Selection of methods and instruments

Abstract

The present invention provides a method and apparatus that can select cultured cell colonies or cultured single cells with desired characteristics from a plurality of heterogeneous cultured cell colonies or cultured single cells in a common culture space. While the cell colonies or single cells are in the common culture space, the cell colonies or single cells are treated so that the functions of the cellular components of those cells with the desired characteristics are altered. The individual cell colonies or individual single cells are then evaluated for markers of altered functions in the common culture space.

Description

[0001] This application is a divisional application of patent application with application number 2013800457186, application date July 2, 2013, and invention name “Selection method and apparatus”.

[0002] This application claims the benefit of European Patent Application No. 12174574.9 filed on July 2, 2012, the entire contents of which are incorporated herein by reference.

[0003] There are many situations in which it is necessary to select (in other words pick) among a heterogeneous population, cell colony or single cell for those possessing desired characteristics.

[0004] An example is to select cell colonies or single cells to form cell lines for cell-based reporter tests. Cell-based reporter tests can, for example, be used to screen candidate compounds for new drugs. In the example of such tests, suitable cell line cultures expressing specific proteins are exposed to candidate compounds, and markers of the required interaction (directly or indirectly) between the indicator candidate compounds and the protein of the cell are evaluated. Cells that are easily cultured can be transfected to make the cells express a functional copy of a transgenic or recombinant polypeptide or protein of interest, thereby generating cell lines for cell-based reporter tests. However, the transfection process results in a heterogeneous mixture of cells, and it is critical to select those cells in which the transfection process has produced the desired characteristics.

[0005] Currently, after transfection, cells are subjected to limiting dilution and plated in multiwell plates so that statistically no well contains more than 1 cell. Many wells are devoid of cells. The clonal cell colonies formed during the incubation are subcultured to produce a variety of different clonal cultures, each with two replicates. For each clonal culture, one of the replicates is tested for the desired characteristics (usually destructively). This reduces the clonal cultures (i.e., the replicates that were not tested) to those that exhibit the desired characteristics. Since this method inevitably requires the cultivation of clones that do not exhibit the desired characteristics, this method is inefficient. SUMMARY OF THE INVENTION

[0006] According to a first aspect of the present invention, a method for selecting cultured cell colonies or cultured single cells having desired characteristics is provided, which comprises: providing a plurality of cultured cell colonies or cultured single cells in a common culture space, wherein the plurality are heterogeneous in terms of the desired characteristics; treating the cell colonies or the single cells while the cell colonies or the single cells are in the common culture space, wherein the treatment induces a functional change in at least one cellular component of the cell colony or the single cell, wherein the functional change indicates that the or each cell colony or single cell that is induced to have a functional change has the desired characteristics; evaluating markers of functional changes in each of the cell colonies or the single cells in the common culture space; selecting at least one but not all of the cell colonies or the single cells, wherein the selection takes into account the evaluation to determine the predicted situation of having the desired characteristics, wherein based on the evaluation, the or each selected cell colony or single cell is predicted to have the desired characteristics; and providing an indication of the spatial position of at least one selected cell colony or single cell.

[0007] Preferably, when the method is performed on cultured cell colonies, the colonies are clonal cell colonies, but the method can also be used on non-clonal cell colonies.

[0008] The desired characteristic may be the presence of a functional copy of a particular polypeptide or protein, preferably a transgenic or recombinant polypeptide or protein. For example, the polypeptide or protein may interact with or form a portion of a signal transduction pathway. More specifically, the desired characteristic may be the presence of a functional copy of a protein (preferably a transgenic or recombinant protein) selected from the group consisting of: G protein coupled receptors, ion channels, kinases, phosphatases, transcription factors, cytoskeletal proteins, cell cycle proteins, proteolytic proteins, and ligases. Alternatively, the desired characteristic may be the presence of a functional signal transduction pathway.

[0009] In a preferred embodiment, the desired characteristic is the presence of a functional copy of a cellular receptor, the treatment comprises adding a chemical that interacts with the cellular receptor, and the interaction directly or indirectly induces a change in function.

[0010] The method described includes providing a plurality of cultured cell colonies or a plurality of cultured single cells in a common culture space. The common culture space is a single continuous culture space. It can include different interrelated regions, but does not include disconnected culture spaces, such as separate holes of a multi-well plate. Therefore, a common culture space can be, for example, a culture space in a single conventional petri dish, a culture space in a single culture bottle, or a culture space in a single hole of a multi-well culture plate. Cell colonies or single cells are cultured together in a common culture space, but usually they are separated from each other in space. The common culture space usually comprises a continuous volume of growth medium, which supports all cultured cell colonies or all single cells. The growth medium can be, for example, a liquid growth medium or a semi-solid growth medium. Preferably, the culture medium is a liquid growth medium, and the cell colonies or single cells are attached. Usually, the precise composition of the culture medium is not important, as long as the culture medium has all the components necessary for supporting the cells.

[0011] Preferably, the cells of the cell colony divide while in culture. However, this is not required, and as used herein, the term "culture" includes situations where the cells remain viable but do not divide.

[0012] Preferably, the method is carried out on cultured cell colonies rather than single cells. Cell colonies are generally provided by preparing a suspension of single cells, wherein the cells are heterogeneous with respect to the desired characteristics; and seeding the cells in a common culture space such that the cells are substantially spatially separated from each other. The cells are then cultured to form spatially separated cell cultures, most of which are clonal. The method preferably uses cell colonies because the growth rate of the colonies can be taken into account when selecting suitable colonies.

[0013] In the same common culture space, there may be a mixture of cell colonies and single cells, and the method described above may be applied to the mixture.

[0014] The heterogeneity exhibited between different cell colonies or between different single cells may be in the form of the presence or absence of a desired characteristic, or in the form of different levels or magnitudes of a desired characteristic, in the latter case including instances where the characteristic is not distinctly present.

[0015] The method includes treating the cell colonies or single cells when the cell colonies or single cells are in a common culture space. In a preferred embodiment, the common culture space is treated in such a way that all cell colonies or all single cells are treated (but possibly with different intensities or concentrations) in a relatively short time (e.g., 10, 20 or 30 seconds). In this case, if the indicator decays, it is preferred that all cell colonies or all single cells are treated in a shorter time than the time when the indicator is observed to decay significantly thereafter. In other preferred embodiments, each cell colony or each single cell is treated at different times. For example, if the treatment is to apply an electric current or voltage, it can be achieved by applying an electric current or voltage to each colony or single cell at different times. For example, another example, in which the treatment is to apply a chemical, the chemical can be included in the growth medium in an inactive form. Subsequently, the chemical can be converted into an active form in a local manner near each cell colony or single cell by applying a suitable local stimulus (e.g., a narrow beam of light).

