WO2020067891A1 - Device for controlled stirring, method for controlled stirring using the device - Google Patents

Abstract

A device (1) for providing controlled stirring comprising: an electromagnetic system (3) arranged for realising a magnetic field; and at least one stirring chamber (5) comprising at least one Suspended Magnetically Actuatable Pillar (7), SMAP (7), suspended in said chamber (5) from a support (9) and configured, in use, in said magnetic field such that said SMAP (7) is magnetically actuated, by said magnetic field, for providing said controlled stirring. A method using said device (1) for controlled stirring in a stirring chamber (5), said method comprising the steps of: realising, by said electromagnetic system (3), said magnetic field; and magnetically actuating, by said magnetic field, said SMAP (7), for realising said controlled stirring in said stirring chamber (5). The invention further relates to the use of a SMAP (7), for controlled stirring in a stirring chamber (5), wherein said SMAP (7) is suspended from a support (9), wherein said SMAP (7) is magnetically actuated, by a magnetic field, for said controlled stirring.

Description

Title: DEVICE FOR CONTROLLED STIRRING, METHOD FOR CONTROLLED STIRRING USING THE DEVICE

Technical field

This invention relates to a device and a method for controlled stirring and to the use of a suspended magnetically-actuatable pillar (SMAP) for controlled stirring.

Background

In most research laboratories, controlled stirring is applied using a standard, commercially available magnetic stirrer plate together with stirrer bars, which are placed at the bottom of the vessel holding the solution to be stirred. Most of the prior art stirring methods are perturbative or require vessels with predefined shapes and sizes. These prior art stirring systems, while simple, are expensive (hundreds to thousands USD) and necessitates relatively large sample volumes in the range of mL to L. These large required volumes are sometimes prohibitive, especially for studies involving precious materials. Such a stirring system is also much less applicable in the context of cell biological research, as the motion of the stirrer bar at the bottom of the flask would disrupt and damage the culture of adherent cells. Other, more specialized alternative stirring methods have been developed and commercialized for cell research, such as spinner flasks and various types of stirred-tank and wheel reactors. These alternative approaches can induce fluid flow in the vessel yet eliminate possible cell damage caused by the stirrer bar, either by effecting whole-vessel movements or by physically separating the cell culture from the impeller. However, all these alternative technologies are costly, involve special equipment and require application- specific vessels with predefined shapes and sizes that are often single-use; in addition these technologies still require large sample volumes (mL to L and kL).

There is a need for a scalable, cost effective solution, especially with the growing appreciation and rapid advance of three-dimensional cultures that mimic in vivo environments. The present invention addresses these problems and aims to provides a novel device and method for controlled stirring that solves one or more the disadvantages above. In addition, it is an aim of the present invention to provide a device and method for controlled stirring of small volumes. In addition, it is an aim of the present invention to provide scalable, non-disruptive stirring device and method that overcome these issues. Summary

The present invention relates in a first aspect to a device for providing controlled stirring comprising: an electromagnetic system arranged for realising a, preferably time-dependent, magnetic field; and at least one stirring chamber comprising at least one Suspended Magnetically Actuatable Pillar, SMAP, suspended in said chamber from a support and configured, in use, in said, preferably time- dependent, magnetic field such that said SMAP is magnetically actuated, by said, preferably time-dependent, magnetic field, for providing said controlled stirring.

An advantageous device is for controlled stirring and comprises: an electromagnetic system arranged for realising a, preferably time-dependent, magnetic field; and at least one stirring chamber comprising at least one Suspended Magnetically Actuatable Pillar, SMAP, suspended in said chamber from a support and provided, in use, in said, preferably time-dependent, magnetic field such that said SMAP is magnetically actuated, by said, preferably time-dependent, magnetic field, for realising said controlled stirring.

It is advantageous if said electromagnetic system comprises a motor having a motor shaft arranged for rotation about a rotation axis, a rotatable plate attached to said motor shaft and a magnet attached to said rotatable plate at a distance from said rotation axis. The magnet can also be mounted on said rotatable plate.

It has certain benefits if said SMAP extends along said rotation axis. For example, the SMAP can be partially aligned with said rotation axis.

In an embodiment, said device further comprises a housing surrounding said motor, said motor shaft, and said rotating plate provided with said magnet, wherein said housing supports said at least one stirring chamber above said rotatable plate.

It is a further possibility that the said at least one stirring chamber is mounted on said housing. For example, said at least one stirring chamber can be placed on said housing via an adhesive.

In a preferred embodiment, said device comprises a multi-well plate provided with a plurality of said stirring chambers, preferably wherein said multi-well plate comprises 24 stirring chambers. For this embodiment, it is advantageous if said support comprises a plurality of said SMAPs, each associated with a stirring chamber of said plurality of at least one stirring chamber. In an embodiment, said support comprises a support plate, preferably a glass plate, wherein said support plate is supported, by said at least one stirring chamber, on an upper open side of said at least one stirring chamber.

It is further advantageous if said SMAP is fabricated from a magnetic precursor material which is composed of thermally curable polydimethylsiloxane and carbonyl iron powder.

It is further possible for said SMAP to have a non-cylindrical shape.

It further has certain benefits if said SMAP is flexible for bending and rotating, by said preferably time-dependent electromagnetic field, said SMAP for realising said controlled stirring.

In an embodiment, said preferably time-dependent magnetic field is controlled by electric current.

In a second aspect, the invention relates to a method for controlled stirring in a stirring chamber, using a device comprising: an electromagnetic system arranged for realising a, preferably time-dependent, magnetic field; and at least one stirring chamber comprising a chamber well, wherein said at least one stirring chamber comprises at least one Suspended Magnetically Actuatable Pillar, SMAP, suspended in said chamber from a support and provided in said, preferably time-dependent, magnetic field such that said SMAP is magnetically actuated, by said, preferably time- dependent, magnetic field, for realising said controlled stirring; said method comprising the steps of: realising, by said electromagnetic system, said, preferably time-dependent, magnetic field; and magnetically actuating, by said, preferably time- dependent, magnetic field, said SMAP, for realising said controlled stirring in said stirring chamber.

