WO2018234257A1 - Method for verifying the success of single cell seeding in nanowell arrays, and device and nanowell array - Google Patents

Abstract

The invention relates to a method for verifying the success of single cell seeding in nanowell arrays (9), comprising the steps: - illumination of a nanowell array (9) seeded with individual biological cells (2), the nanowell array (9) having a number of nanowells (17) in which a maximum of one cell (2) should be contained after the seeding process, by means of at least one illumination- or stimulation beam and spatially-resolved recording, as image data, of a detection beam, caused by the illumination- and/or stimulation beam, - evaluation of the image data with regard to the presence of exactly one cell (2) in each nanowell (17), - evaluation of the image data with regard to the presence of cells (2) in spaces (18) between the nanowells (17), and - storage of the positions of those nanowells (17) in which there is not exactly one cell (2) and those spaces (18) in which cells (2) are present. The invention also relates to a device for carrying out the method and to nanowell arrays (9).

Description

 Method for the verification of the success of single cell seeds in nanowellarravs as well as

Device and Nanowellarrav

The invention relates to a method and apparatus for verifying the success of single cell seeds in nanowellarravs and nanowellarravs.

Many (recombinant) proteins, such as pharmaceutical agents, are produced in eukaryotic cells. In contrast to prokaryotic expression systems are eukaryotic expression systems, such as yeasts, insect cells and

Mammalian cells, i.a. by their ability to make post-translational modifications that are crucial for the biological activity of a variety of proteins.

The first step in the production of (recombinant) proteins on an industrial scale is the generation of stable production cell lines. Common production cells are hybridoma cells (production of monoclonal antibodies) or, for the production of recombinant proteins, genetically modified cells, such as Chinese hamster ovary (CHO) or human embryonic kidney cells (HEK-293 or HEK-293T). Hybridoma cells are fusions of plasma cells and immortalized myeloma cells that produce antibodies specific to the particular plasma cell. The plasma cells are obtained from the spleen of animals infected with an antigen of

Interest were immunized. Following the fusion of plasma cells and myeloma cells, a heterogeneous population of hybridoma cells are obtained which produce antibodies with the desired specificity (against the antigen used for immunization) or other specificity. In addition, the cells differ in their productivity.

For the production of recombinant proteins, a transgene encoding the protein of interest is introduced into suitable cells, eg CHO or 293T (eg by transfection). Integration of the transgene occurs randomly into the genome of the host cell in conventional methods. The site of integration by neighboring endogenous regulatory elements (enhancer, silencer, chromatin structure, etc.) strongly influences the promoter activity and thus the transcription rate of the transgene. In addition, the copy number of the transgene in a cell (number of integrates after transfection or gene amplification) is crucial for its expression level. After transfection, therefore, one obtains a pool of cells of different specific productivity, ie cells which show an individually specific production of the desired product. Was the Transfection unsuccessful or was done so-called gene silencing, none of the desired products are produced and the specific productivity is zero.

In order to achieve the maximum yield in the biotechnological production of the (recombinant) protein, it is essential to select from the pool of hybridoma cells or transgenic cells those cells with high specific productivity as starting material for the generation of a cell line.

Often there is a requirement that the producer cells must be a cell clone, i. cells that have been produced by cell division from a single parent cell and are genetically identical. The cell clone of interest should also be characterized by a high proliferation capacity and the stability of (recombinant) protein expression. According to the current state of the art, the following three methods are mainly used to generate new monoclonal cell lines:

 1) single-cell cloning using limiting dilution,

 2) single-cell cloning in semi-solid medium and

 3) Single cell cloning based on flow cytometry.

Limiting dilution

In this method, a suspension of hybridoma cells, genetically engineered cells (usually after selection for antibiotic resistance) or naturally a protein of interest producing cells is seeded at various dilution levels. The starting point for cell line production is the so-called limiting dilution, in which statistically a single or no cell is present per well of a 96- or 364-well plate. After transfection of the cells in the usual laboratory scale, there are approximately 10 ⁷ cells for distribution. From this it becomes clear which large number of well plates have to be charged, cultivated and evaluated. The individual cells are then allowed to grow under appropriate culture conditions cell clones, which are then characterized in terms of their (specific) productivity, growth rate and stability. The characterization can be carried out for example by an analysis of the cell culture supernatant by means of ELISA. Limiting dilution is very time-consuming and labor-intensive. In addition, a high cost of materials, such as cell culture plates and others

Consumables, media, etc. required. In order for cell line development, how commonly proposed to screen, ie identify and analyze, at least 1000 (genetically modified) single cells or the resulting cell clones, at the usual sowing density of 0.5 cell / well in a first step, for example at least 21 plates with 96 wells or six 384-well plates are filled. Often, a sowing density of 0.1 cell / well is chosen, which increases the number of cell culture plates required accordingly. In addition, sowing usually takes place in parallel in several dilutions. All seeded cells are cultured and propagated in the sequence, since no characterization and selection takes place at the single cell level. Without additional and comprehensible documented microscopic monitoring of cell proliferation from the time of sowing, usually two rounds of limiting dilution must be carried out, for example, to meet the regulatory requirements

To comply with the authorization procedure. This is necessary because individual wells may contain more than one parent cell after seeding the cell suspension and serves to exclude resulting non-monoclonal cell lines.

Single cell cloning in semi-solid medium

 In this approach, cells in semi-solid medium, such as

Methylcellulose, sown. The cells are immobilized in the viscous medium and can grow into colonies, which can then be isolated. However, since a colony may have been formed from a single cell or may be due to two or more closely spaced cells, this method lacks unambiguous evidence of monoclonality. In addition, visualization of single cells on the day of sowing are difficult because, for example, not all cells in the focal plane of a

Success control of sowing used optical device lie.

However, the specific productivity of the producer cells can be easily characterized by suitable immunodiffusion tests. These are due to the precipitation of the product, which diffuses around the producer cell clone into the semi-solid medium. Characterization is possible, for example, using fluorochrome-conjugated antibody. The size of a precipitate ring forming around the producer cell correlates with the amount of secreted product and thus permits one

Evaluation of the productivity of a cell clone considering the size of the cell clone.

Single cell cloning based on flow cytometry

In this method for single-cell cloning, individual cells are selectively isolated from a cell suspension using a sorter and then deposited separately in wells of a target plate. In this case, a single cell is introduced into each well, for example, a 96- or 384-well plate. The target cells are determined by their fluorescence on the Expression of reporter genes or staining of surface antigens with fluorochrome-conjugated antibodies may be differentiated from other cells. In the wells of the target plates, the individual cells then grow up to cell clones. An advantage of this method is the high throughput that can be achieved in the isolation of producer cells by the sorter. Up to 5 x 10 ⁷ output cells can be sorted in 30 min. In addition, a fluorescence-based evaluation of the productivity of the cell and the targeted isolation of cells with desired properties are possible. However, since the method is not as gentle as the previously described alternatives, the viability of isolated cells may be impaired. In addition, cell sorting is not done under sterile conditions, so the use of antibiotics is essential to reduce the risk of contamination. Critical to the success of the process are also the settings made by the sorter used. The settings considered optimal (eg gating, compensation, etc.) are sample-specific and are based on a part of the present sample, for example the cell suspension. The cells in it, possibly also the target cells, are discarded, so that they are not available for isolation or subsequent steps. But even after the initial definition of suitable parameters, cells are irretrievably lost. Loss of possible target cells throughout the sorting process is reported as up to 50%. Another difficulty is to ensure that truly single cells and no aggregates are isolated from two or more cells or multiple single cells in a drop. Proof of successful

Single cell deposition can only be achieved by microscopic examination of the target plates. Without this combination of single cell cloning by flow cytometry with microscopy, no monoclonal detection is possible.

