WO2018019383A1

WO2018019383A1 - Method for analyzing a cell pellet

Info

Publication number: WO2018019383A1
Application number: PCT/EP2016/068115
Authority: WO
Inventors: Christian Mayer; Philipp Hofmann
Original assignee: Curevac Ag
Priority date: 2016-07-28
Filing date: 2016-07-28
Publication date: 2018-02-01

Abstract

The present invention relates to method for analyzing a cell pellet, a device for analyzing a cell pellet and a system for analyzing a cell pellet. Further, several uses as described herein are part of the present invention. The method for analyzing a cell pellet comprises the following steps: a) providing an image comprising the cell pellet, b) determining a radial dimension of the cell pellet based on the image, and c) calculating an amount of cells in the cell pellet based on the determined radial dimension of the cell pellet and a predefined cell type constant.

Description

Method for analyzing a cell pellet

Field of the invention

The present invention relates to a method for analyzing a cell pellet, a device for analyzing a cell pellet and a system for analyzing a cell pellet. Further, several uses described herein are part of the present invention.

Background of the invention

Many industrial and laboratory fermentation and cultivation processes rely on liquid culturing of cells of interest. Cells that are commonly cultured in liquid growth medium are for example bacterial cells, yeast cells and cell types from higher eukaryotes (e.g., mammalian cells, human cells). Efficient cultivation of cells in liquid culture requires a quantification of cell numbers in the liquid culture medium. These quantitative measurements are often required to adjust downstream processes and to optimize yield of the cultured cells. Quantification of cell numbers is also crucial for a proper control and adjustment of critical parameters which may influence the cell growth rate (e.g. temperature, pH value, nutrient levels, etc.) and eventually a final yield of the fermentation process. Moreover, in the context of molecular cloning approaches, it is mandatory to determine cell numbers (e.g., bacterial cells, yeast cells) in liquid cultures for a successful propagation of cloned genetic material, e.g. plasmid DNA. A fast and reliable determination of the cell number in a given cell culture is particularly important in the context of molecular cloning in a high throughput format.

High throughput (HT) cloning, which often uses robot-assisted parallel cloning of various different DNA constructs, e.g. plasmid DNA constructs, is increasingly used to generate cDNA libraries, BAC libraries, genomic DNA libraries, mutant libraries, or other DNA construct libraries. In the context of HT cloning, a handling of a cell culture (containing an undefined number of cells) to generate distinct clones (e.g., yeast clones or bacterial clones carrying the genetic constructs) should ideally be performed in an automated way to save time, resources and labor costs. In HT cloning approaches, several liquid cell cultures (e.g. 6, 12, 24 or 96) are ideally handled in parallel.

Besides the laborious way of manual counting of cells, which is still performed for human cell cultures, some other methods exist to quantify the cell number in liquid cultures. Typically, cell numbers are quantified via light scattering techniques of turbidimetry, laser- or impedance flow cytometry (e.g., free-floating cell cultures) or microfluidic mass sensors.

However, for all these methods, sampling of a liquid cell culture suspension is needed which involves destroying part of the sample. Furthermore, by taking a sample of the cell culture, there is a risk of contaminating the cell culture. Also, the measurement error e.g. in turbidimetry based methods can be very large for cultures with high cell densities and large inhomogeneity. Therefore, such measurements often require additional laborious manual and/or complicated automated processing steps of the samples (e.g., inverting, and/or homogenizing, and/or diluting the cell culture sample etc.).

Summary of the invention

Thus, in view of the above, there is still a need for providing a more economic method for the determination of cell numbers in liquid cell cultures.

This problem is solved by the subject-matter of the independent claims, wherein further embodiments are incorporated in the dependent claims. It should be noted that the features of the invention described in the following apply equally to the method for analyzing a cell pellet, the device for analyzing a cell pellet, the system for analyzing a cell pellet and to the uses described herein. In a first aspect, the present invention is directed to a method for analyzing a cell pellet. The method comprises the following steps: a) providing an image comprising the cell pellet,

b) determining a radial dimension of the cell pellet based on the image, and c) calculating an amount of cells in the cell pellet based on the determined radial dimension of the cell pellet and a predefined cell type constant.

The present invention thereby relates to an image based method for the determination of an amount of cells in a cell pellet. The cell pellet may be formed by pelletizing and in particular by centrifugation of a liquid cell culture. The cell pellet may be arranged in a multi-well plate as container.

The method is based on the surprising finding that a contour of the cell pellet is sufficient information to calculate an amount of cells in the cell pellet and thereby in the liquid culture of cells forming the cell pellet. To determine the contour of a given cell pellet, an image taken e.g. from the bottom of a container and thereby of the cell pellet may be acquired using an imaging device. The contour of the cell pellet may be approximated as a circle. After detection of the cell pellet, a radial dimension as e.g. a radius or a diameter of the cell pellet can be determined. The radial dimension may refer to the largest cross section of the cell pellet, i.a. a base circle of a ball segment. For example, the cell pellet radius can be used to calculate a cell number in the cell pellet and thereby in the liquid culture of cells forming the cell pellet. This calculation depends on a predefined cell type constant as e.g. a density constant of the used liquid cell culture. A height of the cell pellet needs not to be known for the calculation of the cell number.

