Patent Publication Number: US-2021163875-A1

Title: Cell imaging with growth matrices

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation-in-part of and claims benefit to U.S. patent application Ser. No. 15/603,184 filed on May 23, 2017, the entire disclosure of which is hereby expressly incorporated herein by reference. U.S. patent application Ser. No. 15/603,184 claims priority under 35 U.S.C. § 119(a) to GB Application No. 1609508.5, which filed on May 31, 2016. Accordingly, GB Application No. 1609508.5 is hereby incorporated by reference in its entirety. 
    
    
     TECHNICAL FIELD 
     The present disclosure relates to cell imaging. 
     BACKGROUND 
     It is often desirable to be capable of dispensing exactly one cell from a container of cells into a receptacle (such as a well of a microtiter well plate). For example, medical research such as for drug approval might call for the cultivation of a single cell. This process usually involves providing a reservoir containing a growth medium in which a concentration of cells are provided, and using a pump together with a dispensing tube whose width is very slightly larger than a single cell, to take a sample from the reservoir and place it in to its own receptacle. The size of each sample and the concentration of the cells in the growth medium in the reservoir are chosen so that, with some degree of probability, a sample will contain a single cell. Each sample is then cultivated over a period of time (e.g. 72 hours). In this way, a number of cultivations take place and the results can be averaged. However, this process suffers from a number of drawbacks. Firstly, there is no guarantee that any given sample will contain a cell. Such cultivation attempts therefore fail. If the number of successful cultivations is too low, the entire process may be considered a failure and may have to begin again. Secondly, any given sample might contain more than one cell. This is especially true if the concentration is increased in order to inhibit samples from containing zero cells. However, in this case, the cultivation results might be incorrect, since they refer to the cultivation of multiple cells rather than the cultivation of a single cell. Worse still, it may not be possible to determine that multiple cells were dispensed in the first place. It would therefore be desirable to improve the chances of performing cultivation on a single cell. This process can be made more complicated for certain types of tissue cell (e.g. stem cells) where a matrix is required in order to promote growth of the cell culture. 
     SUMMARY 
     Viewed from a first example configuration, there is provided an apparatus comprising: a receptacle configured to receive a sample; a sample dispenser configured to dispense the sample into the receptacle; an image capture device configured to capture an image of the sample in the receptacle; processing circuitry configured to process the image to determine whether the receptacle contains zero cells, exactly one cell, or more than one cell, wherein in response to the processor determining that the receptacle contains zero cells, the processing circuitry causes the sample dispenser to dispense a further sample into the receptacle; and a matrix dispenser configured to dispense a matrix into the receptacle, wherein the matrix is only added to the receptacle after the image capture device has captured the image of the sample in the receptacle and the processing circuitry had processed the image and determined that the receptacle contains exactly one cell. 
     Viewed from a second example configuration, there is provided a machine-implemented method comprising: dispensing a sample into a receptacle; capturing an image of the sample in the receptacle; processing the image to determine whether the receptacle contains zero cells, exactly one cell, or more than one cell; in response to determining that the receptacle contains zero cells, the causing the sample dispenser to dispense a further sample into the receptacle; dispensing a matrix into the receptacle, wherein the matrix is only added to the receptacle after the image of the sample in the receptacle has been captured and after the image has been processed and after it has been determined that the receptacle contains exactly one cell. 