All three standard methods have in common that the monoclonality of the respective cell line can not be clearly established. In addition, the time and material cost of producing monoclonal cell lines is high.

The object of the invention is to propose a method which is improved over the prior art and which is suitable for the production of monoclonal cell lines. In addition, to be proposed with the invention, a device with which the method is executable.

The object is achieved with regard to the method by the subject matter of claim 1 and with respect to the device by the features of claim 12. One in the

Method usable and usable with the device nanowell array is the subject Claim 16. Advantageous developments are the subject of the independent and dependent claims.

The method according to the invention makes it possible to verify the success of

Single cell seeds in nanowell arrays and comprises several steps.

 In one step, a nanowell array seeded with individual biological cells is illuminated, wherein the nanowell array has a number of nanowells in which, immediately after sowing, if appropriate afterward

Washing steps, each containing at most one biological cell. The

Nanowell array is illuminated with at least one illumination and / or excitation radiation. One caused by the illumination and / or excitation radiation

Detection radiation is spatially resolved (2D) recorded as image data. It serves a

Illumination radiation of the illumination of the nanowell array, without changing the cells or their components. An excitation radiation is used to change excitable molecules and / or mixtures and, for example, to emit a

 To excite fluorescence radiation. A detection radiation can be given by reflected portions of the illumination radiation and / or the excitation radiation as well as by radiation which is emitted on the basis of the excitation processes. It is also possible to detect the detection radiation through the sample (transmitted light method).

For the purposes of the description, wells for receiving samples are referred to as nanowells if their volume is smaller than 1 mm ³ (1 μΙ). Wells for receiving samples, such as those formed on 384-well microtiter plates (about 40 μΙ volume per well) or 1536 (about 10 μΙ volume per well), are referred to as wells or macrowells.

Preceded by the method according to the invention may be the steps of sowing individual cells on the nanowell array and rinsing the nanowell array. The latter step serves to remove cells outside the nanowells and / or excess cells in the nanowells.

In one step, so-called production cells as single cells in the

Seeded nanowell array. For example, the production cells may be

Hybridoma cells, by transfection, transduction or transformation genetically modified cells or naturally a protein of interest-expressing cells. The cells are present for sowing in suspension or, in the case of adherent cells, prior to sowing mechanically, enzymatically or with the aid of corresponding buffer detached and brought into suspension. Separation of the cells can also be achieved using sieve-like devices such as nylon nets. All available cells are seeded in the nanowell array. In further embodiments of the method, production cells can also be seeded on several nanowell arrays simultaneously. In this case, a plurality of nanowell arrays can be arranged and seeded and / or the cells washed away by one of the nanowell arrays are seeded on a different nanowell array. The seeding of the cells into the nanowell array can be done manually or with a device and include tilting and pivoting movements, centrifugation steps and washing steps. The device according to the invention described below can be designed for sowing cells on a nanowell array and, for example, via a

Have a pipetting unit and communicate with a cell strainer.

In the respective nanowells, the presence of at least one cell is expected after sowing, so that one can also speak of an expected range in each case. The gaps between the nanowells, which are given for example by webs between the nanowells or by an edge of the nanowell array, do not provide any

Expected areas. In practice, often results in a distribution of the nanowell with a cell, with multiple cells and with empty cells, which by means of a

Probability distribution, such as a Gaussian, can be described. The expected number of one cell per nanowell corresponds to an ideal sowing. If nanowells with more than one cell and / or empty nanowells are present, an expected or desired occupancy of the nanowells with one cell each can subsequently be brought about by means of the correction steps outlined below.

To evaluate which of the nanowells contains only a single cell, the loaded nanowell array is illuminated. The distribution of the cells is detected and analyzed, for example, by means of bright field illumination and, if appropriate, by means of fluorescence images. In the bright field, the nanowells themselves as well as single cells in the nanowells are detected. The image data obtained in this way are compared with respect to the position of the nanowell as well as the

Existence of exactly one cell in the respective nanowells evaluated. In addition, the image data is evaluated for the presence of cells in interstices between the nanowells. Should cells be in the interstices between the nanowells, they will also be detected in the step and adjacent nanowells may be excluded for isolation to prevent the uptake of more than one cell. A spatially resolved detection of the detection radiation refers to a possible

Determination and specification of two-dimensional coordinates of a respective location from which outgoing detection radiation was detected. In particular, it is possible

to capture and indicate two-dimensional coordinates of a detection radiation emitting cell.

The positions of those nanowells in which there is not exactly one cell and those interstices in which cells are present are stored and given an identity (in the following also briefly: ID), for example in the form of a number, a coordinate pair and optionally further ID. Parameters such as the experiment number, name of the cells, laboratory number of the experiment / cell, etc. In addition, the positions of those nanowells can be stored in which exactly one cell and / or no cell is present.

In an advantageous embodiment of the method, nanowells without cells and / or nanowells having more than one cell and / or nanowells adjoining interstices in which cells have been detected are identified, their IDs are recorded and their positions are stored. The nanowells identified in this way are subsequently identified

Process steps excluded. As a result, it is advantageously achieved that neither empty nanowells nor nanowells with more than one cell are used for subsequent steps. Therefore, delivery movements of removal devices to empty nanowells are avoided or no nanowells are approached that certainly do not fulfill the requirements for monoclonality of their contents. With such a configuration, time, energy and consumables such as pipettes are saved. In a possible development of the method, nanowells, in which more than one cell is present after sowing, and / or cells in interspaces, a removal device, for. As a pipette delivered. By means of the removal device and a corresponding control of the same are previously by means of a

Evaluation unit (see below) excess cells, preferably individually, taken from the respective nanowells and / or interstices. With this procedure, incorrectly seeded nanowells and gaps can be detected and the respective sowing error corrected. The removed cells can be discarded. It is better, however, if the extracted cells can continue to be used. For this purpose, it is proposed that after sowing empty nanowells are identified and determined their position. Sampled cells are individually deposited in these hitherto empty nanowells.

The aforementioned embodiments of the method are by appropriate

Control protocols and image and / or video recordings comprehensibly documented and stored so that a subsequent verification of the executed process steps is possible and their respective successes can be clearly understood.

In order to obtain the advantages of the above method variants, it is advantageous if the stored positions of the nanowells without cells and / or nanowells having more than one cell and / or nanowells adjoining interstices in which cells have been detected are used to around depending on these positions

Generate control commands for subsequent process steps or adapt.

