilitated the appearance of air bubbles or the appearance of other artifacts such as scratches on the bag which could drastically alter the precision of the counting method.

[0011] Similarly, the absence of labeling made counting and image analysis more complex (no contrast markers possible). An additional problem arose from the nature of the cells (T lymphocytes) and the cell culture volumes sought for the purpose of automatic design of innovative drugs. Indeed, these cells are non-adherent (in suspension) and as the concentration and culture volume are high, the absence of dilution further complicated the cell counting method at these high concentrations.

[0012] In addition, T lymphocytes can become activated: they tend to form large cell clusters.

[0013] In addition, T lymphocytes have variable morphologies depending on the individual, the culture medium (5.8 to 8.14 pm) but also depending on the stage of the cell cycle and the culture time, which can drastically alter the precision of the automatic counting method. Indeed, the diameter of T lymphocytes doubles before division.

[0014] Finally, the state-of-the-art solutions are not compatible with counting directly in a culture bag which imposes different optical constraints. Solution provided by the invention

[0015] In order to overcome these drawbacks, the present invention relates to a device for contactless counting of the concentration of particles suspended in a fluid comprising a container associated with a stirring means for homogeneously suspending the particles, characterized in that it comprises: - an image acquisition means having a field adapted to form an image comprising a plurality of particles after sedimentation focused on the interior surface of the bottom of said container, - a light source to illuminate the inner surface of the bottom of the container - and a calculator. This calculator runs a computer program to control: - the acquisition of a time-stamped sequence of at least two images (h, h) in the focal plane of sedimentation of said suspended particles - counting, in each of said images (f, h), the number of particles P; - the determination of a set of values ​​(P;, h) - the determination of the concentration CP of particles, at time t of the last image (h, h) acquired by applying a function F to said set of values ​​(P,, h), the function F being a function of the hydrodynamic drag force. Advantageously, said light source is arranged on the side opposite the camera relative to said background, said light source comprising a condenser focused on the surface of said background.

[0016] Advantageously, said light source comprises an LED source positioned at the bottom of a tube having anti-reflection grooves.

[0017] Preferably, the axis of said tube is horizontal, and a mirror returns the light beam at 90°, perpendicular to the plane of a transparent sample support surface.

[0018] According to an alternative embodiment, said image acquisition means comprises a microscope optic with an input lens and an image sensor. The assembly is supported by a motorized stage.

[0019] Detailed description of a non-limiting example of embodiment

[0020] The present invention will be better understood on reading the following description, concerning a non-limiting example of embodiment illustrated by the appended drawings where:

[0021] [Fig-1] [Fig.l] represents a schematic view of a device according to the invention

[0022] [Fig.2] [Fig.2] represents a schematic view of the image processing according to the invention device according to the invention

[0023] [Fig.3] [Fig.3] represents the curve of the variance of the Laplace transform in position function (5M / ml)

[0024] [Fig.4] [Fig.4] represents the cell size distribution curve on 50 images.

[0025] [Fig.5] [Fig.5] represents the steps of the concentration measurement process cellular

[0026] [Fig.6] [Fig.6] represents the accumulation curve of the number of cells in function of time. General principle of the invention

[0027] The invention relates to a method for cell counting on a bag making it possible to monitor the proliferation of T lymphocytes throughout the culture (between 0.01 and 10 million cells per milliliter), which is implemented automatically and in a closed environment, without taking a sub-sample of the cell culture and without applying dilutions and / or cell labeling. The method is based on determining the sedimentation kinetics of the cells contained in the culture medium, and deducing therefrom the concentration of cells - and therefore the number of cells in a reference volume, as a function of the dynamic viscosity of the culture medium and the average diameter of the cells.

[0028] the method for counting cell culture in real time according to the invention: • be fully automated, • be carried out entirely in a closed system directly in a culture bag (without sampling, dilution, cell marking), • alter cellular homeostasis as little as possible, • ensure a counting accuracy of 90 to 95% throughout the culture (for a wide range of T lymphocyte concentrations between 0.01 and 10 million cells per milliliter).

[0029] For this purpose, the invention is based on the measurement of cell proliferation by analysis of sedimentation during time intervals from images acquired using a microscope in a reference plane, generally the bottom of the bag containing the culture medium and the cells to be counted, and implements: • A culture bench containing the culture medium and the cells to be counted, comprising a controllable means of resuspending the cells, for example a flexible bag, with at least one constriction to generate turbulence. • an image acquisition device with a lighting source (100) and a microscope (200) having a motorized adjustable focus and sufficient magnification for viewing the cells and an optical condenser adapted to the culture bench, associated with an image sensor (210). The assembly has a thickness of less than 30 mm. • and a computer running a program controlling the acquisition of images and their processing, and in particular: a. automatic microscope objective focusing control b. lighting control c. bubble detection d. the concentration calculation.