This method allows an improved determination of an amount of cells in a cell pellet comprising a liquid cell culture and in particular, a very economic method for determining an amount of cells in a liquid culture of cells. The method according to the invention may be an in particular fast, easy and safe method for a determination of cell numbers in liquid cell cultures. Further, this method may be an automated, non-destructive and non-invasive method for a determination of an amount of cells in a liquid cell culture. In other words, the method requires no consumption or destruction of the cell culture.

Only a single and simple image may be required to determine the cell number of the cell pellet without any manual processing, i.a. without sampling of the culture and subsequent turbidimetry measurement. This image based method can be used for multi-array cell culture containers, e.g. multi deep-well plates, facilitating the instant and multiplexed calculation of the amount of cells in several cell cultures. As the present method is a non-invasive method, there is no danger of cross-contaminating samples in a multi-array format. The method according to the invention can be integrated into robot-assisted cloning as well as in cultivation and fermentation procedures and allows a streamlining and optimizing of these procedures in particular for high throughput (HT) formats.

Further, the method is operable in highly regulated environments as e.g. current good manufacturing practice and cGMP.

In an embodiment, the cell type constant is a density constant or a mass constant. In an embodiment, the cell type constant is defined using a calibration curve for the cell type of the cell pellet. This step may be introduced in the method before the cell amount calculation step. The cell type constant (density constant and mass constant) may be determined once for a specific cell type. In cases the cell type constant also depends on the pelletizing parameters and/or the culture medium, the cell type constant may be determined once for a specific cell type, the pelletizing parameters as e.g. centrifuge type and/or centrifugation speed and/or container type and/or the culture medium. In a preferred embodiment, the density constant may be determined by an optical density measurement, e.g. an OD600 measurement. In a specific embodiment, several cultures of a specific cell type, e.g., bacteria cells {Escherichia coli) in LB medium may be cultured at 37°C in a suitable incubator. In certain intervals, samples of the liquid culture may be taken and the OD600 value may be determined. In addition, the pellet size after centrifugation may be determined according to the present invention. The obtained values may be used to generate a calibration curve for the respective cell type and to determine the density constant. In a preferred embodiment, the mass constant may be determined by determining weight measurements. In a specific embodiment, several cultures of a specific cell type, e.g., bacteria cells (Escherichia coli) in LB medium may be cultured at 37°C in a suitable incubator. At certain intervals, the weight of the cell pellet after centrifugation (1200g) and discarding of the culture medium may be determined. The obtained values may be used to generate a calibration curve for the respective cell type and to determine the mass constant. In an embodiment, the method further comprises the step of calculating a weight of cells in the cell pellet based on the determined amount of cells in the cell pellet and the mass constant.

In an embodiment, the method further comprises the step of calculating a density of cells in the cell pellet. This calculation may be based on the determined amount of cells in the cell pellet and a volume of the cell pellet. The volume of the cell pellet may be calculated based on the determined radial dimension of the cell pellet and a geometry of a bottom of the container that has been used for pelletizing the cell culture (e.g., round bottom containers, conical bottom containers).

In an embodiment, the container is a single tube or a multi deep well plate. The container may be any container suitable for holding the respective cell type. In an embodiment, the same container is used for cultivating, pelletizing and/or imaging. For the subsequent analysis of the cell pellet, it is preferred to already culture the cells in culturing containers suitable for pelletizing (centrifugation). Alternatively, the cell cultures may be transferred into containers suitable for centrifugation for the pellet analysis. Particularly preferred are containers suitable for high-throughput culturing methods, e.g. multi deep-well plates, e.g. 96 well deep well plates. In particular, it is preferred to use containers suitable for centrifugation that have a round bottom or a conical bottom because the bottom geometries promote the formation of circular pellets during centrifugation. Moreover, round bottom and conical bottom geometries promote the formation of cell pellets positioned in the center of the bottom of the respective container. In an embodiment, the container has a round bottom which may promote cell pellet formation in shape of a spherical cap, so that the calculation of the volume of the cell pellet is based on a hemispherical geometry of the container bottom. In other words, when containers with round bottoms are used for pelletizing, the bottom of each well may have the geometry of a hemisphere. During pelletizing (centrifugation), the cell pellet may be homogeneously formed in this hemisphere with the shape of a spherical cap. A height of the cell pellet then follows by the laws of geometry. With the formula for spherical caps, the volume of the cell pellet can be calculated. This volume, e.g. expressed in voxels, can be used to calculate the cell density of the cell culture.

In another embodiment, the container has a conic bottom which may promote cell pellet formation in shape of a conic cap, so that the calculation of the volume of the cell pellet is based on a cone geometry of the container bottom. In other words, when containers with conic bottoms are used for pelletizing, the bottom of each well has the geometry of a cone. During pelletizing (centrifugation), the cell pellet is homogeneously formed in this cone with the shape of a conic cap. The height of the cell pellet follows by the laws of geometry. With the formula for conic caps, the volume of the cell pellet can be calculated. This volume, e.g. expressed in voxels, can be used to calculate the cell density of the cell culture.

In embodiments in which containers are used that have other bottom geometries, the calculation of the cell pellet volume has to be adapted accordingly. In an embodiment, the method further comprises the step of pelletizing the cell culture to obtain the discrete cell pellet described above. This step may be introduced in the method before the imaging step. Any suitable procedure for pelletizing the liquid cell culture can be used. In an embodiment, the pelletizing is a centrifuging and in particular a centrifuging by means of a swing-out rotor centrifuge. This provides the advantage of a formation of the cell pellet in a center of the container. The conditions for centrifugation as e.g. centrifugation speed, centrifugal force, centrifuge type etc. may be chosen based on e.g. the container type (single tube, multi-well plate) and/or the cell type (bacteria, yeast, other eukaryotic cells) and/or downstream applications (e.g., extraction of nucleic acids/proteins or further cultivation).

