Patent Application
20060166305
The invention relates to an apparatus for and method of detecting confluence in animal cells.
It is common practice to culture animal cells in 96 well plates. However, it is a well known property of such colonies that they display contact inhibition whereby cell division ceases once the cells have grown across the well to fill the available area and touch each other. The degree to which the cells have grown to fill the well or other biological sample container is referred to as confluence, and one speaks of a well, plate or dish being 70% confluent, 80% confluent and so forth. The term subconfluent is also used to refer to a plate in which the cell colony or other cell aggregate has not yet reached confluence. If a colony is grown to high or full confluence this may also damage the experiment. For example, some cells grown at high confluence may lose their adherent phenotype.
Since the cell growth rate is not generally predictable, and since different colonies grow at different rates, the standard practice is for an operator to examine the well plates daily, or at longer or shorter regular intervals, by viewing the plate directly or under a microscope. Based on this visual inspection, the operator makes a decision on whether the cells should be disrupted and re-plated into larger wells, such as a 24 well plate or a 6 well plate. Often, the colonies are re-plated several times into progressively larger wells, e.g. from 96 to 24 to 6 well plates. For example, it is typical that replating will be performed if the cultures approach confluence, for example 65-75% confluence, or a lower degree of confluence, for example 50%, if the cells would be adversely affected if they became confluent.
The manual visual inspection for confluence is highly time-consuming and to a certain extent also non-auditable and non-repeatable in that it relies on human judgement and experience. Typically, it might take an experienced operator an hour to inspect a batch of 10 well plates.
It would therefore be desirable to automate the confluence detection process, in particular in a robot with well plate handling and cell picking capability.
A known method for detecting confluence is electrically using an impedance measurement. For example, extracellular electrode arrays can be used for capacitance measurements of adherent cells growing in colonies. Although possibly automatable, impedance measurement is generally viewed with suspicion, because it is considered undesirable to apply voltages to the cells in case this interferes with the cells in some way. An example of this method is disclosed in De Blasio et al, Biotechniques 2004 April 36(4), pages 650ff “Combining optical and electrical impedance techniques for quantitative measurement of confluence in MDCK-I cell culture” [2].
Phase contrast microscopy is another well known technique which can be used to image cell boundaries and thus detect confluence. However, it would be difficult and expensive to integrate a phase contrast microscope into a suitable robot. In particular, the inherent wavelength dependence of phase contrast microscopy makes it difficult to automate when viewing wells of standard well plates bearing in mind that the colony will often be an adherent one, adhered to the base and lower side walls of the well, which is at a refractive index discontinuity created by the material of the well plate and the liquid or air filling the well.
Although an automated optical approach would be desirable to replicate the manual inspection, the colorless and low-contrast nature of the usual cell boundaries makes this challenging.
The invention provides a process for detecting the degree of confluence of animal cells being cultured in a biological sample container, comprising: arranging a biological sample container in an object position of an imaging station; illuminating the object position with an optical source from below at an oblique angle; collecting an image of the biological sample container arranged in the object position such that the image is taken in a dark field configuration where light from the optical source, if not scattered, does not contribute to the image; and processing the image to determine the degree of confluence of the animal cells in the biological sample container.
The biological sample container may be a well plate or other type of container such as a Petri dish, omni tray, Q-tray etc.
By the simple solution of illuminating from below at an oblique angle, it has been found that many animal cell types can be imaged with sufficient contrast to allow cell identification and consequent cell area computation using image processing techniques, thereby allowing confluence to be determined of animal cells being cultured in well plates or other receptacles. This avoids the need for more complex optical imaging techniques, such as phase contrast microscopy, and in many cases avoids the need for fluorescently tagging the cells or staining the cells. Moreover, this simple solution is amenable to automation in a picking robot with minimum disruption to other design features of a picking robot, such as head design and positioning, and well plate feeding and stacking.
The animal cells could be individual cells, colonies of cells, cell monolayers or other kinds of cell aggregates.
The oblique angle at which the optical source illuminates the object position is preferably between 10 to 50 degrees, or 20 to 40 degrees, to the horizontal, with angles of around 30 degrees (25 to 35 degrees) being optimal for the systems used to date. The angle refers to the optical axis of the illumination.