It is advantageous if said electromagnetic system comprises a motor provided with a motor shaft arranged for rotation about a rotation axis a rotating plate attached to said motor shaft and a magnet attached to said rotating plate at a distance from said rotation axis, wherein, during said step of realising, said magnetic field is realised by rotating said magnet, by said motor, about said rotation axis.

In an embodiment of the second aspect, said device comprises a housing surrounding said motor, said motor shaft, and said rotating plate provided with said magnet, wherein said method further comprises the step of placing said at least one stirring chamber on said housing via an adhesive. In a third aspect, the invention relates to the use of a Suspended Magnetically Actuatable Pillar, SMAP, for controlled stirring in a stirring chamber, wherein said SMAP is suspended from a support, wherein said SMAP is magnetically actuated, by a, preferably time-dependent, magnetic field, for said controlled stirring.

It is advantageous if said SMAP comprises a plurality of magnetic particles in a polymer matrix.

It can be foreseen that said magnetic particles are homogenously distributed in said polymer matrix. It can also be foreseen that said magnetic particles are concentrated near an outer end of said SMAP. Said magnetic particles can further be aligned in said polymer matrix.

In an embodiment, the use is for stirring of mammalian cells provided in said stirring chamber or for mesofluidic mixing for realising at least one of colloid self- assembly, directed assembly and separation of microparticles and biological cells or for generation of cell spheroids for cell biological and drug screening studies.

Embodiments of these aspects and more details are disclosed below.

Brief description of drawings

Fig. 1 shows the fabrication process of different types of SMAPs.

Fig. 2 shows the inventive SMAPs stirring system.

Fig. 3 shows the characterization of SMAPs.

Fig. 4 shows fluid flow velocity profiles during stirring using an SMAP.

Fig. 5 shows stirring-induced clustering of cells.

Detailed description

In this invention, a novel controlled stirring technology based on suspended magnetically-actuatable pillars (SMAPs) is applied. These SMAPs can be constructed with low-cost minimal technical requirements. The advantages of the present system are that it is cost-effective, contactless, simple to fabricate, easily scalable to fit the geometry of the vessel of choice, and requires no specialized equipment. In addition, there are very suitable for stirring small volumes. In addition, multiple stirring chambers may be stirred simultaneously with the present device and method.

From the publication by S. Zhang , Y. Wang, R. Lavrijsen, P. R. Onck and J. M. J. den Toonder, in Sensor Actuat B-Chem, 2018, 263, 614-624 entitled“Versatile microfluidic flow generated by moulded magnetic artificial cilia" a method of fabricating a substrate (from polydimethylsiloxane, also called PDMS) provided with multiple magnetic artificial cilia (MACs) is known. A micro-moulding technique is disclosed in that publication that leads to MACs with well-defined geometries and spatial arrangements on a transparent non-magnetic substrate. The distribution of magnetic particles in the polymer matrix of the MACs can be controlled to be either homogeneously distributed or a concentrated distribution at the tip of the MACs. Moreover, these magnetic particles may be (linearly) aligned by application of a magnetic field during curing of the polymer matrix. The substrates with MACs were used in the fabrication of microfluidic devices; the substrate comprising said MACs was placed on a certain position on the bottom of a channel in which fluid flow is desired. The device is then placed on a rotating disc with an off-centred permanent magnet that - when rotating - sets the MACs in a tilted-cone motion; the MACs extending from the bottom up into the fluid present in the channel.

The present inventors have now found a completely new and inventive controlled stirring system based on the MAC technology and the use of said system for high throughput controlled stirring in e.g. a multi-well configuration. The present inventors have devised suspended magnetically-actuatable pillars (SMAPs) that can be used for stirring systems.

This device and method can be a versatile tool for various applications, such as mesofluidic mixing, colloid self-assembly, directed assembly and separation of microparticles and biological cells, as well as generation of cell spheroids for cell biological and drug screening studies.

“Magnetically actuatable pillar” in the context of the present invention is to be understood as a pillar can be actuated by a magnetic field that is preferably time- dependent, thereby arriving a magnetic actuated pillar. When the device according to the invention is in use, the pillar is magnetically actuated to realise a movement of the magnetically actuated pillar due to the preferably time-dependent magnetic field. The term“suspended” in suspended magnetically-actuatable pillars (SMAPs) means that the MAPs are suspended in a stirring chamber, e.g.“hanging” from a support.

Preparation and deflection of SMAPs

In an embodiment of said device 1 and/or method and/or use according to the invention, said SMAP 7 comprises a plurality of magnetic particles 29 in a polymer matrix.

In an embodiment of said device 1 or method, said SMAP 7 is fabricated from a magnetic precursor material which is composed of thermally curable polymer matrix, e.g. polydimethylsiloxane, and magnetic particles, e.g. carbonyl iron powder. In an embodiment of said device 1 or method, said SMAP 7 is flexible for bending and rotating, by said preferably time-dependent electromagnetic field, said SMAP 7 for realising said controlled stirring.

The pillars may be fabricated by moulding a mixture of a flexible polymer, for example polydimethylsiloxane (PDMS) (or another rubbery or flexible material that can be moulded) and magnetic particles, such as iron particles. The mixture may be mixed with hardener or curing agent, or may be dried out. The dimensions of the mould and therefore the pillars (e.g. height, width and shape) can be adapted depending on the required size of the vessel to be stirred.

In an embodiment of said device 1 and/or method and/or use, said magnetic particles 29 are homogenously distributed in said polymer matrix. This may be obtained by stirring the magnetic particles and the polymer matrix prior to addition to the mould and curing/hardening/drying of the polymer matrix comprising said magnetic particles. More details regarding the method of preparation is disclosed in the above disclosed publication.

