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EP-3751290-B1 - APPARATUS AND METHOD FOR DETECTING CELLS OR PARTICLES IN A LIQUID CONTAINER

EP3751290B1EP 3751290 B1EP3751290 B1EP 3751290B1EP-3751290-B1

Inventors

  • SCHÖNDUBE, Jonas
  • GROSS, ANDRE

Dates

Publication Date
20260506
Application Date
20170619

Claims (10)

  1. Apparatus for detecting cells or particles (16) in a liquid container (10), comprising: a liquid container (10) at least partially filled with a liquid (12); a dispenser (14) configured to dispense a free-flying drop in which at least one cell (16) or at least one particle (16) is encapsulated, wherein the dispenser (14) and the liquid container (10) are arranged such that the free-flying drop is dispensed into a defined sub-volume (18) of the liquid (12) with which the liquid container (10) is at least partially filled, wherein the defined sub-volume is arranged above the bottom of the container; and a detection apparatus (22, 32) configured to perform a detection in the defined sub-volume (18) and/or in one or several sub-volumes (18a-18d) underneath the defined sub-volume (18), wherein the detection apparatus (22, 32) comprises an image capturing apparatus (32) configured to focus on the defined sub-volume (18) and/or the one or several sub-volumes (18a-18d) underneath the defined sub-volume (18) and to capture images of the defined sub-volume (18) and/or the one or several sub-volumes (18a-18d) underneath the defined sub-volume (18), characterized in that the apparatus further comprises a control unit configured to coordinate dispensing the cell or the particle by the dispenser and detecting by the detection apparatus in a timely coordinated manner in order to sense the at least one cell (16) or the at least one particle (16) when entering the liquid (12) and before the at least one cell (16) or the at least one particle (16) reaches the bottom of the liquid container (10) or after entering the liquid (12) and before the at least one cell (16) or the at least one particle (16) reaches the bottom of the liquid container (10), wherein the liquid container (10) is transparent and the image capturing apparatus (22, 32) is arranged underneath the liquid container (10) and/or the apparatus comprises a plurality of liquid containers (10) and a positioning mechanism configured to position the dispenser (14), the detection apparatus (22, 32) and each one of the plurality of liquid containers (10) relative to each another in order to sense the at least one cell (16) or the at least one particle (16) when entering the liquid (12) and before the at least one cell (16) or the at least one particle (16) reaches the bottom of the liquid container (10) or after entering the liquid (12) and before the at least one cell (16) or the at least one particle (16) reaches the bottom of the liquid container (10).
  2. Apparatus according to claim 1, wherein the detection apparatus (22, 32) is configured to sense the at least one cell (16) or the at least one particle (16) no later than 10 seconds after entering the liquid (12), and/or wherein the defined sub-volume (18) comprises an area (A1) which is smaller than the area (A2) of an entry opening of the liquid container (10).
  3. Apparatus according to claim 1, wherein the detection apparatus (22, 32) is configured to perform a detection of the at least one cell (16) or the at least one particle (16) in the defined sub-volume (18) comprising a depth of less than 5mm from the upper surface of the liquid (12).
  4. Apparatus according to claim1 or 2, wherein the detection apparatus (22, 32) is configured to, starting with a sub-volume (18d) in a greater depth, successively perform detections in several sub-volumes (18-18d) comprising a respectively decreasing depth, or, starting with a sub-volume (18) in a lesser depth, successively perform detections in several sub-volumes (18-18d) comprising a respectively increasing depth.
  5. Apparatus according to any one of claims 1 to 4, wherein the liquid container (10) comprises a side wall with an edge, a contact angle of the liquid being adjusted with respect to the side wall such that the liquid (12) comprises a flat surface.
  6. Method for detecting cells or particles (16) in a liquid container (10), comprising: dispensing (50, 60) a free-flying drop in which at least one cell (16) or at least one particle (16) is encapsulated into a defined sub-volume (18) of a liquid (12) with which a liquid container (10) is at least partially filled, wherein the sub-volume is arranged above the bottom of the container, characterized in that the method further comprises performing (52, 62), in a time-coordinated manner with dispensing the at least one cell (16) or the at least one particle (16), a detection in the defined sub-volume (18) and/or in one or several sub-volumes (18a-18d) underneath the defined sub-volume (18) in order to sense the at least one cell (16) or the at least one particle (16) when entering the fluid (12) or after entering the fluid (12) before the at least one cell (16) or the at least one particle (16) reaches the bottom of the liquid container (10), wherein performing a detection comprises focusing on the defined sub-volume (18) and/or the one or several sub-volumes (18a-18d) underneath the defined sub-volume (18) and capturing images of the defined sub-volume (18) and/or of the one or several sub-volumes (18a-18d) underneath the defined sub-volume (18).
  7. Method according to claim 6, wherein sensing the at least one cell (16) or the at least one particle occurs no later than 10 seconds after entering the liquid (12), and/or wherein the defined sub-volume (18) comprises an area (A1) which is smaller than the area (A2) of an entry opening of the liquid container (10).
  8. Method according to claim 6, wherein a detection of the at least one cell (16) or the at least one particle (16) is performed in the defined sub-volume (18) comprising a depth of less than 5mm from the upper surface of the liquid (12).
  9. Method according to any one of claims 6 or 7, wherein, starting with a sub-volume (18d) in a greater depth, detections are successively performed in several sub-volumes (18-18c) comprising a respectively decreasing depth, or, starting with a sub-volume in a lesser depth (18), detections are successively performed in several sub-volumes (18a-18d) comprising a respectively increasing depth.
  10. Method according to any one of claims 6 to 9, comprising positioning a dispenser (14), a detection apparatus (12) and each one of several liquid containers relative to each other in order to sense the at least one cell or the at least one particle when entering the liquid or after entering the liquid before the at least one cell (16) or the at least one particle (16) reaches the bottom of the liquid container.

