A process and system for cell culture automation

Abstract

A method and system are disclosed for releasing adherent cells in a cell container with a plurality of acoustic transducers. The cell container includes a surface. The cell container contains a medium and cells adherent to the surface of the cell container. A first acoustic field is generated by controlling the plurality of acoustic transducers. The first acoustic field generates a first acoustic effect to release a first group of cells from a first portion of the surface of the cell container. A second acoustic field is generated by controlling the plurality of acoustic transducers. The second acoustic field generates a second acoustic effect to release a second group of cells from a second portion of the surface of the cell container.

Description

[0001] A PROCESS AND SYSTEM FOR CELL CULTURE AUTOMATION

[0002] FIELD OF THE INVENTION

[0003] The present invention is in the field of cell culture automation. Specifically, in the field of cell passaging and cell manipulation within cell culture automation.

[0004] BACKGROUND OF THE INVENTION

[0005] Cell passaging is the procedure of harvesting cells from a culture in a container and transferring the cells to one or more culture vessels with fresh growth medium. Released cells may then be used to start new cultures. Released cells may alternatively be put into new containers for assays (which could be used in further assays) or could be put directly into a new assay (e.g. a dye or an instrument such as a mass spectrometer). Cell culture automation may be used to automate the cell passaging procedure. Cell passaging is the procedure of harvesting cells from a culture, transferring the cells to one or more culture vessels with fresh growth medium, and using those cells to start new cultures. It is common for a transfer step to involve the separation of a portion of the cell culture and the placing into one or more additional containers, e.g., aliquoting, or sub-aliquoting. That is, a known step in a cell passaging process is a transfer step to transfer the plurality of cells from the cell container into one or more additional cell containers.

[0006] When growing cells, it is desirable to keep them sterile while moving them from one container to another container. Typical cell passaging process involves multiple manual processes which are difficult to automate. In addition, growing cells often adhere to the surface which they are grown in. Unsticking adherent cells is a challenge in cell culture automation.

[0007] In a typical cell passaging process cells in suspension may be transferred from a dish (i.e., a cell container) to a tube to be inserted into a centrifuge in order to agglomerate cells into a concentrated cluster called a pellet. The existing medium is then removed and new medium added. The cells are then agitated to release them back into suspension. This centrifuge and resuspension process is done before seeding the cells into a new dish. In addition, shaker plates have been used in automation processes, but these are often unsatisfactory. Transferring cells to a tube and moving the tube to a centrifuge both create bottlenecks in the automation process & requires a larger, more complex system. Moving a cell container to an incubator also creates a bottleneck in the automation process & requires a larger, more complex system. Centrifuges can cause vibrations which interfere with robotic systems. Shaker plates are also unsatisfactory as they poorly recreate manual agitation and require optimisation. Cell culture plates are generally open topped meaning there is a limit on the agitation that a shaker plate can provide before spilling media. Shaker plates provide a very different type of agitation to the more effective manual processes. Robot arms are not designed to shake plates or bang culture plates on a lab bench. Manual processes often use vortex mixers, but these are difficult to integrate into a robotic system and can cause similar vibration problems to centrifuges. Sometimes cells grow attached to surfaces. Chemical dissociation reagents may be used to release cells from these surfaces into suspension during the culturing process, prior to the above centrifugation step. Often the reagents alone are not sufficient. Often some agitation is used manually to unstick the cells, but this is difficult to replicate in an automated setting. Manual intervention is often required such as “drumming” on the bottom of a cell culture dish or hitting the cell culture containers against a lab bench to help release the cells. Vigorous pipetting can also be used to help release the cells. These manual processes have a host of problems including unreliability, being time consuming, and being a source of contamination.

[0008] The existing acoustic approaches have many problems but none of them currently offer easy integration to standard cell culture containers and live cell adherence release.

[0009] SUMMARY OF THE INVENTION

[0010] A first aspect of the invention provides a method for releasing adherent cells in a cell container with a plurality of acoustic transducers, wherein the cell container comprises a surface, wherein the cell container contains a medium and cells adherent to the surface of the cell container the method comprising: generating a first acoustic field by controlling the plurality of acoustic transducers, wherein the first acoustic field generates a first acoustic effect to release a first group of cells from a first portion of the surface of the cell container; and generating a second acoustic field by controlling the plurality of acoustic transducers, wherein the second acoustic field generates a second acoustic effect to release a second group of cells from a second portion of the surface of the cell container. The first acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers to generate the first acoustic effect. The second acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers to generate the second acoustic effect. The first acoustic effect is a focus point and the second acoustic effect is a focus point.

[0011] Optionally, the first group of cells is different to the second group of cells. Optionally, the first portion of the surface is different to the second portion of the surface. Acoustic fields of the first aspect enable cells to be released over a large area of the cell container or the whole plate. Acoustic parameters may be tuned to ensure that the cells released have a high viability (i.e. are alive).

[0012] The method of the first aspect is independent of the resonant frequency of the cell container (or dish comprising the cell container). This makes the system more flexible and therefore easily compatible with different types of cell containers or different dishes of the same type if there are manufacturing differences. The method of the first aspect allows the cell container to be easily inserted or removed. The method of the first aspect allows the dish to be in different locations relative to the source of the acoustic field.

[0013] The method of the first aspect allows the removal of cells in a certain portion (i.e., area) of the cell container depending on the requirements. This may be useful for only releasing some of the cells to allow the others to regrow without requiring a new cell container, either to maintain a cell culture at a certain confluency or to test how the remaining cells regrow. This may also be useful for only releasing certain cells if it is desired for them to be removed.

[0014] Advantageously, the method of the first aspect reduces the contamination risk of cells when compared to manual methods of agitation (e.g., scraping). The method of the first aspect reduces the risk of spilling the cell container when compared to manual agitation or a shaker plate. The method of the first aspect removes a requirement for cells to be placed into an incubator of a cell culture automation process (e.g., cell passaging), which removes a bottleneck in automation. The method of the first aspect is at least two times faster than classical incubator approaches. The method of the first aspect can remove the requirement for providing a dissociation reagent, some of which (e.g., enzymes) may damage cell surface receptors and / or alter cell behaviour. Thus, removing or reducing the requirement for providing a dissociation reagent may result in better quality cells.

[0015] Advantageously, the release of cells in a cell container can be carefully controlled and may be easily reprogrammed by a control unit. Specifically, without requiring a complex submerged motion control stage or any other moving parts. Advantageously, the distributions of the acoustic fields or effects across the cell container can be even. The parameters of the method of the first aspect may be easily adapted for different cell container types (either different sizes or different brands of the same size). Controlling the phase of the acoustic transducers may allow for the creation of pattern of cells in the dish for bioengineering, tissue engineering or other cell patterning purposes.

[0016] Optionally, the first acoustic effect is at least one of: an acoustic vortex; an acoustic twin-trap; or, an acoustic bottle field. Optionally, the second acoustic effect is at least one of: an acoustic vortex; an acoustic twintrap; or, an acoustic bottle field. Optionally, the first acoustic field is generated by controlling an amplitude of a first acoustic transducer of the plurality of acoustic transducers to generate the first acoustic effect. Optionally, the second acoustic field is generated by controlling an amplitude of a second acoustic transducer of the plurality of acoustic transducers to generate the second acoustic effect. Optionally, the amplitude of the first acoustic transducer is different to the amplitude of the second acoustic transducer. Optionally, the first acoustic transducer is the second acoustic transducer. Optionally, the first acoustic transducer is different to the second acoustic transducer. Optionally, the first acoustic field is generated by controlling each amplitude of one or more first acoustic transducers of the plurality of acoustic transducers to generate the first acoustic effect, and wherein the second acoustic field is generated by controlling each amplitude of one or more second acoustic transducers of the plurality of acoustic transducers to generate the second acoustic effect. Optionally, the one or more first acoustic transducers and the one or more second acoustic transducers are controlled to have different amplitudes.

[0017] Optionally, the first acoustic field is generated at a different time to the second acoustic field. Optionally, the method further comprising, prior to the generation of the first and second acoustic fields: providing a dissociation reagent to the cell container. The combination of acoustic field and chemical cues (including dissociation enzymes or non-enzymatic chemicals) may be tuned to ensure that the highest possible live cell recovery, i.e., a high cell viability and high recovery of the original number of cells. Optionally, the plurality of acoustic transducers is arranged linearly and in a plane. Optionally, the plurality of acoustic transducers is arranged in a 2-dimensional pattern in the plane. Optionally, the method further comprises generating a third acoustic field by controlling the plurality of acoustic transducers, wherein the third acoustic field generates a third acoustic effect to release a third group of cells from a third portion of the surface of the cell container. Optionally, the plurality of acoustic transducers are arranged to generate a series of acoustic effects upon a plurality of portions of the surface. Optionally, the area corresponding to the plurality of portions of the surface is substantially equal to the total area of the surface. Optionally, the plurality of portions of the surface comprise the first, second, and third portions of the surface. Advantageously, using a large active acoustic area (proportional to the size of the cell container) allows the release of most of the cells in the cell container.

[0018] Optionally, each of the plurality of acoustic transducers are excited continuously between the generation of the first and second acoustic fields. Optionally, the first acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers to generate the first acoustic effect comprising a complex acoustic effect at the first position. Optionally, the second acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers to generate the second acoustic effect comprising the complex acoustic effect at the second position. Optionally, each of the plurality of acoustic transducers is controlled to move the complex acoustic effect between the first and second position. Optionally, each of the plurality of acoustic transducers are turned off between the generation of the first acoustic field and the second acoustic field. Optionally, the method further comprises adjusting the temperature of an acoustic coupling arranged between the plurality of acoustic transducers and the surface of the cell container. Optionally, the acoustic coupling is suitable for transmitting the generated acoustic fields to the surface of the cell container.

[0019] Optionally, the method further comprises capturing an image of the cell container to determine the cell container contains a medium and cells adherent to the surface of the cell container. Optionally, the method further comprises determining that the first portion of the surface of the cell container comprises the first group of cells adherent to the surface of the cell container based on the image. Optionally, the method further comprises generating the first acoustic field in response to determining that the first portion of the surface of the cell container comprises the first group of cells. Optionally, the method comprises observing the behaviour of the cells in response to the acoustic fields and changing the fields in response to the observing of the behaviour. Advantageously, using a dynamically controlled system, calibration for new types of cells may be done easily, e.g., via an iterative method.

[0020] The method of the first aspect may be a computer implemented method.

[0021] Optionally, the method further comprises generating a temporal series of acoustic fields by controlling each of the plurality of acoustic transducers. Optionally, the temporal series of acoustic fields generates a temporal series of acoustic effects upon a plurality of portions of the surface of the cell container suitable for releasing cells from the plurality of portions of the surface of the cell container. Optionally, the temporal series of acoustic fields comprises the first and the second acoustic fields. Optionally, the series of acoustic effects comprises the first and second acoustic effects. Optionally, the plurality of portions of the surface of the cell container comprise the first and second portions of the surface of the cell container.

[0022] A second aspect of the invention provides a system to release adherent cells from a surface, the system comprising: a cell container comprising a surface, wherein the cell container is suitable for containing a medium and cells adherent to the surface of the cell container; and, a plurality of acoustic transducers suitable for generating acoustic fields.

[0023] Optionally, the processor is configured to perform the method of the first aspect. Optionally, the system of the second aspect further comprises an acoustic coupling arranged between the plurality of acoustic transducers and the surface of the cell container, wherein the acoustic coupling is suitable for transmitting the generated acoustic fields to the surface of the cell container.

[0024] Optionally, the acoustic coupling comprises at least one of: a liquid; a hydrogel; a gel; a solid; an acoustic metamaterial or other designed material with a different speed of sound to the rest of the coupling to enable a phase and / or amplitude variation (e.g., an acoustic hologram or lens), or other designed material with a different effective speed of sound to the rest of the coupling to enable a phase and / or amplitude variation. Optionally, the system of the second aspect further comprises a temperature sensor, and a temperature adjusting means to adjust the temperature of the acoustic coupling based on the temperature sensor. Optionally, the plurality of acoustic transducers is arranged linearly and in a plane. Optionally, the plurality of acoustic transducers is arranged in a 2-dimensional pattern in a / the plane. Optionally, each of the plurality of acoustic transducers is arranged to operate at a frequency greater than 1 MHz. Optionally, the cell container comprises a plurality of wells. Optionally, each of the plurality of wells is suitable for containing a medium and cells adherent to the surface of the cell container. Advantageously, control of the acoustic fields of the first and / or second aspects may be dynamically controlled such that only certain portions of the cell container may be released. This may be useful in a multi-well container (i.e., a multi well plate) for only releasing certain wells.