     Viewed from a third example configuration, there is provided an apparatus comprising: means for dispensing a sample into a receptacle; means for capturing an image of the sample in the receptacle; means for processing the image to determine whether the receptacle contains zero cells, exactly one cell, or more than one cell, wherein in response to determining that the receptacle contains zero cells, the means for dispensing the sample is caused to dispense a further sample into the receptacle; means for dispensing a matrix into the receptacle, wherein the matrix is only added to the receptacle after the image of the sample in the receptacle has been captured and after the image has been processed and after it has been determined that the receptacle contains exactly one cell. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
       The present technique will be described further, by way of example only, with reference to embodiments thereof as illustrated in the accompanying drawings, in which: 
         FIG. 1  illustrates an example apparatus in accordance with some embodiments; 
         FIG. 2  shows a receptacle after dispensing a first sample having zero cells and a second sample having one cell; 
         FIG. 3  shows a receptacle after dispensing a first sample having zero cells and a second sample having multiple cells; 
         FIG. 4  shows a flowchart illustrating a method of dispensing in accordance with some embodiments; 
         FIG. 5  shows a flowchart illustrating a method of dispensing in which a user is warned in the event that the concentration of the mixture appears to be incorrect; 
         FIG. 6  illustrates an example apparatus in accordance with some embodiments; 
         FIG. 7  shows a flowchart illustrating a method of dispensing in accordance with some embodiments; and 
         FIG. 8  shows a flowchart illustrating a method of dispensing in accordance with some embodiments. 
     
    
    
     DETAILED DESCRIPTION 
     Before discussing the embodiments with reference to the accompanying figures, the following description of embodiments and associated advantages is provided. 
     In accordance with one example configuration there is provided an apparatus comprising: a receptacle configured to receive a sample; a sample dispenser configured to dispense the sample into the receptacle; an image capture device configured to capture an image of the sample in the receptacle; processing circuitry configured to process the image to determine whether the receptacle contains zero cells, exactly one cell, or more than one cell, wherein in response to the processor determining that the receptacle contains zero cells, the processing circuitry causes the sample dispenser to dispense a further sample into the receptacle; and a matrix dispenser configured to dispense a matrix into the receptacle, wherein the matrix is only added to the receptacle after the image capture device has captured the image of the sample in the receptacle and the processing circuitry had processed the image and determined that the receptacle contains exactly one cell. 
     A sample, containing zero, one, or more than one cell is dispensed into a receptacle. By using an image capture device (e.g. a camera) to capture an image of the sample when it is in the receptacle (e.g. a well of a microtiter well plate), and by subsequently processing the image, it is possible to determine whether the dispensed sample contains zero cells, one cell, or more than one cell. A further sample is dispensed if it is determined that the receptacle contains zero cells. One a cell has been dispensed, a matrix is also dispensed into the receptacle. Note that since the matrix (e.g. in a solution) is only added to the receptacle after it has been determined that the receptacle contains exactly one cell, the matrix itself does not interfere with the imaging process (e.g. by occluding the sample, or by reflecting light for instance). 
     In some examples, the matrix dispenser is configured to dispense a mixture comprising the matrix and media. That is, the matrix and the media could be combined so that they are dispensed at the same time using the same equipment. In other embodiments, the matrix could be provided separately. 
     In some examples, the media comprises conditioned media. Conditioned media can be considered to be media that has previously been used to grow cells (e.g. stem cells). Having been used for cell growth before, such substances have a tendency to encourage cell growth. 
     In some examples, the matrix is a cell culture matrix. A cell culture matrix can be used to provide a ‘scaffolding’ on which cell growth can occur. 
     In some examples, the matrix comprises MatriClone. MatriClone is a type of cell growth matrix (specifically a recombinant laminin) used for Embryonic Stem (ES) or Induced pluripotent stem cell (iPSC) growth. In other embodiments, the matrix uses Matrixome technology developed by Professor Kiyotoshi Sekiguchi. 
     In some examples, the exactly one cell is a tissue cell. For instance, in some embodiments, the exactly one cell is a stem cell. Tissue cells (such as stem cells) benefit greatly from the presence of a cell matrix in order to grow. In particular, the absence of such a matrix could make it difficult or even impossible for cell growth to occur with these types of cells. Cell growth could be achieved by coating the receptacle (e.g. the well) with a matrix such as Vitronectin, and then supplying growth medium and the single cell. However, such coatings can interfere with imaging by either obscuring the contents of the receptacle (particularly if imaging occurs from underneath the receptacle) or by reflecting light. Hence, in these embodiments, the matrix (in the form of a solution) is only added to the receptacle after imaging has been carried out and after it is determined that exactly one cell is within the receptacle. 