For example, the control commands for controlling the removal device are adjusted accordingly. In order to induce or support certain desired physiological responses of the cells, the nanowell array is advantageously maintained under culture conditions which allow the desired physiological responses of the cells. Such physiological reactions are, for example, the survival, growth and / or proliferation of the cells. In addition, or instead, for example, the expression and secretion of certain substances such as peptides, proteins, hormones,

 Growth factors and the like as desired physiological reactions are enabled and supported.

The cells lie after sowing and after a possible separation each alone in a nanowell. The nanowells with the cells can be covered with a medium, for example a nutrient medium, so that communication of the cells with one another is possible. Such communication can advantageously increase the proliferation rate and thus increase the efficiency of the method. The steps of the method may take place in a sterile environment adapted to the needs of the cells, for example by defining desired temperatures Gas contents such as a defined CO2 and / or 02 content and a certain humidity are regulated.

If the isolation of the cells is to be based on their specific productivity, the production of the protein of interest must be visualizable and be able to be assigned to the target cell. In the case of productive transgenic cells, the detection can u.a. based on the additional production of reporter proteins by the target cells. In this method, in addition to the sequence encoding the recombinant protein, the coding sequence of a reporter protein, which is detected later, was previously introduced into the cell. The expression of both genes must correlate.

Alternatively, the presence of the (secreted) protein is detected directly. In this case, surface proteins, for example. B and T cell receptors, directly via immunological

Be detected. In the case of secreted proteins, immobilization of the secreted (recombinant) protein occurs in close proximity to the producer cell (eg by coating the nanowell structure or via antigen-antibody or other protein-protein interactions).

It is possible that substances released from the cell and / or mixtures of substances in the well and / or next to the well are at least partially bound by means of binding partners specific for the substances and / or mixtures to be detected. Proteins can also have a tag that can be bound. The protein of interest can now be detected by immunological methods and results in a distinct, measurable signal (eg fluorescence, luminescence). Such immobilization of the released, for example, secreted, substances or substance mixtures in the spatial proximity of the respective cell makes it possible to evaluate the productivity of the relevant cell or a cell clone which has passed from the cell.

The detection of the specific productivity of single cells may be based on the detection of the (secreted) (recombinant) protein. Suitable for this purpose are, for example, fluorochrome-coupled antibodies such as fluorochrome-coupled detection antibodies or unlabeled detection antibodies and fluorochrome-coupled secondary antibodies. Furthermore, via enzyme-linked antibodies (enzyme-linked detection antibodies or unlabeled detection antibodies and enzyme-linked secondary antibodies); on the interaction of the protein of interest with other binding partners (eg the antigen for which a secreted antibody is specific) and their detection (fluorochrome-coupled Antibodies, enzyme-linked antibodies) as well as other detection cascades, for example biotinylated detection antibodies and enzyme-coupled avidin.

The signal intensity of the cell or of the cell clone or in the nanowell (fluorescence,

Chemiluminescence, a measurable color change upon enzymatic conversion

chromogenic substrates) is detected by an image capture device. The

 Signal intensity allows conclusions to be drawn on the amount of (recombinant) protein produced and is analyzed to distinguish high producers from producers and non-producers. Depending on the selected detection method, fluorescence images can be used

 Detection of target cells that produce the (recombinant) protein. Since the fluorescence signal in this case correlates with the amount of target protein produced, the device can be quantitated by fluorescence signal intensity

Recognize high producers. In the method according to the invention or one of its embodiments, detection and evaluation of fluorescence signals and quantification of the productivity of cells or cell clones can take place shortly after sowing. As a result, an early evaluation of the signals of the seeded cells or the developing cell clones is possible. In further steps of the method, selected cells or cell clones resulting from a single cell are automatically removed from the respective wells and further cultured individually, so that monoclonal cell lines are obtained later.

Such single cells or cell clones are advantageously selected based on their specific productivity, preferably automated, for the isolation process. Subsequently, the selected individual cells or cell clones are selectively isolated from the device and placed in a target vessel. The isolation process is advantageously documented by the recording of images before and after the harvest of the target cell and a harvest video. The target vessel may be, for example, another nanowell array or a conventionally used microtiter plate, for example a 96- or 384-well plate. It is advantageous if the target vessel was examined in advance to see whether it still contains no cells and its nanowells or wells are empty. Such a control can

for example, using an imaging method and documented. If this is not the case, then the wells are not demonstrably empty, the corresponding wells of the target vessel are excluded as target wells. In order to meet the high requirements for the detection of the monoclonality of cell lines, the physiological reactions can be documented, whereby growth and proliferation processes are recorded and measured, for example in the bright field. The cell density and the number and size of the cells or cell clones can be used as parameters. Production of substances by the cell or cell clone may be by labeled antibodies, in particular fluorochrome-labeled antibodies, enzyme-labeled antibodies, specific interactions of produced substances, nanobodies, single chain variable fragment antibodies (scFv), DARPin (Designed Ankyrin Repeat Protein) and / or metabolic processes of the respective cell are detected and evaluated.

The proliferation of the single cell to the cell clone is documented by the acquisition of images at regular intervals (eg daily), for example by an image capture unit. The cell clone is then analyzed by means of suitable analysis methods regarding its specific productivity, the stability of the (recombinant)

Characterized protein production and its growth rate.

The method advantageously allows a comprehensive documentation of all executed steps. So in an automated documentation step in the same

Sample container, in particular in the same nanowell array, image acquisition, detection of monoclonality, the detection of proliferation and production, based on the selection and isolation of single cells or cell clones from the nanowells of the nanowell array and the transfer of the detected and by proliferation and / or Production criteria selected cells in a target vessel. Likewise, the cell delivery into the target vessel and the proliferation of the single cell in the target vessel can be documented. The documentation can be done with the image capture unit or with an additional documentation unit, which is designed for image acquisition and storage of image data. The

Settings of the detection and harvesting device are automatically stored. Pictures of the nanowell array before and after picking (isolation) and the

Target vessel can be recorded before and after cell deposition.

In the case of secreted proteins, the immobilization of the (recombinant) protein in spatial proximity or at the producer cell is indispensable for the detection of the specific productivity of single cells. This can be done via:

the corrugated structure preventing pathway diffusion of the (recombinant) protein from the nanowell; - Overlapping the nanowells with semi-solid medium, which prevents pathway diffusing the (recombinant) protein from the nanowells;

 - coating the nanowells with interaction partners of the (recombinant) protein;

 - antigen or antibody;

- antibody binding proteins such as protein L, protein A, protein G;

 - antigen or antibody (including single-domain antibodies such as nanobodies);

other interaction partners:

 - diverse linkers between secreted protein and producer cell, possibly also after modification of the target cell;

artificial, antigen-binding proteins such as DARPins, scFv fragments, monobodies, affilins, anticalins, avimers, affibodies;

 Biotinylation of the cells and avidin-coupled antibodies to the (recombinant) protein;

 bispecific antibodies that bind an antigen on the cell surface and the secreted protein;

other linkers:

 inducible change between membrane-bound form of the protein and the secreted form, as described, for example, by the publications of Yu et al. (2014;

Antibody-membrane switch (AMS) technology for facile cell line development. Protein Eng Des Sei (10): 309-15) and Chuang et al. (2014; High-Throughput Sorting of the Highest Producing Cells via a Transiently Protein-Anchored System.PLS ONE 9 (7): e102569) is known.