[0025] Optionally, the system of the second aspect further comprises a multi-well plate. Optionally, the cell container is a well of the multi-well plate.

[0026] A third aspect of the invention provides a liquid handling system comprising the system of the second aspect.

[0027] A fourth aspect of the invention provides a cell passaging process, for transferring cells from a cell container containing a medium and the cells, the cell passaging process comprising: a cell concentration step comprising: generating one or more first acoustic fields by controlling a plurality of acoustic transducers; increasing the density of cells in a first portion of the cell container above a density threshold using the one or more first acoustic fields; and, decreasing the density of cells in a second portion of the cell container using the one or more first acoustic fields; and, removing a first amount of the medium from the second portion of the cell container.

[0028] Optionally, the first portion of the cell container is different to the second portion of the cell container. Acoustic fields of the fourth aspect enable cells to be moved within the cell container for the purpose of removing media from the cell container without substantially removing cells.

[0029] The method of the fourth aspect allows the cell container to be easily inserted or removed. The method of the fourth aspect allows the dish to be in different locations relative to the source of the acoustic field. Advantageously, the method of the fourth aspect reduces the contamination risk of cells when compared to manual steps of cell passaging. The method of the fourth aspect reduces the risk of spilling the cell container when compared to some cell passaging steps involving manual agitation or a shaker plate.

[0030] Optionally, the density threshold is at least a cell density of: 0.8 million cells per millilitre (cells / ml), 0.5 million cells / ml, 0.4 million cells / ml, 0.3 million cells / ml, 0.2 million cells / ml, 0.1 million cells / ml, or 0.05 million cells / ml. Optionally, the one or more first acoustic fields comprise: a one or more holding acoustic fields to substantially maintain the density of cells in the first portion of the cell container after the density threshold is reached. Optionally, the one or more first acoustic fields comprise: a one or more holding acoustic fields to substantially maintain the density of cells in the first portion of the cell container after the density threshold is reached against the flow of removing a first amount of the medium. Optionally, the first portion of the cell container comprises a side wall of the cell container. Optionally, the one or more first acoustic fields comprise: a first acoustic field to generate a first acoustic effect to move a first plurality of cells relative to the cell container and towards the first portion of the cell container; and a second acoustic field to generate a second acoustic effect to move a second plurality of cells relative to the cell container and towards the first portion of the cell container. Optionally, the second plurality of cells comprises the first plurality of cells. Optionally, the first plurality of cells is the second plurality of cells. Optionally, the first plurality of cells is different from the second plurality of cells. Optionally, the first acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers to generate the first acoustic effect. Optionally, the second acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers to generate the second acoustic effect. Advantageously, the manipulation of cells in a cell container can be carefully controlled and may be easily reprogrammed by a control unit. Specifically, without requiring a complex submerged motion control stage or any other moving parts. Advantageously, the distributions of the acoustic fields or effects across the cell container can be even. The parameters of the method of the fourth aspect may be easily adapted for different cell container types (either different sizes or different brands of the same size).

[0031] Optionally, the first acoustic effect is at least one of: a focus point (e.g., a focused acoustic line [if a linear acoustic array is used]); an acoustic vortex; an acoustic twin-trap; or, an acoustic bottle field, and / or wherein the second acoustic effect is at least one of: a focus point; an acoustic vortex; an acoustic twin-trap; or, an acoustic bottle field. Optionally, the first acoustic field is generated by controlling an amplitude of a first acoustic transducer of the plurality of acoustic transducers to generate the first acoustic effect. Optionally, the second acoustic field is generated by controlling an amplitude of a second acoustic transducer of the plurality of acoustic transducers to generate the second acoustic effect. Optionally, the amplitude of the first acoustic transducer is different to the amplitude of the second acoustic transducer. Optionally, the first acoustic field is generated at a different time to the second acoustic field. Optionally, operating one or more of the plurality of acoustic transducers at a frequency greater than 1 MHz. Optionally, the plurality of acoustic transducers is arranged linearly and in a plane. Optionally, the plurality of acoustic transducers is arranged in a 2-dimensional pattern and in a plane. Optionally, the first acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers to generate the first acoustic effect comprising a complex acoustic effect at a first location within the second portion. Optionally, the second acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers to generate the second acoustic effect comprising the complex acoustic effect at a second location within the first portion. Optionally, each of the plurality of acoustic transducers is controlled to move the location of the complex acoustic effect from the first location to the second location.

[0032] Optionally, the first acoustic effect is the second acoustic effect. Optionally, the first acoustic field is the complex acoustic effect. Optionally, the first acoustic field is a focus point (or other field) generated by the plurality of acoustic transducers arranged linearly and in a plane (i.e., using linear acoustics). Optionally, the first acoustic field is a focus point (or other field) generated by the plurality of acoustic transducers arranged in a 2-dimensional pattern, and optionally in a plane. Optionally, the fourth aspect further comprises: capturing an image of the cell container to determine a distribution of cells in the cell container; determining that the first portion of the cell container comprises cells below the density threshold based on the image; and, generating the one or more first acoustic fields in response to determining that the first portion of the cell container comprises cells below the density threshold. Optionally, the fourth aspect further comprises: capturing an image of the cell container to determine a distribution of cells in the cell container; determining that the second portion of the cell container comprises cells above a second density threshold based on the image; and, generating the one or more first acoustic fields in response to determining that the second portion of the cell container comprises cells above the second density threshold. Optionally, the second density threshold is based on the density threshold. Optionally, the second density threshold is at least a cell density of: 20000 cell per millilitre (cells / ml), 10000 cells / ml, 5000 cells / ml, 2500 cells / ml, or 1500 cells / ml. Optionally, the volume of the first portion plus the volume of the second portion is equal to the volume of the cell container. A high density of cells may be in the first portion of the cell container after the cell concentration step of the fourth aspect. A low density of cells may be in the second portion of the cell container after the cell concentration step of the fourth aspect. Optionally, the fourth aspect further comprises releasing a plurality of cells adherent to a surface of the cell container. Optionally, the cell disassociation step further comprises generating one or more second acoustic fields by controlling the plurality of acoustic transducers. Optionally, the plurality of cells adherent to the surface of the cell container are released using the one or more second acoustic fields. Optionally, the cell disassociation process further comprises, prior to the cell concentration step and the generation of the one or more second acoustic fields: providing a dissociation reagent to the cell container. Optionally, the fourth aspect further comprises after the cell disassociation step: a first cell mixing step comprising redistributing cells in the medium. Optionally, the first cell mixing step comprises generating one or more third acoustic fields by controlling the plurality of acoustic transducers. Optional ly, the plurality of cells in the medium are distributed using the one or more third acoustic fields.

[0033] Optionally, the fourth aspect further comprises after the cell concentration step: a volume adjustment step, comprising: determining that the total volume of the medium in the cell container is at a predetermined volume. Optionally, the volume adjustment step comprises removing the first amount of the medium from the second portion of the cell container. Optionally, the volume adjustment step comprises providing a second amount of media to the cell container. Optionally, the fourth aspect further comprises after the volume adjustment step, performing a second cell mixing step to redistribute cells within the medium. Optionally, the second cell mixing step comprises generating one or more fourth acoustic fields by controlling the plurality of acoustic transducers. Optionally, the cells in the medium are redistributed using the one or more fourth acoustic fields. Optionally, the fourth aspect further comprises a transfer step comprising: transferring the plurality of cells from the cell container into one or more additional cell containers. Optionally, the transfer step further comprises counting cells in the cell container before transferring the plurality of cells from the cell container into the one or more additional cell containers. Optionally, the transfer step further comprises counting cells in the cell container by removing a sample and calculating the total number of cells. Optionally, the process further comprises adjusting the temperature of an acoustic coupling arranged between the plurality of acoustic transducers and the surface of the cell container. Optionally, the acoustic coupling is suitable for transmitting the generated acoustic fields to the cell container. Optionally, the process further comprises capturing an image of the cell container to determine the distribution of cells in a cell container containing a medium and cells. Optionally, the process further comprises generating the first acoustic field based on the image. Optionally, the process comprises observing the behaviour of the cells in response to the acoustic fields and changing the fields in response to the observing of the behaviour. Advantageously, using a dynamically controlled system, calibration for new types of cells may be done easily, e.g., via an iterative method.

[0034] The process of the fourth aspect may be a computer implemented process.

[0035] A fifth aspect of the invention provides a system to release adherent cells from a surface, the system comprising: a cell container comprising a surface, wherein the cell container is suitable for containing a medium and cells adherent to the surface of the cell container; a plurality of acoustic transducers suitable for generating acoustic fields; and, a processor configured to perform the cell passaging process of the fourth aspect.

[0036] Optionally, the system of the fifth aspect further comprises an acoustic coupling arranged between the plurality of acoustic transducers and a surface of the cell container, wherein the acoustic coupling is suitable for transmitting the generated acoustic fields to the cell container. Optionally, the acoustic coupling comprises at least one of: a liquid; a hydrogel; a gel; a solid; an acoustic metamaterial or other designed material with a different speed of sound to the rest of the coupling to enable a phase and / or amplitude variation (e.g., an acoustic hologram or lens), or other designed material with a different effective speed of sound to the rest of the coupling to enable a phase and / or amplitude variation. Optionally, the system of the fifth aspect further comprises a temperature sensor, and a temperature adjusting means to adjust the temperature of the acoustic coupling based on the temperature sensor. Optionally, the plurality of acoustic transducers is arranged linearly and in a plane. Optionally, the plurality of acoustic transducers is arranged in a 2-dimensional pattern in a / the plane. Optionally, each of the plurality of acoustic transducers is arranged to operate at a frequency greater than 1 MHz. Optionally, the cell container comprises a plurality of wells. Optionally, each of the plurality of wells is suitable for containing a medium and cells. Advantageously, control of the acoustic fields of the fourth and / or fifth aspects may be dynamically controlled. This may be useful in a multi-well container (i.e., a multi well plate) for only performing the process within certain wells. Optionally, the system of the fifth aspect further comprises a multi-well plate. Optionally, the cell container is a well of the multi-well plate.

[0037] A sixth aspect of the invention provides a liquid handling system comprising the system of the fifth aspect.

[0038] BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Embodiments of the invention will now be described with reference to the accompanying drawings, in which: Figure 1 illustrates a side view of a system for performing a cell passaging process.

[0040] Figure 2 illustrates a side view of the system of Figure 1 as an acoustic array generates one or more first acoustic fields.

[0041] Figure 3a illustrates a side view of the system of Figure 1 with a pipette.

[0042] Figure 3b illustrates a side view of the system of Figure 1 with a pipette after a first amount of the medium is removed from a second portion of the cell container.

[0043] Figure 4a illustrates a side view of a cell container with a focus point at a first position which is configured to displace cells at the first position at a first time.

[0044] Figure 4b illustrates a side view of a cell container with a focus point at an intermediate position which is configured to displace cells at the intermediate position at a second time.

[0045] Figure 4c illustrates a side view of a cell container with a focus point at a second position which is configured to displace cells at the second position at a third time.

[0046] Figure 5 illustrates a cell passaging process for use with a plurality of acoustic transducers.

[0047] Figure 6 illustrates a side view of a system to displace cells (for increasing the density of cells at a portion of the cell container) as an acoustic array generates a first acoustic field with phase control.

[0048] Figure 7 illustrates a side view of a system to displace cells (for increasing the density of cells at a portion of the cell container) as an acoustic array generates a first acoustic field with amplitude control.

[0049] Figure 8 illustrates a top view of the system of Figure 1 suitable for the cell passaging process.

[0050] Figure 9 illustrates a top view of a system suitable for the cell passaging process with an acoustic array arranged in a 2-dimensional pattern of transducers.