     In some examples, the imaging process is performed from underneath the receptacle. The presence of the matrix can obfuscate imaging of the receptacle and can make it more difficult to determine the contents of the receptacle—particularly when only a single cell might be present. Consequently, by adding the matrix to the receptacle only after the imaging has been performed, it is possible to accurately determine whether a single cell has been dispensed (as required for regulatory approval) while also providing a matrix for cell growth to occur. 
     In some embodiments, the apparatus further comprises an actuator to move the sample dispenser relative to the receptacle, wherein in response to the processor determining that the receptacle contains zero cells, the actuator moves the receptacle relative to the sample dispenser to dispense a further sample into the receptacle. The movement performed by the actuator is very slight and continues to position the dispenser such that samples will be dispensed into the same receptacle. However, by performing such a slight movement, it is possible to reduce the chances of multiple samples being dispensed on top of each other. Accordingly, it is less likely that one cell will eclipse another and so it is more likely that the processing circuitry will correctly determine whether the receptacle contains zero cells, one cell, or more than one cell once the further samples has been dispensed into the receptacle. For example, the movement might be such that the dispenser&#39;s original location as adjusted by the movement would still cause the further sample to be dispensed into the same receptacle. 
     In some embodiments, in response to the processing circuitry determining that the receptacle contains more than one cell, the processing circuitry is configured to perform at least one of: notifying a user that the receptacle comprises more than one cell, and storing data to indicate that the receptacle comprises more than one cell. As previously explained, it is undesirable for a receptacle to contain more than one cell, since this can produce erroneous results. However, if it is known, ahead of time, that a receptacle contains more than one cell, then an error action can be taken in order to mitigate the effect of multiple cells in a single receptacle. In some examples, the error action comprises notifying a user that the receptacle comprises more than one cell. The user can therefore take appropriate action to either disregard that particular receptacle or perhaps clear the receptacle so that a further attempt at dispensing a sample having a single cell can be made. In other examples, the error action comprises storing data to indicate that the receptacle comprises more than one cell. Such information could be reported to the user at the end of the process. Alternatively, if further operations are performed on the receptacle, the stored information could be used as an internal reference to disregard results or not perform operations on the receptacle in question. It will be appreciated that the error action may comprise a number of sub-actions including those mentioned here, together with others that would occur to the skilled person. 
     In some embodiments, the apparatus further comprises an actuator to move a plurality of receptacles, including the receptacle, relative to the sample dispenser; in response to the processing circuitry determining that the receptacle contains one or more cells, the processing circuitry is to cause relative movement between the sample dispenser and the plurality of receptacles so that the sample dispenser dispenses subsequent samples into a different receptacle in the plurality of receptacles. In this way, when it is determined that a receptacle contains zero cells, a further sample is dispensed into the receptacle. However, when it is determined that a receptacle contains one or more cells, the dispenser is moved so that future samples are dispensed into a different receptacle. Hence, a number of receptacles such as the wells in a microtiter well plate can be efficiently filled in such a manner that cultivation is more likely to occur using a single cell in each of the receptacles. 
     In some embodiments, in response to the processing circuitry determining that a first predefined proportion of dispensed samples each contain zero cells, the processing circuitry is configured to perform at least one of: notifying a user, and causing the volume of each dispensed sample to be increased. In such cases, if a predefined proportion of dispensed samples contain zero cells, then it is likely that the concentration of cells is inappropriate (e.g. too low). It can therefore be helpful to alert the user, who can thereby confirm whether or not the concentration is appropriate and correct the concentration if necessary. The predefined proportion could be based on an overall percentage (e.g. 85%), a fixed number (e.g. 5), a sliding window (6 out of the last 10), an average number of cells (0.7) dispensed per sample, or another statistical representation that will be known to the skilled person. Alternatively, or as well as alerting the user, the volume of each dispensed sample can be increased. As a consequence of increasing the volume of each dispensed sample, it is more likely that a given sample will contain a cell. 