In an embodiment, the centrifuging is done by means of a swing-out rotor centrifuge in a range of 800 to 8000g for about 5 to 15 minutes to form the cell pellet in a center of the container. In another embodiment, the centrifuging is done by means of a swing-out rotor centrifuge in a range of 100 to 300g for about 5 to 15 minutes to form the cell pellet in a centre of the container. In embodiments where the pelletized cells are not used for extraction methods (e.g., nucleic acid extraction, protein extraction), that is where cells after pelletizing have to be viable, a gentle

centrifugation conditions may be chosen.

In an embodiment, the image is taken from the top or the bottom of the container. After pelletizing (centrifugation) and optionally discarding the growth medium, an image of the cell pellets formed at the bottom of the container is generated. Any suitable imaging device may be used to acquire an image or multiple images (e.g. an image stack). In an embodiment, a single image is taken from the bottom of the container to acquire an image of the cell pellet or the cell pellet array. In an embodiment, the cell pellet is identified in the image by means of image segmentation. In other words, the captured images may be processed or analyzed to detect or identify cell pellets. Any image processing method suitable for image segmentation can be used to identify or segment the cell pellets. For example, thresholding based segmentation, clustering based segmentation, compression-based segmentation, histogram-based segmentation, or edge-detection based segmentation may be used. In an embodiment, segmentation of the pellets is based on thresholding of the acquired image. In an embodiment, a mask is projected onto the image to define margins of the container. The mask can be used and projected onto the segmented image to define margins of the container (e.g., wells in a multi-well plate). In an embodiment, where the container is a multi-well deep-well plate, e.g. a 6 well deep-well plate, a 12 well deep-well plate, a 24 well deep-well plate or a 96 well deep-well plate, a mask comprising 6-, 12-, 24-, or 96 wells can be used to determine the margins of each individual well. Such a virtual mask may be adapted to the respective container type used for cultivation and/or pelletizing (centrifugation) of the cells. In an embodiment, the cell pellet is verified before the cell pellet volume is calculated. For example, it is desired that the detected cell pellets are positioned in the center of the container, that the detected pellets are not smaller than a defined minimum size (to exclude false positive pellets), that the detected pellets are not larger than a defined maximum size, and/or that the shape of the pellet meets a circularity requirement.

In an embodiment, the cell pellets are verified based on a distance between a center of the cell pellet and a center of the container. In other words, the distance of the cell pellet center and the distance of a mask center (e.g., center of the container) may be compared, wherein the determined distance should be smaller than a predefined maximum value. Then, only detected pellets that are positioned in the center of the mask are considered for further calculations.

In an embodiment, the cell pellets are verified based on a circularity of the cell pellet. Then, detected pellets that are non-circular are not considered for further

calculations. In an embodiment, the cell pellets are verified based on a minimum size of the cell pellet. Then, detected pellets that are smaller than a predefined minimum value are not considered for further calculations. In an embodiment, the cell pellets are verified based on a maximum size of the cell pellet. Then, detected pellets that are larger than a predefined maximum value are not considered for further calculations.

In an embodiment, the cell culture is a free floating cell culture in a liquid growth medium, i.e. the cells do not adhere to the tissue culture container. The cell culture may comprise microorganism and/or cells. The cell culture may be at least one of a group of a yeast cell culture, a bacterial cell culture (e.g., Escherichia coli), a eukaryotic cell, and a non-adherent suspension cell culture (e.g., hematopoietic cells).

The liquid growth medium is adapted to the method, in which the device or system for analysing a cell pellet is used. This e.g. relates to medium suitable for the growth of the specific microorganisms or cells used e.g. in high throughput cloning or suitable for the growth of the microorganisms or cells to be detected.

In an embodiment, the method further comprises the step of cultivating the cell culture in the container. In an embodiment, the method further comprises the step of discarding the growth medium of the cell culture. The discarding may be done after pelletizing (centrifugation). In embodiments where the cells after pelletizing are further cultivated, the growth medium may not be discarded and/or fresh growth medium may be added.

In a second aspect, the present invention is directed to a device for analyzing a cell pellet. The device comprises a provision unit and a processing unit.

The provision unit is configured to provide an image comprising the cell pellet. The processing unit is configured to determine a radial dimension of the cell pellet based on the image. The processing unit is further configured to calculate an amount of cells in the cell pellet based on the determined radial dimension of the cell pellet and a predefined cell type constant.

In an embodiment, the container is a single tube or a multi deep well plate. The container may be any container suitable for holding the respective cell type. In an embodiment, the same container is used for cultivating, pelletizing and/or imaging. For the subsequent analysis of the cell pellet, it is preferred to already culture the cells in culturing containers suitable for pelletizing (centrifugation). Alternatively, the cell cultures may be transferred into containers suitable for centrifugation for the pellet analysis. Particularly preferred are containers suitable for high-throughput culturing methods, e.g. multi deep-well plates, e.g. 96 well deep well plates. In particular, it is preferred to use containers suitable for centrifugation that have a round bottom or a conical bottom because the bottom geometries promote the formation of circular pellets during centrifugation. Moreover, round bottom and conical bottom geometries promote the formation of cell pellets positioned in the center of the bottom of the respective container.