[0016] The treatment may include, for example, adding one or more chemicals to a common culture space so that the chemicals come into contact with the cell colonies or single cells in the common culture space. The added chemicals may be biological chemicals in nature, such as biochemical small molecules, peptides, proteins, or non-peptide macromolecules; or alternatively, the chemicals may be non-biological chemicals in nature. Instead of or in addition to chemical treatment, the treatment may include non-chemical intervention, an example of which is adjusting the temperature to the temperature at which the cell colonies or single cells are exposed in the common culture space. Another non-chemical treatment is the application of an electric current or voltage. The treatment may include two or more stages, such as adding a first chemical in a first stage and adding a second chemical in a subsequent second stage.

[0017] In cases where the treatment includes exposure to a chemical and the cell colonies or single cells are cultured in a liquid growth medium, all of the cell colonies or single cells can be exposed to the chemical in a relatively short period of time by adding a volume of liquid containing the chemical (more than the volume of the liquid medium) to the medium. For example, the volume of liquid added can be between 20% and 50% of the volume of the liquid medium, or even more liquid can be added if the cells have not separated. In cases where the culture medium is a semi-solid culture medium, the cell colonies or single cells can be exposed to the chemical in a relatively short period of time by spraying a mist of the chemical solution onto the surface of the semi-solid culture medium. Alternatively, in cases where the culture medium is a semi-solid culture medium, the treatment chemical can be included in the culture medium in an inactive form and activated simultaneously by applying a suitable activation stimulus to the entire culture medium.

[0018] The concentration of chemical to which a cell colony or a single cell is exposed need not be uniform.

[0019] In addition to the treatment described, other interferences can be performed on cell colonies or single cells. An example of this interference is to perform when other interferences are needed to enable detection of markers of functional changes caused by the treatment described. For example, a cultured cell colony or a cultured single cell can be first incubated with a reporter chemical, which enters the cells of the colony or a single cell. After the interference, the common culture space is processed, and functional changes in the cell components of the cell colony or single cell with the desired characteristics are triggered. The internalized reporter chemical enables markers (or signals) of functional changes to be detected. A more specific example is that when a colony or a single cell is first incubated with a fluorescent or luminescent reporter chemical, these fluorescent or luminescent reporter chemicals enter the cells of the colony or a single cell, thereby enabling measurement of the calcium concentration of the cytosol. Then, a colony or a single cell is subjected to a process that can trigger functional changes in the cell components, which causes the calcium concentration of the cytosol to change.

[0020] Ideally, the treatment and any other perturbation should not irreversibly change the cell colonies or single cells with the desired characteristics. Preferably, the treatment is substantially or completely non-toxic to the cell colonies or single cells with the desired characteristics.

[0021] The described treatment has caused the change of the function of the cell component. The change of the function of the cell component indicates that the cell colony or single cell in which the function is changed has the desired characteristics. In fact, the desired characteristics can be (but not necessarily have to be) the presence of a component in which the function is changed. The change of the function can be, for example, a change in the magnitude of the function, a change from inactive to active or active to inactive, or a change from one function to a different function. For example, the functional change can be a change in the catalytic function between an active and inactive state, or a change in the magnitude of the catalytic function. Another example of functional change is a change in the configuration of a membrane channel, such as a change between an open and closed configuration. The change of the function does not necessarily have to involve any change in the structure or configuration of the cell component itself. For example, the change of the function can be caused by a change that is not caused by the cell component itself, but by a change from inactive to active or active to inactive caused by the cell component moving from one subcellular position to another subcellular position.

[0022] The component can be any cellular component that can undergo a functional change indicating the presence of a desired characteristic. For example, the component can be an enzyme, a cell receptor, a membrane channel, or a transcription factor. By way of example, the desired characteristic can be the presence of a specific membrane receptor (e.g., a G protein coupled receptor (GPCR)), the treatment can be the addition of a chemical (e.g., a ligand) that can interact with the receptor, the component can be a component (e.g., phospholipase C) of a signal transduction pathway (e.g., a phosphatidylinositol signaling pathway) controlled by the receptor, and the functional change can be a change in the catalytic activity of the component caused by the interaction of the chemical with the receptor. Activation of phospholipase C results in an increase in the calcium concentration of the cytosol, so in this example, a change in the calcium concentration of the cytosol can serve as a marker for a change in cellular function and (indirectly) the presence of a desired characteristic.

[0023] In alternative illustrations, the desired feature may be the presence of a functional copy of a specific transcription factor. The cellular component may be the same transcription factor. The treatment may be the addition of a ligand to the transcription factor. The functional change may be a change in the activity of the transcription factor that occurs during the migration of the transcription factor from the cytoplasm to the nucleus. In this illustration, the functional change and (indirectly) the presence of a marker for a functional transcription factor may be a detectable change in the location of the transcription factor.

[0024] The method comprises evaluating each cell colony or single cell for a marker of functional change in a common culture space. The evaluation may simply involve determining whether a marker can be detected in each cell colony or cell evaluated. Alternatively, the evaluation may include estimating the magnitude of the marker in each cell colony or single cell evaluated. In the case of estimating the magnitude of the marker (as opposed to just being present or lacking), a cell colony or single cell may still exhibit an undetectable marker.

[0025] It may not be necessary to evaluate all cell colonies or single cells for markers. For example, selection of cell colonies or single cells may be based on the presence of two or more different desired characteristics. It is possible that a cell colony or single cell may be excluded because it does not have a first desired characteristic, in which case it may not be necessary to evaluate the colony or cell for markers indicating the presence of a second desired characteristic.

[0026] The marker is preferably an intracellular marker.

[0027] The marker can be any marker that indicates a change in function and can be detected. A preferred marker is a change in cytosolic calcium concentration. Another preferred marker is a change in membrane potential, which can be detected by a membrane potential dye. A third preferred marker is a change in the activity of a specific enzyme. The marker and the change in the function of a cell component can be the same thing. For example, the marker and the change in function can be the same change in the activity of the same cell enzyme.

[0028] The markers are assessed while the cell colonies or single cells are in a common culture space. The common culture space is not subdivided into discrete culture spaces (eg, circular clones). Preferably, the cell colonies or single cells are still in culture medium when the assessment is performed.