[0051] Figure 10 illustrates a top view of a system suitable for the cell passaging process with an acoustic array arranged in a 2-dimensional pattern of transducers.

[0052] Figure 1 1 illustrates a side view of a system suitable for the cell passaging process, with optional features. Figure 12 illustrates a side view of a system suitable for the cell passaging process, with optional features. Figure 13 illustrates a side view of a system suitable for the cell passaging process, with optional features. Figure 14 illustrates a side view of a system suitable for the cell passaging process, with optional features. Figure 15 illustrates a cell passaging process for use with a plurality of acoustic transducers.

[0053] Figure 16 illustrates a side view of a system to release adherent cells from a surface of a cell container.

[0054] Figure 17 illustrates a side view of the system of Figure 16 as an acoustic array generates a first acoustic field and a second acoustic field.

[0055] Figure 18 illustrates a side view of a system to release adherent cells as an acoustic array generates a first acoustic field with phase control.

[0056] Figure 19 illustrates a side view of a system to release adherent cells as an acoustic array generates a first acoustic field with amplitude control.

[0057] Figure 20a illustrates a side view of a cell container with a focus point at a first position which is configured to release adherent cells at the first position at a first time.

[0058] Figure 20b illustrates a side view of a cell container with a focus point at an intermediate position which is configured to release adherent cells at the intermediate position at a second time.

[0059] Figure 20c illustrates a side view of a cell container with a focus point at a second position which is configured to release adherent cells at the second position at a third time.

[0060] Figure 21 illustrates a top view of the system of Figure 16 to release adherent cells from a surface of a cell container.

[0061] Figure 22 illustrates a top view of a system to release adherent cells from a surface of a cell container with an acoustic array arranged in a 2-dimensional pattern of transducers.

[0062] Figure 23 illustrates a top view of a system to release adherent cells from a surface of a cell container with an acoustic array arranged in a 2-dimensional pattern of transducers.

[0063] Figure 24 illustrates a side view of a system to release adherent cells, with optional features.

[0064] Figure 25 illustrates a side view of a system to release adherent cells, with optional features.

[0065] Figure 26 illustrates a side view of a system to release adherent cells, with optional features.

[0066] Figure 27 illustrates a method for releasing adherent cells in a cell container with a plurality of acoustic transducers.

[0067] DETAILED DESCRIPTION OF EMBODIMENT(S)

[0068] A Process and System for Cell Passaging

[0069] By way of a non-limiting overview, an array of acoustic elements is used to project one or more acoustic beam(s) into an easily removable cell culture container to perform one or more steps of a cell passaging process, such as a cell concentration step. An acoustic cell concentration step may be used in place of a centrifuge and may advantageously avoid manual steps in a typical cell passaging process such as transferring cells from a dish to a tube, and the use of a centrifuge.

[0070] Figure 1 shows a side view of a system 10 for cell passaging from a cell container 14. The system 10 includes an acoustic array 16 of acoustic transducers 16a, 16b, 16c, ... 16h and the cell container 14. The cell container 14 comprises a plurality of surfaces: a base surface 14a; one or more side surfaces 14b; and optionally a top surface 14c. The cell container 14 may be any vessel in which cells 12 may grow. As shown in Figure 1 , the cell container 14 contains a medium 18 and cells 12.

[0071] The cell container 14 may be any container suitable for containing cells 12, such as a plate, a petri dish, a well in a multi-well plate (e.g., a 24-well SBS plate), a single layer flask / cell culture flask, a multi-layer flask, a standard bioreactor, a microfluidic container (including bioreactors), a fibrous bioreactor, custom consumable sterile plastic, or a roller bottle.

[0072] The medium 18 may be any medium suitable for cell culture, such as, cell culture media, water, agar, Trypsin, Ethylenediaminetetraacetic acid (EDTA), foetal bovine serum, other brand name dissociation or loosening reagents, or any combination thereof.

[0073] The acoustic array 16 is shown in Figure 1 to comprise 8 acoustic transducers 16a, 16b, 16c, ..., 16h for simplicity. It will be understood that the acoustic array 16 may contain any number of acoustic transducers, which may be arranged in any suitable plan.

[0074] A step of the cell passaging process is a cell concentration step. In known cell passaging systems this requires: 1 ) transferring cells from a dish to a tube; 2) the use of a centrifuge to cluster cells together (i.e., a high density of cells).

[0075] Figure 2 shows the system 10 of Figure 1 as the acoustic array 16 generates one or more first acoustic fields 20 as part of an acoustic cell concentration step. The acoustic array 16 generates each of the one or more first acoustic fields 20 by controlling two or more of the plurality of acoustic transducers 16b, 16c, ..., 16h. The system 10 is used to increase the density of cells in a first portion 24a of the cell container 14 above a density threshold using the one or more first acoustic fields 20. This generates a high density group of cells 12a (i.e., agglomerated cells). The density threshold may be at least a cell density of 0.4 million cells per millilitre.

[0076] The one or more acoustic fields 20 move cells 12 to agglomerate them together. The agglomeration of cells 12 via the one or more acoustic fields 20 may be done in many ways such as by the generation of multiple acoustic effects sequentially in time or a single continuous acoustic effect (e.g., complex acoustic effect). The acoustic effect(s) may be at least one of: a standing wave node or nodes; a focus point; an acoustic vortex; an acoustic twin-trap; or, an acoustic bottle field. Each acoustic effect may also induce fluid movement in the medium 18, which may be known as acoustic streaming (e.g., bulk motion of the medium 18). A single continuous acoustic effect may be steered or moved to induce a movement of cells 12 relative to the cell container 14. Alternatively, multiple acoustic effects sequentially in time may have the same effect: to induce a movement of cells 12 relative to the cell container 14. The movement of cells 12 generates two volumes which differ in their cell density: the first portion 24a, and the second portion 24b.

[0077] As a result of increasing the density of cells 12 in a first portion 24a of the cell container 14, the density of cells in a second portion 24b of the cell container 14 is decreased using the one or more first acoustic fields 20. With the cells substantially (e.g., 60%, 70%, 80%, 90%, 95%, 97%, 100%) positioned in the first portion 24a, a first amount of the medium 18 is removed (e.g., aspirated) from [a location in] the second portion 24b of the cell container. Thus, the first amount of the medium 18 may be removed without removing the cells positioned in the first portion 24a. One or more holding acoustic fields may be used to substantially maintain the density of cells in the first portion 24a of the cell container 14 while the first amount of the medium 18 is removed.

[0078] Each one of the first acoustic fields 20 may generate a corresponding acoustic effect. That is, each acoustic field is generated by one or more transducers 16a-16h. A complex acoustic effect may be the effect of the generated acoustic field. For example, a high pressure region in 3D space (i.e., a focus point) may be the acoustic effect of a corresponding acoustic field.

[0079] In the example, the first portion 24a of the cell container 14 comprises a side wall 14b of the cell container. Thus, the one or more first acoustic fields 20 move the cells 12 to the edge of the cell container 14. Advantageously, this simplifies control of the transducer array, and distances cells in the first portion 24a from a tool (e.g., a pipette) used to remove media 18 from the cell container 14.

[0080] The system 10 of Figures 3a and 3b show all of the features of the system 10 of Figure 1 , in addition to certain optional features. The same reference numerals are used to denote the same / corresponding features in relation to Figure 1 and will not be described in detail again below.

[0081] Figures 3a and 3b show a volume adjustment step of a cell passaging process, following the agglomeration of cells 12 in the first portion 24a of the cell container 14. Figures 3a and 3b show a pipette 22 arranged to remove a first amount of the medium 18 from the cell container 14. The tip of the pipette 22 is inserted into the medium 18 at the second portion of the cell container 14. Thus, a first amount of the medium 18 is removed from the cell container 14 from a location in the second portion 24b of the cell container 14. Optionally as the first amount of the medium 18 is removed (e.g., aspirated), the one or more first acoustic fields comprise: one or more holding acoustic fields to substantially maintain the density of cells in the first portion 24a of the cell container 14 after the density threshold is reached.

[0082] Figure 3a shows a volume amount (V1 ) of the medium 18 in the cell container 14. The volume adjustment step comprises removing a first volume amount of the medium 18. Figure 3b shows a volume amount (V2) of the medium 18 in the cell container 14, after the first amount of the medium 18 is removed. Thus, the first volume amount of the medium 18 removed is equal to the V1 -V2. Additionally, the volume adjustment step may further comprise: providing a second amount of media to the cell container 14, and determining that the total volume of the medium 18 in the cell container 14 is at a predetermined volume for a transfer step.

[0083] Figures 4a, 4b, 4c show a series of acoustic effects, specifically focus points, over a time period which result in cells 12 being moved to a first side 28a of the cell container 14. Each of the plurality of acoustic transducers (not shown for conciseness) may be excited continuously over a time period to produce an acoustic effect between first and second sides of the cell container 28a, 28b.

[0084] Figure 4a shows a first acoustic field generated by controlling the phase of each of the plurality of acoustic transducers of the acoustic array 16 to generate the focus point 32a starting from the second side 28b of the cell container 14. The first acoustic field generates the focus point 32a to move a first plurality of cells 31 a relative to the cell container 14 and towards the first portion 24a of the cell container 14. Figure 4c shows the second acoustic field generated by controlling the phase of each of the plurality of acoustic transducers of the acoustic array 16 to generate the focus point 32a ending at the first side 28a of the cell container 14. The second acoustic field generates the focus point 32a to move a second plurality of cells 31 b relative to the cell container 14 and towards the first portion 24a of the cell container 14. In an example, the second plurality of cells 31 b comprises the first plurality of cells 31 a.

[0085] Figure 4b shows the intermediate state between Figures 4a and 4c, where each of the plurality of acoustic transducers of the acoustic array 16 is controlled to move the focus point 32a from the second side 28b of the cell container 14 to the first side 28a of the cell container 14 via an intermediate position. The movement of the focus point 32a (i.e., a high pressure region) “pushes” cells (to a low pressure region and) towards the first portion 24a as it moves from the second side 28b of the cell container 14 to the first side 28a of the cell container 14.

[0086] The phase of each acoustic transducer of the acoustic array 16 may be continuously modified (e.g., at an update rate) to result in the moving focus point 32a which may push cells from the second side 28b to the first side 28a of the cell container 14 as it moves continuously between a start position and an end position over the time period. Therefore, the focus point 32a may be swept across the cell container 14 by phase control of the array 16 (i.e., beam steering) or by amplitude control of the array (i.e., moving the peak amplitude of an acoustic effect). Depending on the size of the focus point 32a and the length of the time period, not all of the cells may be moved to the first portion 24a of the cell container 14, however, the density of cells in the first portion 24a will be greater than the density of cells in the second portion 24b. The system may be configured to repeat the sweep of the focus point 32a from the second side 28b to the first side 28a of the cell container 14 to further increase the density of cells in a first portion 24a of the cell container 14 until a desired density threshold is reached.

[0087] In an alternative example, each of the plurality of acoustic transducers 16a-16h may be operated non- continuously. Each of the plurality of acoustic transducers 16a-16h may be turned off (e.g., receive no control signal from the control unit 30) between the generation of the subsequent acoustic fields (each acoustic field may correspond to a respective acoustic effect). That is, an initial acoustic effect is generated, then no acoustic effect is generated, then another acoustic effect is generated, then no acoustic effect is generated, etc. Thus, the system 10 may be configured to pulse (i.e., turn off and on) the acoustic array 16 between each respective acoustic field / effect to generate pulsing acoustic fields / effects. The system 10 may comprise one or more processors, computers, or controllers. The system 10 is configured to control the plurality of acoustic transducers of the acoustic array 16 to perform at least part of a cell passaging process. Such a cell passaging process, as shown in Figure 5, for transferring cells from a cell container 14 using a plurality of acoustic transducers may comprise one or more of the following steps:

[0088] S1 : a cell disassociation step comprising releasing a plurality of cells adherent to a surface of the cell container.

[0089] S2: a first cell mixing step comprising redistributing cells in the medium 18 of the cell container 14. The cells may be redistributed within the medium 18 to result in a homogeneous cell suspension.

[0090] S3: a cell concentration step to increase the density of cells in a first portion of the cell container above a density threshold using the one or more first acoustic fields as described with reference to Figures 1 to 4c.

