Acoustic release of adherent cells

Abstract

The disclosed invention relates to a method and system 10 for releasing adherent cells 12 in a cell container 14 with a plurality of acoustic transducers 16a, 16b, 16c, 16h. The method comprises: gene

Description

FIELD OF THE INVENTION The present invention is in the field of cell culture automation. Specifically, in the field of cell dissociation and manipulation within cell culture automation. BACKGROUND OF THE INVENTION Cell passaging is the procedure of harvesting cells from a culture, 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 would be used in further assays) or could be put directly into a new assay (e.g. a dye). Cell culture automation may be used to automate the cell passaging procedure. When growing cells, it is desirable to keep them sterile while moving them from one container to another container. Growing cells often adhere to the surface which they are grown in. Unsticking adherent cells is a challenge in cell culture automation. Chemical dissociation reagents may be used to release cells from these surfaces during the culturing process, but this is often 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. Incubators and shaker plates have been used in automation processes, but these are often unsatisfactory. Moving a cell container to an incubator creates a bottleneck in the automation process &requires a larger, more complex system. Shaker plates are also unsatisfactory as they poorly recreate manual agitation and require a lot of 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 (moving side to side is not the same as force applied from the bottom). Robot arms are not designed to bang culture plates on a lab bench. Scrapers are difficult to automate and can destroy cells. 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. SUMMARY OF THE INVENTION 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. 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). 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. 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. 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. 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 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. Optionally, the first acoustic effect is at least one of: a focus point; an acoustic vortex; an acoustic twin-trap; or, an acoustic bottle field. Optionally, the second acoustic effect is at least one of: a focus point; 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. 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. 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. 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. The method of the first aspect may be a computer implemented method. 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. 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. 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. 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 1MHz. 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. 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. A third aspect of the invention provides a liquid handling system comprising the system of the second aspect. BRIEF DESCRIPTION OF THE DRAWINGS 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 to release adherent cells from a surface of a cell container. Figure 2 illustrates a side view of the system of Figure 1 as an acoustic array generates a first acoustic field and a second acoustic field. Figure 3 illustrates a side view of a system to release adherent cells as an acoustic array generates a first acoustic field with phase control. Figure 4 illustrates a side view of a system to release adherent cells as an acoustic array generates a first acoustic field with amplitude control. Figure 5a 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. Figure 5b 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. Figure 5c 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. Figure 6 illustrates a top view of the system of Figure 1 to release adherent cells from a surface of a cell container. Figure 7 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. Figure 8 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. Figure 9 illustrates a side view of a system to release adherent cells, with optional features. Figure 10 illustrates a side view of a system to release adherent cells, with optional features. Figure 11 illustrates a side view of a system to release adherent cells, with optional features. Figure 12 illustrates a side view of a system to release adherent cells, with optional features. Figure 13 illustrates a method for releasing adherent cells in a cell container with a plurality of acoustic transducers. DETAILED DESCRIPTION OF EMBODIMENT(S) 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. Figure 1 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 1, 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. 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. 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. 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. Figure 2 shows the system 10 of Figure 1 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. 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. The system 10 of Figures 3 and 4 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. Figures 3 and 4 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. Figures 3 and 4 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. 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 3 and 4 includes a top surface 14c. Alternatively, the cell container 14 may be any suitable cell container 14. 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. Figure 3 shows the control unit 30 configured to generate and transmit a phase control signal to $8 to 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. Although not shown in Figures 3 for conciseness, the second acoustic field may be generated by controlling the phase 0 of each of the plurality of acoustic transducers 16a-16h to generate the second acoustic effect similarly to the first acoustic effect 32a. 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 3 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. In an example, the acoustic effect may be a focus point, and the phase for each transducer may be calculated with equation (1) below: <Pi = k(ri-rj) (1) Where is the phase for a transducer j, k is the wavenumber for the system 10 and r is calculated by equation (2) below: rj = J(xj ~ xz)2 + zf W Where xj is the x axis location of the centre of transducer j, xf is the x axis location of the desired focus point and zf 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. Figure 4 shows the control unit 30 configured to generate and transmit an amplitude control signal a1 to a8 to 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. 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. Transducers 16b-16h of the acoustic array 16 are each controlled to generate at least two unique amplitudes. As shown in Figure 4 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 4. Although not shown in Figure 4 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. In both examples shown in Figures 3 and 4, 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). 