In a third aspect, the present invention is directed to a system for analyzing a cell pellet. The system comprises a device for acquiring an image comprising the cell pellet and the device for analyzing the cell pellet as described above. The device for analyzing the cell pellet as described above comprises a provision unit and a processing unit. The provision unit is configured to provide an image comprising the cell pellet, whereby the image is acquired by the device for acquiring an image. In a fourth aspect, the present invention is directed to a use of the device or system for analyzing a cell pellet in the method for analyzing a cell pellet of the present invention. In a fifth aspect, the present invention is directed to a use of the device or system for analyzing a cell pellet in a method for fermentation and cultivation.

In a sixth aspect, the present invention is directed to a use of the device or system for analyzing a cell pellet in a method for high throughput cloning.

In an embodiment, the method is an automated method. The term "automated" as used herein refers to a situation where it is not necessary to carry out steps of the method by hands, i.e. manually. To this aim, in particular a suitable device (such as a robot) may be used.

It shall be understood that the method for analyzing a cell pellet, the device for analyzing a cell pellet, the system for analyzing a cell pellet and the uses described herein according to the independent claims have similar and/or identical preferred embodiments, in particular, as defined in the dependent claims. It shall be understood further that a preferred embodiment of the invention can also be any combination of the dependent claims with the respective independent claim.

These and other aspects of the present invention will become apparent from and be elucidated with reference to the embodiments described hereinafter.

Brief description of the drawings

The Figures shown in the following are merely illustrative and shall describe the present invention in a further way. These figures shall not be construed to limit the present invention thereto.

Figure 1 shows a flow chart of a method for analyzing a cell pellet. Figure 2 shows another embodiment of the method for analyzing a cell pellet.

Figure 3 shows a cell pellet detection and verification exemplified on four different scenarios.

Figure 4A shows an image of bacterial cell pellets after centrifugation in a 96 deep well culture plate container.

Figure 4B shows an image after applying an algorithm to identify and verify cell pellets.

Figure 5A shows a geometrical drawing of a spherical cap which is needed for a calculation of a cell pellet volume when using round-bottom containers.

Figure 5B shows a geometrical drawing of a spherical cone when using cone- bottom containers.

Figure 6 shows a graph of a comparative analysis of calculated normalized pellet area and spherical cap volume using a pellet quantifier and a corresponding optical density experimentally determined by an

OD600 measurement.

Figure 7 shows a system and device for analyzing a cell pellet. Definitions

For the sake of clarity and readability the following definitions are provided. Any technical feature mentioned for these definitions may be read on each and every embodiment of the invention. Additional definitions and explanations may be specifically provided in the context of these embodiments.

As used in the specification and the claims, the singular forms of "a" and "an" also include the corresponding plurals unless the context clearly dictates otherwise. It needs to be understood that the term "comprising" is not limiting. For the purposes of the present invention, the term "consisting of is considered to be a preferred embodiment of the term "comprising of. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also meant to encompass a group which preferably consists of these embodiments only.

The term "microorganisms" as used herein refers to microorganisms capable of forming colonies on solid medium, in particular to bacteria, fungi (e.g. yeasts) and single-celled eukaryotes. Most preferred microorganisms are selected from the group consisting of bacteria (e.g. E. coli), fungi (e.g. S. cerevisiae) and protists (e.g. xenic strains of Acanthamoeba).

The term "cells" as used herein in particular refers to eukaryotic cells, preferably human cells, mouse cells, monkey cells or insect cells. Said eukaryotic cells may also be stem cells and in particular undifferentiated stem cells. Further, said eukaryotic cells may also be cancer cells.

The medium used depends on the method carried out using the device or system according to the invention. For high throughput cloning, it is in particular preferred to use microorganisms which are routinely used in laboratories for carrying out standard procedures. The media suitable for growth of the following microorganisms or cells is known to the skilled person from text books and such media may be used in the context of the method, device or system according to the invention. Protists as microorganisms: A protist is generally selected from the group consisting of the Amoebozoa (e.g. Tubulinae, Flabellinea, Stereomyxida, Acanthamoebidae, Entamoebida, Mastigamoebidae or Eumycetozoa), Archaeplastida (e.g.

Glaucophyta, Rhodophyceae or Chloroplastida), Chromalveolata (e.g.

Cryptophyceae, Haptophyta or Stramenopiles), Exavata (e.g. Fornicata, Parabasalia, Preaxostyla, Jakobida, Heterolobosea or Euglenozod), Rhizaria (e.g. Cercozoa, Haplospodidia, Foraminifera or Radio laria), and Opisthokonta (e.g. Mesomycetoza, Choanomonada ox Metazoa). It can be preferred that said protists are selected from the group consisting of Chlorella; Chlamydomonas; Dunaliella;

Haematococcus; Chorogonium; Scenedesmus; Euglena; xenic strains of

Acanthamoeba, Naegleria, Hartmannella and Willaertia; and xenic strains of Vannella, Flabellula, Korotnevella, Paramoeba, Neoparamoeba, Platyamoeba and Vexillifera. Microorganisms: Preferred bacteria are Escherichia coli, Corynebacterium (e.g. Corynebacterium glutamicum), Pseudomonas fluorescens, and Streptomyces (e.g. Streptomyces lividans). Most preferred can be Escherichia coli strains XL 1 -Blue, XLIO-Gold, DH10B, DH5a, SURE, Stbll-4, TOP10 and Machl . Alternatively, said microorganisms are fungi, particularly yeasts, wherein Arxula adeninivorans (Blastobotrys adeninivorans), Yarrowia lipolytica, Candida boidinii, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenula polymorpha (Pichia angusta), Pichia pastoris, Aspergillus (e.g. Aspergillus oryzae), Trichoderma (e.g.