     In some embodiments, a plurality of initially dispensed samples including the first predefined proportion of dispensed samples is dispensed into a subset of the plurality of receptacles, wherein the subset of the plurality of receptacles is less than the first predefined proportion of dispensed samples. Consequently, a small number of receptacles (e.g. one) is used in order to determine the concentration of the mixture and take action if the concentration is too low. In particular, the size of the subset is less than the first predefined proportion of dispensed samples such that fewer receptacles are used as compared to the number of samples that are dispensed, thereby reducing the number of receptacles required to determine if action need be taken. 
     In some embodiments, in response to the processing circuitry determining that a second predefined proportion of dispensed samples each contain more than one cell, the processing circuitry causes a user to be alerted. Another indicator that the concentration of cells is incorrect is if a (second) predefined proportion of dispensed samples each contain more than one cell. In this case, this would suggest that the concentration was too high. The second predefined proportion could be based on an overall percentage (e.g. 85%), a fixed number (e.g. 5), a sliding window (6 out of the last 10), an average number of cells (2.7) dispensed per sample, or another statistical representation that will be known to the skilled person. Alternatively, or as well as alerting the user, the volume of each dispensed sample can be decreased. As a consequence of decreasing the volume of each dispensed sample, it is less likely that a given sample will contain a cell. 
     In some embodiments, a plurality of initially dispensed samples including the second predefined proportion of dispensed samples is dispensed into a subset of the plurality of receptacles, wherein the subset of the plurality of receptacles is less than the second predefined proportion of dispensed samples. Consequently, a small number of receptacles (e.g. one) is used in order to determine the concentration of the mixture and take action if the concentration is too high. In particular, the size of the subset is less than the second predefined proportion of dispensed samples such that fewer receptacles are used as compared to the number of samples that are dispensed, thereby reducing the number of receptacles required to determine if action need be taken. 
     It will be appreciated that in the above embodiments, the same dispensed samples can be used to both determine if the first predefined proportion of dispensed samples each contain zero cells and/or if the second predefined proportion of dispensed samples each contain more than one cell. In other words, a number of samples could be dispensed into a subset of the receptacles (less than the number of samples) in order to determine whether the number of cells being dispensed is too high or too low, and thereby alert the user and/or adjust the volume of each dispensed sample to compensate. 
     In some embodiments, the apparatus further comprises output circuitry to output the image. Examples of such output circuitry could include a printer, a hard disk, or other form of storage. Accordingly, the image can be used to illustrate what the resulting cultivation started from. In the case where the image is of a single cell, this can be used as evidence that the resulting cultivation occurred from a single cell. Note that in some cases, the image can be stored as part of the error action that takes place if more than one cell is detected in the well. 
     In some embodiments, the apparatus further comprises a reservoir to hold a plurality of samples, connected to the sample dispenser; and an agitator to agitate the plurality of samples in the reservoir such that cells within the plurality of samples are distributed. The agitator could, for example, be an oscillator or simply a mixing apparatus to continually distribute cells in the reservoir. This can be used to prevent the cells from settling to the bottom of the reservoir and helps to maintain an equal distribution/dispersal of cells such that the probability of a sample containing a cell will be approximately in accordance with the concentration of cells in the reservoir. 
     Particular embodiments will now be described with reference to the figures. In the following detailed description of embodiments of the invention, numerous specific details are set forth in order to provide a more thorough understanding of the invention. However, it will be apparent to one of ordinary skill in the art that the invention may be practiced without these specific details. In other instances, well-known features have not been described in detail to avoid unnecessarily complicating the description. 