The object is further achieved with the device already mentioned. This serves to verify the success of single cell seeds in nanowell arrays and is intended to be used in a method of generating monoclonal cell lines. The device comprises an image acquisition unit which is designed to acquire image data of a nanowell array arranged in a sample area. Furthermore, a lighting unit for providing at least one illumination and / or excitation radiation and means for controlled illumination of the sample with the at least one illumination and / or excitation radiation are part of the device. The

Image capture unit is configured such that on the nanowell array

Expected areas with an expected presence of each sample, in particular of each cell, can be defined or verified by being able to evaluate acquired image data of the respective expected areas by means of an evaluation unit of the device. This includes the automatic detection of nanowells as areas of expectation. In addition, the image capture unit is designed in such a way that gaps can be defined, detected or verified on the nanowell array in which no samples are expected. After illuminating the nanowell array, a spatially resolved detection of one caused by the illumination and / or excitation radiation

Detection radiation take place, for which purpose the image acquisition unit has a detector, in particular a two-dimensionally resolved detector, for example a matrix detector, a CCD or a CMOS camera.

The image acquisition unit is further configured to acquire captured image data regarding the presence of exactly one cell in the respective nanowell and with regard to the

 Existence of cells in interstices between the nanowells to evaluate and store. For this purpose, the device may have an evaluation unit, which is connected to the image acquisition unit in a manner suitable for the exchange of data. In addition, it is possible to store the positions of those nanowells in which there is not exactly one cell and those gaps in which cells are present.

In further exemplary embodiments, the image acquisition unit can be designed to identify a nanowell array with regard to its type and to determine its position and spatial orientation or to detect all nanowells independently of the array. Each well of a nanowell array can be assigned an ID by means of the image acquisition unit and / or the evaluation unit.

For this purpose, the image acquisition unit can be connected to a database in which data of customary nanowell arrays of various manufacturers and types are stored. On the basis of at least one image of a nanowell array arranged in the sample space, for example on a sample table, its type and manufacturer can be determined by comparing the image data captured by the nanowell array. At the same time, the relative

Positioning of the relevant nanowell array can be determined in the sample space. With the type of nanowell array and its relative positioning in the sample space, the positions of the expected ranges and the positions and, if appropriate, the courses of the gaps can also be determined. For example, according to known positions and data about nanowells without cell, with more than one cell, and nanowells adjacent to cells located in a space, control commands may be generated for a pipetting unit that will respectively harvest the selected cells in the selected nanowells. Alternatively, by means of an evaluation unit connected to the image acquisition unit based on image-analytical detection steps, the dimensions and positions of all existing nanowells independent of a database more common

Nanowell arrays are detected. In this case, the evaluation unit supplies a list of all recognized nanowells, including their size, position, outlines, edge regions, etc., so that no prior definition of expected ranges is necessary. Each nanowell is assigned a one-unambiguous ID, which enables subsequent assignment and retrieval. Based on the position and outlines of the detected nanowells can then be an assignment of the position of the detected cells and thus conclusions about e.g. the number of cells in the area of a nanowell take place.

In order to allow a documentation of all process steps and relevant events during the process, the device is advantageous for documenting the

Nanowellarrays and / or formed of selectable process steps and process stages. For this purpose, the device advantageously, for example, a camera, a

Storage unit and optionally additional documentation storage on. In addition, there may be a connection to an evaluation unit or computer unit.

The device can also have a unit by means of which selected cells can be transferred from an expected region, in particular a well, of the nanowell array into an area, for example a well, of a target vessel. Such a unit may be designed as a pipette unit.

By means of the device, in particular by means of the removal device, a previously determined monoclonal cell clone can be deposited separately in a delimited nanowell or well of a target vessel and further grow there.

Alternatively, by means of the device, a selected individual cell can also be deposited separately in a delimited nanowell or well of a target vessel and grow there to a defined cell clone. Since the device stored in this case only one cell in a nanowell or well, it may be only a cell clone in the resulting cell line in the nanowell or well.

It is also possible that the device interacts with other units. Such units may be modularly associated with the device and, for example, modules for

Liquid handling, picker, incubator etc. include, preferably in one

Clean room housing. The method according to the invention is advantageously carried out using a nanowell array. Such a nanowell array is advantageously also used during operation of the device.

The nanowell arrays can be present in a practically usable plate format, for example according to the SBS standard.

A nanowell in possible embodiments of nanowell arrays is only slightly larger than the target cell itself, thus ensuring that only a single cell, but not several cells, can fit in a nanowell. The mean diameters of the cells are determined as 95% intervals of the cell diameter measured by FACS or microscopy in a new-build chamber or by means of the Casy Cell Counter. For various large cell types, therefore, nanowell arrays with nanowell size matched to the cell size are used.

In addition to the geometry of the nanowell array are also cell number and the

Loading protocol, for example, including the washing steps to be performed, etc., tuned so that there is generally only one cell or no cell per nanowell. The geometry of the nanowell is designed such that the cell is also held in the nanowell during optional washing steps. The nanowell arrays may also have functionalized surfaces. For example, interaction partners for immobilization of secreted proteins or adhesion preventing

Coating be present in strongly adherent cells. The nanowell arrays may, for example, have a polyHEMA coating. Their effect prevents cells from adhering to the sidewalls of the nanowells and instead sinks into the nanowells and comes to lie approximately in the middle of the respective nanowell. In addition, the nanowells can be designed as round bottom wells.

In further embodiments, the wells may contain sieves on which the cell or cell clone is maintained by the action of a negative pressure. For example, the flow of a medium through one of the sieves creates a vacuum that can suck cells into the nanowell.

While nanowells tuned to the size of the production cells have advantages in ensuring only one cell per nanowell, nanowell arrays with nanowells of different sizes must be kept in stock when used in a laboratory

to work in different sized production cells. The invention therefore also encompasses nanowell arrays and the use of such nanowell arrays having nanowells with a diameter that allows for the uptake of multiple cells. The advantages are that it allows the cell to grow into small clones within the nanowell. In addition, only one type of nanowell array needs to be kept.

In order to increase the probability that only one cell per nanowell is present after sowing and the subsequent washing steps, in one possible embodiment of a nanowell array according to the invention at the bottom of each of the nanowells is one

Anchor structure for binding only one cell present.

Such nanowells may, for example, have a diameter of 50 μm or more. The anchor structure may be formed, for example, in the form of a surface structuring of the material forming the nanowell and / or a coating with anchor molecules. Anchor molecules may for example be suitable proteins, lipids,

Carbohydrates and / or polymers or mixtures thereof.

Such an anchor structure results in an improved binding of the cell to the nanowell and reduces the risk of the cell being erroneously removed in a washing step. In addition, the anchor structure is advantageously dimensioned such that only one cell can bind to it, for example can adhere. Other cells, despite the fact that they find room in the nanowell, do not bind to the material surface of the nanowell and are washed away. This effect is aided by a coating on the inside of the nanowell that prevents adhesion of cells to the inner wall of the nanowell.