In an embodiment of said device 1 and/or method and/or use, said magnetic particles 29 are concentrated near an outer end 27 of said SMAP 7. This may be obtained by stirring the magnetic particles and the polymer matrix prior to addition to the mould and allowing the magnetic particles to settle in the mould over a time period of several hours to several days prior to curing/hardening/drying of the polymer matrix comprising said magnetic particles. More details regarding the method of preparation is disclosed in the above disclosed publication.

In an embodiment of said device 1 and/or method and/or use, said magnetic particles 31 are aligned in said polymer matrix. This may be obtained by applying a magnetic field prior and/or during curing/hardening/drying of the polymer matrix comprising said magnetic particles. More details regarding the method of preparation is disclosed in the above disclosed publication.

Figure 1 shows the fabrication process of different types of SMAPs 7; each with a different distribution of the magnetic particles 29. Figure 1A shows a SMAP 7 with a homogeneous (“h”) magnetic particle distribution (called hSMAP). Figure 1 B shows a SMAP 7 with an aligned (“a”) magnetic particle distribution (called aSMAP) obtained by applying a magnetic field during curing. Figure 1 C shows a SMAP 7 with a concentrated (“c”) magnetic particle distribution at the tip 27 (called cSMAP) obtained by storing the uncured SMAPs prior to curing, allowing the heavier magnetic particle 29 to sink to the bottom of the mould prior to curing.

The SMAPs 7 are produced in a micro mould 31 , e.g. of polycarbonate (PC), comprising one or more indentations 33, e.g. cylindrical depressions having a certain diameter and height. In an embodiment, the length of the pillars is greater than the diameter of the pillars. In an embodiment, the diameter of the pillars 7 (diameter of the depression in the mould) is 1 mm. In an embodiment, the length of the pillars 7 (depth of depression in the mould) is 7 mm. In an embodiment, the spacing between the tip 27 of the SMAP and the bottom of the stirring chamber 5 is > 0 mm.

Figure 3 shows the characterization of SMAPs 7. Figures 3A, 3B and 3C respectively show optical microscopy images of the fabricated hSMAP, aSMAP, and cSMAP. The scale bars in these figures are set to 1 mm. The larger magnification images show the distributions of magnetic particles 29 within the SMAPs 7. The SMAPs 7 are shown to be cylindrically shaped with a pointed tip 27, reflecting the shape of the mould 31 used in the fabrication process, with a diameter of 1 mm and a length of 7 mm. For the hSMAPs, the magnetic particles 29 are homogeneously distributed as expected, with only a few randomly-located regions of high or low magnetic particle concentrations (Fig. 3A). For the aSMAPs 7, the application of the magnetic field during the fabrication process results in an ordering of magnetic particles 29 into linear chains oriented along the length of the pillars 7 (Fig. 3B). The slow curing step in the fabrication of the cSMAPs 7 successfully leads to a concentrated region of magnetic particles 29 at the tip 27 of the pillar 7, with a clear boundary between the section rich in iron particles (e.g., carbonyl iron powder, CIP) and the pure PDMS section at the root of the pillar (Fig. 3C). With the present fabrication protocol, the average length ratio between these two sections is found to be 1 :2.

To assess how the variations in the magnetic particle distribution influence the effectiveness of SMAPs 7 as a stirring system, the different types of SMAP 7 were actuated with an actuation setup 3 and the motion of the SMAPs 7 were recorded. The projected tip 27 deflections of the SMAPs 7 are shown in Fig. 3D. Figure 3D shows the lateral tip 27 deflections of the different types of SMAPs 7 when these are magnetically actuated. The data are mean ± standard deviation from measurements of 3 pillars for each SMAP type.

The inventors have found that the deflection of the aSMAPs is significantly larger than the deflection of the other two types of SMAPs. This is consistent with the theoretical prediction that aligned magnetic particle distribution enhances the overall magnetic susceptibility. The deflection of the cSMAPs is the smallest and is close to that of the hSMAPs. This can be explained by the prediction that the magnetic forces acting on the pillars are proportional to the weight of the magnetic particles 29, as the cSMAPs contain only 20% of the magnetic particles 29 compared to hSMAPs and aSMAPs, resulting in a decreased tip deflection. The aSMAPs show the best actuation performance, indicated by the largest tip 27 deflection, and they are used in the remaining examples.

The magnetic susceptibility and the resulting deflection of the pillars 7 can be tuned in multiple ways: by modifying the distribution of the magnetic particles 29 in the pillars 7, by changing the dimensions and/or stiffness of the pillars 7, by using magnets 17 with different flux densities, or by varying the distance between the pillar 7 and the actuating magnets 17.

Moreover, it is straightforward to modulate the resulting flow by taking into account the geometries of the SMAP7 and the vessel 5 and to parallelize the system 1 to perform high-throughput experiments.

These strategies give flexibility to adjust the design of the stirring system 1 based on the exact experimental setup, and therefore makes the system 1 scalable. The main strengths of this system 1 are that it is cost-effective, contactless, simple to fabricate, easily scalable to fit the geometry of the vessel 5 of choice, and it requires no specialized equipment. Moreover, it is straightforward to modulate the resulting flow by considering the geometries of the pillars 7 and the vessel 5 and to parallelize the system 1 to perform high-throughput stirring, e.g. in a multi-well configuration.

In an embodiment of said device 1 or said method according to the invention, said device 1 comprises an electromagnetic system 3 arranged for realising a preferably time-dependent magnetic field. In an embodiment, said electromagnetic system 3 comprises a motor 1 1 provided with a motor shaft 13 arranged for rotation about a rotation axis r, a rotating plate 15 attached to said motor shaft 13 and a magnet, preferably a permanent magnet 17, attached to said rotating plate 15 at a distance d from said rotation axis r. In a specific embodiment of said device 1 or said method, said SMAP 7 extends along said rotation axis r.