The process can be applied iteratively to scan across all the wells of a well plate. For example, the optical source and the detector can be iteratively realigned relative to the well plate so that images of a sequence of wells in the well plate are collected and processed, whereby the degree of confluence of the animal cells is determined in a plurality of wells across the well plate. This can be achieved by mounting the optical source and detector on a common platform and mounting the platform on an xy-positioning system which is driven to move the optical source and detector together from well to well. Alternatively, the optical source and detector can remain static, and the well plate can be moved. This can be achieved by providing a well plate mounting platen or other form of carrier on the bed of the apparatus which is coupled to its own xy-positioning system. In any given apparatus, either one or both of these two xy-positioning systems could be provided.
The optical source can conveniently comprise a plurality of directional light emitting units arranged to emit beams having optical axes lying on the surface of a common cone, the point of which is coincident with the object position. Most conveniently, the directional light emitting units are arranged in a ring.
In some embodiments, the optical source comprises a plurality of directional light emitting units arranged to emit beams having optical axes lying on the surface of at least two cones whose points are coincident with each other and the object position. According to this design alternative, most conveniently the directional light emitting units are arranged in multiple concentric rings. This can provide a greater illumination power when all rings are illuminated simultaneously. Perhaps more importantly, each ring has its own characteristic illumination angle which is a key determinant for contrast of the cell perimeters in the dark field image, so that the ring that provides the greatest contrast in the image can be used for the confluence determination.
The light emitting units are LEDs in the main embodiment described below, but in other embodiments could be superfluorescent LEDs, lasers, in particular semiconductor lasers, or lamp sources, such as a Xenon lamp. The LEDs used in the main embodiment are white LEDs, but other embodiments could use UV LEDs or single color LEDs, or groups of single color LEDs of different color to produce broader band emission, such as white light. Groups of LEDs or other sources of two different colors may also be a useful combination for optimizing contrast or other purposes.
The image is preferably collected from below the object position. This provides a very convenient design, since both the illumination and collection optics are then arranged below the well plate, leaving the entire half space above the well plate free for plate handling mechanisms, cell picking head movement and other activities. The mechanical design of the cell picking and confluence detection functions can then be done largely separately, greatly simplifying the automation.
The optical source is preferably formed such that an open light path exists downwardly from the object position, and the light is collected via this open light path. A detector, such as a CCD camera, can thus be positioned to collect light scattered downwardly from the object position, and the light can be collected by the detector via this open light path. Alternatively, the image can be captured from above rather than below, so that light scattered upwardly from the object position is collected. For example, a CCD camera or other detector can be housed above the main bed of the apparatus in the roof or suspended from a gantry.
The degree of confluence can be determined by an automated cell count which is translated into an area by multiplication of the cell count by an area representing an average area for the cell type being cultured. Alternatively, the degree of confluence is determined by processing the image to: establish cell boundaries, compute the area of each cell from the cell boundary, and sum the cell areas. Image processing software, or alternatively any mixture of software, firmware and hardware, can be used to perform the image processing.
The cell boundaries can be determined directly by contrast from the plasma membrane, or from the extent of the cytoplasm, or possibly in some cases from contrast provided by an extracellular fluid in which the cell is located. Although testing to date has indicated that no fluorescence staining is necessary in many cell types of interest, modifying the cells by inclusion of a fluorescent tag may be performed, e.g. to image other cell parts, such as the nucleus, or to assess the physiological state of the cell, such as cell cycle. For example, red lectin can be used to tag the cell membranes. Nuclear tags that do not kill the cells may also be suitable to provide contrast in the case that the aggregate area of the cells is determined by cell counting rather than by direct cell area calculation. Whole cell stains may also be considered, such as Phalloidin FM4-64. A variety of suitable dyes are known and can be selected, for example, from the Molecular Probes catalog.
The invention also provides an apparatus for detecting the degree of confluence of animal cells being cultured in a biological sample container, comprising: an imaging station where a biological sample container can be arranged in an object position; an optical source arranged to illuminate the object position from below at an oblique angle; a detector arranged to collect an image of the biological sample container arranged in the object position such that the image is taken in a dark field configuration where light from the optical source, if not scattered, does not contribute to the image; and an image processing unit for processing images to determine the degree of confluence of animal cells culturing in the biological sample container.