[0029] Preferably, an imaging system is used to assess the marker. More preferably, the imaging system detects fluorescence or luminescence, which indicates the presence or magnitude of the indicator in a separate cell colony or a separate single cell. In this case, the imaging system can measure the intensity of fluorescence or luminescence for a separate cell colony or a separate single cell.

[0030] Where the marker comprises a change in cytosolic calcium concentration and the marker is detected using a fluorescence or luminescence based system for detecting cytosolic calcium concentration, it is preferred that the imaging system provide the intensity level of fluorescence or luminescence for each individual cell colony or single cell being assessed at least at a first time point prior to treatment and at a second time point after the change in cytosolic calcium concentration has occurred. It may be desirable to measure intensity at more than two time points or substantially continuously in real time.

[0031] As mentioned above, a preferred marker is a change in cytosolic calcium concentration. Changes in cytosolic calcium concentration can be assessed, for example, using calcium ligands whose fluorescent properties change when they bind to calcium. An example of a fluorescence-based detection system for detecting changes in cytosolic calcium concentration is the FLIPR TM Calcium5Assay Kit TM, available from Molecular Devices of Sunnyvale, California, USA. In addition, changes in cytosolic calcium levels can also be detected by luminescent systems such as those using Aequeorin.

[0032] When using FLIPR TM Calcium 5 TM It can be used together with probenecid as a reagent. Probenecid reduces the concentration of Calcium 5 TM The reagent disappears from the cell and increases the fluorescence inside the cell.

[0033] As an alternative to using fluorescence or luminescence imaging to assess markers of cell colonies or single cells, Raman spectroscopy can be used. Raman spectroscopy is particularly well suited to detecting the presence of specific chemicals in microscopic systems and can be used to detect specific chemicals within living cells.

[0034] The method includes selecting at least one but not all cell colonies or single cells, thereby predicting that the selected ones have the desired characteristics. The selection takes into account the evaluation of markers to determine whether each cell colony or single cell may have the desired characteristics associated with the marker. In some embodiments, the selection takes into account the relative magnitude of the markers exhibited by the cell colony or single cell; and this generally means selecting colonies or cells that exhibit relatively high levels of markers or levels that meet predetermined criteria (e.g., fall within a predetermined range). However, the selection may take into account other factors, so it is not necessary to select cell colonies or single cells with the desired characteristics. For example, if a cell colony or cell with the desired characteristics does not have another desired characteristic, such a cell colony or cell may not be selected. Preferably, the selection is performed by a processor. In fact, it is preferred that the described method is implemented in an automated manner by an automatic operating instrument.

[0035] The method further comprises providing an indication of the spatial location of at least one of the selected cell colonies or single cells.

[0036] Preferably, the spatial position is a position specified with reference to a common culture space or culture container. In this manner, the culture container can be moved, and by determining the change in position of the culture container, the new position of the cell colony or single cell can be determined. This is useful because it allows the cell colony or single cell to be picked up after the culture container has been moved.

[0037] Indication of spatial location can take various forms. The basic mode of indicating the spatial location of selected colony or single cell is to simply mark or indicate the location adjacent to the location on the culture container that provides a common culture space. For example, if the common culture space is the culture space of a petri dish, then the position adjacent to the cell colony or single cell can be marked on the bottom surface of the culture dish. The mark can be a circle around the colony or cell, or a circle that tracks the colony border. Such a mark is carried out so that the colony or single cell can be picked up manually. For example, the mark can be used to place a ring or cylinder to seal against the bottom of the culture dish and around the colony or cell. Then a trypsin solution can be used in the ring to peel off the cell colony or single cell.

[0038] As used herein, the term "picking" refers to taking a cell colony or a single cell from the culture space in such a way that no other cell colonies or single cells are taken in the same picking action. When picking a cell colony, it is not necessary to take all the cells that make up the colony.

[0039] However, it is more preferred that the spatial position of the selected cell colony or single cell is indicated by generating coordinates identifying the position. The coordinates may be XY coordinates or XYZ coordinates associated with a common culture space. This approach is particularly useful if picking is performed in an automated manner.

[0040] Preferably, the method further comprises picking up at least one selected cell colony or single cell.

[0041] The method may include determining the respective boundaries of individual cell colonies or single cells. For example, in the case where the cell colonies are attached to a horizontal surface, the determined boundaries may be the perimeter of the horizontal surface area covered by the colonies. Determining the boundaries of the cell colonies is useful because the size of the colonies or the number of cells in the colonies can be estimated. This is also useful because it allows the magnitude of the estimated colony markers to be normalized for the size of the colonies or the number of cells in the colonies. This is also useful for determining the boundaries of the cell colonies or single cells to facilitate picking up the colonies or cells.

[0042] According to a second aspect of the present invention, there is provided a computer program comprising instructions for directing an automated operating apparatus to implement the method according to the first aspect of the present invention. The computer program may be provided on a computer readable medium.

[0043] According to the third aspect of the present invention, there is provided an automatic operating instrument, comprising a processor and a storage medium, wherein the storage medium has the computer program according to the second aspect of the present invention stored thereon, and the processor is connected to the storage medium for running the computer program on the processor.

[0044] According to a fourth aspect of the present invention, there is provided an automatic operating instrument for selecting cultured cell colonies or cultured single cells having desired characteristics, comprising: an imaging station in which a culture container may be provided; an imaging system adapted to generate at least one image covering a field of view, the field of view comprising a plurality of cell colonies or single cells contained in a common culture space of a culture container placed in the imaging station; a processing device programmed to analyze at least one image to evaluate markers indicating functional changes in cellular components of each cell colony or single cell included in the field of view, the functional changes indicating the presence of desired characteristics; the processing device programmed to select at least one but not all of the cell colonies or single cells, wherein the selection takes into account the evaluation to determine a predicted situation of having the desired characteristics, wherein based on the evaluation, the or each selected cell colony or single cell is predicted to have the desired characteristics; and the processing device programmed to extract an indication of the spatial position of at least one selected cell colony or single cell.