In an embodiment, said device 1 further comprises a housing 19 surrounding said motor 1 1 , said motor shaft 13, and said rotating plate 15 provided with said magnet 17, wherein said housing 19 supports said at least one stirring chamber 5 above said rotating plate 15. Said housing 19 may for example be formed as a safety box. In a specific embodiment said at least one stirring chamber 5 is placed on said housing 19 via an adhesive 21. In a specific embodiment said at least one stirring chamber 5 is placed on said housing without an adhesive.

In an embodiment of said device 1 or method, said support 9 comprises a support plate 25, preferably a glass plate, wherein said support plate 25 is supported, by said at least one stirring chamber 5, on an upper open side of said at least one stirring chamber 5. When a multiwell plate 23 is used, a support plate 25 covering the upper open side of (part of) the multiwell plate 23 may be used, with a SMAP“hanging” in each of the wells (stirring chambers 25) of said multiwell plate 23. In other words, the support plate 25 is acting as a lid for the multiwell plate 23. In that case, each of the wells in the multiwell plate 23 is simultaneously stirred by the present device 1 and method by using one single electromagnetic system 3.

In an embodiment of the method, said electromagnetic system 3 comprises a motor 1 1 provided with a motor shaft 13 arranged for rotation about a rotation axis r, a rotating plate 15 attached to said motor shaft 13 and a magnet, preferably a permanent magnet 17, attached to said rotating plate 15 at a distance d from said rotation axis r, wherein, during said step of realising, said preferably time-dependent magnetic field is realised by rotating said magnet, by said motor 1 1 , about said rotation axis r. In a specific embodiment of the method, said device 1 comprises a housing 19 surrounding said motor 1 1 , said motor shaft 13, and said rotating plate 15 provided with said magnet 17, wherein said method further comprises the step of placing said at least one stirring chamber 5 on said housing via an adhesive 21. The type and amount of adhesive may be determined by a person skilled in the art and should be such that the stirring chamber(s) 5 are attached in a removable manner so that the stirring chamber(s) may be removed after stirring has completed.

The system 1 according to the invention distinguishes a flexible magnetic pillar 7 (SMAP = Suspended Magnetically Actuatable Pillar), a chamber well 5, a motor 1 1 , a permanent magnet 17, and optionally a safety box 19. The well plate may be placed on the box 19 with or without adhesion. Without adhesion there is considerable flexibility in scalability of the chambers 5 of the well plate. The permanent magnet 17 is placed off-axis on a supporting plate 15, which is fixed to the rotating axis 13 of the motor 1 1. An element, such as a bar, is fixed to the motor. The pillar 7, held by e.g. a glass plate 25, suspends in a chamber well 5, which is located above the rotating magnet 17. In an embodiment, the pillar 7 is aligned to the rotating axis r of the motor 1 1 and it is actuated by attraction to the magnet 17. The motor movement results in rotation of the pillar 7, which generates a controlled flow in the chamber 5.

In an embodiment of said device 1 and/or method and/or use, said use is for stirring of mammalian cells provided in said stirring chamber 5 or for mesofluidic mixing for realising at least one of colloid self-assembly, directed assembly and separation of microparticles and biological cells or for generation of cell spheroids for cell biological and drug screening studies.

Embodiments of the invention provide a versatile tool for various applications, such as mesofluidic mixing, colloid self-assembly, directed assembly and separation of microparticles and biological cells, as well as generation of cell spheroids for cell biological and drug screening studies. Generally speaking embodiments could be used in chemical applications (e.g., mixing, particle assembly and separation), biological applications (e.g., 3D cell culture, spheroid and organoid formation and maintenance), to daily products (e.g., food processing, fluid and polymer flow control).

Embodiments could be varied, for example, to enhance versatility the rotating permanent magnet 17 could be replaced by an electromagnetic system 3 of which the preferably time-dependent magnetic field is controlled by electric current. To enhance the effect of the rotating pillar 7 on the flow, non-cylindrical shapes of the pillar 7 may be conceived that increase the flow generation effectivity.

In an embodiment of said device or method according to the invention, said SMAP has a non-cylindrical shape.

In an embodiment of said device or method according to the invention, said preferably time-dependent magnetic field is controlled by electric current.

In an embodiment of the method and/or the use, the stirring speed is from 200 to 300 ppm, preferably from 200 ppm to 250 ppm. This has the advantage that it yields an optimal cluster size.

Stirring system

The use of the SMAPs 7 allows an actuation setup to control the motion of the pillars 7 remotely. These pillars 7 can be seen as artificial cilia, and have the added advantage of being compatible with biological fluids and cells. The system 1 ensures that magnetic actuation results in robust bending of the pillars 7 and large-scale fluid flow in the stirring chambers 5, e.g. in a multiwell plate 23.

Quantitative analysis using computational fluid dynamics modelling indicates that the flow profile in a stirring chamber, e.g. a well, can be tuned by modulating the applied magnetic field and the geometries of the stirring chamber 5 and the pillar 7.

In an embodiment, at least one permanent magnet 17 is fixed at a radial distance d from the rotation axis r of the motor 1 1. In an embodiment, at least one permanent magnet 17 is fixed at a vertical distance h from the tip 27 of the SMAP. The rotation of the support plate 15 results in vertical cone stirring motion by the SMAP 7 whereas the distance h results in bending of the SMAP 7. In an embodiment, multiple magnets are used on top of each other (i.e., at the same radial distance) on the rotating disc. In a specific embodiment, the four magnets were used on top of each other on the rotating disc. The number of magnets to be used depends i.a. on the strength of the magnet. By varying the number of magnets and their strength, a larger range of vertical distance h can be accessed.