     In the following description of  FIGS. 1-8 , any component described with regard to a figure, in various embodiments of the invention, may be equivalent to one or more like-named components described with regard to any other figure. For brevity, descriptions of these components will not be repeated with regard to each figure. Thus, each and every embodiment of the components of each figure is incorporated by reference and assumed to be optionally present within every other figure having one or more like-named components. Additionally, in accordance with various embodiments of the invention, any description of the components of a figure is to be interpreted as an optional embodiment, which may be implemented in addition to, in conjunction with, or in place of the embodiments described with regard to a corresponding like-named component in any other figure. 
       FIG. 1  illustrates an example apparatus  100 . The apparatus  100  includes a well  105  (an example of a receptacle), which forms part of a microtiter well plate  110  together with a plurality of other wells. The well  105  is able to move along a track  115  by virtue of the microtiter well plate moving along the track  115 , the microtiter well plate being placed on a carriage connected to an actuator such as a screw turned by a motor controlled by a control system. A reservoir  120  contains a mixture  125  comprising a number of cells mixed together with a growth medium. A biological agitator  130  (an example of the claimed agitator) agitates the mixture  125  so as to evenly distribute the cells within the growth medium and discourage the cells from settling in the reservoir  120 . A pump  135  is provided to extract a small quantity of the mixture  125  (a sample) from the reservoir and to dispense the sample through a tube  140  into a well  105 . In this embodiment, the well  105  is located beneath the tube  140 . The tube  140  is such that it is just wide enough to pass one of the cells. Accordingly, for a given concentration of cells in the mixture  125  and for a given sample size, there is a probability with which a sample will contain a single cell  145 . In this example, the pump  135  and tube  140  collectively make up a dispenser. An image capture device in the form of a camera  150  captures an image of the sample in the well  105  once the sample has been dispensed into the well  105 . Processing circuitry  155  processes the image and determines, through image analysis, whether the sample or samples in the well  105  contain zero cells, one cell, or more than one cell. The action that is subsequently taken depends on which of these three conditions is met. 
     In the event that zero cells are detected, the well  105  and the tube  140  are moved relative to one another while still keeping a position such that a subsequent sample will be dispensed into the well  105 . In other words, the well  105  and the tube  140  are moved relative to each other such that a subsequent sample will be dispensed into a different part of the well  105 . In this example, the relative movement occurs by the microtiter well plate  110  being moved slightly along the track  115 . 
     In the event that one cell is detected, growth medium  160  stored in a secondary reservoir  165  is provided by using a secondary pump  170  and a secondary dispensing tube  175 . Sufficient growth medium is provided to the well  105  in order to encourage cultivation of cells whilst not over-filling the well  105 . By providing the majority of the growth medium  160  after it has been established that the well  105  includes a single cell, it is possible to dispense numerous samples without the well  105  overfilling. Furthermore, due to the limited amount of growth medium in the well, it is less likely that a cell will be carried to one of the walls of the well  105 , which would make accurate image analysis more difficult or even impossible. Once the growth medium  160  has been dispensed, the microtiter well plate  110  is moved so that a subsequent sample will be dispensed into an unused well, i.e. a well that has not had any samples dispensed into it during the process. Furthermore, the image of the sample in the well  105  is stored in a storage medium  180  for later retrieval by the user. 
     In the event that more than one cell is detected, an error action is performed. In this embodiment, the error action includes making note of the particular well  105  into which the sample was dispensed. For example a number or other identifier that uniquely identifies the well  105  in the microtiter well plate  110  is made. At the end of the overall process, the user is informed of those wells that were marked. In this embodiment, the image of the well  105  having more than one cell is stored in a storage medium  180  for later retrieval by the user. 
     In any of the above cases, unless the last well has been filled, the process is repeated, with another sample being dispensed. 