[0045] Preferably, the automated instrument comprises an automated dispenser for dispensing fluid into the common culture space of the culture container of the imaging station, the automated dispenser being controlled by the processing device. In this case, the processing device of the automated instrument is preferably programmed to implement the steps in the following order:

[0046] Manipulating the imaging system to obtain a first image of a field of view containing a plurality of cell colonies or a single cell in a common culture space of a culture container located at the imaging station;

[0047] operating the automatically operated dispenser to dispense a fluid treatment agent comprising one or more treatment chemicals into the common cultivation space of the cultivation container;

[0048] The imaging system is operated to obtain a second image of a field of view containing a plurality of cell colonies or a single cell in the common culture space of the culture container located at the imaging station.

[0049] When the processing device is programmed to perform these steps, the analysis of the markers by the processing device includes comparing the first image and the second image to determine changes between the two images corresponding to each of the cell colonies or single cells.

[0050] The automatic operating instrument of the fourth aspect of the present invention preferably includes a detector for detecting fluorescence or luminescence emitted by a cell colony or a single cell in the field of view. The detector preferably includes an sCMOS image sensor. Preferably, the detector includes a sensing area of ​​at least 100mm 2image sensor.

[0051] The robot of the fourth aspect of the present invention preferably comprises a robot cell picker for picking up a separate cell colony or a separate single cell. The robot cell picker is preferably controlled by a processing device. In this case, the processing device is programmed to manipulate the robot cell picker to pick up a cell colony or a single cell selected by the processing device and having extracted an indication of its spatial position.

[0052] Preferably, the automatic operation instrument of the fourth aspect of the present invention includes a culture container having a plurality of cultured cell colonies or cultured single cells in a common culture space, wherein the plurality are heterogeneous in terms of desired characteristics, and the common culture space contains at least one treatment chemical for inducing functional changes in at least one cellular component of the cell colony or the single cell, wherein the functional changes indicate that the or each cell colony or single cell in which the functional changes are induced has the desired characteristics.

[0053] According to a fifth aspect of the present invention, there is provided a method for selecting a cultured cell colony or a cultured single cell having a desired characteristic, comprising: providing a plurality of cultured cell colonies or cultured single cells in a common culture space, wherein the plurality is heterogeneous in terms of the desired characteristic; treating the cell colonies or the single cells while the cell colonies or the single cells are in the common culture space, wherein the treatment induces a change in the cytosolic calcium concentration of at least one of the cell colonies or the single cells, the change in the cytosolic calcium concentration indicating that the or each cell colony or single cell in which the cytosolic calcium concentration change is induced has the desired characteristic; detecting a change in the cytosolic calcium concentration in at least one of the cell colonies or single cells while the cell colonies or single cells are in the common culture space; and providing an indication of the spatial position of at least one cell colony or single cell in which the cytosolic calcium concentration change is detected. Preferably, the change in cytosolic calcium concentration is detected using fluorescence or luminescence emitted from a position within the cell.

[0054] For all aspects of the invention, the cultured cell colony of the fifth aspect of the invention is preferably a clonal cell colony.

[0055] Where the context permits, the preferred features and descriptions given above in relation to the first aspect of the invention also apply to the fifth aspect of the invention.

[0056] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

[0057] Embodiments of the present invention are described in more detail below by way of example with reference to the accompanying schematic drawings, in which:

[0058] Figure 1 A perspective view of an automatic operating instrument;

[0059] Figure 2 Show Figure 1 Imaging systems for automated instrumentation;

[0060] Figure 3A and 3B For use Figure 2 White light images of cell colonies obtained by an imaging system;

[0061] Figure 4 for Figure 1 A perspective view of a cell picking head of a robotic instrument;

[0062] Figure 5 Shown in using FLIPR TM Calcium 5 TM White light images of CHO K1 cell colonies expressing AT1 at two different time points after treatment and without treatment;

[0063] Figure 6 To show FLIPR TM Calcium 5 TM A graph showing the effect of agents on cell growth of CHO K1 cells expressing AT1;

[0064] Fig. 7A and 7B Fluorescence images of cell colonies before and after treatment are shown;

[0065] Figure 8 is a graph showing the change in fluorescence intensity over time; and

[0066] Fig. 9 Fluorescence images of cells before and after treatment are shown.

[0067] Figure 1 The robot 10 is shown with its outer shell removed to reveal its internal components. The operation of the components of the robot 10 is controlled by a computer 12 (which is located in the Figure 2 ) control as described below.

[0068] The robot 10 has a main platform 14 on which a culture plate containing cultured cells is manipulated. Figure 2As can be seen most clearly, the main platform 14 is provided with an aperture 16. A transparent glass plate 18 is located above the aperture 16. The upper surface of the transparent glass plate 18 forms an imaging station 20 in which a culture plate can be placed so that cells cultured in the culture plate can be visualized, as described below.

[0069] In addition, the imaging station 20 includes positioning guides (not shown) that hold standard configuration culture plates in precise positions.

[0070] like Figure 1 As shown, the robotic instrument 10 includes an imaging system 22 located below the main stage 14, and more specifically, below the aperture 16. The imaging system 22 is mounted (described in more detail below) on an optical stage 24, which is also fixedly mounted relative to the main stage 14.

[0071] See now Figure 2 , the imaging system 22 includes a first light source 26, a second light source 28, and a camera 30 for obtaining images of cells in a culture plate located in the imaging station 20. In very general terms, the first light source 26 is used with the camera 30 to obtain white light images of the cells using a dark field configuration. This can be used to determine the location of cell colonies and cells, determine the boundaries of cell colonies, and determine the rate of colony growth by taking consecutive images. In addition, in general terms, the second light source 28 is used with the camera 30 to obtain images of fluorescence or luminescence emitted by the cells. The imaging system 22 and its use will now be described in more detail.

[0072] The first light source 26 includes a plurality of white light emitting diodes (LEDs) 32. The white light LEDs 32 are mounted on a mounting ring 34 having a central aperture 36. The optical axes of the LEDs 32 are located on the surface of a common virtual shared phantom cone (not shown), so that the light beams emitted by the LEDs 32 converge at a convergence point 38 at the top of the phantom cone. Figure 2 In FIG. 8 , oblique arrows 40 show the light paths from the two LEDs 32 to the convergence point 38 .

[0073] For illustration purposes, Figure 2 A six-well culture plate 42 is shown placed on the imaging station 20. However, it should be understood that different types of culture containers can also be used. Each well of the six-well culture plate 42 is identified by a number 44 and is directly above the imaging system 22. The well 44 has a horizontal culture surface 46 on which a plurality of adherent cell colonies 48 grow. Liquid growth medium (not shown) is provided in the well 44, which covers the cell colonies 48.