Description

The present invention concerns apparatuses and methods for detecting cells or particles dispensed by a dispenser into a defined sub-volume of a fluid located in a fluid container. After inserting cells or particles into a fluid container, it is generally often necessary to sense if the cell or the particle is actually located in the fluid container. For example, monoclonal antibodies and other proteins, which are subsequently called products, are prepared by means of so-called monoclonal cell lines. These are populations of cells originating from a single cell. This ensures to the best extent that all cells of the population comprise approximately the same genotype and, thus, generate a product which is as equal as possible. In order to generate a monoclonal cell line, cells are individually transferred into so-called microtiter plates and multiply there in a controlled manner until the desired population size is reached. Depositing single cells in the microtiter plates occurs by free-jet printing methods or by pipetting single cells into the single bowls or cavities of the microtiter plate, which are herein after referred to as "wells". These wells represent containers. When manufacturing therapeutic products from cell cultures, it has to be demonstrated for regulatory reasons that indeed only one cell was located in the well at the beginning of the process. It is important for the well bottom to be sufficiently large, i.e., significantly larger than a cell, in order to allow the population to grow to the required size. Ultimately, from a series of a few hundreds to thousands of such clone populations, the one that produces the desired product in the most stable manner and in the greatest quantity is transferred to manufacturing. Methods for sensing cells in fluid containers, for example, the wells of a microtiter plate, are known from the prior art. In "Assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging", 2015, American Institute of Chemical Engineers, Biotechnol. Prog., vol. 00, No. 00, http://doi.org/10.1002/btpr.2145, K. Evans et al describe a process for producing monoclonal cell lines. Cells are transferred into the well of a microtiter plate by means of a so-called FACS apparatus (FACS = fluorescent activated cell sorting). After that, the same is centrifuged in order to transport the cells to the bottom. Successively, the entire well bottom is examined under the microscope, typically by means of a so-called imager, and single cells are searched for therein, which is effected by the user. In this case, it is extremely difficult to recognize a single cell in the large observation volume. Flow cytometry represents a known method for analyzing cells passing an electric voltage or a light ray. For example, US 3,380,584 A describes a method for separating particles in which a printing method comprising a continuous jet is employed, which has the disadvantage of drops being continuously generated without being able to interrupt the drop stream in a controlled manner. In selectively sorting cells or particles by means of this technique, it is therefore necessary to deposit the drops at different positions according to content. This occurs by an electrostatic deflection during flight. The higher the number of positions and the required deposition accuracy (e.g., in 96 or 384 well plates), the more difficult and technically complex the process. From EP 0 421 406 A2, apparatuses and methods for separating paticles are known, in which a thermal printing head is used in order to dispense particles The particles are arbitrarily arranged in the reservoir and are optically analyzed after ejection during flight. The above-described methods allow for depositing cells individually but cannot achieve an efficiency of 100 percent. Therefore, the microtiter plates have to be examined under the microscope afterwards by means of so-called imagers. From US 7,310,147 B2, EP 1 686 368 A2, US 8,417,011 B2 and US 8,795,981 B2, apparatuses and methods for sensing cells and particles in microtiter plates are known. US 7,646,482 B2 describes a method for automatically finding the right focal plane in order to, e.g., examine cells at a well bottom under a microscope. In the course of this, the method detects patterns in the sensor signal, while the microscope focuses through the bottom of the plate. US 8,383,042 B2 describes an imager comprising a vacuum holder. The vacuum holder sucks in the microtiter plate in order to maintain the bottom of the microtiter plate in a plane manner and, in this way, provides a lower variance of the distance of the well bottom to the objective. From WO2011/154042A1, apparatus and methods for dispensing a cell or a particle in a free-flying droplet are known. From US2015253223A1, apparatus and methods for detecting that a free-flying droplet has been dispensed in a microtiter plate are known. A plurality of tec