[0091] S4: a volume adjustment step to adjust the total volume of the medium in the cell container 14 to a predetermined volume. Media may be removed and provided via any suitable means, such as, a pipette.

[0092] S5: a second cell mixing step comprising redistributing cells in the medium 18 of the cell container 14. The cells may be redistributed within the medium 18 to result in a homogeneous cell suspension.

[0093] S6: a transfer step comprising transferring the plurality of cells from the cell container 14 into one or more additional cell containers.

[0094] The system 10 of Figures 6 and 7 show all of the features of the system 10 of Figure 1 , in addition to certain optional features. The same reference numerals are used to denote the same / corresponding features in relation to Figure 1 and will not be described in detail again below.

[0095] Figures 6 and 7 show the system 10 including a control unit 30. The control unit 30 is configured to be communicatively coupled to each transducer of the plurality of acoustic transducers 16a-16h which make up the acoustic array 16.

[0096] Figures 6 and 7 show the system 10 comprises an acoustic coupling 34 arranged between the plurality of acoustic transducers 16a-16h of the acoustic array 16 and the surface 14a of the cell container 14. The acoustic coupling 34 is suitable for transmitting the generated acoustic fields 20 to the medium 18 in the cell container 14. The acoustic coupling 34 may comprise at least one of: a liquid; a hydrogel; a gel; a solid; and an acoustic metamaterial or other designed material with a different speed of sound to the rest of the coupling to enable a phase and / or amplitude variation. For example, the acoustic coupling 34 may be a combination of a solid material and a thin layer of gel and / or rubber. In the example, the cell container 14 is a closed container or a sealed container, such as a microfluidic chip, bioreactor, or sealed flask. That is, the cell container 14 in the example of Figures 6 and 7 includes a top surface 14c. Alternatively, the cell container 14 may be any suitable cell container 14.

[0097] The control unit 30 may transmit a control signal to each transducer 16a-16h of the acoustic array 16 to control the phase, amplitude, and / or frequency of the acoustic field generated by each respective transducer 16a-16h. The first acoustic field 20 may be the sum of each acoustic field generated by each respective transducer 16a-16h.

[0098] Figure 6 shows the control unit 30 configured to generate and transmit a phase control signal <p±to <p8to each transducer 16a-16h of the acoustic array 16. Although, it will be understood that in other examples the control unit may also be configured to control the amplitude and / or frequency of the transducers 16a- 16h in addition to the phase of the transducers 16a-16h.

[0099] The first acoustic field 20a is generated by controlling the phase <p of each of the plurality of acoustic transducers 16a-16h to generate the focus point 32a (or other complex acoustic effect) at a first location within the second portion 24b. The first acoustic field 20a is arranged to displace the first plurality of cells 31 a from the second portion 24b of the cell container 14. In the example, the first acoustic effect 32a is shown in 2D as a focus point.

[0100] Although not shown in Figures 6 for conciseness, a second acoustic field may be generated by controlling the phase <p of each of the plurality of acoustic transducers 16a-16h to generate the focus point 32a (or other complex acoustic effect) at a second location within the first portion 24a. The second acoustic field may be arranged to displace a plurality of cells. Each of the plurality of acoustic transducers 16a-16h is controlled to move the location of the focus point 32a (i.e., complex acoustic effect) from the first location (e.g., second side 28b) to the second location (e.g., first side 28a), as shown in Figures 4a-4c.

[0101] The control unit 30 may generate at least two unique phase signals, such that two or more transducers 16a-16h may receive the same phase. As shown in Figure 6 by the angle of each arrow from the corresponding transducer 16a-16h, each transducer 16a-16h are controlled to generate unique phases. Optionally, one or more transducers 16a-16h may not receive a control signal.

[0102] In an example, the acoustic effect may be a focus point, and the phase for each transducer may be calculated with equation (1 ) below: is the phase for a transducer j, k is the wavenumber for the system 10 and r is calculated by equation (2) below:

[0103] Where Xj is the x axis location of the centre of transducer j, xfis the x axis location of the desired focus point and z is the z axis location of the desired focus point. A skilled person may reconfigure this system to produce a focus point (or other field) in different locations using linear acoustics.

[0104] Figure 7 shows the control unit 30 configured to generate and transmit an amplitude control signal to a8to each transducer 16a-16h of the acoustic array 16. Although, it will be understood that in other examples the control unit may also be configured to control the phase and / or frequency of the transducers 16a-16h in addition to amplitude of the transducers 16a-16h.

[0105] The first acoustic field 20b is generated by controlling the amplitude a of one or more of the acoustic transducers 16a-16h to generate the first acoustic effect 32b at a first location in the second portion 24b of the cell container 14. The first acoustic field 20b is arranged to displace the first plurality of cells 31 a from the second portion 24b of the cell container 14.

[0106] Transducers 16b-16h of the acoustic array 16 are each controlled to generate at least two unique amplitudes. As shown in Figure 7 by the length of each arrow from the corresponding transducer 16a-16h, each transducer 16a-16h are controlled to generate unique amplitudes. Optionally, one or more transducers 16a-16h may not receive a control signal, or receive a control signal indicating an amplitude of 0, such as transducer 16a in Figure 7.

[0107] Although not shown in Figure 7 for conciseness, the second acoustic field may be generated by controlling the amplitude of one or more of the plurality of acoustic transducers 16a-16h to generate the second acoustic effect similarly to the first acoustic effect 20b. Optionally, the amplitude of one acoustic transducer 16a is different to the amplitude of another acoustic transducer 16b.

[0108] In both examples shown in Figures 6 and 7, the first acoustic effect 32a, 32b may indirectly result in the displacement of the first plurality of cells 31 a from a second portion 24b of the cell container 14, e.g., via acoustic streaming (the fluid flow of the medium induced by the first acoustic effect 32a, 32b). Alternatively, the first acoustic effect 32a, 32b may be applied to a location in the second portion 24b to directly displace the first plurality of cells 31 a from the location. In some examples, the first acoustic effect 32, 32b may be generated by at least two, four, eight of the transducers 16a-16h of the acoustic array 16 (or more based on the number of transducers of the acoustic array).

[0109] In an alternative example, the transducer array 16 may be an irregular array with sectoral transducers for each cell container to create Bessel beams to ‘push’ the cells outwards such that the first portion of the cell container is the outer perimeter of the cell container, and the second portion of the cell container is the inner area of the cell container. Figure 8 shows a top view of the system 10 of Figure 1 . Figure 8 shows the plurality of acoustic transducers 16a-16h is arranged linearly and in a plane (e.g., the x-y plane as shown in Figure 8). In an example, the control unit 30 is configured to control the phase of each transducer 16a-16h (i.e., a phased array system), and the first acoustic effect 32a may be a focus point, specifically, a focus line.

[0110] Figure 9 shows a top view of a system 10a. The system 10a a comprises all of the features of the system 10 of Figures 1 and 8, in addition to certain optional features. The same reference numerals are used to denote the same / corresponding features in relation to Figures 1 and 8, and will not be described in detail again below. Figure 9 shows the plurality of acoustic transducers 16a-16 / ? arranged in a 2-dimensional pattern in the plane (e.g., the x-y plane). The acoustic array 16 is shown in Figure 9 to comprise 64 acoustic transducers, wherein the acoustic transducer 16 / ? represents the final (i.e., 64th) transducer in the acoustic array 16. In an example, the control unit 30 is configured to control the phase of each transducer 16a-1 Q / 3 (i.e., a phased array system). A 2-dimensional pattern of acoustic transducers 16a-1 Q / 3 enables an acoustic effect 32c to be at least one of: a focus point; an acoustic vortex; an acoustic twin-trap; or an acoustic bottle field.

[0111] Figure 10 shows a top view of a system 10b. The system 10b comprises all of the features of the system 10 of Figure 1 , in addition to certain optional features. The same reference numerals are used to denote the same / corresponding features in relation to Figure 1 and will not be described in detail again below. Figure 10 shows the plurality of acoustic transducers 16a-16 / ? arranged in a 2-dimensional pattern in the plane (e.g., the x-y plane). The acoustic array 16 is shown in Figure 10 to comprise 36 acoustic transducers, wherein the acoustic transducer 16 / ? represents the final (i.e., 36th) transducer in the acoustic array 16. In an example, the control unit 30 is configured to control the phase of each transducer 16a-1 Q / 3 (i.e., a phased array system). In this example, the plurality of acoustic transducers may be orientated inward towards the cell container, e.g., 30, 45, 90 degrees relative to the plane, e.g. the x-y plane.

[0112] Figure 10 also shows a multi-well plate 40 comprising a plurality of cell containers 14, 41 , 42, 43, 44, 45, known as wells. Each of the plurality of cell containers 14, 41 , 42, 43, 44, 45 is suitable for containing the medium 18 and cells adherent to the surface of each cell container 14, 41 , 42, 43, 44, 45. In an alternative example, the system 10b may comprise a cell container 14 as shown in Figures 8 and 9, in place of the multi-well plate 40.

[0113] Figure 11 shows a side view of a system 10c. The system 10c comprises all of the features of the system 10 of Figures 1 and 8, in addition to certain optional features. The same reference numerals are used to denote the same / corresponding features in relation to Figure 1 and will not be described in detail again below. Figure 1 1 shows a plurality of acoustic transducers 16a-16I of an acoustic array 16. Figure 1 1 shows the cell container 14 is a well of the multi-well plate 40. The multi-well plate 40 also includes two additional cell containers 41 , 42. The system 10c comprises an acoustic coupling 34. The acoustic coupling 34 comprises a first acoustic coupling component 34a and second acoustic coupling component 34b. The first acoustic coupling component 34a is different to the second acoustic coupling component 34b. The first acoustic coupling component 34a may comprise at least one of: a liquid; a hydrogel; a gel; or, a solid. The second acoustic coupling component 34b may comprise an acoustic lens, acoustic hologram, acoustic kinoform, or other acoustic metamaterial (or other designed material with a different speed of sound to the rest of the coupling to enable a phase and / or amplitude variation). The second acoustic coupling component 34b may be time variant and electrically controlled (by the control unit 30) for dynamic control of the system 10c. In an alternative example, each of the first and second acoustic coupling components 34a, 34b, may comprise at least one of: a liquid; a hydrogel; a gel; a solid; an acoustic lens, acoustic hologram, acoustic kinoform, or other acoustic metamaterial (or other designed material with a different speed (or relative speed) of sound to the rest of the coupling to enable a phase and / or amplitude variation).

[0114] Figure 12 shows a side view of a system 10d. The system 10d comprises all of the features of the system 10c of Figure 11 , in addition to certain optional features. The same reference numerals are used to denote the same / corresponding features in relation to Figure 11 and will not be described in detail again below. In the example, the multi-well plate 40 is coupled to the acoustic array via a first acoustic coupling component 34a. In addition, each cell container 14, 41 , 42 corresponds to a respective second acoustic coupling component 34b.

[0115] Figure 13 shows a side view of a system 10e. The system 10e comprises all of the features of the system 10 of Figures 1 to 8, in addition to certain optional features. The same reference numerals are used to denote the same / corresponding features in relation to Figure 1 and will not be described in detail again below. The system 10e may be a liquid handling system.

[0116] In the example of Figure 13, the acoustic coupling 34 comprises a first acoustic coupling component 34a and second acoustic coupling component 34b. In the example of Figure 13, the cell container 14 comprises a means of changing liquids, such as an input pipe 50 and an outlet pipe 51 (suitable for the volume adjustment step S4). Alternatively, the means of changing liquids may be via a pipette 22, or via a single pipe. A pipe 50 or a pipette may be configured to provide a dissociation reagent to the cell container 14.

[0117] Figure 13 shows a temperature sensor 52, and a temperature adjusting means 54 (e.g., a heater) to adjust the temperature of the acoustic coupling 34, 34a, 34b based on the temperature sensor 52. The temperature sensor 52 may be in communication with the medium 18, or may be a laser thermometer or thermal camera. The control unit 30 may receive as an input a reading from the temperature sensor 52 and generate a temperature control signal for operating the temperature adjusting means 54. The temperature adjusting means may heat or cool the system depending on the requirements. In a cell passaging process, the temperature may be advantageously controlled within a temperature range to promote desired cell activity. The temperature adjusting means 54 may be operated to maintain the temperature within an operating temperature range, or be operated to maintain the temperature at an operating temperature set point, e.g., 32°C or 37°C.