[0074] The focusing point 38 of the light beam emitted by the LED 32 is slightly above the culture surface 46 and the cell colony 48 .

[0075] The power supplied to the LED 32 is controlled by the computer 12, as shown schematically by control line 50. Thus, the illumination of the LED 32 is controlled by the computer 12.

[0076] In addition, the imaging system 22 also includes a semi-silvered mirror 52, which is at an angle of 45° to the horizontal and is located below the central aperture 36 of the mounting ring 34. In a known manner, most of the light moves vertically downward, as shown by the downward arrow 54, and directly passes through the semi-silvered mirror 52 without any change of direction. On the other hand, most of the light moves horizontally in the direction shown by the horizontal arrow 56 toward the inclined upper surface of the semi-silvered mirror 52, and is reflected vertically upward by the semi-silvered mirror 52, and the incident angle and the reflection angle are both 45°.

[0077] In addition, the imaging system 22 includes an autofocus zoom lens 58. One commercially available zoom lens that has been found to be particularly useful is the Leica McaroFluo Z6APOA zoom lens, but other lenses may also be used. The zoom lens 58 receives light that passes vertically downward in the direction of the downward arrow 54 after passing through the semi-silvered mirror 52. The zoom lens 58 focuses the light for reception by the camera 30. The focal length of the zoom lens 58 can be changed, which is controlled by the computer 12, as schematically shown by the control line 60. The optical axis of the zoom lens 58 is aligned with the convergence point 38.

[0078] The camera 30 receives the light focused by the zoom lens 58. The camera generates an image from the light and transmits the image to the computer 12 via the control line 62.

[0079] The camera 30 is capable of producing images from relatively low intensity light, such as fluorescence or luminescence emitted by cells on a culture plate placed on the imaging station 20. Preferably, the camera 30 is also capable of covering a relatively large field of view of the culture surface 46 of the culture plate 42 (which is placed on the imaging station 30) while producing images from such low intensity light sources. Preferably, the image produced by the camera 30 covers a plurality of separate cell colonies 48 (or single cells) attached to the culture surface 46.

[0080] One commercially available camera that has been found to be particularly suitable is the Hamamatsu ORCA Flash 4.0. This camera uses an sCMOS image sensor that provides a higher sensitivity. In addition, the sensing area of ​​the sCMOS image sensor used in the Hamamatsu camera is relatively large in size (13.312 mm x 13.312 mm), which allows the camera 30 to obtain images covering a larger field of view. Another advantage of the Hamamatsu camera is that it has a relatively large number of pixels (2048 x 2048). If necessary, a relatively large number of pixels can have a higher resolution. Optionally, when sensitivity is more important than resolution, the signal obtained by a large number of adjacent pixels can be passed in to increase sensitivity.

[0081] In addition, the following cameras were evaluated and found to be suitable: Apogee ALTA U260; Andor Neo-sCMOS; and Andor Neo-sCMOS oem. The dimensions of the sensing area of ​​the image sensors of these cameras are: 10.2mm x 10.2mm; 16.6mm x 14mm; and 16.6mm x 14mm, respectively. From these studies, it was concluded that the sensing area of ​​100mm 2 Cameras of 400 mm or larger are preferred.

[0082] Furthermore, cameras with image sensors other than sCMOS image sensors may also be used. For example, cameras with conventional CMOS or CCD image sensors may be used, but the results obtained may not be particularly good.

[0083] The imaging system 22 includes a first filter 64. The maximum passband of the filter is 516 nm. The first filter 64 can be moved out of the optical path between the zoom lens 58 and the camera 30 by a suitable motor (not shown) under the control of the computer 12. The first filter 64 is used for the purpose described below.

[0084] like Figure 2 As shown, the second light source 28 is placed at the same horizontal position as the half-silvered mirror 52 and directs light toward the inclined upper surface of the half-silvered mirror 52 in the direction of the horizontal arrow 56. The illumination of the second light source 28 is controlled by the computer 12 via the control line 68.

[0085] One commercially available light source found to be suitable for use as the second light source 28 is the Sola 6-LCR SA white light source, although other commercially available light sources may be used.

[0086] The second filter 70 with a maximum passband of 495 nm is placed between the second light source 28 and the semi-silvered mirror 52 .

[0087] The operation of imaging system 22 will now be described.

[0088] As described above, the first light source 26 (LED 32) is used in conjunction with the camera 30 to obtain a white light image of cells and / or cell colonies 48 growing on the culture surface 46 of the culture plate 42 (located on the imaging station 20). The white light obtained by the LED 32 is directed upward through the aperture 16 of the main platform 14, through the glass plate 18, through the bottom wall of the culture plate 42, and toward the focusing point 38. Light scattered downward by the cell colonies 48 and / or single cells returns downward through the culture plate 42, through the glass plate 18, through the aperture 16, through the center aperture 36, and toward the semi-silvered mirror 52, where most of the scattered light passes straight through the semi-silvered mirror 52 to the auto-focusing zoom lens 58. The zoom lens 58 focuses the light, thereby allowing the camera 30 to produce an image of the cell colonies 48 and / or single cells on the culture surface 46.

[0089] When an image is acquired in the manner described using the first light source 26 and the camera 30, the first filter 64 is removed from the optical path and has no effect.

[0090] The images generated by the camera 30 are transmitted to the computer 12 for analysis.

[0091] Figure 3A and 3B 4 is a white light image of a cell colony 48 obtained in this manner. Figure 3A is the original image, Figure 3B The same image is shown after using a known electronic variance filter to increase the contrast between the cell colony 48 and the background.

[0092] The second light source 28 can be used in combination with the camera 30 to obtain a fluorescent image of a cell colony 48 or a single cell on the culture plate 42. The first filter 64 and the second filter 70 are selected according to the specific fluorescence system. In this specific example, the first filter 64 and the second filter 70 are selected to be compatible with the FLIPR discussed above. TM Calcium 5 TM The reagent is used together with a fluorescent reagent. This reagent is used to indicate the concentration of cytosolic calcium. The reagent penetrates into the cell and binds to cytosolic calcium. When the reagent is used, the binding of calcium to the reagent increases the fluorescence response of the reagent, so the increased cytosolic calcium level will lead to an increase in fluorescence in the cell. As discussed above, the maximum bandpass of the second filter 70 is 495nm, which is the maximum bandpass of calcium 5 TM The maximum excitation of the reagent. The maximum bandpass of the first filter 64 is 516nm, which is equivalent to Calcium 5 TMThe maximum fluorescence emission of the reagent. Obviously, when other fluorescent systems are used, the first filter 64 and the second filter 70 described above can be replaced with other filters having different maximum bandpasses suitable for the fluorescent system under investigation. The first filter 64 and the second filter 70 can be carried by a filter wheel, so as to be used to replace the filter under the control of the computer 12.