For full automation, a well plate feeder/stacker, or feeder/stacker for other type of biological sample container, is preferably provided to supply each of a plurality of well plates from a feed, typically a well plate storage cassette, to the imaging station, and return them to a stack, which is typically a further well plate storage cassette. Moreover, a biological sample container feeder/stacker, which may be well plate feeder/stacker, is preferably also provided to automate the replating operations. The biological sample container feeder/stacker is operable to supply each of a plurality of biological sample containers from a further feed to a replating station and return them to a further stack. For some applications, multiple feeder/stackers for replating may be provided.
For some classes of application, full automation may not be required. In particular, automated well plate feeding and stacking may not be needed. For example, when the source well plate has a large number of wells (96 or more) dispensing into 4 destination well plates also with a large number of wells, the transfer operation from that single well plate may take several hours including incubation periods. For such applications, manual placement of well plates on the bed of the robot may be adequate.
To perform the replating there is provided a cell picking head provided having at least one hollow pin for aspirating animal cells, and a head position system operable to move the cell picking head to allow replating of animal cells from a target well plate to a destination sample container.
The invention thus envisages use of a robot equipped with an animal cell picking head comprising at least one hollow pin for aspirating animal cells and replating them by moving them from a target well plate, or other biological sample container to a destination biological sample container under the control of a head positioning system, the use comprising: providing a well plate in which animal cells are being cultured; arranging the well plate at an imaging station and detecting the degree of confluence in each well by repeatedly: (i) illuminating a selected well from below at an oblique angle; (ii) acquiring an image of the illuminated well; and (iii) processing the image of the well to determine the degree of confluence; and dependent on the degree of confluence, either replating the animal cells out of the well plate, or leaving them to continue to culture.
In an embodiment, the robot is equipped with at least one automated well plate supply mechanism, and the use comprises: providing a plurality of well plates in which animal cells are being cultured; supplying each well plate in turn to an imaging station, and at the imaging station detecting the degree of confluence in each well by repeatedly: (i) illuminating a selected well from below at an oblique angle; (ii) acquiring an image of the illuminated well; and (iii) processing the image of the well to determine the degree of confluence; and dependent on the degree of confluence, either replating the animal cells out of the well plate in which they are located, or leaving them to continue to culture.
The replating may be decided upon on individually for each well based on the degree of confluence of the well exceeding a confluence threshold. Alternatively, the replating may be decided upon on a well plate specific basis, in which replating is performed if a threshold number of wells exceed a confluence threshold, which may be 1, or a higher number.
For a better understanding of the invention and to show how the same may be carried out reference is now made by way of example to the accompanying drawings in which:
The apparatus may be considered to be a picking robot with integrated confluence detection optics. The apparatus can be subdivided notionally into two half spaces existing above and below a main bed 5 which is supported by a frame 94.
Above the main bed 5, the apparatus appears as similar to a conventional picking robot. A cell picking head 118 is provided that comprises a plurality of hollow pins for aspirating animal cells. The cell picking head 118 is movable over the main bed 5 by a head position system made up of x- y- and z-linear positioners 98 connected in series and suspended from a gantry 96. A wash/dry station 102 is also provided on the main bed 5 for cleansing the pins. The whole upper half space of the apparatus will typically be enclosed in a housing (not shown) including a hinged door extending over one side and part of the top of the apparatus.
Below the main bed 5, an optics sub-assembly 110 is provided to accommodate confluence detecting optics system which is mounted on a tray 90 suspended from the main bed 5 by pillars 92. The under-slung optics system is arranged to view well plates placed on the imaging station 100.
The main bed 5 is provided with two main working stations, namely an imaging station 100 and a replating station 104, each of which is positioned at the end of a respective well plate feed lane. Each well plate feed lane has a well plate feeder/stacker. The well plate feeder/stacker 107 for the imaging station 100 has a well plate feed storage cassette 106 and well plate (re-)stack storage cassette 108. A stack of well plates are held in the feed storage cassette 106, fed in turn down the lane via a delidder (not shown) to the imaging station 100, returned back along the lane, relidded and passed into the rear storage cassette 108. A similar well plate feeder/stacker 113 is used for the other lane to supply well plates from the storage cassette 112 to the replating station 104 and back along the lane to the (re-)stack storage cassette 114.
The well plate feeder/stacker mechanisms including delidding are described fully in EP-A-1 293 783 [2], the contents of which are incorporated herein by reference.