[0118] Figure 13 shows a camera sensor 56. The camera sensor 56 may be configured to capture one or more images of the cell container 14 to determine that the total volume of the medium 18 in the cell container 14 is at a predetermined volume. The camera sensor 56 may be configured to capture one or more images of the cell container 14 to determine the density of cells in ta first and second portion of the cell container 14, and / or the number of cells in the cell container 14 before transferring.

[0119] In an example, the camera sensor 56 may capture an image of the cell container to determine a distribution of cells in the cell container. The control unit 30 may determine that the first portion 24a of the cell container 14 comprises cells below the density threshold based on the image. The control unit 30 may then operate the plurality of transducers to generate the one or more first acoustic fields in response to determining that the first portion 24a of the cell container comprises cells below the density threshold.

[0120] In the example of Figure 13, the camera sensor 56 is positioned above the cell container 14. In an alternative example, the camera sensor 56a may be positioned below the acoustic array 16 as shown in Figure 14. The camera sensor 56 may image the cell container 14 through gaps in the acoustic array 16. The camera sensor 56 may image the cell container 14 through a transparent acoustic array 16. In an alternative example, the camera sensor 56 may be integrated amongst the acoustic array 16 to image the cell container 14. The camera sensor 56 may be a microscope. The system 10e may include fluorescence sensors (if the cells are treated to generate a florescent effect) or other cell analysis sensors.

[0121] The control unit 30 may comprise one or more processors, computers, or controllers. The control unit 30 is configured to control the plurality of acoustic transducers 16 to perform at least part of a cell passaging process. With the system 10, 10a, 10b, 10c, 10e many steps of the cell passaging process, as shown in Figure 15, may be automated using one or more acoustic fields.

[0122] Such a cell passaging process, as shown in Figure 15, may comprise one or more of the following steps:

[0123] The cell disassociation step S1 comprises releasing a plurality of cells adherent to a surface 14a of the cell container 14. The cell disassociation step S1 may comprise one or more of the following optional steps:

[0124] P1 : providing a dissociation reagent to the cell container. The amount of dissociation reagent required may be less than the amount of dissociation reagent required if no acoustic fields were used. The amount of dissociation reagent required may be the same amount of dissociation reagent required if no acoustic fields were used (advantageously, this may release cells at a faster speed).

[0125] P2: capturing an image of the cell container 14.

[0126] P3: determining (with the control unit 30) if a portion of the surface of the cell container 14 comprises a group of cells adherent to the surface of the cell container based on the image. If no (or an acceptable number of) cells are adherent to the surface of the cell container 14 (i.e., determining if the surface of the cell container is free (or below an acceptance threshold) from adhered cells based on the image), then the method may move to step S2. If a group of cells are adherent to the surface of the cell container based on the image, then the method moves to step P4. In an alternative example, if no cells are adherent to the surface of the cell container 14 (i.e., determining if the surface of the cell container is free from adhered cells based on the image), then the process may move to step S2, or move to step P5 if a dissociation reagent is used.

[0127] P4: Generating one or more second acoustic fields by controlling the plurality of acoustic transducers (e.g., transducers 16a, 16b, ...). The acoustic fields generate a respective acoustic effect to release a respective group of cells from a respective location of the surface of the cell container 14. Each acoustic field may be operated for a respective time period.

[0128] Each acoustic effect may be focused on one or more portions in order to release one or more corresponding group of cells. In an example, a first of the one or more second acoustic fields may be a focus point directed at the surface 14a of the cell container 14 and configured to release a corresponding group of cells at the location of / near the focus point. In an alternative example, the one or more group of cells may be controllably released via reflected forces, acoustic streaming, and / or bulk motion of the medium 18. The bulk motion of the medium may be as a result of any stage of the acoustic field application or release. Therefore, the acoustic effect may be directed anywhere above or below the bottom of the cell container 14 to obtain a maximum force (to release one or more cells) if all acoustic parameters (particularly reflections) are considered.

[0129] In an example, the acoustic array 16 may generate a plurality of acoustic fields comprising at least, a first acoustic field, a second acoustic field, and a third acoustic field. Each acoustic field may generate a respective acoustic effect to release a respective group of cells from a respective portion of a surface (or multiple surfaces) of the cell container 14.

[0130] In an example similar to that shown in Figures 4a, 4b, and 4c, a series of acoustic effects over a time period may result in all cells 12 being released from the base surface 14a of the cell container 14. Each of the plurality of acoustic transducers may be excited continuously over a time period to produce an acoustic effect between the first side 28a and the second side 28b of the cell container 14.

[0131] Similar to Figure 4a, a first acoustic field of the one or more second acoustic fields may be generated by controlling the phase of each of the plurality of acoustic transducers of the acoustic array 16 to generate a focus point at a first location (e.g., a location at / near the second side 28b) to release cells (at or around the location) from the surface 14a of the cell container 14. Similar to Figure 4c, the second acoustic field of the one or more second acoustic fields may be generated by controlling the phase of each of the plurality of acoustic transducers of the acoustic array 16 to generate the focus point at a second location (e.g., a location at / near the first side 28a) to release cells (at or around the second location) from the surface 14a of the cell container 14. Similar to Figure 4b, an intermediate state where each of the plurality of acoustic transducers of the acoustic array 16 is controlled to move the focus point between the first location and the second location via an intermediate position 24c. The phase of each acoustic transducer of the acoustic array 16 may be continuously modified (e.g., at an update rate) to result in the moving focus point which may release cells from the surface 14a of the cell container 14 as it moves continuously between a start position (i.e., the first location) and an end position (i.e., the second location) over the time period. Therefore, the acoustic effect 32a may be swept across the surface 14a of the cell container 14 by phase control of the array 16 (i.e., beam steering) or by amplitude control of the array (i.e., moving the peak amplitude of the acoustic effect 32b).

[0132] In an alternative example, each of the plurality of acoustic transducers 16a-16h may be operated non- continuously. Thus, the system 10 may be configured to pulse (i.e., turn off and on) the acoustic array 16 between each respective acoustic field / effect to generate pulsing acoustic fields / effects. The system 10 may be further configured to pulse the acoustic array 16 to generate the same acoustic field / effect multiple times in the same portion of the surface of the cell container 14. Thus, at each position the corresponding adhered cells may be loosened by repetitive cycles of the acoustic field until the cells are released. This may advantageously loosen one or more cells of a portion over a series of pulses (over time), which may reduce stresses on the one or more cells and improve cell viability.

[0133] In an example, the plurality of acoustic transducers 16a-16h may be arranged to generate a series of acoustic effects upon a plurality of locations of the cell container 14. The area corresponding to the plurality of portions of the surface 14a may be substantially equal to the total area of the surface 14a of the cell container 14. That is, the acoustic array 16 may be arranged to release cells from the total area of the surface 14a (and / or other surfaces 14b, 14c) of the cell container 14. The plurality of portions of the surface 14a of the cell container 14 may comprise the first portion 24a, the second portion 24b, and further portions of the surface 14a. The first portion 24a, the second portion 24b, and further portions of the surface 14a may cover the total surface 14a of the cell container 14. The total surface may be the base surface 14a of the cell container 14, or the area of a surface of the cell container 14 in which cells may adhere to (e.g., the base surface 14a, and the side surface 14b). An excitation area of the acoustic array 16 may be arranged to be substantially equal to (e.g., 70%, 80%, 90%, 95%, 97%, 100% of) the total area of the surface 14a of the cell container 14. The excitation area of the acoustic array 16 may be the area of a vibrating surface of the plurality of transducers 16a-16h.

[0134] P5: The cells 12 within the cell container 14 may now be in suspension within the cell container 14 (in the medium 18). If a dissociation reagent was added to the cell container 14, a media may be added to the cel container to quench reaction.

[0135] Steps P6, P7, and P8 may be operated independently from Steps S1 to S6. Steps P6 to P8 are optional and may only be operated if the system comprises a temperature sensor 52, and a temperature adjusting means 54.

[0136] P6: Receiving (at the control unit 30) a temperature measurement from a temperature sensor 52. The temperature measurement corresponding to the temperature of an acoustic coupling arranged between the plurality of acoustic transducers and the surface of the cell container 14 or the temperature of the cell media itself. The acoustic coupling 34 is suitable for transmitting the generated acoustic fields to the surface of the cell container 14.

[0137] P7: Determining if the temperature of the acoustic coupling 34 is within an acceptable range of temperatures or operating at a temperature set point. If it is not, then the method may move to step P8. If the temperature is within the acceptable range of temperatures or operating at the temperature set point, then the method may return to step P6.

[0138] P8: Adjusting the temperature of the acoustic coupling 34, optionally with a temperature adjusting means 54 (e.g., a heater).

[0139] The first cell mixing step S2 comprising redistributing cells in the medium 18 of the cell container 14. The cells may be redistributed within the medium 18 to result in a homogeneous cell suspension. The first cell mixing step S2 may comprise the following optional step:

[0140] P9: generating one or more third acoustic fields by controlling the plurality of acoustic transducers of the acoustic array 16. The plurality of cells in the medium are distributed using the one or more third acoustic fields. For example, via moving a focus point throughout the medium 18 of the cell container 14.

[0141] The cell concentration step S3 increases the density of cells in a first portion of the cell container above a density threshold using the one or more first acoustic fields as described with reference to Figures 1 to 14. The cell concentration step S3 may comprise the following steps:

[0142] P10: Optionally, capturing an image of the cell container 14.

[0143] P11 : Determining (with the control unit 30) the distribution of cells in the cell container 14. Specifically, determining that the first portion 24a of the cell container 14 comprises cells below the density threshold (e.g., based on the image, or other means). If the density of cells in the first portion 24a of the cell container 14 is above a density threshold, then the method may move to step S4. If the density of cells in the first portion 24a of the cell container 14 is determined to be less than or equal to a density threshold (e.g., the density of cells in the second portion 24b of the cell container 14 may also be greater than or equal to a second density threshold), then the method may move to step P12. The first density threshold may be 0.4 million cells / ml.

[0144] In an alternative example, if the density of cells in the second portion 24b of the cell container 14 is below a second density threshold, then the method may move to step S4. If the density of cells in the second portion 24b of the cell container 14 is determined to be greater than or equal to the second density threshold (e.g., the density of cells in the first portion 24a of the cell container 14 may also be less than or equal to the [first] density threshold), then the method may move to step P12. The second density threshold may be 2500 cells / ml. P12: Generating one or more first acoustic fields by controlling the plurality of acoustic transducers (e.g., transducers 16a, 16b, ...). The plurality of acoustic transducers are controlled to increase the density of cells in a first portion 24a of the cell container and decrease the density of cells in a second portion 24b of the cell container 14 using the one or more first acoustic fields.

[0145] The second cell mixing step S5 comprises redistributing cells in the medium 18 of the cell container 14. The cells may be redistributed within the medium 18 to result in a homogeneous cell suspension. The second cell mixing step S5 may comprise the following optional step:

[0146] P13: generating one or more fourth acoustic fields by controlling the plurality of acoustic transducers of the acoustic array 16. The plurality of cells in the medium are distributed using the one or more fourth acoustic fields. For example, via moving a focus point throughout the medium 18 of the cell container 14.

[0147] The transfer step S6 comprises transferring the plurality of cells from the cell container 14 into one or more additional cell containers. The transfer step S6 may comprise one or more of the following optional steps:

[0148] P14: Optionally, capturing an image of the cell container 14 and counting cells in the cell container 14.

[0149] P15: transferring the plurality of cells from the cell container 14 into one or more additional cell containers based on the number of cells counted, e.g., re-seeding. In an alternative example, instead of re-seeding cell containers the cells may be cryopreserved, optionally, with the addition of a cryopreservation agent. In an alternative example, instead of being re-seeded the cells may be subjected to some form of analysis to determine how they grew and developed due to the impact of other factors such as growth media, an external agent or growth conditions. In an alternative example, instead of being re-seeded into different cell containers the cells may be used for an ongoing process such as the construction of a tissue culture or being gathered for administration of a cell therapy.