Figure 2 shows an embodiment of the inventive SMAPs stirring system 1 . Figure 2A shows a schematic of the stirring system 1 , including an actuation setup 3 and the SMAP 7 suspended on the lid of a 24-well plate as a stirrer. The actuation setup 3 includes permanent magnets 17 placed off-axis on a support plate 15 that is controlled by a motor 1 1 , enclosed in a safety box 19. In this setup, d is for instance 4 mm and h is for instance 20 mm. Figure 2B shows a photograph of the actuation setup.

Figure 4 shows the simulated fluid flow velocity profiles during stirring using a 7 mm pillar 7. The pillar 7 is actuated with angular rotations of 150, 200, 250, and 300 rpm. Figure 4A shows the resulting flow profile measured at different heights: 0.2, 2, 4, and 6 mm from the bottom of the well 5. Figure 4B shows the vertical cross-sectional flow profile at the middle of the well 5. Figure 4C shows the distributions of the flow directions, as shown by the arrows. The colours correspond to the magnitude of the flow velocity as indicated in the colour bar. Figures 4D and 4E show the flow velocities plotted as a function of radial distance from the centre of the well (r = 0) at the height of the pillar tip 27 and at 0.2 mm above the bottom of the well 5, respectively. The profiles at different stirring speeds are indicated by different colours.

The fluid flow was visualized by adding microparticles as fiducial markers in the fluid and recording their motion in water resulting from SMAP motion. The SMAP rotating motion was found to be strong enough to generate flow in the whole well. It can be seen that the flow is faster near the tip 27 of the pillars 7, compared to further away from the pillar tip 27.

To obtain more insights into the flow induced by the motion of the SMAPs 7, Circular Fluid Dynamics simulations of the SMAP stirring were performed. Furthermore, the effect of different stirring speeds on the flow was tested by applying angular frequencies in the range of 150-300 rpm. This range of stirring speeds lies within the range accessible for most commercial stirrer plates as well as the present magnetic actuation setup 3. The flow velocity profiles generated by the stirring motion of a 7 mm pillar 7 in a well 5 of the same dimensions as that used in the experiments according to the invention are shown in Fig. 4. Consistent with the experimental observation, the simulations indicate that the pillar motion can effectively induce large- scale flow in the well 5, even at heights more than 2 mm below from the tip 27 of the pillars 7(Fig. 4A). The flow is highest at the trajectory of the pillar 7, reaching 40-90 mm/s, and decays with increasing distance from the pillar 7 (Fig. 4B). Increasing the rotation speed results in higher fluid flow at all locations in the well 5. The vertical cross-sectional flow profiles suggest that circumferential flow is maintained away from the pillar (Figs. 4B,C), reaching -40% of the maximum flow at the opposite face of the well (Fig. 4D). Moreover, the stirring motion generated a vortex along the rotation axis of the pillar 7, characterized by a local drop in the flow velocity. The flow profile near the bottom of the wells 5 show a clear dependence both on the rotation speed and on the radial location (Figs. 4C,E). At the centre and near the edge of the well, the flow in the radial direction is close to zero, while at other locations there is a counter-current directed towards the centre of the well.

Multiwell plate

In an embodiment of the device 1 according to the invention, said device comprises a multi-well plate 23 provided with a plurality of said stirring chambers 5, preferably wherein said multi-well plate comprises 24 stirring chambers. In a specific embodiment, said support 9 comprises a plurality of said SMAPs 7, each associated with a stirring chamber 5 of said plurality of at least one stirring chamber 5.

Multiwell plate 23, also called microplate, multi-chamber plate, microtiter plate, a cell-culture plate or microwell plate is a flat plate with multiple "wells" each of which is used as a small test chamber or tube. In the present invention a stirring chamber 5 may be a well of a multiwell plate 23. It is a standard tool in analytical research and clinical diagnostic testing laboratories. A multiwell plate 23 typically has 6, 12, 24, 48, 96, 384 or 1536 or even 3456 or 9600 sample wells, usually arranged in a 2:3 rectangular matrix. Multiwell plates are available in various materials, such as polystyrene or polypropylene. Multiwell plates are available in various styles, colours, formats as well as surface treatments.

Clustering of cells

By employing the stirring system 1 according to the invention in a standard cell culture plate, a controlled clustering of cells can be obtained. The SMAP stirring system 1 is therefore a promising cost-effective and scalable stirring approach for various types of studies involving colloids as well as soft and biological materials.

The device 1 and method according to the invention can be used in a 24-well culture place to effectively induce 3D spatial clustering of cells, such as mammalian cells.

Figure 5 shows stirring-induced clustering of cells. Figure 5A shows a setup for cell stirring, wherein the stirring chambers 5 are wells in a multiwell plate 23 in which the SMAP 7 is suspended from a support plate 9, such as a glass plate, that can be seen as a lid of the multiwell plate. The cell culture medium is stirred in the wells 5. Figure 5B shows the spatial distribution of cells at the bottom of the well 5. Figures 5C, 5E and 5G show microscopic images in the centre of the well 5 after stirring at, respectively, 200, 250 and 300 rpm for 20 hours. Figures 5D, 5F and 5H show the wall shear stress (WSS) profile at the bottom of the well 5 in a stirring speed of, respectively, 200, 250 and 300 rpm, with the stirring direction indicated by the arrow. The colour coding is indicative of the WSS, as shown in the colour bar. The dashed circles indicate the central areas where WSS < 10 mPa. Scale bar = 500 pm.

The rotation-speed-dependent radial flow suggests that tuning the stirring speed can be an approach to control the shear stresses at the bottom of the wells 5, which in turn can regulate the spatial deposition of particles. This may offer a way to promote controlled clustering of cells without labelling. A stirring experiment was performed in cell-seeded wells with different stirring speeds (Fig. 5A), and the distribution of MDA-MB-231 cells at the bottom of the well after 20 hours of culturing was observed (Fig. 5B). The MDA-MB-231 breast cancer cell line, cultured either as single cells, clusters, or spheroids, is commonly used as a tumour model for studying cancer invasion and testing drug efficacy.