The cell picking head 118 can thus be moved from the imaging station to the replating station to allow replating of animal cells from a target well plate to a destination well plate. In the illustrated embodiment, there is only one destination lane. However, it may be desirable in some cases to have 2, 3 or 4 destination lanes. This may be useful when it is desired to split the animal cells from a given target well into multiple destination wells. The feeder/stacker mechanism is fully modular, so the number of well plate feed lanes can be increased without difficulty.
The optical sub-assembly 110 comprises an illumination part and a collection part. The illumination part is formed of a plurality of white light emitting diodes (LEDs) 24 arranged to form an LED ring 26 located in a collar 28 with a central aperture 25 with the optical axes of the LEDs lying on the surface of a common cone, the point of which is coincident and labeled as the object position O in the figure. An apertured top plate 20 lying above the LED ring 26 is also illustrated. This is a structural component and has no significance for the optical design. The collection part of the optical sub-assembly is made up of a zoom lens 30 with autofocus. The optical axis is vertical and coincident with the object position O. A semi-silvered mirror 32 is also illustrated. This is for integrating a second illumination source (not shown) from the side onto the sample in order to perform fluorescence measurements.
The well to be imaged is thus aligned laterally with the optical axis of the collection optics and laterally and vertically with the center point of illumination, whereby the center point of illumination is around the base of the well or slightly higher as illustrated. The LEDs 24 thus illuminate a well 12 arranged in the object position O at an oblique angle from below so that an image of the well 12 is taken in a dark field configuration where light from the LEDs, if not scattered, does not contribute to the well image gathered by the collection lens 30.
To determine confluence, first the image is divided into background and foreground by extracting the background. This is done by applying a large Gaussian blur filter to the image, then subtracting this from the original image before adding the mean of the original image to each pixel. After this operation, pixels with intensities close to the mean of the resulting image are considered background, the remainder are considered foreground. The closeness to the mean is adjustable to accommodate variations in lighting etc. A segmented binary image is then generated by assigning foreground pixels to white and background pixels to black.
We envisage measuring the degree of confluence in one of two ways.
The first way involves counting the cells, and then assuming a value for cell area. This is usually reliable, since the variance in average cell area of a given cell type is usually small. The degree of confluence is then calculated to by the number of cells multiplied by the cell area divided by the available area of the well or other substrate, plate or dish.
The second way is to directly measure the aggregate area of all the cells by image processing of each individual cell to determine its boundary and thus area. The area of the cells can then be scaled up by a packing factor, e.g. assuming hexagonal close packing, before being divided by the available area of the well to arrive at a degree of confluence.
In the flow diagram, the image processing step, Step S4, is shown in the same loop as the image acquisition step, Step S3. It will be understood that these two steps need not be coupled in the same loop. For example the image processing can be done decoupled from the image acquisition, either one after the other, or in parallel.
The optics sub-assembly 110 is now described in more detail.
The previously described collar-mounted LED ring 24, 26, 28 is evident in all three figures. The LED collar 28 is cantilevered out on a side bracket from a vertical mounting plate 65 (
The collection lens 30 is held vertically in a mounting tube 66 (see
The optical components are thus all mounted directly or indirectly on the base plate 62. The base plate 62 is carried by a linear positioner 82 which is in turn carried by a linear positioner 74 to provide xy-motion for the whole optical set-up. In the illustration, the x-positioner 74 is at the bottom with the y-positioner mounted on top of it. However, it will be appreciated this choice is arbitrary. It will also be appreciated that a parallel mechanism xy-positioner could be provided instead of two piggy-backed linear positioners. The x-positioner 74 comprises a motor 76, lead screw 78 and a pair of sets of guide bearings 80. The y-positioner 82 is the same, comprising a motor 84, lead screw 86 and a pair of sets of guide bearings 88.
As an alternative to having colored LED of different colors arranged in filter positions on a filter wheel as described above, it is possible to have concentric rings of different colors of LED in a single mounting. For example, the white light LED ring could be exchanged or supplemented with a number of LED rings of different colors. In principle an arbitrary arrangement of LEDs of different colors would provide the same functionality so long as LEDs of different colors could be driven independently, but would be a less elegant design. It would also be possible to use a single group of broadband LEDs in combination with filtering. However, this approach would tend to provide less illumination power than using different colors of LED. It will also be appreciated that other optical sources could be used including superfluorescent LEDs or diode lasers. Fixed wavelength or tunable diode lasers may be used.