[0093] In operation, the white light obtained by the second light source 28 is filtered by the second filter 70 so that the light with the maximum wavelength of 495 nm is reflected upward by the half-silvered mirror 52. The reflected light passes through the central aperture 36, the aperture 16, the glass plate 18 and the bottom of the culture plate 42 to reach the cell colony 48 or cells in the culture plate 42. This light excites the Calcium 5 TM Reagents: Intracellular Calcium 5 TM The emitted light from the reagent passes back downward through the bottom of the culture plate 42, the clear glass plate 18, the aperture 16, the central aperture 36, and reaches the half-silvered mirror 52. Most of this emitted light passes straight through the half-silvered mirror 52 and enters the zoom lens 58, which focuses the light onto the camera 30. In this case, the first filter 64 is located in the light path and filters the emitted light as it passes between the zoom lens 58 and the camera 30. The fluorescent image obtained by the camera 30 is transmitted to the computer 12 for analysis.

[0094] When the imaging system 22 using the first light source 26 is used to obtain a white light image of a cell colony or a single cell, or when the imaging system using the second light source 28 is used to obtain a fluorescent image, the automatic focusing zoom lens 58 can be controlled by the computer 12 to zoom in on a specific cell colony 48 or a specific single cell. This is useful, for example, when it is necessary to determine the location within a cell that emits fluorescence.

[0095] can be carried out by an optical transport 72 (see Figure 1 ) The imaging system 22 is mounted on the optical platform 24, wherein the optical transport 72 allows the entire imaging system 22 to be moved in any horizontal direction. The optical transport 72 is connected to the computer 12 so that the movement of the imaging system 22 is controlled by the computer 12. The movement of the imaging system 22 in this manner enables the imaging system 22 to obtain images of other areas (fields of view) of the culture plate 42 (located on the imaging station 20).

[0096] The position of the optical transport 72 is correlated by the processor to an x, y, z coordinate system that is common to the various robotic components of the robotic instrument 10 .

[0097] As described above, the culture plate 42 is held on the imaging station 20 by a positioning guide (not shown) at a known precise position. The x, y, z coordinates of this position (using a common x, y, z coordinate system) are stored in the computer 12. Therefore, when the cell colony 48 or a single cell is imaged by the imaging system 22, the computer 12 can use the position of the colony or cell in the image, the position of the optical transport 72 when the image was taken, and the known geometry of the culture plate 42 on the imaging station 20 and the known position of the culture plate 42 to calculate the position of the cell colony or single cell on the culture plate 42. This approach is useful because it allows the culture plate 42 to be moved to another known position, and as long as the orientation of the culture plate 42 at the new position is known, the common x, y, z coordinate system can be used to determine the precise position of the cell colony or single cell. This approach can be useful if, for example, it is necessary to pick up cells from the culture plate 42 at a different position on the imaging station 20 as described below.

[0098] See also Figure 1 The robot 10 also has a cell picking head 74 located above the main platform 14. The cell picking head 74 is similar to the one described in EP1686380 for ClonePix TM 2 Robotic Instrument (from Molecular Devices, New Milton, UK) is similar to the known cell picking head.

[0099] As described in EP1686380, the cell picking head 74 is movable on the main platform 14 by a head positioning conveyor 76, which is composed of x, y and z linear position regulators connected in series and suspended on a gantry 78. The head positioning conveyor 76 is controlled by the computer 12 to move the cell picking head above the main platform 14. The processor 12 relates the position of the cell picking head 74 to a common x, y, z coordinate system.

[0100] The cell picking head 74 is of conventional design and is shown in FIG. Figure 4 In. Figure 4 As shown, the cell picking head 74 has eight hollow picking needles 80, 81, 82, 83, 84, 85, 86, 87. The internal open space of each hollow picking needle 80, 81, 82, 83, 84, 85, 86, 87 is fluidly connected to a corresponding control port 88, 89, 90, 91, 92, 93, 94, 95. Each control port 88 to 95 is connected to a control pump (not shown) through a control tube (not shown).

[0101] In conventional manner, each of the eight picking needles 80-87 can be independently operated to pick up cell colonies or single cells from the culture plate 42 by operating a control pump associated with each picking needle to cause the picking needle to aspirate the cell colonies or single cells. The cell colonies or cells can be attached or suspended in a semi-solid culture medium.

[0102] However, each of the eight hollow pick-up needles 80-87 also has a secondary function, namely, dispensing liquid into the culture plate. Each of the eight hollow pick-up needles 80-87 is connected to a respective liquid reservoir through its respective control tube and respective control pump. Liquid from each of these liquid reservoirs can be pumped into the corresponding pick-up needle for dispensing. Each pick-up needle 80-87 can dispense liquid independently of the other pick-up needles.

[0103] The operation of the hollow picking needles 80 - 87 picking up cell colonies and single cells and the operation of the picking needles 80 - 87 dispensing liquids are all controlled by the computer 12 .

[0104] The robot 10 also includes many other automated features known in the art and not particularly relevant to the present invention. For example, the robot 10 includes a device for stacking and storing culture plates and for incubating the stored culture plates at a desired temperature. The robot 10 also includes a washing station for cleaning the hollow pick-up needles 80, 81, 82, 83, 84, 85, 86, 87. In addition, the robot 10 includes a device for re-seeding cells on a new plate and for replacing the culture medium on the culture plate. In addition, a transport device for moving the culture plates around the chamber of the robot is provided.

[0105] The following example is provided to illustrate how the robotic instrument 10 described above may be used to select and optionally pick cultured clonal cell colonies or cultured single cells having desired characteristics.

[0106] Example 1

[0107] The following examples utilize a fluorescent Calcium 5 reagent, allowing for the assessment of cytosolic calcium levels using fluorescent imaging. The purpose of this example is simply to demonstrate that the Calcium 5 reagent is not overtly toxic to the cells used in the following examples.

[0108] This example utilizes cultured Chinese hamster ovary cells KI (CHO-KI) expressing angiotensin II type 1 (AT1) receptors. AT1 receptors are G protein-coupled receptors (GPCRs). They bind to the ligand angiotensin II. Activated AT1 receptors bind to G q / 11 Binds, in turn activates phospholipase C, which in turn increases cytosolic calcium concentration.