Stirring using SMAP 7 at 200, 250, and 300 rpm was found to induce clustering of cells in the centre of the well 5 (Figs. 5C,E,G) and, to a lower extent, at the edge (i.e., close to the wall) of the well 5 (Figs. S2B-D in the ESI). Moreover, the stirring speed influenced the size of the cluster: increasing the stirring speed from 200 to 250 and 300 rpm resulted in monotonically decreasing cluster size. The clusters remained stable even after 2 days of further culture and change of medium without stirring. This indicates that the clusters were not only aggregates but the cells actually formed stable cell-cell adhesions that support 3D cell spheroid, as is expected for MDA-MB-231 cells. In contrast, a control experiment at 0 rpm did not result in any cluster formation (results not shown), whereas stirring at 150 rpm only generated clusters at the edge, but not in the middle, of the well (results not shown).

Without wishing to be bound by theory, the inventors believe that the formation of cell clusters in the middle and at the edge of the well 5 is associated with SMAP- induced radial flow of medium (Fig. 4E) and the associated wall shear stress (WSS) at the bottom of the well. The WSS profiles are remarkably consistent with the locations of the cluster formation: areas of low WSS are associated with cluster formation for all stirring speeds (Figs. 5D,F,H), including at the edge of the well 5. This suggests that radial flow near the bottom of the well 5 pushes the cells towards the centre and the edge of the well 5, where flow and WSS are low or even close to zero. When the stirring speed is too low (150 rpm), the WSS is not strong enough to redistribute the cells towards the centre of the well 5 and cluster formation only happens at the edge of the well 5. Comparison between the experimental and simulation results suggests a threshold WSS around 10 mPa (dashed circles in Figs. 5D,F,H), below which local cluster formation occurs.

The SMAPs 7 are fabricated by moulding a mixture of PDMS and magnetic particles 29. The dimensions of the mould 31 and therefore the SMAPs 7 can be adapted depending on the required size of the vessel 5 to be stirred. The use of SMAPs 7 , together with a home-built magnetic actuator 3, as a stirring system in a standard 24-well culture plate is demonstrated here. The magnetic susceptibility and the resulting deflection of the SMAPs 7 can be tuned in multiple ways: by modifying the distribution of the magnetic particles 29 in the SMAPs 7 (as shown through the comparison of hSMAP, aSMAP, and cSMAP), by changing the dimensions and/or stiffness of the pillars 7, by using magnets 17 with different flux densities, or by varying the distance between the SMAP 7 and the actuating magnets 17. These strategies give flexibility to the skilled person to adjust the design of the stirring system 1 based on the exact experimental setup, and therefore makes the system 1 scalable. The stirring motion of the SMAP 7 is able to generate large-scale fluid flow, which can be characterized numerically using Computational Fluid Dynamics (CFD) analysis. Computational analysis was performed assuming a non-deformable pillar.

The present examples demonstrate that the flow generated by the SMAP motion can be exploited for controlling spatial distribution of particles. More specifically, comparison between experimental and computational data suggests that the radial flow and wall shear stress at the bottom determine the deposition of particles. As one potential application of the SMAP stirring device 1 and/or method according to the invention, it was shown that it is able to produce controlled multicellular clustering, which is an important first step to obtain 3D cell culture, in standard 24-well cell culture plates. Comparison between cluster formation at different stirring speeds indicates that the size of the cluster depends on the stirring speed: cluster size decreases with increasing stirring speed. This trend is consistent with previous reports of flow-induced cell clustering obtained using commercial orbital rotary shaker or spinner flasks. It is worth noting that the magnitude of the angular velocities in prior art studies is significantly lower than that used in the present examples (200-300 rpm), because of the large vessel size necessitated by these methods. Moreover, it was found that a 150 rpm stirring speed fails to produce cell clusters, suggesting that there may be an optimal window of flow speed in the well that enables cell clustering.

3D cell culture is increasingly recognized to better mimic in vivo conditions than conventional 2D cell culture. To date, various methods to obtain 3D cell culture have been developed, but these methods have several important drawbacks. For example, agitation-based approaches such as spinner-flask bioreactors require specialized equipment and consumables and are commercially available only in predefined shapes and size; the hanging-drop and forced-floating methods often suffer from limitations in the size and variability of cell spheroids and are either labour- intensive (when custom-prepared) or expensive (with commercial products); contactless cell manipulation such as the magnetic levitation method inherently involves magnetization of cells that raises toxicity and epigenetics questions; whereas microfluidics-based and scaffolds-based approaches require expertise in microfabrication or polymer chemistry and entail difficult sample retrieval and analysis.

The SMAP stirring system 1 of the present invention circumvents some of these drawbacks by allowing the experimenter to steer the organization of cells with any standard culture plate with simple scaling of the SMAP geometry. The stirring procedure is contactless, label-free, and single-step, thereby making the strategy time- and labour-efficient. Another advantage of the SMAP stirring system is that in principle it requires no specialized equipment. The SMAP 7 can also be actuated using common laboratory stirrers with only minimal modification: a magnet should be affixed off-axis on the rotator of the stirrer.

Examples

Fabrication of suspended magnetic pillars

The magnetic precursor material used to fabricate the SMAPs 7 was composed of thermally curable polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning, Moerdijk, the Netherlands; 10: 1 base to curing agent weight ratio) and carbonyl iron powder (CIP, 5 pm diameter, 99.5%, Sigma-Aldrich, Zwijndrecht, the Netherlands). The weight ratio between PDMS and CIP was kept at 1 : 1 , except for the pillars with concentrated magnetic particles at the tips (see below), for which the weight ratio was 5: 1. The magnetic precursor material was prepared by vigorously mixing PDMS and CIP by hand for 10 min.