In the present embodiment, the inner pin 164 has an inside diameter of 0.7 mm an outside diameter of 1.07 mm. The outer pin 162 is 35 mm long and has a 5 mm outer diameter, tapering to 4.2 mm at its end, and a 3.2 mm inner diameter. These dimensions are suitable for picking cell colonies or other cell aggregates of average size circa 0.5 mm.
In the position illustrated, the motor 160 is actuated to oscillate the end of the inner pin 164, thereby creating turbulence in the liquid 172 in which the cells are being cultured. An oscillation frequency of around 100 Hz has been successfully used. Other frequencies would probably also work. The forces induced by this motion have been found sufficient to detach the cells and allow aspiration of the detached cells into the hollow pin, which as mentioned above forms the end of a capillary 170. The inner pin 164 is constrained by a flange 168 which fits into the top of the outer pin 162 and has a central through hole through which the inner pin 164 passes in a push fit.
Another way of assisting detachment mechanically is by tapping, knocking or otherwise applying a mechanical shock to the well plate to dislodge cells, wherein this mechanical shock may be applied manually or through automation.
As an alternative to, or in combination with, mechanical detachment methods, adherent cells may be detached chemically, for example using buffers, salt solutions, detergents or biological materials, such as enzymes. Example media that can be placed in the wells to increase the efficiency by which cells can be dislodged are either an isotonic buffer containing different concentrations of divalent ions, or a buffer containing enzymes such as trypsin or proteases for releasing the cells from solid substrate. These media can be dispensed by a tube on the robotic head from the reservoir. After a period of incubation, normally between 5 and 20 minutes, a further medium may be added from another tube on the robotic head to stop the dissociation process. This isotonic medium may contain protein or divalent cations. The cell suspension can then be aspirated by a further tube on the robotic head and a measured aliquot of the cells dispensed into one or more wells in a destination well plate or multiple destination well plates. To assist incubation of an enzyme used to promote detachment, the well plate can beneficially be provided with a heated carrier element, such as a platen. For example, trypsin can be maintained at around 37 degrees Celsius to speed up its activity.
The above description has taken the example of an adherent cells. It will be understood that when cells are not adherent, a simplified form of the same process can be carried out with the steps associated with detaching an adherent cells being omitted. It will also be understood that some of the parts of the apparatus are redundant in the case that mechanical detachment of adherent cells is not needed and these parts could be omitted. For example, the outer pin could be omitted as well as the motor and associated drive parts.
In use, the valve 185 is controlled as follows. When the valve 185 is in its rest state with no inputs, the N.O. port 193 is open and the N.C. port 189 is closed. This connects the reciprocating pump 183 to the fluid vessel 103 so that it can draw liquid out of the reservoir by suitable downward motion of the pump piston in its cylinder. On the other hand, when the valve 185 is in its actuated state with an energizing input signal, the N.O. port 193 is closed and the N.C. port 189 is open. This connects the reciprocating pump 183 to the pin 126 allowing the liquid column in the fluid path formed by elements 126, 128, 197 and 195 to be moved in either direction by motion of the pump cylinder. This provides the fine control for the aspiration and expulsion of animal cells shown schematically in
This completes Step S7.
It will be understood that well plates with no wells identified as having reached threshold confluence will be returned to an incubator. This may be performed manually or in a semi- or fully automated way.
The process can be implemented as an expansion process to populate multiple well plates from a single well plate, optionally in multiple stages, such as 1 to 4, 4 to 16 etc. At each stage the well size may be increased, for example by using 96-well well plates in stage 1, 24-well well plates in stage 2, 6-well well plates in stage 3 and a single-well well plate or Petri dish (or other biological sample container) in stage 4. It can also be implemented as a consolidation process whereby positives (i.e. wells measured to be above threshold confluence) from a number of well plates are transferred into a sub-set of well plates, perhaps only a single well plate. It can also be implemented as a stratification process, whereby wells measured to be above threshold confluence are transferred to different target well plates depending on how rapidly they reached threshold, or depending on some other parameter. This can be used to separate fast, medium and slow growing cells or colonies of cells for example.
It will be appreciated that reference to well plates should be construed to include any receptacle in which the concept of confluence as described above is relevant.
It will be appreciated that although particular embodiments of the invention have been described, many modifications/additions and/or substitutions may be made within the spirit and scope of the present invention.