Trichoderma reesei), and Myceliophthora thermophila are particularly preferred. Depending on the purpose, it can be most preferred to use Saccharomyces cerevisiae strains optimized for the analysis of interactions, such as e.g. yeast-two-hybrid- analyses.

The media may be media suitable for the growth of microorganisms. Exemplary microorganisms are listed in the following, and the skilled person is aware of corresponding media for growth to be used in the method, device or system according to the invention.

Bacteria as microorganisms: Bacillus (e.g. Bacillus cereus); Bartonella (e.g.

Bartonella henselae, Bartonella quintana); Bordetella (e.g. Bordetella pertussis); Borrelia (e.g. Borrelia burgdoferri, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis); Brucella (e.g. Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis); Campylobacter (e.g. Campylobacter jejuni); Chlamydia and

Chlamydophila (e.g. Chlamydia pneumoniae, Chlamydia trachomatis,

Chlamydophila psittaci); Clostridium (e.g. Chlostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostdridium tetani); Corynebacterium (e.g.

Corynebacterium diphtheriae); Enterococcus (e.g. Enterococcus faecalis,

Enterococcus faecium); Escherichia (e.g. Escherichia coli); Francisella (e.g.

Francisella tularensis); Haemophilus (e.g. Haemophilus influenzae); Heliobacter (e.g. Heliobacter pylori); Legionella (e.g. Legionella pneumophila); Leptospira (e.g. Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii); Listeria (e.g. Listeria monocytogenes); Mycobacterium (e.g.

Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans); Mycoplasma (e.g. Mycoplasma pneumoniae); Neisseria (e.g. Neisseria gonorrhoeae, Neisseria meningitidis); Pseudomonas (Pseudomonas areuginosa); Rickettsia (Rickettsia rickettsii); Salmonella (Salmonella typhi, Salmonella typhimurium); Shigella (e.g. Shigella sonnei); Staphylococcus (e.g. Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus); Streptococcus (e.g. Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes); Treponema (e.g. Treponema pallidum); Ureaplasma (e.g. Ureaplasma urealyticum); Vibrio (e.g. Vibrio cholerae); Yersinia (e.g. Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis).

Examples of suitable growth media used for the liquid culturing of bacteria comprise nutrient broth (NB), nutrient agar (NA), Luria-Bertani broth (LB), LB Miller broth, LB Lennox broth, YT medium, 2x YT medium, Mueller-Hinton cation-adjusted broth (MH), a sporulation broth, eosin- methylene blue agar (EMB), yeast and mold (YM), blood agar, MacConkey agar, Hektoen enteric agar (HE), mannitol salt agar (MSA), Terrific Broth (TB), xylose lysine deoxycholate (XLD), SOC medium, SOB medium, RPMI-1640 media, molasses-based media (e.g., beet and cane molasses), minimal media, synthetic media, selective media, or any combinations or

modifications thereof. Fungi as microorganisms: Candida (e.g. Candida species); Aspergillus (e.g.

Aspergillus fumigatus, Aspergillus flavus, Aspergillus clavatus); Cryptococcus (e.g. Cryptococcus neoformans, Cryptococcus laurentii, Cryptococcus albidus,

Cryptococcus gattii); Histoplasma (e.g. Histoplasma capsulatum); Stachybotrys (e.g. Stachybotrys chartarum).

The detection of mold on a corresponding medium is also possible. A mold is a fungus that grows in the form of multicellular filaments called hyphae. In contrast, fungi that can adopt a single celled growth habit are called yeasts. Molds are a large and taxonomically diverse number of fungal species where the growth of hyphae results in discoloration and a fuzzy appearance, especially on food. The network of these tubular branching hyphae, called a mycelium, is considered a single organism. Further relevant fungi are: Acremonium, Dematiaceae, Phoma, Alternaria, Eurotium, Rhizopus, Aspergillus, Fusarium, Scopulariopsis, Aureobasidium, Monilia,

Stachybotrys, Botrytis, Mucor, Stemphylium, Chaetomium, Mycelia sterilia,

Trichoderma, Cladosporium, Neurospora, Ulocladium, Paecilomyces, Wallemia, and Curvularia Penicillium.

Yeasts are eukaryotic microorganisms classified as members of the fungus kingdom with 1,500 species currently identified and are estimated to constitute 1% of all described fungal species. Yeasts are unicellular, although some species may also develop multicellular characteristics by forming strings of connected budding cells known as pseudohyphae or false hyphae. Most yeasts reproduce asexually by mitosis, and many do so by the asymmetric division process known as budding. Yeasts do not form a single taxonomic or phylogenetic grouping. The term "yeast" is often taken as a synonym for Saccharomyces cerevisiae, but the phylogenetic diversity of yeasts is shown by their placement in two separate phyla: the

Ascomycota and the Basidiomycota. The budding yeasts ("true yeasts") are classified in the order Saccharomycetales. The species are: Arxula adeninivorans (Blastobotrys adeninivorans), Candida boidinii, Schizosaccharomyces pombe, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces uvarum, Candida utilis, Candida albicans, Saccharomyces boulardii, Brettanomyces bruxellensis, Hansenula polymorpha (Pichia angusta), Pichia pastoris, Kluyveromyces lactis, Yarrowia lipolytica, and Malassezia furfur.