[0030] The principle of dynamic cell counting proposed by the invention is based on the sedimentation rate and Stockes' law. The method implemented consists of agitating the culture medium in a bag in order to resuspend the cells so as to have a homogeneous distribution of the cells in suspension.

[0031] A microscope focused on the bottom of the bag makes it possible to visualize only the cells that settle there by sedimentation. Immediately after resuspension by stopping the agitation, the kinetics of cell sedimentation are measured to estimate the cell concentration as a function of the number of cells at the bottom of the bag as a function of time. Optical device

[0032] The optical device consists of a miniaturized microscope of low height, less than 30 millimeters, the architecture of which is illustrated in [Fig.l]. The device consists of a light source (100) and an image sensor (200).

[0033] The light source (100) comprises an LED source (110) positioned at the bottom of a tube (120) having anti-reflection corrugations (130). The axis of the tube (120) is horizontal, and a mirror (140) returns the light beam at 90°, perpendicular to the plane of a transparent sample support surface (150). The output of the illumination source comprises an anti-bubble dome (160) and creates a hollow on the top of the pocket to facilitate the removal of bubbles from the camera field.

[0034] The image acquisition block comprises a microscope optic (220) with an input lens (230), and an image sensor (210). The assembly is supported by a motorized stage (240).

[0035] Program controlling the operation of the device

[0036] A computer controls the execution of the control programs of the optical block for automatic detection of sedimented cells as well as the resuspension of the cells in the culture medium.

[0037] The control of the optical unit includes:

[0038] • a self-illumination function

[0039] • an auto-focus function.

[0040] The self-illumination function controls the automatic adjustment of the power of the LED (110) in order to systematically have the same average gray level for each count. Indeed, the higher the concentration, the stronger the absorbance of the solution. The function consists of sweeping the power of the LED from 5 to 100% and then applying a polynomial regression for an estimation of the LED power at a set gray level. Visually, an average gray level of 130 (out of 256) allows good contrast, a parameter which was tested with a concentration of 0.1 to 100M / ml of T lymphocytes,

[0041] The auto-focus function (10) allows the automatic adjustment of the focus of the objective (230) of the microscope (220). A first step controls the initialization of the position of the camera (210) by retracting to the maximum and then goes back to a defined value. The camera (210) is moved to the area to be scanned. Then for each step, an image is taken and the variance of the Laplace transform is calculated. The position, for which the variance is maximum is then the position of best focus. The area to be scanned must exclude the outer wall of the container (bag) which risks being detected as the optimal focal point if few cells are present at the bottom of the bag. [Fig.3] represents the curve of the variance of the Laplace transform as a function of the position (for a concentration of 5M / ml).Curve (1) represents the variation of the Laplace transform after sedimentation, as a function of the stage setting, in number of microsteps (inner surface of the cell-coated bag). Curve (2) represents the variation of the Laplace transform without sedimentation, as a function of the stage setting, in number of microsteps (outer surface of the bag).

[0042] As shown in [Fig.3], at high concentration, the maximum of the Laplacian allows to find the best focus automatically. Optionally, a Gaussian filtering step (kernel 7,7) level 2, allows to reduce the noise and facilitates the measurement of the best focus for low cell concentrations

[0043] The dynamic counting and image processing algorithm is repeated several times, 6 times for example, with different kinetics, to smooth the result and retain the value corresponding to the median of the successive dynamic counts.

[0044] The kinematics is determined by the time and speed of agitation for resuspension of the cells. With relatively high agitation, the disaggregation of cell clusters is promoted.

[0045] The time between stopping the stirring and initiating the kinetics measurement is typically between 10 seconds and 30 seconds.

[0046] The acquisition of images to measure the sedimentation kinetics is done every 2 seconds for 100 seconds (i.e. 50 images per kinetic), this acquisition time can be reduced for high concentrations in order to avoid having too many sedimented cells and making image analysis (cell counting) difficult, or conversely can be extended in the case of low concentration to increase the number of cells which sediment to make the counting more reliable.

[0047] The algorithm for processing automatic image analysis and cell counting is based on image processing by binarization (conversion to gray level, Gaussian filter (Kernel (7,7), level 5), extraction of edges by Scharr Filter (gradient by first derivative of the two axes x and y then fusion), thresholding of the gray level (>100 non-critical value), dilation (Kernel (7,7), level 1), closing of the contours by elliptic morphological transformation of kernel (15,15) then recording of the image).