The SMAPs 7 were fabricated using a moulding method that was adapted from the micro-moulding approach as described in S. Zhang , Y. Wang, R. Lavrijsen, P. R. Onck and J. M. J. den Toonder, in Sensor Actuat B-Chem, 2018, 263, 614-624 for the fabrication of magnetic artificial cilia. Briefly, it consisted of 6 main steps (Fig. 1A). Step 1 , a polycarbonate (PC) mould 31 was fabricated using micro-milling (Mikron wf 21C), featuring wells with a diameter of 1 mm and a height of 7 mm. Step 2, the prepared magnetic precursor material was poured onto the mould 31 and degassed using a vacuum pump at a vacuum pressure of 2 mbar to remove air bubbles. Step 3, the excess precursor material outside the wells was erased with a scraper and cleanroom tissues (Technicloth TX606, Texwipe). Step 4, pure PDMS solution (i.e. , without the CIP) was poured onto the mould. This layer will function as a transparent substrate for suspending the magnetic pillar. Note that, in order to minimize the vertical distance between the SMAP 7 and the actuating magnet 17 in the setup 1 (described in the next section), the thickness of this PDMS substrate was set to 6 mm. Step 5, the mixture was cured in an oven for 2 h at 65°C. Step 6, the cured PDMS structure was peeled off the PC mould with the help of isopropanol to facilitate the release of the structure. Finally, the magnetic pillars 7, supported by a transparent PDMS substrate, were obtained. This protocol results in magnetic pillars 7 with a homogeneous distribution of magnetic particles (hSMAP).

In addition to the SMAP 7 with random distribution of magnetic particles 29, also pillars 7 with an aligned magnetic particle 29 distribution (aSMAP) and pillars with a concentrated magnetic particle 29 distribution at the tips 27 (cSMAP) were fabricated.

The fabrication processes for aSMAP 7 and cSMAP 7 were based on that of hSMAP 7. To fabricate the aSMAP 7, a permanent magnet (10x 10x5 mm³) with a remnant flux density of 1.3 T was placed under the mould during step 5 to align the magnetic particles along the magnetic field (Fig. 1 B). The cSMAP 7 was fabricated making use of the relatively high mass density of the magnetic particles compared to that of the PDMS solution (PCIP: PPDMS ^¾ 8: 1). Directly after step 4, the sample was placed in a fridge at 5°C for 3 days to slow down the curing of the PDMS, offering enough time for the magnetic particles to settle at the bottom of the mould (Fig. 1 C). After these modified steps, the pillars 7 can be released from the mould to obtain aSMAP 7 and cSMAP 7.

Actuation setup

A simple home-built magnetic actuation setup 3 was used to actuate the SMAPs 7 (Fig. 2A). The setup 3 consists of a unipolar stepper motor 1 1 (Wantai Nema 17) with an aluminium supporting plate 15 for the stacked three pieces of permanent magnets 17. The setup 3 was fixed in the centre of a transparent safety box 19 made from poly (methyl methacrylate) (PMMA) (Fig. 2B). Each magnet 17 has a geometry of 20x20x5 mm³ with a remnant flux density of 1.3 T. The top surface of the safety box 19 contains a circular opening of 40 mm in diameter, which allows the magnet 17 to rotate freely and minimizes the vertical distance h between the magnets and the SMAP 7 on top of the safety box 19. In this setup, h was set to be 20 mm, close to the height of a 24-well plate used in the cell experiment. The SMAP 7, supported by a glass plate which was used as the lid of the well 5, was aligned with the rotating axis r of the motor 1 1 with the help of a stereo microscope (Olympus SZ61 , Leiden, the Netherlands) for capturing the flow in the well 5. The magnets 17 were placed with an offset distance d of 4 mm with respect to the rotation axis r of the motor 11 to induce bending of the pillar 7 towards the stronger magnetic field and thus actuate the SMAP 7 to perform a vertical cone motion. Note that this actuation scheme, in which the magnetic gradient force actuates the pillar 7, differs from that used previously for magnetic artificial cilia, where the cilia tend to align with the applied magnetic field due to the magnetic torque acting on them. To control the motor 1 1 , a microcontroller board (Arduino Uno), a dual H-bridge drive controller board (L298N), an analog potentiometer to tune the motor speed, and a LED screen to display the speed were used.

Characterization of pillar motion and medium flow

A high-speed camera (Phantom V9, Vision Research, Bedford, UK) mounted on the stereo microscope was used to capture the top-view motion of the SMAPs and the generated liquid flow. The tip deflection of the SMAPs 7, projected on the x-y (horizontal) plane, was measured from static snapshots and averaged for each type of SMAP 7. To characterize the flow generated by the SMAP 7 one well 5 of a 24-well plate was filled with deionized water seeded with 30 pm polylactic acid particles (micromod Partikeltechnologie GmbH, Rostock, Germany) as fiducial markers, and then the SMAP was placed onto this well 5. Subsequently the SMAP 7 was actuated by the magnetic setup 3 to perform the vertical cone motion. The high-speed camera was used to capture the generated flow at a frame rate of 150 fps. The generated flows close to the SMAP tip 27 were measured by a Manual Tracking analyses in Image.