Larger nanowells are used, possibly in conjunction with a

Coating which ensures that cells settle only in the center of the nanowells, this has a number of advantages. Here, as in previous methods by statistical

 Dilution of the cell suspension seeded and confirmed by image analysis and documented that only nanowells with only one cell for all further investigations and

Selection steps are observed. The background is the economically sensible production and use of larger wells. If the cells are in the middle of each well, the optical detection and analysis is also supported. In further possible embodiments of the nanowell array, at least one coating is additionally present inside and / or outside the wells in order to at least partially bind substances and / or mixtures released from the cell and / or a detection reaction for detecting the presence of the substance or mixture of substances and / or or to initiate the detection of a signal intensity of a detection radiation.

The bottom of the nanowell may be optically transparent (transparent) in order to inversely screen the content of the nanowell, for example to be able to scan it. It can the

Separation of the cells tested and the transport of individual cells are documented. The

Nanowell arrays may be, for example, plastic, glass, ceramic or silicon (chips), which may be formed by processes such as injection molding, etching, laser or

 Electron beam machining can be brought into the desired geometric shape.

 Optional coatings permit binding of the expected expressed compounds to the wall or bottom of the nanowell and their detection by means of imaging and image processing techniques.

Separating the individual cells by placing them in the nanowells ensures that the cells remain identifiable throughout the process. In cell culture, it is known that the growth and viability of cells are also largely determined by the direct cell contact to the neighboring cells and by communication of the cells via the surrounding media. Unlike previously

Although the microtiter plates used can not be used for direct cell contact due to the use of the described nanowells, they do, however, have a connection via a medium which overlays the nanowell, as has already been described above.

In order to enable unimpeded communication between the cells, for example via an over-coated medium, a nanowell array can also be designed in other embodiments without nanowells, wherein this then has only the anchor points and is otherwise essentially a plate. The plate may be, for example, the bottom of a petri dish or a slide. The anchor points are located on at least one of the surfaces of the nanowell array and are dimensioned so that only one cell can bind per anchor point. In other embodiments, nanowells are present, but have a depth that is about equal to or less than the

Diameter of the cell is. The advantages of the method and the device as well as the nanowell array are manifold. Clear evidence of monoclonality is achieved by sowing in nanowell arrays designed to have only one cell in a nanowell of the array. Different cell lines can be seeded in nanowell arrays with geometry adapted to them. In addition, the separation performed by means of imaging techniques in conjunction with an image processing is checked. A removal of a cell or a cell clone from a nanowell is documented by means of images before and after the harvest and / or a video from the harvesting process. In addition, a documentation can be made that a nanowell or well of a target vessel is empty before a harvested cell or a cell clone (in the following, for simplicity, only one cell is spoken) is placed in it.

Identification of high producers is possible without additional genetic modification of the cells. The identification can take place via the detection of surface proteins or the immobilization of the secreted proteins close to the producer cell and their detection, for example analogously to an ELISA in prepared and coated nanowells. This is especially important for biopharmaceuticals in human therapy and theirs

Approvals (national regulatory agencies, EMA, FDA, etc.). The cell does not have to express reporter genes, as is customary in flow cytometric methods, and can therefore be unmodified or modified only with the expression cassette of the recombinant protein (transgenic production cells).

The totality of all (genetically modified) cells is examined for their specific productivity. Specific productivity is quantified and all cells can

be compared to each other. Based on this, suitable high and medium producers are selected from all cells. There is no danger of losing productive single cells or cell clones.

In contrast, limiting dilution causes cell losses because not all cells can be sown and analyzed (material and space requirements too large). In flow cytometric methods, losses of cells occur during the definition of optimal sorting settings and during the sorting process.

It can also be achieved optimal culture conditions during cell handling. All work with the device is done under sterile conditions. Culture conditions (parameters) such as temperature, humidity, CO2 and 02 content can be adapted to the needs of the cells. A reduction in material cost is achieved since each nanowell array has significantly more nanowells than the number of wells on conventionally used 96 and 384 well plates, respectively. Furthermore, less cell culture medium is consumed (μΙ / ml instead of I) and less space needed in incubators. Furthermore, a reduction in the time required is achieved because several nanowell arrays can be processed in parallel.

Identification of high producers can already take place on the single cell level. Only promising single cells are cultivated further. A repetition of processes, such as in limiting dilution, is not necessary, since the monoclonality can be detected properly.

The method is used for cells which are eukaryotic cells, for example cells of yeasts, insect cells or mammalian cells. The cells are in a suspension for sowing as single cells. If necessary, the yeast cells are brought into suspension by means of suitable methods. The cells are genetically modified (transfected, transduced, transformed) in such a way that they contain an expression cassette with the sequence which codes for the target protein (optionally together with other sequences which can, for example, code for reporter proteins or selection markers). You can also

be antibody-producing hybridoma cells. Also possible are cells that

Of course, without genetic modification, produce a protein of interest. These may be secreted proteins, but also surface proteins such as B or T cell receptors. Furthermore, the method can be carried out in the culture of tumor cells (eg, circulating tumor cells, CTCs). The CTC are covered with a common medium. The method and the apparatus as well as the nanowell array can also be used, for example, for cultivating stem cells, self-cells (autologous cells) of genetically modified cells, tumor cells and for

Interaction studies between cells are used. The invention will be explained in more detail with reference to figures and embodiments. Show it:

Fig. 1 is a table showing a comparison of single-cell cloning methods and the method of the invention;

2 shows a schematic overview of the working steps up to the monoclonal cell line using the example of recombinant protein production in various methods; Fig. 3 is a schematic overview of the method steps in the use of

Nanowells with a capacity for several cells (top) or for only one cell (bottom);

4 shows a schematic representation of a corrugated plate in a clean-room housing during a first optional method step;

Fig. 5 is a schematic representation of a corrugated plate during a second

 optional method step;

Fig. 6 is a schematic representation of a corrugated plate during a third

 optional method step; Fig. 7 is a schematic representation of a corrugated plate during a fourth

 optional method step;

Fig. 8 is a schematic representation of a corrugated plate during a fifth

 optional method step;

9 is a schematic representation of a corrugated plate during a sixth optional method step;

10 is a schematic representation of a detail of a nanowell array with

 cells;

1 1 is a schematic representation of a nanowell array in plan view;

12 is a schematic representation of a first embodiment of a device according to the invention;

13 is a schematic representation of a second embodiment of a device according to the invention; 14 is a schematic representation of a section of an embodiment of a nanowell array with anchor points in each nanowell and Fig. 15 is a schematic representation of another embodiment of a

Nanowell arrays with anchor points and without nanowells.

The figures show schematically and simply elements of the devices and illustrate process steps. In this case, the same reference numerals designate the same technical elements or the same method steps.

In Fig. 1, the prior art methods for generating single cell clones, already described above, are tabulated against the method of the invention in terms of some of its economic parameters. The process sequences of the methods are summarized and compared in FIG.

