Ultrasonic device and method for generating three-dimensional cell spheroids

Abstract

The invention relates to an ultrasonic device and an associated method for generating three-dimensional cell spheroids. The device comprises a polymer structure (2) of acoustically soft material with acoustic impedance similar to that of a liquid medium to be treated, which in turn comprises a set of containers (3) coupled to an ultrasonic actuator (1) on the bottom part thereof. The device allows three-dimensional cell spheroids to be generated from liquid suspensions in short periods of time of a few minutes and is also valid for particles or other micro-elements, with applications in research or analysis in tumour biology, tissue engineering and clinical analysis and research concerning molecular targets in personalised medicine, thus replacing the use of animals.

Description

[0001]ULTRASONIC DEVICE AND METHOD FOR GENERATING THREE-DIMENSIONAL CELLULAR SPHEROIDS OBJECT OF THE INVENTION The present invention belongs to the field of acoustofluidics, combining aspects of millifluidics and acoustophoresis. Specifically, the invention relates to an ultrasonic device and an associated method for generating three-dimensional cellular spheroids in short periods of time, just a few minutes, from liquid suspensions, and is also valid for particles or other microelements. BACKGROUND OF THE INVENTION Currently, the use of three-dimensional (3D) in-vitro models represents an effective bridge to animal models. These 3D models mimic characteristics of the in-vivo environment and are presented as a tool for studying cellular, tissue, organ, and tumor behavior.They represent a key tool for transdisciplinary research in cancer biology, tissue engineering, and the search for optimized drugs for personalized medicine. The advantages of these three-dimensional in vitro models, and particularly tumor spheroids or 3D models, are: - cell morphology and signaling are usually more physiological than routine 2D cell culture, - they allow rapid experimental manipulations and hypothesis testing, and - they allow higher quality real-time and / or fixed imaging by microscopy than in animals. Many 3D models have been developed from isolated cells, such as cell lines, dissociated tissues, or stem cells. A widely used strategy is the implantation of cultured cells in 3D scaffold structures as tissue-like aggregates. Tumor spheroids, on the other hand, consist of 3D aggregates of cells from monocultures or c-cultures.Spheroids can be formed from hanging droplets, in bioreactors, or in 3D matrices, and can be created from either single-cell populations or co-cultures, or from multicellular mixtures of tumor, stromal, and immune system cells. It is widely known that these aggregates can mimic tumor behavior more effectively than 2D cultures because spheroids contain well-oxygenated and hypoxic cells that secrete tumor-associated cytokines. These spheroids can be valuable for the development of efficient drugs, replacing the use of animals in testing. Spheroids can also be used to study the adherent properties of tumor cells and allow the analysis of their individual and collective behavior. However, long laboratory times are required to induce tumor spheroid formation using conventional systems, ranging from 14 hours to several days.It is also well known that epithelial cells, and particularly tumor cells exposed to ultrasound, acquire oscillatory movements when exposed to an acoustic wave. At certain amplitudes below the threshold of cellular damage, they are exposed to a radiation force that drives them toward acoustic pressure nodes established under standing wave conditions, known as acoustophoresis. Over the past few decades, multiple devices based on this mechanism have been developed for sorting and separation applications, but only for the formation of 3D cell spheroids. First, a device consisting of a silicon wafer containing 10x10 receptacles, with dimensions of 0.3x0.3 mm, is known. 2each one. This device allows the establishment of an acoustic pressure node in the center of each receptacle at a frequency close to 2.5 MHz, where it is established thanks to the rigidity of the walls, and which allows the establishment of a standing wave ^ for acoustic pressures between 0.5-1 MPa. However, this device presents certain disadvantages associated with the high manufacturing cost required by silicon microfluidic devices, which also require the use of clean rooms and restrictive conditions in the manufacturing process. For this reason, their mass production for clinical or biomedical use has never occurred. Furthermore, these devices are unviable for any application that requires disposability, not complying with the sustainability requirements of the European Union.Another well-known device for 3D cell aggregation consists of a conventional culture dish exposed from below to standing waves at a frequency of 110 kHz and voltages of 70 V peak-peak applied