[0150] Acoustic Release of Adherent Cells

[0151] By way of a non-limiting overview, an array of acoustic elements is used to project one or more acoustic beam(s) into an easily removable cell culture container to release large areas of live adherent cells for future culturing or analysis.

[0152] Figure 16 shows a side view of a system 10 to release adherent cells 12 from a surface of a cell container 14. The system 10 includes an acoustic array 16 of acoustic transducers 16a, 16b, 16c, ... 16h and the cell container 14. The cell container 14 comprises a plurality of surfaces: a base surface 14a; one or more side surfaces 14b; and optionally a top surface 14c. The cell container 14 may be any vessel in which cells may grow. As shown in Figure 16, the cell container 14 contains a medium 18 and cells 12 adherent to the base surface 14a of the cell container 14. The cell container 14 may be any container suitable for containing cells 12, such as a plate, a petri dish, a well in a multi-well plate (e.g., a 24-well SBS plate), a single layer flask / cell culture flask, a multi-layer flask, a standard bioreactor, a microfluidic container (including bioreactors), a fibrous bioreactor, custom consumable sterile plastic, roller bottle.

[0153] The medium 18 may be any medium suitable for cell culture, such as, cell culture media, water, agar, Trypsin, Ethylenediaminetetraacetic acid (EDTA), foetal bovine serum, other brand name dissociation or loosening reagents, or any combination thereof.

[0154] Alternatively, the cells 12 may adhere to any surface 14a, 14b, 14c of the cell container 14, and the system 10 may release the adherent cells 12 from any surface.

[0155] The acoustic array 16 is shown in Figure 16 to comprise 8 acoustic transducers 16a, 16b, 16c, ..., 16h for simplicity. It will be understood that the acoustic array 16 may contain any number of acoustic transducers, which may be arranged in any suitable plan.

[0156] Figure 17 shows the system 10 of Figure 16 as the acoustic array 16 generates a first acoustic field 20 and a second acoustic field 22. The acoustic array 16 generates the first acoustic field 20 by controlling two or more of the plurality of acoustic transducers 16b, 16c, ..., 16h. The first acoustic field 20 generates a first acoustic effect to release a first group of cells 12a from a first portion 24a of the surface 14a of the cell container 14. The acoustic array 16 generates the second acoustic field 22 by controlling two or more of the plurality of acoustic transducers 16b, 16c, ..., 16h. The first acoustic field 22 generates a second acoustic effect to release a second group of cells 12b from a second portion 24b of the surface 14a of the cell container 14. The first acoustic field 20 may be generated at a different time to the second acoustic field 22.

[0157] The first acoustic effect may be at least one of: a focus point; an acoustic vortex; an acoustic twin-trap; or, an acoustic bottle field. The second acoustic effect may be at least one of: a focus point; an acoustic vortex; an acoustic twin-trap; or, an acoustic bottle field. Each acoustic effect may induce fluid movement in the medium 18, which may be known as acoustic streaming. Each acoustic effect may be focused on the first and / or second portion 24a, 24b in order to release the first and / or second group of cells 12a, 12b respectively. Alternatively, each acoustic effect may be focused on other locations in order to release the first and / or second group of cells 12a, 12b. For example, the first and / or second group of cells 12a, 12b may be controllably released via reflected forces, acoustic streaming, and / or bulk motion of the medium 18. The bulk motion of the medium may be as a result of any stage of the acoustic field application or release. In an example, the first acoustic effect 20 may be a focus point directed at the surface 18a of the medium 18 (or, if present, the top surface 14c of the cell container 14), which releases the first group of cells 12a via reflected forces. In an example, surface deformation of the medium 18 may be used to induce effects within the cell container 14 to release the adherent cells 12. Therefore, the acoustic effect may be directed anywhere above or below the bottom of the cell container 14 to obtain a maximum force (to release one or more cells) if all acoustic parameters (particularly reflections) are considered. In an example, the acoustic array 16 may generate a plurality of acoustic fields comprising at least, the first acoustic field 20, the second acoustic field 22, and a third acoustic field. The third acoustic field may be generated similarly to the first and second acoustic fields 20, 22, that is, by controlling the plurality of acoustic transducers 16a-16h. The third acoustic field may generate a third acoustic effect to release a third group of cells from a third portion of the surface of the cell container 14. Further acoustic fields of the plurality of acoustic fields may be generated by controlling the plurality of acoustic transducers 16a-16h. Each acoustic field may generate a respective acoustic effect to release a respective group of cells from a respective portion of a surface (or multiple surfaces) of the cell container 14.

[0158] The system 10 of Figures 18 and 19 show all of the features of the system 10 of Figure 16, in addition to certain optional features. The same reference numerals are used to denote the same / corresponding features in relation to Figure 16 and will not be described in detail again below.

[0159] Figures 18 and 19 show the system 10 including a control unit 30. The control unit 30 is configured to be communicatively coupled to each transducer of the plurality of acoustic transducers 16a-16h which make up the acoustic array 16.

[0160] Figures 18 and 19 show the system 10 comprises an acoustic coupling 34 arranged between the plurality of acoustic transducers 16a-16h of the acoustic array 16 and the surface 14a of the cell container. The acoustic coupling is suitable for transmitting the generated acoustic fields 20, 22 to the surface 14a of the cell container 14. The acoustic coupling 34 may comprise at least one of: a liquid; a hydrogel; a gel; a solid; and an acoustic metamaterial or other designed material with a different speed of sound to the rest of the coupling to enable a phase and / or amplitude variation. For example, the acoustic coupling 34 may be a combination of a solid material and a thin layer of gel and / or rubber.

[0161] In the example, the cell container 14 is a closed container or a sealed container, such as a microfluidic chip, bioreactor, or sealed flask. That is, the cell container 14 in the example of Figures 18 and 19 includes a top surface 14c. Alternatively, the cell container 14 may be any suitable cell container 14.

[0162] The control unit 30 may transmit a control signal to each transducer 16a-16h of the acoustic array 16 to control the phase, amplitude, and / or frequency of the acoustic field generated by each respective transducer 16a-16h. The first acoustic field 20 may be the sum of each acoustic field generated by each respective transducer 16a-16h.

[0163] Figure 18 shows the control unit 30 configured to generate and transmit a phase control signal <p±to <p8to each transducer 16a-16h of the acoustic array 16. Although, it will be understood that in other examples the control unit may also be configured to control the amplitude and / or frequency of the transducers 16a- 16h in addition to the phase of the transducers 16a-16h. The first acoustic field 20a is generated by controlling the phase <p of each of the plurality of acoustic transducers 16a-16h to generate the first acoustic effect 32a to release the first group of cells 12a from the first portion of the surface 14a of the cell container 14. In the example, the first acoustic effect 32a is shown in 2D as a focus point.

[0164] Although not shown in Figures 18 for conciseness, the second acoustic field may be generated by controlling the phase <p of each of the plurality of acoustic transducers 16a-16h to generate the second acoustic effect similarly to the first acoustic effect 32a.

[0165] The control unit 30 may generate at least two unique phase signals, such that two or more transducers 16a-16h may receive the same phase. As shown in Figure 18 by the angle of each arrow from the corresponding transducer 16a-16h, each transducer 16a-16h are controlled to generate unique phases. Optionally, one or more transducers 16a-16h may not receive a control signal.

[0166] In an example, the acoustic effect may be a focus point, and the phase for each transducer may be calculated with equation (1 ).

[0167] Figure 19 shows the control unit 30 configured to generate and transmit an amplitude control signal to a8to each transducer 16a-16h of the acoustic array 16. Although, it will be understood that in other examples the control unit may also be configured to control the phase and / or frequency of the transducers 16a-16h in addition to amplitude of the transducers 16a-16h.

[0168] The first acoustic field 20b is generated by controlling the amplitude a of one or more of the acoustic transducers 16a-16h to generate the first acoustic effect 32b to release the first group of cells 12a from the first portion of the surface 14a of the cell container 14.

[0169] Transducers 16b-16h of the acoustic array 16 are each controlled to generate at least two unique amplitudes. As shown in Figure 19 by the length of each arrow from the corresponding transducer 16a-16h, each transducer 16a-16h are controlled to generate unique amplitudes. Optionally, one or more transducers 16a-16h may not receive a control signal, or receive a control signal indicating an amplitude of 0, such as transducer 16a in Figure 19.

[0170] Although not shown in Figure 19 for conciseness, the second acoustic field may be generated by controlling the amplitude of one or more of the plurality of acoustic transducers 16a-16h to generate the second acoustic effect similarly to the first acoustic effect 20b. Optionally, the amplitude of one acoustic transducer 16a is different to the amplitude of another acoustic transducer 16b.

[0171] In both examples shown in Figures 18 and 19, the first acoustic effect 32a, 32b may indirectly result in the release the first group of cells 12a from a first portion 24a of the surface of the cell container 14, e.g., via acoustic streaming (the fluid flow of the medium induced by the first acoustic effect 32a, 32b). Alternatively, the first acoustic effect 32a, 32b may be applied to the first portion to directly release the first group of cells 12a from a first portion 24a of the surface 14a of the cell container 14. In some examples, the first acoustic effect 32, 32b may be generated by at least two, four, eight of the transducers 16a-16h of the acoustic array 16 (or more based on the number of transducers of the acoustic array).

[0172] In an example, the plurality of acoustic transducers 16a-16h may be arranged to generate a series of acoustic effects upon a plurality of portions of the surface 14a of the cell container 14. The area corresponding to the plurality of portions of the surface 14a may be substantially equal to the total area of the surface 14a of the cell container 14. That is, the acoustic array 16 may be arranged to release cells from the total area of the surface 14a (and / or other surfaces 14b, 14c) of the cell container 14. The plurality of portions of the surface 14a of the cell container 14 may comprise the first portion 24a, the second portion 24b, and further portions of the surface 14a. The first portion 24a, the second portion 24b, and further portions of the surface 14a may cover the total surface 14a of the cell container 14. The total surface may be the base surface 14a of the cell container 14, or the area of a surface of the cell container 14 in which cells may adhere to (e.g., the base surface 14a, and the side surface 14b). An excitation area of the acoustic array 16 may be arranged to be substantially equal to (e.g., 70%, 80%, 90%, 95%, 97%, 100% of) the total area of the surface 14a of the cell container 14. The excitation area of the acoustic array 16 may be the area of a vibrating surface of the plurality of transducers 16a-16h.

[0173] Figures 20a, 20b, 20c show a series of acoustic effects over a time period which result in all cells 12 being released from the base surface 14a of the cell container 14. Each of the plurality of acoustic transducers (not shown for conciseness) may be excited continuously over a time period to produce an acoustic effect between the first and second portions of the cell container 24a, 24b.

[0174] Figure 20a shows the first acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers of the acoustic array 16 to generate the focus point 32a at the first position 24a. Figure 20c shows the second acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers of the acoustic array 16 to generate the focus point 32a at the second position 24b. Figure 20b shows the intermediate state between Figures 20a and 20c, where each of the plurality of acoustic transducers of the acoustic array 16 is controlled to move the focus point 32a between the first position 24a and the second position 24b via an intermediate position 24c. The phase of each acoustic transducer of the acoustic array 16 may be continuously modified (e.g., at an update rate) to result in the moving focus point 32a which may release cells from the surface 14a of the cell container 14 as it moves continuously between a start position (i.e. , first position 24a) and an end position (i.e., second position 24b) over the time period. Therefore, the acoustic effect 32a may be swept across the surface 14a of the cell container 14 by phase control of the array 16 (i.e., beam steering) or by amplitude control of the array (i.e., moving the peak amplitude of the acoustic effect 32b).

[0175] In an alternative example, each of the plurality of acoustic transducers 16a-16h may be operated non- continuously. Each of the plurality of acoustic transducers 16a-16h may be turned off (e.g., receive no control signal from the control unit 30) between the generation of the first acoustic field, the second acoustic field, subsequent acoustic fields, and / or intermediate acoustic fields (each acoustic field may correspond to a respective acoustic effect). That is, a first acoustic effect is generated, then no acoustic effect is generated, then another acoustic effect is generated, then no acoustic effect is generated, etc. Thus, the system 10 may be configured to pulse (i.e., turn off and on) the acoustic array 16 between each respective acoustic field / effect to generate pulsing acoustic fields / effects. The system 10 may be further configured to pulse the acoustic array 16 to generate the same acoustic field / effect multiple times in the same portion of the surface of the cell container 14. Thus, at each position the corresponding adhered cells may be loosened by repetitive cycles of the acoustic field until the cells are released. This may advantageously loosen one or more cells of a portion over a series of pulses (over time), which may reduce stresses on the one or more cells and improve cell viability.