[0048] The performance of cell detection by the shape detection algorithm is superior to that by circle detection.

[0049] The calculator then determines the measurement of the average size of the cells of the 50 images by kinetics then median of the 6 averages obtained for the 6 repeated experiments (detection of the contours of the cells by vertical / horizontal / diagonal compression, sorting of the contours by extraction of the surface and calculation of the diameter and selection of the unit cell diameters and sorting by form factor (see [Fig.4]). The curve of the statistical distribution in size of the cells on a series of images makes it possible to determine the median diameter.

[0050] Relationship between sedimentation kinematics, median cell diameter, viscosity of culture medium and cell number

[0051] The above-mentioned treatments make it possible to determine two parameters: - The evolution over time of the number of cells Nc(t) deposited on the bottom of the surface S of the pocket, allowing the determination of the sedimentation rate vsed - The median diameter dc of the cells - The cell concentration C in the solution in M ​​per mL

[0052] Image processing measures the median diameter of the cells and then counts the number of cells. The diameter allows us to go back to the sedimentation rate which, coupled with the number of cells, allows us to go back to the concentration

[0053] The number Nc of cells deposited on the bottom of the pocket is a function of time t, according to a formula:

[0054] Nc—tCS V^e(|+ initial Nc

[0055] Nc initial number of cells at t=0 S imaged surface

[0056] Furthermore, the sedimentation rate is a function of the difference between the density Pc of the cells and the density P; of the culture medium (in kg:m3), the diameter dc (in m) of the cells and the dynamic viscosity pi of the medium (in Pascal per second).

[0057] This relationship is for example determined by Stokes' law ~ d c2 * ( / ¾ - A) * 18.p, z

[0058] The usual culture medium for the culture of Car-T is for example RPMI + 8% HSA. The proliferation of T lymphocytes in culture is carried out by CDC3 / CD28 activation in the presence of interleukin 2 (IL-2), or other serum-free culture media better suited to the manufacture of Car-T. Functioning

[0059] The measurement cycle includes a succession of treatments, some of which are iterative. • Camera connection • Step (10) of positioning the plate (230) for focusing the optical block • Start of a dynamic counting cycle (20) • Step (21) Agitation to resuspend and break the clusters • LED power adjustment • Recursive bubble detection: • LED power threshold • Evacuation of bubbles if necessary (raising / lowering pallet) • Delta threshold (max-min gray level) and gray mean threshold • Evacuation of bubbles if necessary (raising / lowering pallet) • Step (22) Taking images, for example every 10 seconds • Step (23) determination of the median diameter of the cells and calculation of the Vsed parameter • Step (24) Determination of the number of cells • Step (25) calculation of cell concentration • Camera disconnection Step (26) of focusing adjustment Return for a new dynamic counting cycle (20), six iterations for example Step (30) Calculation of a statistical value of the number of cells

Claims

Claims

1. Device for contactless counting of the concentration of particles suspended in a fluid comprising a container associated with a stirring means for homogeneously suspending the particles, characterized in that it comprises: - an image acquisition means (210) having a field adapted to form an image comprising a plurality of particles after sedimentation focused on the inner surface of the bottom of said container, - and a light source (100) for illuminating the inner surface of the bottom of the container - a computer controlling • the acquisition of a time-stamped sequence of at least two images (h, h) in the focal plane of sedimentation of said particles in suspension • the counting, in each of said images (h, h), of the number of particles P;• the determination of a set of values ​​(P,, f) • the determination of the concentration CP of particles, at the time t of the last image (f, f ) acquired by the application of a function F to said set of values ​​(P,, h), the function F being a function of the hydrodynamic drag force.;

2. Device for non-contact counting of the concentration of particles suspended in a fluid according to claim 1 characterized in that said light source (100) is arranged on the opposite side of said image acquisition means (210) relative to said bottom, said light source (100) comprising a condenser focused on the surface of said bottom.

3. Device for contactless counting of the concentration of particles suspended in a fluid according to claim 1 characterized in that said light source (100) comprises an LED source (110) positioned at the bottom of a tube (120) having anti-reflection grooves (130).

4. Device for non-contact counting of the concentration of particles suspended in a fluid according to claim 3 characterized in that the axis of said tube (120) is horizontal, and a mirror (140) returns the light beam at 90°, perpendicular to the plane of a transparent sample support surface (150).

5. Device for contactless counting of the concentration of particles suspended in a fluid according to claim 1 characterized in that said image acquisition means (210) comprises a microscope optic (220) with an input lens (230), and an image sensor, this assembly being supported by a motorized plate (240).