by a Langevin transducer. At such a low frequency, the wavelength is approximately 15 mm, which is two orders of magnitude larger than that of the previous work. This generates radiation forces ten times lower than those produced by a wave applied at 1 MHz, which also results in cell conduction times and collection at pressure nodes that are approximately an order of magnitude longer.In view of the above, a sustainable and low-cost device is needed for the rapid formation of cellular spheroids for the purposes of research in tumor biology, tissue engineering, clinical analysis and the search for molecular targets in personalized medicine, allowing the replacement of the use of animals and complying with European regulations on the sustainability of technological developments. DESCRIPTION OF THE INVENTION The object of the present invention is an ultrasonic device and associated method for generating three-dimensional cellular spheroids in short times, just a few minutes, from liquid suspensions, also being valid for particles or other microelements.Regarding the applications of the present invention, it is applicable, for example, to research or analysis purposes in tumor biology, tissue engineering and clinical analysis and search for molecular targets in personalized medicine, allowing the replacement of the use of animals. The device comprises a disposable polymeric structure that in turn comprises a set (array) of receptacles or wells. The receptacles comprise a base and four sides, and are intended to contain a liquid medium to be treated, in which the spheroids will be generated. The polymeric structure has an acoustic impedance similar to that of the liquid medium to be treated. The acoustic impedance Z is the product of the density of the medium and the speed of propagation of the wave through it (^ = ^^). The impedance in plastics is Zplastics ^2.1 - 2.4 MRayls and while the impedance in a liquid suspension is Z. susp liquida^1.5-1.7 MRayls. Preferably, the base of each receptacle of the polymeric structure is a square section, with sides between 2 and 4 mm in length, and the four sides are flat walls between 8 and 12 mm in height. In addition, the device comprises an ultrasonic (piezoelectric) actuator concentrically coupled below the receptacles, in contact with their base. The device further comprises a signal generator to supply sinusoidal electrical waves to the ultrasonic actuator. The ultrasonic actuator is preferably a piezoelectric ceramic (piezoelectric transducer), resonant in thickness mode at a certain frequency, mechanically coupled under the bases of the receptacles. Once it receives the electrical signal from the signal generator at the desired frequency, it transforms it into mechanical vibrations that it transfers to the structure of the set of receptacles to which it is coupled.The polymeric structure with the set of receptacles can be made of a transparent material, and as indicated, its acoustic impedance must be similar to that of the liquid medium to be treated, with a difference of less than 60% between the two media: liquid suspension and the plastic material of the structure. An example of a polymeric material that can be used is polyethylene. The physical properties of the polymers used in the polymeric structure are very different from those of the ultrasonic actuator, which is preferably a piezoelectric ceramic, whose acoustic impedance is much higher than that of the plastic material Z. piezoelectrico=17.3 MRayls, approximately seven times higher, so their acoustic response is very different and closer to that of a liquid medium. Furthermore, polymers are highly absorbent media, in which a plane wave quickly transforms into three-dimensional waves. In this way, instead of establishing standing waves between the side walls of each of the receptacles, a 3D wave is generated in each of them produced by three-dimensional vibrations established in the plastic structure of the array or set of receptacles. The properties of these polymeric materials make it very difficult to achieve standing wave conditions inside the receptacles, but in return they have the advantages of being easy to manufacture, low cost, and easy to handle.Furthermore, there are several frequencies with optimal acoustic pressure distributions for particle or cell collection, rather than a single frequency that imposes the resonant frequency of the receptacles at a frequency determined by their dimensions, which the signal generator applies to the ultrasonic actuator. The three-dimensional mechanical vibrations established in the polymeric structure with the array of receptacles are specific to each frequency, and at the interfaces of each receptacle with the contained liquid medium, they are transferred to the receptacle, generating a complex 3D distribution of acoustic pressures that differ in each receptacle and frequency.Once the wave is established in the liquid phase contained in each receptacle, a