     As a consequence of the above, it is possible to reduce the number of occasions in which a well contains zero cells. Furthermore, since the majority of the growth medium is only added if and when a well contains a single cell, it is possible to add a large number of samples to an individual well without the well overflowing. Consequently, the concentration of the mixture can be lowered as compared to any similar systems since the scenario of a sample containing zero cells can be easily corrected for by dispensing further samples and the scenario of a sample containing more than one cell (which may not be easily corrected) will occur more rarely. 
       FIG. 2  shows a receptacle, e.g. well  105  after dispensing a first sample  205  having zero cells and a second sample  210  having one cell  215 . In this example, the well  105  has an area of 2.7 mm by 2.7 mm and a volume of 110 microlitres. The volume of a sample is approximately 10 nl and the concentration of the mixture is about 0.1 million per ml. After the first sample  205  is dispensed, the processing circuitry  155  determines that the sample in the well  105  contains no cell. Consequently, the dispenser and the well  105  are moved relative to each other such that a subsequent sample  210  will be dispensed in a different part of the well  105 . When the second sample  210  is dispensed, the processing circuitry  155  again processes an image of the well  105  and determines that the well  105  contains exactly one cell  215 . Accordingly, additional growth medium  160  can be provided to partly fill the well  105 . Additionally, the image of the well  110  containing a single cell is output. For example, the image can be output to a storage medium  180  or can be printed on a printer. The microtiter well plate  110  is also moved such that further samples will be dispensed into a different well. Alternatively, if the current well  105  is the last well in the microtiter well plate  110  then the process stops. 
       FIG. 3  shows a receptacle, e.g. well  105  after dispensing a first sample  305  having zero cells and a second sample  310  having two cells  315 ,  320 . In this example, again, the well  105  has an area of 2.7 mm by 2.7 mm and a volume of 110 microlitres. After the first sample  305  is dispensed, the processing circuitry  155  determines that the sample in the well  105  contains no cell. Consequently, the dispenser and the well  105  are moved relative to each other such that a subsequent sample  310  will be dispensed in a different part of the well  105 . In this example, the two samples overlap slightly. When the second sample  310  is dispensed, the processing circuitry  155  again processes an image of the well  105  and determines that the well  105  now contains more than one cell (e.g. two cells  315 ,  320 ). Although the two cells  315 ,  320  slightly overlap each other, the processing circuitry  155  determines that there is not only one cell in the well  105 . The well  105  is marked. For example, the processing circuitry  105  can immediately inform the user or alternatively can keep track of an ID number of the particular well and inform the user at the end of the process that the well should be disregarded due to having more than one cell. In any event, unless this is the final well in the microtiter well plate  110 , the microtiter well plate is moved such that subsequent samples are dispensed into a different well. 
       FIGS. 4, 5, 7, and 8  show flowcharts in accordance with one or more embodiments of the disclosure. While the various steps in the flowcharts are presented and described sequentially, one of ordinary skill will appreciate that some or all of these steps may be executed in different orders, may be combined or omitted, and some or all of the steps may be executed in parallel. In one embodiment of the invention, the steps shown in  FIGS. 4, 5, 7, and 8  may be performed in parallel with any other steps shown in  FIGS. 4, 5, 7, and 8  without departing from the invention. 
       FIG. 4  illustrates a flow chart  400  that shows a method of dispensing. In a step  405 , a sample is dispensed into the current well  105 . In a step  410 , an image of the sample in the well  105  is captured. In a step  415 , the image is analysed. It is then determined, at a step  420 , whether or not the image contains zero cells. If so, then at step  425 , the dispenser and the current well  105  are moved relative to each other such that a subsequent sample is dispensed into a different portion of the same well  105 . Otherwise, at step  430 , the image is stored for later retrieval by a user and the process proceeds to step  435 . At step  435 , it is determined whether or not the image contains more than one cell. If not, then the process proceeds to step  440 . Otherwise, at step  445 , the current well  105  is marked before the process proceeds to step  440 . At step  440 , it is determined whether or not the current well  105  is the last well in the microtiter well plate  110 . If so, then at step  450 , the process ends. Otherwise, at step  455 , the microtiter well plate  110  is moved so that subsequent samples are dispensed into a different well. The process then returns to step  405  where a sample is dispensed. 