[0109] A suspension of CHO-KI cells expressing AT1 in liquid culture medium was plated in six wells of a standard six-well culture plate. The plate was incubated until the cells formed small, dispersed colonies. Next, Calcium 5 TM Add the reagents in the same liquid medium to 3 of the 6 wells. Add an equivalent volume of liquid medium to the other 3 wells. Add Calcium 5 TM In the wells containing reagents, Calcium 5 TM The concentration of the reagent is the concentration recommended by the manufacturer. The cells are then incubated with the reagent at 37°C for 1 hour. After incubation, the cells are incubated for another 1 hour, this time at room temperature. The purpose of this second incubation is to simulate the environmental conditions when the cells are imaged and picked up in the robot 10. After the second incubation, the liquid culture medium is removed from all 6 wells and replaced with fresh liquid culture medium. Finally, the culture plate is returned to 37°C and the cells are allowed to grow for 7 days.

[0110] During the 7-day incubation, CloneSelect TM Imager (obtained from Molecular Devices (NewMilton) Ltd) periodically monitors the growth of cells in the six wells. Similarly, the growth of cells can be monitored using the imaging system 22 of the automated instrument 10 described above. In the latter case, the first light source 26 and camera 30 are used to generate white light images of the cells, and these images are analyzed by the computer 12 to determine the percentage of the culture surface 46 covered by the cells.

[0111] Images obtained on days 0 and 7 of incubation are shown in Figure 5 In. Figure 5 The top two images represent the results of the experiment on day 0 and day 7 respectively without the use of Calcium 5 TM The following two images represent the coverage of cells in the wells treated with Calcium 5 TM Cell coverage in reagent-treated wells. As shown, cell growth was similar in untreated and treated wells.

[0112] Figure 6 The experimental results are shown in a graph, where the y-axis shows the untreated cells and the cells treated with Calcium 5 TM No significant differences were observed in the average growth rates of cells treated with the agents.

[0113] This shows that using Calcium 5 TMTreatment of cells with the agents did not result in significant growth inhibition.

[0114] Example 2

[0115] This example demonstrates that the imaging system 22 of the robotic instrument 10 is sensitive enough to detect the presence of Calcium 5 TM Reagents are used to detect fluorescence emitted within cells, thereby allowing the detection of increases in cytosolic calcium concentrations.

[0116] This example utilizes CHO-K1 cells that express the AT1 receptor and CHO-K1 cells that do not express the receptor.

[0117] CHO-K1 cells expressing AT1 and CHO-K1 cells not expressing AT1 were plated and grown in separate culture plates using appropriate commercially available liquid culture media. The culture plates were incubated until the cells were observed to form small, dispersed, attached colonies, and the following steps were performed on each culture plate to treat the cells expressing AT1 and the cells not expressing AT1 uniformly.

[0118] First, as described in Example 1 above, appropriate culture plates were incubated with Calcium 5 TM The reagent was incubated for 1 hour to load the cell colonies with Calcium 5 TM Reagents: Calcium 5 TM The reagent enters the cultured cells.

[0119] The following steps are performed on the processed culture plate in the imaging station 20 .

[0120] Loading Calcium 5 TM After the reagents are added, the cell colonies are imaged using the first light source 26 and the camera 30 to produce a white light image, which is transmitted to the computer 12 for analysis. During the analysis, the computer 12 determines the location of the imaged colonies in the culture plate and the boundaries of the colonies.

[0121] After white light imaging, the cell colonies on the culture plate are imaged using a second light source 28 and a camera 30. These images are transmitted to a computer 12 and stored. The images obtained at this stage show intracellular localization of Calcium 5 in the cell colonies. TM The baseline level of fluorescence emitted by the reagent.

[0122] Then, the computer 12 manipulates the head positioning conveyor 76 so that the fifth hollow pick-up needle 84 is positioned above the culture plate (which has been previously covered). The hollow pick-up needle 84 is connected to a reservoir containing a solution of angiotensin II in a culture medium through a corresponding control tube and a corresponding control pump. Then, the computer 12 manipulates the control pump so that the hollow pick-up needle 84 dispenses the angiotensin II solution into the culture plate. The volume of the dispensed solution is approximately one-fourth of the volume of the culture medium in the culture plate before dispensing. Dispensing a relatively large volume of solution (e.g., 1 / 4 of the volume of the culture medium in the culture plate) allows angiotensin II to reach all cell colonies in the culture plate at approximately the same time. It is acceptable that the concentration of angiotensin II varies in different areas of the culture plate.

[0123] Immediately after dispensing the angiotensin II solution, the imaging system 22 is operated to obtain continuous fluorescent images of the culture plate showing intracellular Calcium 5 TM This involves using the second light source 28 and second filter 70 to illuminate the colonies and the camera 30 and first filter 64 to detect the emitted light, and operating the imaging system 22 as described above.

[0124] The final average concentration of angiotensin II in the culture plates was 10.5 mM.

[0125] Details of the fluorescence images obtained in this experiment are shown in Fig. 7A and 7B middle. Fig. 7A The 2 images show the results for CHO-K1 cells that do not express the AT1 receptor. Fig. 7A Each of the 2 images covers the same cell colony. Figure 7B The 2 images show the results for CHO-K1 cells expressing AT1. Figure 7B , the left hand image is a baseline fluorescence image covering a cell colony taken before the addition of angiotensin II. A relatively small amount of intracellular fluorescence is evident in this image. Figure 7B The right-hand image is a fluorescent image of a cell colony of the same clone as the left-hand image and was taken approximately 16 seconds after the addition of angiotensin II. The right-hand image shows a large number of light spots. Each light spot represents the Calcium 5 TM The fluorescence level is significantly higher than the baseline image on the left hand side. Therefore, angiotensin II triggers an increase in cytosolic calcium concentration in CHO-K1 cells expressing AT1, and this can be detected using Calcium 5 TMThe reagents are easily detected by the imaging system 22 of the automated instrument 10 .

[0126] See now Fig. 7A , the left-hand image is a baseline image before the addition of angiotensin II, and the right-hand image is an image taken approximately 16 seconds after the addition of angiotensin II. In fact, these images are consistent with what is predicted. In the absence of AT1 receptors, the addition of angiotensin II does not lead to an increase in cytosolic calcium concentration.