FIG. 3 further illustrates essential method steps and processes of a method using nanowells 17 (see FIGS. 7 and following) with a large one

Diameter, which allows the inclusion of several cells 2 (upper row) and of

 Nanowells 17, whose diameter allows only the inclusion of a cell 2 (bottom row). The steps of detection after a sowing of incorrectly occupied nanowells 17 take place after the identification of nanowells 17 with individual cells (denoted by EZ). FIG. 4 shows a corrugated plate 1 located in a clean room housing 5, for example a conventional 96-well plate in SBS format. In the corrugated plate 1 are biological cells 2, which are covered by a medium 3. By means of a suction and / or pipetting unit 4, the medium 3 is sucked off (step A). Fig. 5 shows the corrugated plate 1 into which a washing solution in the form of, for example, a PBS solution (not shown) was placed. The corrugated plate 1 is pivoted and washed (symbolized by arrows), then the PBS solution is aspirated again (step B). It can also be added to a washing solution and removed again. In Fig. 6, the corrugated sheet 1 can be seen, in which the cells 2 with a prepared

Suspension 6 are covered. After addition of an enzyme to cells 2, a medium is added by which the enzymatic reaction is stopped. This is done again by means of the pipetting unit 4 in a step C. The step D shown in FIG. 7 schematically shows the functionalization of a

Nanowell Arrays 9 Four regions are formed on the nanowell array 9, each having a number of nanowells 17. The steps E and F illustrated in FIG. 8 comprise the steps of transferring cells 2 (not shown) from the corrugated sheet 1 to a nanowell array 9 (step E). The cells 2 are filtered by means of a filter 7 to obtain a single cell suspension.

Subsequently, their cell count is determined by a measuring device 8 for measuring the optical density. The cells 2 are seeded onto the nanowell array 9. The single cell suspension is evenly distributed by pivoting movements (symbolized by the arrows) before it is sucked off again. The nanowell array 9 or a number of submerged nanowell arrays 9 can or can be centrifuged in a step G by means of a centrifuge 10 in order, for example, to move the cells 2 into the nanowells 17 of the nanowell array 9 by the action of gravity / centrifugal force (FIG. 9). A lateral section through a section of a nanowell array 9 is shown in FIG. 10. In each case one cell 2 is contained in the two nanowells 17 shown. In one

Interspace 18 between the two nanowells 17 is also a cell 2, which is sucked by means of the pipette 4 in a step H as excess cell 2. At the bottom of each nanowell 17 an anchor point 1 1 is optionally formed, to which the cell 2 is bound. The respective excess cell 2 can be deposited by means of the pipette 4 in a nanowell 17, which was previously recognized as empty.

As a result, a nanowell array 9 is obtained in whose nanowells 17 ideally only one cell 2 is contained in each case and whose interspaces 18 do not contain any cells 2.

Such a nanowell array 9 is shown by way of example in FIG. 11.

In a step I, the nanowell array 9, or as shown in FIG

Nanowellarrays 9, by means of an image acquisition unit 14, which can also be viewed as a microscope, illuminated with an illumination and / or excitation radiation and a resulting detection radiation spatially resolved by means of a suitable detector (not shown) detected as image data. The spatial resolution refers to the possibility of indicating a position of a respective location, for example a nanowell 17, from which a detection radiation is detected as an event in the XY plane.

The image capture unit 14 is configured such that it can be used to determine whether a respective nanowell 17 contains exactly one cell 2, whether cells 2 in FIG Gaps 18 are located and what degrees of specific productivity cells 2 in the respective nanowells 17 show, in which there is only one cell 2 and which are not adjacent to a gap 18 with a cell 2. The evaluation of the image data takes place by means of an evaluation unit 19. Depending on the evaluation, control commands are generated and the pipetting unit 4 is activated so that only cells 2 of selected nanowells 17 are picked up and transferred to a target vessel or a targetwell plate, for example in the form of a target nanowell array 15 ( Step I).

The evaluation unit 19 is connected to a lighting unit 13 and with a

Storage unit 12 in conjunction. The illumination unit 13 provides illumination and / or excitation radiation and permits targeted illumination of the nanowell array 9, for example in the form of a scanning movement. This can be the

Lighting unit 13 include a scanner. The memory unit 12 serves, in particular, for storing the documentation of the method steps, the detected ID and associated productivities. The evaluation unit 19 is also designed to

Generate control commands and, for example, the pipette 4 and the

Lighting unit 13 to control.

The cells 2 transferred to one of the target nanowell arrays 15 by means of the pipette unit 4 are further incubated and their growth, survival and / or production or

Secretion of specific substances or mixtures monitored and documented (step J). The incubation takes place in an incubator 16 (FIG. 13).

An exemplary embodiment of a nanowell array 9 is shown in FIG. 14 as a detail and in a side sectional view. The nanowells 17 are substantially larger than the cells 2 and optionally have an anchor point 11 at their respective bottom. The anchor points 1 1 are each dimensioned so that only one cell 2 can bind to them. Excess cells 2 can therefore be washed away. The anchor points 1 1 are formed for example by a suitable coating, a local surface roughening or structuring.

Another embodiment of a nanowell array 9 is shown in FIG. On one of the surfaces anchor points 1 1 are formed. The nanowell array 9 has none

Nanowells 17, so that at the anchor points 1 1 bound cells 2 via a superimposed medium (not shown) can communicate with each other unhindered. In further embodiments of the nanowell array 9, shallow depressions may be provided as nanowells 17, in which the anchor points 11 are introduced. The wells have a depth that is less than a diameter of the cells 2. Embodiment - Generation, Identification and Isolation of a

high producers

 The following outline is intended to illustrate the individual steps necessary for the isolation of an interferon-gamma (IFN-γ) high producer using the example of CHO-K1 cells adapted to suspension growth:

- Transfection

 CHO-K1 cells are classically transfected with a plasmid having an IFN-γ expression cassette. Subsequently, cells 2 are cultured for a few hours, for example at least 12 hours, for a maximum of 48 hours to allow expression of the transgene.

- Selection of the nanowell array

 A nanowell array 9 is selected in which the individual nanowells 17 of the nanowell array 9 are only slightly larger than the cells 2: With an average size of a CHO-K1 cell of approximately 12 μm to 15 μm, a nanowell array 9 with round 18 is μηη nanowells 17 suitable. The nanowells 17 are 18 μηη high. The array design thus ensures that only one cell 2 can be located in a nanowell 17. - Prepare the nanowell array

 For the detection of IFN-v high producers, the nanowell array 9 (analogous to a classic sandwich ELISA) is coated with capture antibody. Unspecific binding sites are blocked. The nanowell array 9 is then thoroughly washed three times with PBS.

- Sowing the cells in nanowell arrays

 The transfected cells 2 are passed through a 40 μm cell strainer (filter 7) to dissolve any cell aggregates that may be present and one

 To obtain single cell suspension.

The single cell suspension is applied to the prepared nanowell array 9 by means of the device and distributed evenly by tilting movements. If necessary, several nanowell arrays 9 are connected in series or in parallel with the

transfected cells 2 equipped. After an incubation of five minutes, the nanowell array 9 is centrifuged for three minutes at 800 rpm. Subsequently, the nanowell array 9 is washed with fresh cell culture medium to remove cells 2 that are not in nanowells 17 from the gaps 18. These cells 2 are seeded in succession in a second nanowell array 9, so that they too are available for detection and isolation and are not lost.

Cells 2 located in a nanowell 17 are not removed from it by the washing step.