[0176] Figure 21 shows a top view of the system 10 of Figure 16. Figure 21 shows the plurality of acoustic transducers 16a-16h is arranged linearly and in a plane (e.g., the x-y plane as shown in Figure 21. In an example, the control unit 30 is configured to control the phase of each transducer 16a-16h (i.e., a phased array system), and the first acoustic effect 32a may be a focus point, specifically, a focus line.

[0177] Figure 22 shows a top view of a system 10a. The system 10a a comprises all of the features of the system 10 of Figures 16 and 21 , in addition to certain optional features. The same reference numerals are used to denote the same / corresponding features in relation to Figures 16 and 18, and will not be described in detail again below. Figure 22 shows the plurality of acoustic transducers 16a-16 / ? arranged in a 2-dimensional pattern in the plane (e.g., the x-y plane). The acoustic array 16 is shown in Figure 22 to comprise 64 acoustic transducers, wherein the acoustic transducer 16 / ? represents the final (i.e., 64th) transducer in the acoustic array 16.

[0178] In an example, the control unit 30 is configured to control the phase of each transducer 16a-16 / ? (i.e., a phased array system). A 2-dimensional pattern of acoustic transducers 16a-16 / ? enables an acoustic effect 32c to be at least one of: a focus point; an acoustic vortex; an acoustic twin-trap; or an acoustic bottle field.

[0179] Figure 23 shows a top view of a system 10b. The system 10b comprises all of the features of the system 10 of Figure 16, in addition to certain optional features. The same reference numerals are used to denote the same / corresponding features in relation to Figure 16 and will not be described in detail again below. Figure 23 shows the plurality of acoustic transducers 16a-16 / ? arranged in a 2-dimensional pattern in the plane (e.g., the x-y plane). The acoustic array 16 is shown in Figure 23 to comprise 36 acoustic transducers, wherein the acoustic transducer 16 / ? represents the final (i.e., 36th) transducer in the acoustic array 16. In an example, the control unit 30 is configured to control the phase of each transducer 16a-1 Q / 3 (i.e., a phased array system). In this example, the plurality of acoustic transducers may be orientated inward towards the cell container, e.g., 30, 45, 90 degrees relative to the plane.

[0180] Figure 23 also shows a multi-well plate 40 comprising a plurality of cell containers 14, 41 , 42, 43, 44, 45, known as wells. Each of the plurality of cell containers 14, 41 , 42, 43, 44, 45 is suitable for containing the medium 18 and cells adherent to the surface of each cell container 14, 41 , 42, 43, 44, 45. In an alternative example, the system 10b may comprise a cell container 14 as shown in Figures 21 and 22, in place of the multi-well plate 40.

[0181] Figure 24 shows a side view of a system 10c. The system 10c comprises all of the features of the system 10 of Figures 16 and 21 , in addition to certain optional features. The same reference numerals are used to denote the same / corresponding features in relation to Figure 16 and will not be described in detail again below. Figure 24 shows a plurality of acoustic transducers 16a-16I of an acoustic array 16. Figure 24 shows the cell container 14 is a well of the multi-well plate 40. The multi-well plate 40 also includes two additional cell containers 41 , 42. The system 10c comprises an acoustic coupling 34. The acoustic coupling 34 comprises a first acoustic coupling component 34a and second acoustic coupling component 34b. The first acoustic coupling component 34a is different to the second acoustic coupling component 34b. The first acoustic coupling component 34a may comprise at least one of: a liquid; a hydrogel; a gel; or, a solid. The second acoustic coupling component 34b may comprise an acoustic lens, acoustic hologram, acoustic kinoform, or other acoustic metamaterial (or other designed material with a different speed of sound to the rest of the coupling to enable a phase and / or amplitude variation). The second acoustic coupling component 34b may be time variant and electrically controlled (by the control unit 30) for dynamic control of the system 10c. In an alternative example, each of the first and second acoustic coupling components 34a, 34b, may comprise at least one of: a liquid; a hydrogel; a gel; a solid; an acoustic lens, acoustic hologram, acoustic kinoform, or other acoustic metamaterial (or other designed material with a different speed (or relative speed) of sound to the rest of the coupling to enable a phase and / or amplitude variation).

[0182] Figure 25 shows a side view of a system 10d. The system 10d comprises all of the features of the system 10c of Figure 24, in addition to certain optional features. The same reference numerals are used to denote the same / corresponding features in relation to Figure 16 and will not be described in detail again below. In the example, the multi-well plate 40 is coupled to the acoustic array via a first acoustic coupling component 34a. In addition, each cell container 14, 41 , 42 corresponds to a respective second acoustic coupling component 34b.

[0183] Figure 26 shows a side view of a system 10e. The system 10e comprises all of the features of the system 10 of Figures 16 to 21 , in addition to certain optional features. The same reference numerals are used to denote the same / corresponding features in relation to Figure 16 and will not be described in detail again below. The system 10e may be a liquid handling system.

[0184] In the example of Figure 26, the acoustic coupling 34 comprises a first acoustic coupling component 34a and second acoustic coupling component 34b. In the example of Figure 26, the cell container 14 comprises a means of changing liquids, such as an input pipe 50 and an outlet pipe 51. Alternatively, the means of changing liquids may be via a pipette, or via a single pipe. A pipe 50 or a pipette may be configured to provide a dissociation reagent to the cell container 14.

[0185] Figure 26 shows a temperature sensor 52, and a temperature adjusting means 54 (e.g., a heater) to adjust the temperature of the acoustic coupling 34, 34a, 34b based on the temperature sensor 52. The temperature sensor 52 may be in communication with the medium 18, or may be a laser thermometer or thermal camera. The control unit 30 may receive as an input a reading from the temperature sensor 52 and generate a temperature control signal for operating the temperature adjusting means 54. The temperature adjusting means may heat or cool the system depending on the requirements. In a cell culture process, the temperature may be advantageously controlled within a temperature range to promote desired cell activity. The temperature adjusting means 54 may be operated to maintain the temperature within an operating temperature range, or be operated to maintain the temperature at an operating temperature set point, e.g., 32°C or 37°C.

[0186] Figure 26 shows a camera sensor 56. The camera sensor 56 may be configured to capture one or more images of the cell container 14 to determine whether the cell container 14 contains a medium 18 and cells 12 adherent to the surface of the cell container 14. The control unit 30 may be configured to determine that the first portion of the surface of the cell container 14 comprises the first group of cells adherent to the surface of the cell container 14 based on the image. The control unit 30 may generate the first acoustic field in response to determining that the first portion of the surface of the cell container 14 comprises the first group of cells. Thus, the camera sensor 56 and the control unit 30 may identify a portion where cells which are adherent to a surface of the cell container 14 and generate one or more acoustic fields to release the cells from the portion of the surface of the cell container 14. Advantageously, a camera sensor 56 captures the behaviour of the cells as they stick to the cell container 14, so that the system 10e can be controlled in real time or the output optimised for future operations.

[0187] In the example of Figure 26, the camera sensor 56 is positioned above the cell container 14. In an alternative example, the camera sensor 56a may be positioned below the acoustic array 16 as shown in Figure 14. The camera sensor 56 may image the cell container 14 through gaps in the acoustic array 16. The camera sensor 56 may image the cell container 14 through a transparent acoustic array 16. In an alternative example, the camera sensor 56 may be integrated amongst the acoustic array 16 to image the cell container 14. The camera sensor 56 may be a microscope. The system 10e may include fluorescence sensors (if the cells are treated to generate a florescent effect) or other cell analysis sensors.

[0188] The control unit 30 of any described example may be one or more processors, computers, or controllers. The control unit 30 is configured to control the plurality of acoustic transducers 16 to release adherent cells in a cell container 14. The cell container contains a medium 18 and cells 12 adherent to the surface 14a of the cell container 14. Such a method, as shown in Figure 27, to release adherent cells in a cell container 14 with a plurality of acoustic transducers may comprise the following steps:

[0189] T1 : Optionally, providing a dissociation reagent to the cell container. The amount of dissociation reagent required may be less than the amount of dissociation reagent required if no acoustic fields were used. The amount of dissociation reagent required may be the same amount of dissociation reagent required if no acoustic fields were used (advantageously, this may release cells at a faster speed).

[0190] T2: Optionally, capturing an image of the cell container 14. T3: Optionally, determining (with the control unit 30) if a portion of the surface of the cell container 14 comprises a group of cells adherent to the surface of the cell container based on the image. If no cells are adherent to the surface of the cell container 14 (i.e., determining if the surface of the cell container is free from adhered cells based on the image), then the method may return to step T2. If a group of cells are adherent to the surface of the cell container based on the image, then the method moves to step T4.

[0191] In an alternative example, if no cells are adherent to the surface of the cell container 14 (i.e., determining if the surface of the cell container is free from adhered cells based on the image), then the method may end, or move to step T5 if a dissociation reagent is used.

[0192] T4: Generating one or more acoustic fields (e.g., acoustic fields 20, 20a, 20b, 22) by controlling the plurality of acoustic transducers (e.g., transducers 16a, 16b, ...). The acoustic fields generate a respective acoustic effect (e.g., acoustic effect 32, 32a, 32b) to release a respective group of cells from a respective portion of the surface of the cell container 14. Each acoustic field may be operated for a respective time period.

[0193] Optionally, some acoustic fields may generate one or more acoustic effects on a first portion of the surface of the cell container 14. For example, the group of cells of the first portion may not be instantly released from the container 14, so multiple cycles may be used to release all cells from the first portion. That is, for each portion of the cell container 14, the system may generate two or more acoustic effects to release a group of cells from the single portion of the surface of the cell container 14.

[0194] T5: The cells 12 within the cell container 14 may now be in suspension within the cell container 14 (in the medium 18). If a dissociation reagent was added to the cell container 14, a media may be added to the cel container to quench reaction.

[0195] Steps T6, T7, and T8 may be operated independently from Steps T 1 to T5. Steps T6 to T8 are optional and may only be operated if the system comprises a temperature sensor 52, and a temperature adjusting means 54.

[0196] T6: Receiving (at the control unit 30) a temperature measurement from a temperature sensor 52. The temperature measurement corresponding to the temperature of an acoustic coupling arranged between the plurality of acoustic transducers and the surface of the cell container 14. The acoustic coupling 34 is suitable for transmitting the generated acoustic fields to the surface of the cell container 14.

[0197] T7: Determining if the temperature of the acoustic coupling 34 is within an acceptable range of temperatures or operating at a temperature set point. If it is not, then the method may move to step T8. If the temperature is within the acceptable range of temperatures or operating at the temperature set point, then the method may return to step T6. T8: Adjusting the temperature of the acoustic coupling 34, optionally with a temperature adjusting means 54 (e.g., a heater).

[0198] General

[0199] The acoustic transducers described herein may be piezoelectric transducers, electromechanical speakers, laser excitation, Capacitive Micromachined Ultrasound Transducers (CMUT), Surface Acoustic Wave transducers, and Piezoelectric Micromachined Ultrasound Transducers (PMUT).

[0200] The acoustic array 16 may comprise two or more acoustic transducers. Each of the plurality of acoustic transducers is arranged to operate at a frequency greater than 1 MHz, e.g., 2.25MHz.

[0201] In an example, a dissociation reagent may be at least one of: Tryspin; Phosphate-buffered saline (PBS); cell culture media; Collagenase; Ethylenediaminetetraacetic acid (EDTA); Loosening chemicals; water; dilutions or solutions of the above or other liquids.

[0202] In an example, the acoustic array 16 is positioned below the cell container 14 (e.g., Figures 1 and 16). In an alternative example, the acoustic array 16 may be positioned above the cell container 14. The acoustic array 16 may be in communication with the medium 18 of the cell container 14 either directly, or via a sterile barrier. The sterile barrier, acoustic coupling, or acoustic array may be disposable.