     It is possible to keep track of the results of the image analysis over a period of time. Using this information, it is possible to make particular inferences regarding the mixture from which samples are being taken.  FIG. 5  illustrates a flowchart  500  in which such information is used. The flowchart  500  shows the process that occurs in parallel with the flowchart  400  shown in  FIG. 4 . Some of the steps, are common between the two flowcharts, as discussed below. 
     At step  505 , two counters (dispensed and cells) are initialised to zero. The counter ‘dispensed’ counts the number of samples that are dispensed and the counter ‘cells’ counts the number of cells that are detected. At step  510 , a sample is dispensed as previously shown with reference to step  405  in the flowchart  400  shown in  FIG. 4 . Additionally, the counter dispensed is incremented. At step  515 , the image is analysed as per step  415  in the flowchart  400  of  FIG. 4 . At step  520 , the counter ‘cells’ is incremented according to the number of cells detected within the current well  110 . At step  525 , it is determined whether the number of samples that have been dispensed is greater than 5. If not, the process returns to step  510 . This step helps to ensure that the results are given a chance to average out before any inferences are made. At step  530 , it is determined whether or not the ratio dispensed/cells is below a predetermined threshold, e.g.  0 . 2 . In an ideal case, each well will have a single cell placed within it. However, the disadvantage of having multiple cells in a single well is significantly worse than having zero cells in a well because in the former case, the well must be marked and disregarded whereas in the latter case, further samples can be inserted. Accordingly, even though it would be desirable to always have exactly one cell per sample, the concentration of the mixture is generally chosen so that, on average, there is substantially fewer than 1 cell per sample. For example, there might only be a 37% chance of a sample containing a cell. Accordingly, the predetermined threshold may be very low. The exact selection of the threshold could be settable by a user and will, in any event, be chosen having regard to the tradeoff of desired sensitivity to the concentration being used versus the risk of a false positive of incorrectly informing the user that the concentration seems to be incorrect. In any event, if the ratio is below the threshold then at step  540 , the user is warned. If not, then at step  535 , it is determined whether or not the ratio dispensed/cells is above a predetermined threshold, e.g.  1 . 1 . If so, then at step  540 , the user is warned. Otherwise the process returns to step  510 . 
     Of course, there are other ways that the system could infer that the concentration of the mixture is incorrect. For example, in another embodiment, the system could simply count the number of times that more than one cell is detected in a well. If this number were to exceed a threshold, or if a ratio of the number of times that more than one cell is detected in a well divided by the number of dispensed samples were to exceed a threshold, then the user could be warned. A second example could be to perform the steps outlined in  FIGS. 4 and 5  but to arrange that all the samples are dispensed in the same receptacle, by omitting step  455  and moving to a new position in the same receptacle. In this manner, a large number of dispenses can be made and analysed in a single receptacle, purely for the purposes of determining the concentration of cells in the reservoir. This approach has the advantage that only a small number of receptacles, in this case one, is required to determine whether the correct concentration of cells is present in the reservoir. Further, if it is determined that the concentration of cells present in the reservoir is incorrect, it may be possible to repeat the process but adjusting the volume of sample dispensed in order to attempt to correctly dispense a single cell at a time. At that time, single samples can be dispensed into the remaining receptacles (if the target rate is achieved) or the user can be alerted (if the target rate cannot be achieved). The volume can be adjusted either by running the pump  135  for longer or by dispensing more than one drop of the mixture for each sample. 