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. Figures 5a, 5b, 5c 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. Figure 5a 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 5c 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 5b shows the intermediate state between Figures 5a and 5c, 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). 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. Figure 6 shows a top view of the system 10 of Figure 1. Figure 6 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 6). 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. Figure 7 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 6, in addition to certain optional features. The same reference numerals are used to denote the same / corresponding features in relation to Figures 1 and 6, and will not be described in detail again below. Figure 7 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 7 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-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. Figure 8 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 8 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 8 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-16 / ? (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. Figure 8 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 6 and 7, in place of the multi-well plate 40. Figure 9 shows a side view of a system 10c. The system 10c comprises all of the features of the system 10 of Figures 1 and 6, 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 9 shows a plurality of acoustic transducers 16a-161 of an acoustic array 16. Figure 9 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). Figure 10 shows a side view of a system lOd. The system lOd comprises all of the features of the system 10c of Figure 9, in addition to certain optional features. The same reference numerals are used to denote the same / corresponding features in relation to Figure 9 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. Figure 11 shows a side view of a system lOe. The system lOe comprises all of the features of the system 10 of Figures 1 to 6, 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 lOe may be a liquid handling system. In the example of Figure 11, the acoustic coupling 34 comprises a first acoustic coupling component 34a and second acoustic coupling component 34b. In the example of Figure 11, 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. Figure 11 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. Figure 11 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 lOe can be controlled in real time or the output optimised for future operations. In the example of Figure 11, 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 12. 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 lOe may include fluorescence sensors (if the cells are treated to generate a florescent effect) or other cell analysis sensors. 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 13, to release adherent cells in a cell container 14 with a plurality of acoustic transducers may comprise the following steps: SI: 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). S2: Optionally, capturing an image of the cell container 14. S3: 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 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 S4. 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 S5 if a dissociation reagent is used. S4: 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. 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. S5: 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. Steps S6, S7, and S8 may be operated independently from Steps SI to S5. Steps S6 to S8 are optional and may only be operated if the system comprises a temperature sensor 52, and a temperature adjusting means 54. S6: 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. S7: 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 S8. If the temperature is within the acceptable range of temperatures or operating at the temperature set point, then the method may return to step S6. S8: Adjusting the temperature of the acoustic coupling 34, optionally with a temperature adjusting means 54 (e.g., a heater). General 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). 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 1MHz, e.g., 2.25MHz. 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. In an example, the acoustic array 16 is positioned below the cell container 14 (e.g., Figure 1). 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. 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 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. 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. 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. 5 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 10 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. 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; andgenerating 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.

2. The method of claim 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; 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.

3. The method of claim 2, 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 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.

4. The method of any one of claims 1 to 3, 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.

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

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

7. 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.

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

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

10. 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.

11. The method of claim 10, 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.

12. 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.

13. The method of claim 12, 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.

14. The method of any one of claims 1 to 11, wherein each of the plurality of acoustic transducers are turned off between the generation of the first acoustic field and the second acoustic field.

15. 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.

16. 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.

17. 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.

18. The system of claim 17, 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.

19. The system of claim 18, wherein the acoustic coupling comprises at least one of:a liquid;a hydrogel;a gel;a solid; andan 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.

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

21. The system of any one of claims 17 to 20, wherein the plurality of acoustic transducers is arranged linearly and in a plane, or, the plurality of acoustic transducers is arranged in a 2-dimensional pattern and in a plane.

22. The system of any one of claims 17 to 21, wherein each of the plurality of acoustic transducers is arranged to operate at a frequency greater than 1MHz.

23. The system of any one of claims 17 to 22, 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.

24. The system of any one of claims 17 to 22, further comprising: a multi-well plate, wherein the cell container is a well of the multi-well plate.

25. A liquid handling system comprising the system of any one of claims 17 to 24.

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