Computational fluid dynamics (CFD) analysis

Flow patterns in the well were quantified by numerically solving the Navier- Stokes equations using ANSYS CFX (Ansys Inc., Canonsburg, PA, USA). An accurate computer-aided design model of the well in a 24-well plate culture chamber, as used in the experimental setup (height = 16.5 mm, bottom diameter = 15.7 mm, top diameter = 16.28 mm), was constructed using SolidWorks (Dassault Systemes, Velizy- Villacoublay, France). The fluid domain and the magnetic pillar were meshed using 9,633 hexahedral and 14,048 tetrahedral elements, respectively, using ANSYS Meshing (Ansys Inc., Canonsburg, PA, USA). To keep the computational cost affordable, it was assumed that the magnetically-actuated bending of the SMAPs 7 and any effect of hydrodynamic forces achieve a steady state, after which there is no further deformation of the pillar 7. This allowed for modelling of the magnetic pillar 7 as a rigid-body spinning at a constant rotation, thus circumventing the need to couple a finite-element-based structural solver to compute the motion of the magnetic pillar. As a result, ANSYS CFX’s build-in immersed solid method was used to resolve the fluid-structure interaction. Furthermore, two assumptions were made: 1) The flow is laminar. This assumption is always satisfied within the range of angular rotations that we experimentally investigated in this study (150-300 rpm; Reynold’s number < 1 ). 2) The fluid is single phase, isothermal, incompressible, and Newtonian. Flow profiles generated by a 7 mm magnetic pillar in water at 37°C were visualized at four actuation speeds: 150, 200, 250, and 300 rpm. To model stirring using the aSMAPs 7, the geometry of the deformed pillar 7 during stirring was constructed to capture the experimental tip deflection of the aSMAPs 7. No-slip boundary conditions were prescribed on the domain walls while the fluid motions was resolved by prescribing a constant rpm to the magnetic pillar 7.

Cell stirring experiments

MDA-MB-231 cells were cultured in RPMI medium containing 10% FBS and 1 % penicillin-streptomycin at 37°C and 5% CO2. The SMAP 7, attached to a cover slip, was fixed to the lid of a 24 well-plate with tape and sterilized with 70% ethanol and UV exposure for 5 minutes. For the stirring experiments, 5x 10^s cells were seeded into a 24-well plate culture chamber (Greiner Bio-One CELLSTAR, 662160, Alphen a/d Rijn, the Netherlands). The lid containing the SMAP was placed on top of the 24-well plate, and the sample was transported to the actuation setup in an incubator at 37°C and 5% CO2. After 20 hours of stirring, micrographs of the cells were taken with an EVOS XL Core Cell Imaging System (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a 4* objective lens.

The present invention is further elucidated by the following claims.

Claims

1. A device (1 ) for providing controlled stirring comprising:

an electromagnetic system (3) arranged for realising a magnetic field;

at least one stirring chamber (5) comprising at least one Suspended Magnetically ctuatable Pillar (7), SMAP, suspended in said stirring chamber (5) from a support (9) and configured, in use, in said magnetic field such that said SMAP (7) is magnetically actuated, by said magnetic field, for providing said controlled stirring.

2. Device (1 ) according to claim 1 , wherein said electromagnetic system (3) comprises a motor (1 1) having a motor shaft (13) arranged for rotation about a rotation axis (r), a rotatable plate (15) attached to said motor shaft (13) and a magnet (17) attached to said rotatable plate (15) at a distance (d) from said rotation axis (r).

3. Device (1) according to claim 2, wherein said SMAP extends along said rotation axis (r).

4. Device (1) according to claim 2 or 3, wherein said device (1) further comprises a housing (19) surrounding said motor (1 1), said motor shaft (13), and said rotating plate (15) provided with said magnet (17), wherein said housing (19) supports said at least one stirring chamber (5) above said rotatable plate (15).

5. Device (1) according to claim 4, wherein said at least one stirring chamber (5) is mounted on said housing (19).

6. Device (1) according to any one of the preceding claims, wherein said device (1 ) comprises a multi-well plate (23) provided with a plurality of said stirring chambers (5), preferably wherein said multi-well plate comprises 24 stirring chambers.

7. Device according to claim 6, wherein said support comprises a plurality of said SMAPs, each associated with a stirring chamber of said plurality of at least one stirring chamber.

8. Device (1) according to anyone to the preceding claims, wherein said support (9) comprises a support plate (25), preferably a glass plate, wherein said support plate (25) is supported, by said at least one stirring chamber (5), on an upper open side of said at least one stirring chamber (5).

9. Device (1) according to anyone of the preceding claims, wherein said SMAP (7) is fabricated from a magnetic precursor material which is composed of thermally curable polydimethylsiloxane and carbonyl iron powder.

10. Device according to anyone of the preceding claims, wherein said SMAP has a non-cylindrical shape.

1 1. Device (1) according to anyone of the preceding claims, wherein said SMAP (7) is flexible for bending and rotating, by said \ electromagnetic field, said SMAP (7) for realising said controlled stirring.

12. Device (1) according to claim 1 , wherein said magnetic field is controlled by electric current.

13. Method for controlled stirring in a stirring chamber (5), using a device (1) comprising:

an electromagnetic system (3) arranged for realising a magnetic field;

at least one stirring chamber (5), wherein said at least one stirring chamber (5) comprises at least one Suspended Magnetically Actuatable Pillar (7), SMAP, suspended from a support (9) in said stirring chamber (5) and provided in said magnetic field such that said SMAP (7) is magnetically actuated, by said magnetic field, for realising said controlled stirring;

said method comprising the steps of:

realising, by said electromagnetic system (3), said magnetic field;

magnetically actuating, by said magnetic field, said SMAP (7), for realising said controlled stirring in said stirring chamber (5).

14. Method according to claim 13, wherein said electromagnetic system (3) comprises a motor (1 1 ) provided with a motor shaft (13) arranged for rotation about a rotation axis (r) a rotating plate (15) attached to said motor shaft (13) and a magnet (17) attached to said rotating plate (15) at a distance (d) from said rotation axis (r), wherein, during said step of realising, said magnetic field is realised by rotating said magnet (17), by said motor (1 1 ), about said rotation axis (r).

15. Method according to claim 14, wherein said device (1) comprises a housing (19) surrounding said motor (1 1), said motor shaft (13), and said rotating plate (15) provided with said magnet (17), wherein said method further comprises the step of placing said at least one stirring chamber (5) on said housing (19) via an adhesive (21 ).