Detection of high producers in nanowell arrays

 The transfected cells 2 are incubated after the seeding described above for at least 30 minutes in the nanowell array 9 to allow the secretion of IFN-γ in the coated nanowells 17. Subsequently, the nanowell array 9 is optionally washed with fresh cell culture medium to remove unbound IFN-γ from the nanowell 17. Nanowell array 9 is then probed with an Alexa Fluor 488-conjugated antibody against IFN-γ (direct detection) or with an IFN-γ detection antibody (after a washing step) followed by a directed Alexa Fluor 488-conjugated secondary antibody (indirect detection for signal amplification ). After an optional washing step with fresh cell culture medium, the nanowell array 9 is scanned with the image capture unit 14 of the device. The images are taken in brightfield and fluorescence (Alexa Fluor 488). The cells 2 in the nanowells 17 are identified on the basis of the bright field images. In addition, the device checks whether cells 2 are present outside nanowells 17. If this is the case, neighboring

Nanowells 17 are excluded from the isolation process in order to prevent further cells 2 are isolated in addition to the desired single cell in the same step. Bright field detection also makes it possible to make fundamental statements about the viability of a cell 2 in order to exclude dead or dying cells 2 from the isolation process. The fluorescence images serve to detect and quantify the specific productivity of a single cell 2, since secreted IFN-γ is bound by an Alexa Fluor 488-conjugated antibody (directly via detection antibody or indirectly: detection antibody and antibody

Secondary antibody). All images are taken at the same exposure time, so that the signal intensity reflects the amount of secreted IFN-γ. The signals are sorted by fluorescence intensity to identify high, medium and low producers or cells 2 without specific productivity. After the washing steps, possibly taking Färbesch rode (analogous to the washing steps), takes place in a further imaging step by the

Image acquisition unit 14 to those defined in a preselection

Nanowells 17, for example, a detection of a fluorescence or

 Luminescence signal of a respective nanowell 17 and the cell 2 therein. In a further step, the detection of the cells 2 in the bright field to identify multiple cells 2 in a nanowell 17 can be. This control can be in two images or by switching between z. B. Fluorescence and bright field channel in a recording position. Likewise, additional detection levels are through

Multifluoreszenzkanäle applicable.

Isolation of high producers from nanowellarravs and deposition in target structures

Identified high producers and some middle producers are from the

Device automatically selected for isolation and confirmed by the user.

Single cells 2 or cell clones are individually isolated from the nanowells 17 of the nanowave array 9 with the aid of the pipetting unit 4. This is documented by taking pictures before and after the harvest, as well as a video of the actual harvesting process. In addition, the device checks image-analytically whether only one cell 2 was recorded at each individual isolation step. The cells 2 are dispensed separately into nanowells 17 of a 384-well plate. The device checks before cell delivery whether the target nanowells 17 are empty and ensures that the

Cell delivery takes place exclusively in empty target nanowells 17. After the cell delivery, the device checks image-analytically that only one single cell per target nanowell 17 has been deposited.

By means of the pipetting unit 4, which has suitable capillaries, for example made of glass, ceramic or plastic, selected nanowells 17 are approached. The selection of the nanowells 17 can be based on the respective signal intensities of

Detection radiation and / or done by a user. The pipetting unit 4 picks up the contents of the nanowell 17 or a cell 2 from a gap 18 and delivers them to a target nanowell 17. A documentation of the process by the image acquisition unit 14 is possible to provide a complete proof of the

To obtain monoclonality. - Propagation of the single cell to the cell clone

 The proliferation of the high-producing single cells 2 to the cell clone is documented by the image acquisition unit 14 by the recording of images. The

 Device can image analytically determine the confluence of the culture and inform the user if the cell clone should be transferred or split into a larger well format. This process can be automated using the device.

For this purpose, the target nanowell arrays 15 can be removed from the device for longer incubation capped. Additional imaging scans or screenings at defined times can document the cell growth and expression level of a transgene, or the productivity of the resulting clones, and allow further selection of the most productive clones. These steps can be carried out within an appropriately optionally equipped device (integrated incubator 16) and the existing image capture unit 14. The transport of selected clones can also take place in the pipetting unit 4 of the device.

- Test specific productivity cell clone

Subsequently, the specific productivity (IFN-γ secretion) of the individual cell clones is analyzed in a classical ELISA. These assays are performed repeatedly to assess the stability of IFN-v production of a cell clone over time. Stable high producers are identified in this way.

reference numeral

 1 corrugated plate

 2 cell

 3 medium

 4 pipetting unit

 5 clean room housings

 6 cell suspension

 7 filters

 8 measuring device

 9 nanowell array

 10 centrifuge

 1 1 anchor point

 12 storage unit

 13 lighting unit

 14 image capture unit

 15 target nanowell array

 16 incubator

 17 Nanowell

 18 gap

 19 evaluation unit

A suction nutrient medium

 B Washing and aspirating the washing solution

 C addition of enzyme and medium

D Filtering process and cell count

 E coating, functionalization

 F rearrangement in nanowell array

 G centrifugation

 H optional wash step

I image acquisition, evaluation and rearrangement in destination vessel

J Monitoring of the producer cells

Claims

claims

1. A method for verifying the success of single cell seeds in nanowell arrays (9), comprising the steps of:

 Illuminating a nanowell array (9) seeded with individual biological cells (2), the nanowell array (9) having a number of nanowells (17) in which, after sowing, at most one biological cell (2) is to be contained, with at least an illumination and / or excitation radiation and spatially resolved detection of a detection radiation caused by the illumination and / or excitation radiation as image data,

Evaluating the image data for the presence of exactly one cell (2) in the respective nanowells (17),

 Evaluating the image data for the presence of cells (2) in

Interspaces (18) between the nanowells (17), and

- Storing the positions of those nanowells (17) in which not exactly one cell (2) is present and those spaces (18) in which cells (2) are present.

2. The method according to claim 1, characterized in that nanowells (17), in which after sowing more than one cell (2) is present, and / or cells (2) in

Gaps (18) a sampling device (eg., Pipette) delivered and excess cells (2) (individually) are removed.

3. The method according to claim 2, characterized in that after sowing empty nanowells (17) identified and their position is determined and removed cells (2) are stored individually in these nanowells (17).

4. The method according to claim 1, characterized in that nanowells (17) without

Cells (2) and / or nanowells (17) with more than one cell (2) and / or nanowells (17) adjoining interstices (18) in which cells (2) have been detected, whose positions are stored and excluded from subsequent process steps.

5. The method according to any one of the preceding claims, characterized in that in dependence of the stored positions control commands for subsequent

Process steps are generated or adapted.

6. The method according to any one of the preceding claims, characterized in that the nanowell array (9) is maintained under culture conditions, wherein the culture conditions allow desired physiological reactions of the cells (2).

7. The method according to claim 6, characterized in that the physiological

 Reactions are documented, wherein growth and proliferation processes are detected and measured on the basis of a signal intensity of the detection radiation or change in cell morphology in the image and / or production of substances by the cell (2) using labeled antibodies, in particular fluorochrome-labeled antibodies, enzyme-labeled Antibodies, bispecific antibodies, specific interactions of produced substances and / or metabolic processes of the respective cell (2) are recorded and evaluated.