Examples of suitable growth media used for the liquid culturing of yeast cells comprise YEHD medium, HJW medium, YPD Broth, YPD phosphate depleted, standard minimal medium (SD), YPG broth, YPAD broth, minimal media, synthetic media, selective media, or any combinations or modifications thereof.

Single-celled eukaryotes (may also be referred to as protozoans): Entamoeba histolytica; Plasmodium; Giardia lamblia; and Trypanosoma brucei.

Detailed Description of the findings underlying the present invention

Figure 1 shows a flow chart of a method for analyzing a cell pellet. The method comprises the following steps: SI) providing an image comprising the cell pellet,

52) determining a radial dimension of the cell pellet based on the image, and

53) calculating an amount of cells in the cell pellet based on the determined radial dimension of the cell pellet and a predefined cell type constant. Before step S 1 , liquid cell cultures may be cultivated in a multi-well container. The liquid cell cultures may comprise microorganisms and/or cells. Discrete cell pellets may be formed in each well of the multi-well container by pelletizing and in particular by centrifugation of the liquid cell culture. The liquid cell cultures and the cell pellets derived therefrom may be present in the same container, e.g. a multi-well plate, i.e. the liquid cell cultures are not transferred to another container before the cell pellets are prepared. An image of e.g. a bottom of the cell pellet may then be acquired and provided in step S 1. After detection of a cell pellet in the image by means of e.g. image segmentation, a radial dimension as e.g. a radius of the cell pellet is determined in step S2. The cell pellet is thereby approximated as a circle. The cell pellet radius is then used in step S3 to calculate an amount of cells or a cell number in the cell pellet and thereby in the liquid cell culture forming the cell pellet. This calculation depends on a predefined cell type constant as e.g. a measured density constant of the used liquid cell culture. The cell type constant may be determined by using a calibration curve for the specific cell type of the cell pellet.

This method for analyzing a cell pellet can be integrated into robot assisted cloning as well as in cultivation and fermentation procedures and allows a streamlining and optimizing of these procedures in particular for high throughput (HT) formats.

Figure 2 shows another embodiment of the method for analyzing a cell pellet, wherein the image is used to calculate a cell density of the cell culture. This calculation may be based on the determined amount of cells in the cell pellet and a volume of the cell pellet. The volume of the cell pellet may be calculated based on the determined radial dimension of the cell pellet and a geometry of a bottom of the container that has been used for pelletizing the cell culture (e.g., round bottom containers, conical bottom containers). As shown in Figure 2, the method for analyzing a cell pellet in view of the cell density of the cell culture comprises an image acquisition of cell pellets, a computer assisted detection and optional verification of a cell pellet, a determination of a contour and/or a radius of the cell pellet, a calculation of a volume of the cell pellet using e.g. a spherical cap or cone equation, and a conversion of the cell pellet volume into a cell culture density via a density constant.

Figure 3 shows a cell pellet detection and verification exemplified on four different scenarios. Schematics of a bottom of a container 1 after pelletizing (centrifugation) are shown in panels A to D. Panel A shows a cell pellet 2 which is usually located in a centre of the container 1. The centre of the pellet marked by a star is in close proximity to the centre of the container 1 marked by an x, which fulfils a centrality requirement. Other criteria such as circularity of the pellet are also fulfilled, leading to an automatic determination of a cell pellet radius marked by a dashed line around the cell pellet 2, and, depending on a geometry of container 1 bottom (round bottom, cone bottom, etc.), to a determination of a cell density (via a spherical cone equation or a spherical cap equation, respectively). Panel B illustrates a scenario where the obtained cell pellet 2 is not located in the centre of the container 1. As the prediction of the cell pellet volume depends on a geometry of the bottom of the container 1, such cell pellets cannot be used to reliably calculate the cell density and are automatically rejected. Panel C illustrates a scenario where a shape of the obtained pellet is not circular. As the prediction of the cell pellet volume depends on a radius of an approximately circular cell pellet 2, such cell pellets cannot be used to reliably calculate the cell density and are automatically rejected. Panel D illustrates a scenario where the cell culture is overgrown and a size of the obtained cell pellet 2 equals a size of a container 1 diameter. As the prediction of the cell pellet volume depends on a height of the cell pellet 2, calculated e.g. via spherical cone equation or spherical cap equation, such cell pellets cannot be used to calculate the cell density and are automatically rejected.

Figure 4A shows an image 3 of bacterial cell pellets 2 after centrifugation in a 96 deep well culture plate container 1. The image 3 of the cell pellets is taken to verify bacterial growth and to determine a cell density of the original cell culture. Here, a 3x8bit, RGB, 300dpi image 3 is taken from a bottom of the 96 deep well plate. The dashed square indicates a section of the well plate that was further analyzed as shown in Figure 4B.

In Figure 4B, an image 3 after applying an algorithm to identify and verify cell pellets 2 is shown. Such algorithm is e.g. described in Example 1. To outline container or well margins, a predefined mask is projected on the image 3. For each segmented cell pellet 2, a unique identifier was allocated. Such images 3 of the cell pellets 2 are used to determine a radius of the cell pellet 2 and to calculate a volume of the cell pellet 2 as described below.