       FIG. 6  schematically illustrates an apparatus  600  that is used for the dispensing of tissue cells such as stem cells. The growth of stem cell cultures usually involves the provision of a cell culture matrix in the receptacle (e.g. the well  105 ) prior to the cell being dispensed. For instance, a matrix coating could be added to the well plate, and the well plate incubated at 37 degrees Celsius for an hour. The solution can then be removed from the plate (with some residue of the matrix remaining behind—coating the plastic) and the growth medium and cells can then be added to the wells. However, such a technique can be problematic in the previously described techniques because the matrix can interfere with the imaging process—either by obfuscating the view of the sample in the receptacle or by causing reflection that makes any image difficult to see. 
     Accordingly, in the present technique when tissue cells such as stem cells are being dispensed, no matrix is added to the receptacle prior to any samples being dispensed. After a sample is dispensed (A), the imaging process takes place (B) in order to determine whether exactly one cell has been dispensed or not. If no cells have been dispensed then the dispensing process (A) could be repeated until the receptacle  105  contains multiple cells (in which case the error action takes place as previous described) or until exactly one cell is within the receptacle  105 . At this point, the matrix (e.g. in the form of a solution) can be added (C). In this example, the secondary reservoir  665  contains a mixture  660  of both growth medium and cell growth matrix, and these can be added to the well  105  via the secondary pump  170  and the secondary dispensing tube  175 . In other embodiments, the medium and the matrix can be provided via different dispensers. Since the matrix is only dispensed after the imaging process has taken place and after a determination as to the number of cells has taken place, the matrix does not interfere with the imaging process. 
     There are a number of possibilities for the matrix that is provided in the combined solution  660 . In some examples, the matrix is MatriClone. In other examples, the matrix uses another substance that utilises Matrixome technology developed by Professor Kiyotoshi Sekiguchi. The media could be conditioned (“dirty”) media, which has previously been used in the growth of tissue cells. Such media can be ‘friendly’ to encouraging tissue cell growth. 
     This process is illustrated in the form of a flowchart  700  in  FIG. 7 . The process begins at step  705  where a sample is dispensed into a container/receptacle/well  105 . At a step  710 , the contents of the container/receptacle/well  105  is imaged and at a step  715 , it is determined whether exactly one tissue (stem) cell has been dispensed. If not, then the process returns to step  705 . Otherwise, at a step  720 , the matrix is dispensed into the container/receptacle/well  105 . 
       FIG. 8  illustrates a similar process in the form of a flowchart  800 . Steps  805 ,  810 ,  815 , and  820  correspond with steps  705 ,  710 ,  715 , and  720  as previously described. At a step  825 , it is determined whether a given number of cell divisions have occurred. This could be based on an estimation of the cell culture that has developed (e.g. by imaging the cell culture), or could be based on a period of time that has elapsed since the single cell was dispensed. If sufficient cell division has occurred, then the process proceeds to step  830  where further imaging of the contents of the container/receptacle/well  105  takes place in order to demonstrate that cell division occurred and that a culture of cells was generated. If insufficient divisions have occurred (e.g. if insufficient time has elapsed) then the process returns to step  825  for further time to elapse. 
     In the present application, the words “configured to . . . ” are used to mean that an element of an apparatus has a configuration able to carry out the defined operation. In this context, a “configuration” means an arrangement or manner of interconnection of hardware or software. For example, the apparatus may have dedicated hardware which provides the defined operation, or a processor or other processing device may be programmed to perform the function. “Configured to” does not imply that the apparatus element needs to be changed in any way in order to provide the defined operation. 
     Although illustrative embodiments of the invention have been described in detail herein with reference to the accompanying drawings, it is to be understood that the invention is not limited to those precise embodiments, and that various changes, additions and modifications can be effected therein by one skilled in the art without departing from the scope and spirit of the invention as defined by the appended claims. For example, various combinations of the features of the dependent claims could be made with the features of the independent claims without departing from the scope of the present invention.