8. The method according to any one of the preceding claims, characterized in that emitted by the cell (2) substances and / or mixtures in the nanowell (17) and / or next to the nanowell (17) by means of the substances to be detected and / or mixtures Binding partners are at least partially bound.

9. The method according to any one of the preceding claims, characterized in that selected cells (2) or from a single cell (2) resulting cell clones are isolated from the respective nanowells (17) and further cultured individually, so that monoclonal cell lines are obtained.

10. The method according to any one of the preceding claims, characterized in that automated

 - Nanowells (17) are detected with exactly one cell (2) and the positions of these nanowells (17) are stored,

 the nanowells 17 are cultivated over several days and observed on the system and analyzed by image analysis,

- based on the evaluations based on proliferation / vitality data of the cells (2),

 optionally a detection of secreted proteins / surface proteins or internal markers,

- the nanowells (17) containing monoclonal, well-proliferating, high-production clones are automatically selected on the basis of the combination of the data obtained, - From these nanowells (17) for the purpose of further expansion of the cell lines is automatically picked and

 - all these steps are documented.

1 1. A method, characterized in that, preferably automated,

 Image data of a nanowell array (9) arranged in a sample area with an image acquisition unit (14) and a lighting unit (12) for providing at least one illumination and / or excitation radiation and means for controlled illumination of the nanowell array (9) with the at least one illumination and /or

Excitation radiation are detected;

 - By evaluation of these image data nanowells (17) in size, shape and / or position within the arranged in a sample area nanowell array (9) are detected;

- Identify the positions of these detected nanowells (17) with an identity and

be stored;

- Based on the image data, all cells (2) within the nanowell array (9) arranged in a sample area are independently detected;

 - The positions and image-analytically recorded parameters (shape, spectral data, outlines, morphology) of these recognized cells (2) are stored;

 a 2D overlay (X / Y) of the positions and shapes of the stored nanowells (17) and of the detected cells (2) is performed, an association of nanowells (17) with the cells (2) contained in the nanowells (17) is enabled and stored

image-analytical parameters of the cells (2) are assigned to the previously stored data of the nanowells (17) and a resulting list of the nanowells (17) and the image-analytical parameters is created;

optionally, the resulting list is filtered on nanowells (17) containing exactly one cell (2);

 - The nanowells (17) are cultivated with the cells (2) over several days and observed on the system and analyzed by image analysis;

 - generated based on the evaluations based on proliferation and / or vitality data of the cells (2);

 optionally a detection of secreted proteins and / or surface proteins and / or internal markers takes place;

 - selecting from the combination of the data obtained those nanowells (17) containing monoclonal, well-proliferating, high-production clones; - From these selected nanowells (17) for the purpose of further expansion of the cell lines, preferably automated, is picked, and

- All these steps, preferably automated, documented.

12. An apparatus for verifying the success of single cell seeds in nanowell arrays (9), comprising an image acquisition unit (14) for acquiring image data of a nanowell array (9) arranged in a sample area and a lighting unit (12) for providing at least one illumination and / or excitation radiation and means for controlled illumination of the nanowell array (9) with the at least one illumination and / or excitation radiation, wherein the image capture unit (14) is configured such that

 - Expected areas with expected presence of each cell (2), can be set, detected or verified on the nanowell array (9) by being able to evaluate acquired image data of the relevant expected areas by means of an evaluation unit (19) of the device;

 - can be fixed, detected or verified on the nanowell array (9) spaces (18) in which no cells are expected;

- After the illumination of the nanowell array (9) a spatially resolved detection of a caused by the illumination and / or excitation radiation detection radiation can be carried out and

 Evaluating the image data for the presence of exactly one cell (2) in the respective nanowells (17),

Evaluating the image data for the presence of cells (2) in

Interspaces (18) between the nanowells (17), and

 - Storing the positions of those nanowells (17) in which not exactly one cell (2) is present as well as those spaces (18) in which cells (2) are present, is possible.

13. The apparatus according to claim 12, characterized in that the

Image detection unit (14) is adapted to identify a nanowell array (9) in terms of its type and to determine its position and spatial orientation.

14. The apparatus according to claim 12 or 13, characterized in that this for

Documentation of the nanowell array (9) and / or of selectable process steps and process stages is formed.

15. Device comprising

- An image detection unit (14) for detecting image data of a nanowell array (9) arranged in a sample area and a lighting unit (12) for providing at least one illumination and / or excitation radiation and means for controlled Illumination of the nanowell array (9) can be detected with the at least one illumination and / or excitation radiation;

 an evaluation unit (19) configured to evaluate said image data and to detect nanowells (17) with regard to their size, shape and position within the nanowell array (9) arranged in a sample area;

 - wherein the evaluation unit (19) is configured such that

 the positions of these detected nanowells (17) are provided with an ID and stored,

 - Using the same image data all cells (2) within the in one

 Sample area arranged nanowell arrays (9) are recognized independently,

 - The positions and all image-analytically acquired parameters, in particular form, spectral data, outlines and / or morphology of these recognized cells (2) are stored

 - By 2D superposition (X / Y) of the positions and shapes of the stored nanowells (17) and the detected cells (2) an assignment of nanowells (17) with the cells (2) contained in the nanowells (17) is enabled and the

 stored image analytical parameters of the cells (2) are assigned to the previously stored data of the nanowells (17)

 Optionally, the resulting list may be filtered on nanowells (17) containing exactly one cell (2)

 - The nanowells (17) are cultivated over several days and observed on the system and analyzed by image analysis,

 - based on the evaluations based on proliferation and / or vitality data of the cells (2),

 optionally a detection of secreted proteins and / or surface proteins and / or internal markers takes place,

 - Automated by the combination of the data obtained

 Nanowells (17) containing monoclonal, well-proliferating, high-production clones,

 - A pipetting unit (4) is present, by means of which these nanowells (17) for the purpose of further expansion of the cell lines can be picked automatically, all of these steps are automatically documented.

16. Nanowell array (9) comprising nanowells (17) with a diameter that allows the uptake of multiple cells (2) and at the bottom of each of the nanowells (17)

Anchor structure (1 1) for binding only one cell (2) is present.

17. nanowell array (9) according to claim 16, characterized by an anchor structure (1 1) in the form of a surface structuring and / or a coating with anchor molecules.

18. nanowell array (9) according to claim 16 or 17, characterized in that additionally at least one coating inside and / or outside of the nanowell (17) is present in order to at least partially from the cell (2) secreted / secreted substances and / or mixtures to bind and / or initiate a detection reaction to detect the presence of the substance or the mixture of substances and / or the detection of a signal intensity of a detection radiation.

19. Nanowellarray (9) according to any one of claims 16 to 18, characterized in that the bottom of the nanowell (17) is designed as a sieve or permeable membrane to a negative pressure suction of the cells (2) in the nanowells (17) support.

20. nanowell array (9) without nanowells (17) with anchor structures (1 1) on at least one of its surfaces, wherein by each of the anchor structures (1 1) only one cell (2) can be bound.

21. Use of the device according to one of claims 12 to 15 and / or a nanowell array (9) according to one of claims 16 to 20 for culturing stem cells, self-cells, genetically modified cells or tumor cells.