Figure 5A shows a geometrical drawing of a spherical cap which is needed for a calculation of a cell pellet volume when using round-bottom containers or wells. When containers with round bottoms are used for pelletizing, the bottom of each well may have the geometry of a hemisphere. During pelletizing (centrifugation), the cell pellet may be homogeneously formed in this hemisphere with a shape of a spherical cap. A height of the cell pellet then follows by the laws of geometry and as shown in below Example. With the formula for spherical caps, the volume of the cell pellet can be calculated. This volume can be used to calculate the cell number and/or the cell density of the cell culture.

Figure 5B shows a geometrical drawing of a spherical cone when using cone-bottom containers or wells. When containers with conic bottoms are used for pelletizing, the bottom of each well has the geometry of a cone. During pelletizing (centrifugation), the cell pellet is homogeneously formed in this cone with the shape of a conic cap. The height of the cell pellet follows by the laws of geometry. With the formula for conic caps, the volume of the cell pellet can be calculated. This volume can be used to calculate the cell number and/or the cell density of the cell culture.

Figure 6 shows a graph of a comparative analysis of calculated normalized pellet area and spherical cap volume using a pellet quantifier and a corresponding optical density experimentally determined by an OD600 measurement. Figure 6 is further explained in the following Example

Figure 7 shows a system 5 and a device 10 for analyzing a cell pellet. The system 5 comprises the device 13 for acquiring an image comprising the cell pellet and the device 10 for analyzing the cell pellet. The device 13 for acquiring an image may be a plate reader (e.g., Micronics Scanner (Epson Perfection V33)), a digital camera (e.g., Axiocam (Zeiss), a monochrome USB cameras etc.

The device 10 for analyzing the cell pellet comprises a provision unit 11 and a processing unit 12. The provision unit 11 is configured to provide an image comprising the cell pellet, whereby the image is acquired by the device 13 for acquiring an image.

The processing unit 12 may be a processor or a computer and is configured to determine a radial dimension of the cell pellet based on the image. The processing unit 12 is further configured to calculate an amount of cells in the cell pellet based on the determined radial dimension of the cell pellet and a predefined cell type constant.

It has to be noted that embodiments of the invention are described with reference to different subject matters. In particular, some embodiments are described with reference to method type claims whereas other embodiments are described with reference to the device type claims. However, a person skilled in the art will gather from the above and the following description that, unless otherwise notified, in addition to any combination of features belonging to one type of subject matter also any combination between features relating to different subject matters is considered to be disclosed with this application. However, all features can be combined providing synergetic effects that are more than the simple summation of the features.

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. The invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing a claimed invention, from a study of the drawings, the disclosure, and the dependent claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are re-cited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

Example

The following Example is merely illustrative and shall describe the present invention in a further way. The Example shall not be construed to limit the present invention thereto.

Determination of bacterial pellet volume:

1. Preparation of 96 deep well culture plates with bacterial cell pellet For the present example, bacterial cells {Escherichia coli) were grown in 1500 μΐ LB-medium in a (square) 96 deep-well block with round well bottoms (Macherey- Nagel, REF: 740481.24) for 21 h, at 750 rounds per minute at 37°C. Then bacterial cells were centrifuged using a swing-out rotor (Hettich, model 420R, swing-out rotor S/N 005867) at lOOOg for 10 minutes and the supernatant was discarded. Obtained cell pellets of the 96 deep well culture plate were scanned with the Micronics Scanner (Epson Perfection V33) (see Figure 4A). The acquired image was used for the detection and analysis of cell pellets. 2. Detection of bacterial pellets per well via image analysis

To detect the cell pellets and to (optionally) preclude false positive or false negative results, an algorithm was developed. First, a black & white (b&w) image of the 96 deep well plate was taken using the Micronics Scanner. For the present 96 deep well plate, a pre-defined 96 well model (mask) to outline the margins of the different wells was projected onto the image. For each well, bacterial pellets were detected as follows:

1. Extract b&w image of well (=mask) and set mask (x, y) to white if Pixel (x; y) > MINIMUM INTENSITY OF PELLET PIXELS

2. Detect all connected components in mask

3. Sort detected components in decreasing order of their enclosed area

4. For each component C:

- IF (C) exists AND

- Dist(Center(C)), Center(W)) < MAXIMUM DISTANCE TO

WELL CENTER AND

- area(C) > MINIMUM PELLET AREA TO COUNT AND

- circularity(C) < MAXIMUM NON CIRCULARITY THEN

- register C as pellet for W

5. Generate control image result file (see Figure 4B) For the calculation, relevant parameters were defined as follows:

MINIMUM INTENSITY OF PELLET PIXELS = 50.0

MAXIMUM DISTANCE TO WELL CENTER = 27 pixels

MINIMUM PELLET ARE A TO C OUNT = 10 pixels

MAXIMUM NON CIRCULARITY = 25.0

Using the above described algorithm, cell pellets were segmented and also analyzed. If the cell pellet is positioned in the center of the well, and if the cell pellet has a circular shape, the pellet can reliably be used for further calculations.