[0127] Figure 8 FIG. 2 shows the fluorescence intensity levels of four randomly selected cell colonies of cells expressing AT1 as determined by computer 12 by analyzing images obtained by imaging system 22. Figure 8 In the graph, each point on the graph represents the fluorescence intensity level measured for a single cell colony of a single fluorescence image obtained by the imaging system 22. The fluorescence level at t=0 is the fluorescence level in the baseline image. After the addition of angiotensin II, images were acquired every 8 seconds, and the graph shows the relative fluorescence intensity of each of the four colonies obtained from each of the consecutive images. Figure 8 It can be seen that the intracellular fluorescence reached its peak approximately 16 seconds after the addition of angiotensin II, and then decreased relatively steadily over the next 140 seconds.

[0128] Example 3

[0129] In Example 2, by comparing the fluorescence image with the white light image, it is apparent that all or substantially all of the cell colonies expressing AT1 show increased intracellular fluorescence after the addition of angiotensin II. This is predictable because the AT1-expressing CHO-K1 cells used in Example 2 are from a cell line that stably expresses AT1.

[0130] However, it should be understood that the same method can be performed on a heterogeneous mixture of cell colonies (or single cells), and the method can be used to select cell colonies (or single cells) having desired characteristics from the mixture.

[0131] The following is an example of how to implement the above method.

[0132] A gene construct designed to express AT1 is used to transfect a cell line that does not express the AT1 receptor. As is well known, the transfection process is inefficient. After transfection, many cells do not express AT1 at all, and for those cells that express AT1, the degree of expression varies greatly.

[0133] The suspension of transfected cells is plated and incubated to form dispersed cell colonies. Prior to plating, the cell suspension is diluted sufficiently to ensure that most, if not all, colonies grown on the plate are derived from a single cell and are therefore clonal. The same method used in Example 2 is applied to the culture plates containing clonal cell colonies.

[0134] In this process, the relative fluorescence intensity obtained from each clonal colony after the addition of angiotensin II solution was compared with the relative fluorescence intensity obtained from the same clonal colony before the addition of angiotensin II (i.e., baseline level). An increase in intracellular fluorescence indicates an increase in cytosolic calcium concentration, which is in turn a marker for the expression of functional AT1 receptors.

[0135] Those clonal cell colonies that demonstrate increased intracellular fluorescence following the addition of angiotensin II are selected by computer 12, and computer 12 determines the location of these colonies on the culture plate using white light imaging.

[0136] Knowing the locations of cell colonies exhibiting increased intracellular fluorescence, those colonies can be picked up using cell picking head 74 under the control of computer 12, and cells in each picked colony can be subcultured and further analyzed to determine whether the clone exhibits the desired AT1 receptor expression.

[0137] Colonies that did not demonstrate increased intracellular fluorescence were not picked and not further examined. This saves a lot of time and resources that could be spent in analyzing unsuitable clones.

[0138] Example 4

[0139] This example utilizes cells derived from a single colony of CHO-K1 cells expressing AT1, wherein the colony was picked up by the cell picking head 74 following the procedure of Example 2. Cells from the picked colony were suspended and plated in a new culture plate with culture medium. The process of Example 2 was then repeated for the newly plated cells. Images obtained during the process of Example 4 are shown in Fig. 9 In. Fig. 9 In the figure, the upper image is a fluorescence baseline image obtained before the addition of angiotensin II. The lower image is a fluorescence image obtained about 20 seconds after the addition of angiotensin II. As shown, the fluorescence level in the lower image is significantly higher than the fluorescence level in the upper image.

[0140] This example demonstrates that one can expose cells to the Calcium 5 reagent, stimulate them with Angiotensin II, and pick them up, and that the cells are still responsive to Angiotensin II, and also demonstrates an increase in intracellular fluorescence after reseeding.

[0141] Example 5

[0142] With any imaging system of the type described above, there is a trade-off between sensitivity to fluorescence from individual colonies or cells and the field of view. The field of view can be increased (e.g., reduced) as sensitivity to individual colonies or cells decreases, and conversely, sensitivity can be increased (e.g., enlarged) as the field of view decreases. If the field of view does not need to cover the entire culture surface of the culture plate in order to obtain the desired sensitivity, the optical transport 72 can move the imaging system 22 so that the entire culture surface 46 can be imaged by taking a series of images at different positions of the optical transport 72.

[0143] In the above experiments using the robot 10, a single well 44 of a standard six-well culture plate was imaged using nine separate overlay images arranged in a 3x3 matrix. It was found that the nine overlay images could be generated in a total time of 13 seconds, including the exposure time of 200 ms for each image, the time taken to download the images to the computer 12, and the time taken for the optical transport 72 to move to the desired position.

Claims

1. A computer readable medium having a computer program stored thereon, the computer program comprising instructions for directing an automated instrument to implement a method for selecting a cultured cell colony or a cultured single cell having a functional copy of a transcription factor, the method comprising the following steps: providing a plurality of cultured cell colonies or cultured single cells in a common culture space, wherein the plurality of cultured cell colonies or cultured single cells are heterogeneous with respect to the presence of functional copies of transcription factors; When the cell colony or the single cell is in the common culture space, a ligand of a transcription factor is added to the cell colony or the single cell, wherein the addition of the ligand triggers a change in the activity of the transcription factor in at least one of the cell colony or the single cell during the process of migrating from the cytoplasm to the nucleus, and the activity change indicates that the at least one cell colony or the single cell that is induced to change its activity has a functional copy of the transcription factor; assessing each of said cell colonies or said single cells for detectable changes in the location of transcription factors in said common culture space; selecting at least one but not all of said cell colonies or said single cells, wherein said selecting takes into account said evaluating to determine said predicted presence of a functional copy of a transcription factor, wherein based on said evaluating, said selected at least one but not all of said cell colonies or single cells are predicted to have a functional copy of a transcription factor; and An indication of the spatial location of the selected at least one cell colony or at least one single cell is provided.

2. The computer readable medium of claim 1, wherein the evaluating comprises measuring fluorescence or luminescence.

3. The computer readable medium according to claim 1 or 2, wherein the method further comprises the steps of: picking up at least one selected cell colony or single cell from the common culture space; and culturing the picked cell colony or single cell.

4. An automatic operating instrument, comprising a processor and a computer-readable medium according to any one of claims 1 to 3, wherein the processor is connected to a storage medium for running the computer program on the processor.

Images