[0203] In an example, the temperature adjusting means 54 may adjust the temperature of the acoustic coupling 34 and the cell container 14. In an example, a system of the examples of Figures 1 to 12, 14, and 16 to 26 may not comprise an acoustic coupling as sound waves may pass directly from the acoustic array 16 to the cells 12 or medium 18 via air.

[0204] Any example may be integrated into a liquid handling system. A liquid handling system may comprise a liquid handling robot or other liquid exchange system which may be used to add and remove liquids from the cell container 14 automatically.

[0205] In this document, the terms “a” or “an” are used, as is common in patent documents, to include one or more than one, independent of any other instances or usages of “at least one” or “one or more.” In this document, the term “or” is used to refer to a nonexclusive or, such that “A or B” includes “A but not B,” “B but not A,” and “A and B,” unless otherwise indicated. In the appended claims, the terms “including” and “in which” are used as the plain-English equivalents of the respective terms “comprising” and “wherein.” Also, in the following claims, the terms “including” and “comprising” are open-ended, that is, a system, device, article, or process that includes elements in addition to those listed after such a term in a claim are still deemed to fall within the scope of that claim. Moreover, in the following claims, the terms “first,” “second,” and “third,” etc. are used merely as labels, and are not intended to impose numerical requirements on their objects. Method examples described herein can be machine or computer-implemented at least in part. Although the invention has been described above with reference to one or more preferred embodiments, it will be appreciated that various changes or modifications may be made without departing from the scope of the invention as defined in the appended claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

Claims

1. CLAIMS1. A method for releasing adherent cells in a cell container with a plurality of acoustic transducers, wherein the cell container comprises a surface, wherein the cell container contains a medium and cells adherent to the surface of the cell container the method comprising: generating a first acoustic field by controlling the plurality of acoustic transducers, wherein the first acoustic field generates a first acoustic effect to release a first group of cells from a first portion of the surface of the cell container; and generating a second acoustic field by controlling the plurality of acoustic transducers, wherein the second acoustic field generates a second acoustic effect to release a second group of cells from a second portion of the surface of the cell container, wherein the first acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers to generate the first acoustic effect, wherein the second acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers to generate the second acoustic effect, wherein the first acoustic effect is a focus point, and the second acoustic effect is a focus point.

2. The method of claim 1 , wherein the first acoustic effect is at least one of: an acoustic vortex; an acoustic twin-trap; or, an acoustic bottle field, and / or wherein the second acoustic effect is at least one of: an acoustic vortex; an acoustic twin-trap; or, an acoustic bottle field.

3. The method of any one of claims 1 or 2, wherein the first acoustic field is generated by controlling an amplitude of a first acoustic transducer of the plurality of acoustic transducers to generate the first acoustic effect, and wherein the second acoustic field is generated by controlling an amplitude of a second acoustic transducer of the plurality of acoustic transducers to generate the second acoustic effect.

4. The method of claim 3, wherein the amplitude of the first acoustic transducer is different to the amplitude of the second acoustic transducer.

5. The method of any preceding claim, wherein the first acoustic field is generated at a different time to the second acoustic field.

6. The method of any preceding claim, further comprising, prior to the generation of the first and second acoustic fields: providing a dissociation reagent to the cell container.

7. The method of any preceding claim, wherein the plurality of acoustic transducers is arranged linearly and in a plane.

8. The method of any preceding claim, wherein the plurality of acoustic transducers is arranged in a 2-dimensional pattern in a plane.

9. The method of any preceding claim, further comprising: generating a third acoustic field by controlling the plurality of acoustic transducers, wherein the third acoustic field generates a third acoustic effect to release a third group of cells from a third portion of the surface of the cell container.

10. The method of claim 9, wherein the plurality of acoustic transducers are arranged to generate a series of acoustic effects upon a plurality of portions of the surface, wherein the area corresponding to the plurality of portions of the surface is substantially equal to the total area of the surface, wherein the plurality of portions of the surface comprise the first, second, and third portions of the surface.11 . The method of any preceding claim, wherein each of the plurality of acoustic transducers are excited continuously between the generation of the first and second acoustic fields.

12. The method of claim 1 1 , wherein the first acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers to generate the first acoustic effect comprising a complex acoustic effect at the first position, wherein the second acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers to generate the second acoustic effect comprising the complex acoustic effect at the second position, wherein each of the plurality of acoustic transducers is controlled to move the complex acoustic effect between the first and second position.

13. The method of any one of claims 1 to 10, wherein each of the plurality of acoustic transducers are turned off between the generation of the first acoustic field and the second acoustic field.

14. The method of any preceding claim, further comprising: adjusting the temperature of an acoustic coupling arranged between the plurality of acoustic transducers and the surface of the cell container, wherein the acoustic coupling is suitable for transmitting the generated acoustic fields to the surface of the cell container.

15. The method of any preceding claim, further comprising: capturing an image of the cell container to determine the cell container contains a medium and cells adherent to the surface of the cell container; determining that the first portion of the surface of the cell container comprises the first group of cells adherent to the surface of the cell container based on the image; and,generating the first acoustic field in response to determining that the first portion of the surface of the cell container comprises the first group of cells.

16. A system to release adherent cells from a surface, the system comprising: a cell container comprising a surface, wherein the cell container is suitable for containing a medium and cells adherent to the surface of the cell container; a plurality of acoustic transducers suitable for generating acoustic fields.

17. The system of claim 16, further comprising: an acoustic coupling arranged between the plurality of acoustic transducers and the surface of the cell container, wherein the acoustic coupling is suitable for transmitting the generated acoustic fields to the surface of the cell container.

18. The system of claim 17, wherein the acoustic coupling comprises at least one of: a liquid; a hydrogel; a gel; a solid; and an acoustic metamaterial or other designed material with a different speed of sound to the rest of the coupling to enable a phase and / or amplitude variation, or other designed material with a different effective speed of sound to the rest of the coupling to enable a phase and / or amplitude variation.

19. The system of any one of claims 17 or 18, further comprising a temperature sensor, and a temperature adjusting means to adjust the temperature of the acoustic coupling based on the temperature sensor.

20. The system of any one of claims 16 to 19, wherein the plurality of acoustic transducers is arranged linearly and in a plane, and optionally, the plurality of acoustic transducers is arranged in a 2-dimensional pattern in the plane.

21. The system of any one of claims 16 to 20, wherein each of the plurality of acoustic transducers is arranged to operate at a frequency greater than 1 MHz.

22. The system of any one of claims 16 to 21 , wherein the cell container comprises a plurality of wells, wherein each of the plurality of wells is suitable for containing a medium and cells adherent to the surface of the cell container.

23. The system of any one of claims 16 to 21 , further comprising: a multi-well plate, wherein the cell container is a well of the multi-well plate.

24. A liquid handling system comprising the system of any one of claims 16 to 23.

25. A cell passaging process for transferring cells from a cell container containing a medium and the cells, the cell passaging process comprising: a cell concentration step comprising: generating one or more first acoustic fields by controlling a plurality of acoustic transducers; increasing the density of cells in a first portion of the cell container above a density threshold using the one or more first acoustic fields; and, decreasing the density of cells in a second portion of the cell container using the one or more first acoustic fields; and, removing a first amount of the medium from the second portion of the cell container.

26. The cell passaging process of claim 25, wherein the density threshold is at least a cell density of 0.4 million cells per millilitre.

27. The cell passaging process of any one of claims 25 or 26, wherein the one or more first acoustic fields comprise: a one or more holding acoustic fields to substantially maintain the density of cells in the first portion of the cell container after the density threshold is reached.

28. The cell passaging process of any one of claims 25 to 27, wherein the first portion of the cell container comprises a side wall of the cell container.

29. The cell passaging process of any one of claims 25 to 28, wherein the one or more first acoustic fields comprise: a first acoustic field to generate a first acoustic effect to move a first plurality of cells relative to the cell container and towards the first portion of the cell container; and a second acoustic field to generate a second acoustic effect to move a second plurality of cells relative to the cell container and towards the first portion of the cell container.

30. The cell passaging process of claim 29, wherein the first acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers to generate the first acoustic effect; and / or, wherein the second acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers to generate the second acoustic effect.31 . The cell passaging process of claim 30, wherein the first acoustic effect is at least one of: a focus point; an acoustic vortex; an acoustic twin-trap; or, an acoustic bottle field, and / or whereinthe second acoustic effect is at least one of: a focus point; an acoustic vortex; an acoustic twintrap; or, an acoustic bottle field.

32. The cell passaging process of any one of claims 29 to 31 , wherein the first acoustic field is generated by controlling an amplitude of a first acoustic transducer of the plurality of acoustic transducers to generate the first acoustic effect, and wherein the second acoustic field is generated by controlling an amplitude of a second acoustic transducer of the plurality of acoustic transducers to generate the second acoustic effect.

33. The cell passaging process of claim 32, wherein the amplitude of the first acoustic transducer is different to the amplitude of the second acoustic transducer.

34. The cell passaging process of any one of claims 29 to 33, wherein the first acoustic field is generated at a different time to the second acoustic field.

35. The cell passaging process of any one of claims 29 to 34, wherein the plurality of acoustic transducers is arranged linearly and in a plane; or arranged in a 2-dimensional pattern and in a plane.

36. The cell passaging process of any one of claims 29 to 35, wherein the first acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers to generate the first acoustic effect comprising a complex acoustic effect at a first location within the second portion, wherein the second acoustic field is generated by controlling the phase of each of the plurality of acoustic transducers to generate the second acoustic effect comprising the complex acoustic effect at a second location within the first portion, wherein each of the plurality of acoustic transducers is controlled to move the location of the complex acoustic effect from the first location to the second location.

37. The cell passaging process of any one of claims 25 to 36, wherein the density threshold is a first density threshold, the cell passaging process further comprising: capturing an image of the cell container to determine a distribution of cells in the cell container; determining that the second portion of the cell container comprises cells above a second density threshold based on the image; and, generating the one or more first acoustic fields in response to determining that: the second portion of the cell container comprises cells above the second density threshold.

38. The cell passaging process of any one of claims 25 to 37, further comprising: a cell disassociation step comprising releasing a plurality of cells adherent to a surface of the cell container.

39. The cell passaging process of claim 38, wherein the cell disassociation step further comprises: generating one or more second acoustic fields by controlling the plurality of acoustic transducers, wherein the plurality of cells adherent to the surface of the cell container are released using the one or more second acoustic fields.

40. The cell passaging process of any one of claims 38 or 39, wherein the cell disassociation process further comprises, prior to the cell concentration step and the generation of the one or more second acoustic fields: providing a dissociation reagent to the cell container.41 . The cell passaging process of any one of claims 38 to 40, further comprising, after the cell disassociation step: a first cell mixing step comprising redistributing cells in the medium.

42. The cell passaging process of claim 41 , wherein the first cell mixing step comprises: generating one or more third acoustic fields by controlling the plurality of acoustic transducers, wherein the plurality of cells in the medium are distributed using the one or more third acoustic fields.

43. The cell passaging process of any one of claims 25 to 42, further comprising, after the cell concentration step: a volume adjust step, comprising: determining that the total volume of the medium in the cell container is at a predetermined volume; removing the first amount of the medium from the second portion of the cell container; and optionally, providing a second amount of media to the cell container.

44. The cell passaging process of claim 43, further comprising, after the volume adjustment step: performing a second cell mixing step to redistribute cells within the medium.

45. The cell passaging process of claim 44, wherein the second cell mixing step comprises: generating one or more fourth acoustic fields by controlling the plurality of acoustic transducers, wherein the cells in the medium are redistributed using the one or more fourth acoustic fields.

46. The cell passaging process of any one of claims 25 to 45, further comprising, a transfer step comprising: transferring the plurality of cells from the cell container into one or more additional cell containers.

47. The cell passaging process of claim 46, wherein the transfer step further comprises counting cells in the cell container before transferring.

48. A system for transferring cells from a cell container comprising: a cell container comprising a medium and cells; a plurality of acoustic transducers suitable for generating one or more acoustic fields; and, a processor configured to perform the cell passaging process of any one of claims 25 to 47.

49. A liquid handling system comprising the system of claim 48.

Images