3. Estimation of bacterial cell pellet volume per well

The volume of a bacterial pellet per well was calculated as follows:

1. Compute theoretical radius (a) of cycle given the pellet area.

a = max(R, (A/Pi)^A(l/2)), in which the radius is limited by R = 52 pixels, which correlates to the radius of a well (see Figure 4B)

2. Use the spherical cap formula (see Figure 5):

in which R = 52 pixels and h = R - (R^A2 - a^A2)^A(l/2)

3. Normalize with maximum cap volume (a = R).

4. Normalization of pellet area and spherical cap volume to OD600

To assess if the calculated pellet area as well as the spherical cap volume of a specific bacterial culture correlate to the corresponding optical density, OD600 measurements of the bacterial cultures were determined. As shown in Figure 6, there is indeed a strong correlation between the optical density (OD600) and the calculated pellet area and the spherical cap volume. This shows that with the development of the Pellet Quantifier it is possible to determine an amount of cells and/or a density of the cell culture in a nondestructive and quick way.

Claims

C L A I M S

A method for analyzing a cell pellet (2), comprising the steps of:

- providing an image (3) comprising the cell pellet (2),

determining a radial dimension of the cell pellet (2) based on the image

(3), and

calculating an amount of cells in the cell pellet (2) based on the determined radial dimension of the cell pellet (2) and a predefined cell type constant.

Method according to claim 1, further comprising the step of:

calculating a density of cells in the cell pellet (2) based on the determined amount of cells in the cell pellet (2) and a volume of the cell pellet (2) calculated based on the determined radial dimension of the cell pellet (2).

Method according to claim 1 or 2, wherein the container has a round bottom and the cell pellet (2) has a shape of a spherical cap, so that the calculation of the volume of the cell pellet (2) is based on a hemispherical geometry of the container bottom.

Method according to claim 1 or 2, wherein the container has a conic bottom and the cell pellet (2) has a shape of a conic cap, so that the calculation of the volume of the cell pellet (2) is based on a cone geometry of the container bottom.

Method according to one of the preceding claims, wherein the cell type constant is defined using a calibration curve for the cell type of the cell pellet (2)·

6. Method according to the preceding claim, wherein the cell type constant is a density constant or a mass constant.

Method according to the preceding claim, further comprising the step of: calculating a weight of cells in the cell pellet (2) based on the determined amount of cells in the cell pellet (2) and the mass constant.

Method according to one of the preceding claims, further comprising the step of:

pelletizing a cell culture to obtain the cell pellet (2) before providing the image (3) comprising the cell pellet (2).

Method according to claim 8, wherein the pelletizing is configured to obtain the cell pellet (2) essentially in a center of a container (1).

Method according to claim 8 or 9, wherein the pelletizing is a centrifuging.

Method according to the preceding claim, wherein the centrifuging is done by means of a swing-out rotor centrifuge.

Method according to one of the preceding claims, wherein the image (3) is taken from the bottom of the container.

Method according to one of the preceding claims, wherein the cell pellet (2) is identified in the image (3) by means of image segmentation.

Method according to one of the preceding claims, wherein a mask is projected onto the image (3) to define margins of the container.

15. Method according to one of the preceding claims, wherein the cell pellet (2) is verified based on a distance between a center of the cell pellet (2) and a center of the container. 16. Method according to one of the preceding claims, wherein the cell pellet (2) is verified based on a circularity of the cell pellet (2).

17. Method according to one of the preceding claims, wherein the cell pellet (2) is verified based on a minimum size of the cell pellet (2).

18. Method according to one of the preceding claims, wherein the cell pellet (2) is verified based on a maximum size of the cell pellet (2).

19. Method according to one of the preceding claims, wherein the cell culture is a free floating cell culture in a liquid growth medium.

20. Method according to one of the preceding claims, wherein the cell culture comprises microorganisms or cells. 21. Method according to one of the preceding claims, wherein the cell culture is at least one of a group of a yeast cell culture, a bacterial cell culture, and an eukaryotic cell.

22. Method according to one of the preceding claims, wherein the cell culture is a non-adherent suspension cell culture.

23. Method according to one of the preceding claims, further comprising the step of:

cultivating the cell culture in the container.

24. Method according to one of the preceding claims, further comprising the step of:

discarding a growth medium of the cell culture. 25. Method according to one of the preceding claims, wherein the method

requires no consumption or destruction of the cell culture.

26. A device (10) for analyzing a cell pellet (2), comprising:

- a provision unit (11), and

- a processing unit (12),

wherein the provision unit (1 1) is configured to provide an image (3) comprising the cell pellet (2),

wherein the processing unit (12) is configured to determine a radial dimension of the cell pellet (2) based on the image (3), and

wherein the processing unit (12) is further configured to calculate an amount of cells in the cell pellet (2) based on the determined radial dimension of the cell pellet (2) and a predefined cell type constant.

27. Device (10) according to the preceding claim, wherein the container is a single tube or a multi deep well plate.

28. Device (10) according to claim 26 or 27, wherein the same container is used for cultivating, pelletizing and/or imaging. 29. A system (5) for analyzing a cell pellet (2), comprising:

- a device (13) for acquiring an image (3) comprising the cell pellet (2), and

- a device (10) for analyzing the cell pellet (2) according to one of the preceding claims,

wherein the device (10) for analyzing the cell pellet (2) comprises a provision unit (11) configured to provide an image (3) acquired by the device (13) for acquiring an image (3).

30. Use of a device (10) or a system according to one of the claims 26 to 29 in a method according to one of the claims 1 to 25.

31. Use of a device (10) or a system according to one of the claims 26 to 29 in a method for fermentation and cultivation. 32. Use of a device (10) or a system according to one of the claims 26 to 29 in a method for high throughput cloning.

33. Use according to one of claims 30 to 32, wherein the method is automated.