nonlinear interaction occurs between the incident wave and the scattering wave created by each cell in the suspension, generating a time-independent hydrodynamic radiation force that acts on the cell and guides it individually toward defined acoustic equilibrium zones within each receptacle, corresponding to pressure nodes. Particles or cells drawn by the radiation force toward these positions collect in short times of less than five minutes. Once collected at the acoustic pressure nodes within the receptacles, the cells aggregate, forming stable cellular spheroids, in which the cells remain alive during subsequent culture processes.Regarding the specific operation and method of the invention, first, the signal generator activates the ultrasonic actuator, converting the electrical signal into a mechanical vibration, with maximum amplitude at a resonance frequency of its thickness mode. To identify the optimal actuation frequencies, an experimental electrical characterization is performed. This is due to the large number of parameters involved in these processes: dimensions and geometry of the polymeric structure and the receptacles, quantity and spatial distribution of the receptacles, concentration of cells in the suspension, typology of the cells in suspension, and location of the ultrasonic actuator with respect to the polymeric structure.Furthermore, when identifying the preferred resonant frequencies, the following requirements must be taken into account: - formation and consolidation of the cell aggregate in the minimum time to optimize acoustic energy consumption, - compaction of the cell aggregates, and - generation of cell aggregates in the maximum number of receptacles. Since the device uses an ultrasonic transducer, it is advisable to search within the range of singular frequencies determined in the electrical conductance measurements and directly verify the formation of cell aggregates by visual observation in the receptacles over time. Therefore, sinusoidal electrical waves are applied to the receptacles of the polymeric structure using the ultrasonic actuator and generated in the signal generator at a variable frequency between 450 kHz and 520 kHz.Next, a measurement of the ultrasonic actuator's conductance is obtained for each of the applied frequencies, and at least one working frequency for which the conductance is maximum is determined. Returning to the device's operation, secondly, the standing wave at the working frequency established in the ultrasonic actuator is transmitted to the polymeric structure, where complex three-dimensional vibration modes are established. The three-dimensional mechanical vibrations established in the polymeric structure are specific to each frequency and are transferred to the liquid sample through the sides (interfaces) of each receptacle, generating a three-dimensional acoustic pressure distribution specific to each receptacle and frequency, with spatial amplitude gradients within each receptacle.Once the wave is established in the liquid phase contained in each receptacle, a non-linear interaction occurs between the incident wave and the scattering wave created by each cell in the suspension, generating a radiation force ^^. ^ Independent of time, it acts on the cell and drives it individually toward defined acoustic equilibrium zones within each receptacle, corresponding to pressure nodes. This is a hydrodynamic force proportional to the volume and shape of each cell or particle in suspension, the frequency of the applied acoustic wave, the spatial distribution of pressure in each receptacle, as well as the viscoelastic properties of the cells (or particles) and the liquid medium in which they are immersed. The radiation force drives each cell or particle toward the nearest established acoustic equilibrium positions and is calculated for a standing wave as: where ^(^ ^, ^ ^ , ^ ^ , ^ ^ ) = ^^ ^ ^^^ ^ ^ ^ ^ ^^ ^ - ^ ^ It is defined as the acoustic contrast factor, "^", which defines the relationship between densities and adiabatic compressibilities of particles or cells (^ ^ , ^ ^ ) and the liquid medium in which they are immersed (^ ^ , ^ ^), respectively. The distance to the nearest pressure node is defined by “^”, and “^” represents the length of the acoustic wave in the medium. ^ indicates the direction of movement of the particles or cells, either towards the pressure nodes when it is positive (^ > 0) or towards the antinodes when it is negative (^ < 0), where the acoustic pressure has maximum amplitude. The acoustic contrast factor is positive for both particles of the receptacle material and epithelial cells, so they are driven towards the acoustic pressure nodes.The conduction process from their initial positions within the receptacle to the node requires different times and trajectories depending on the initial position of the cells (or distance from the pressure nodes). This means that this process is not instantaneous, but rather requires a time interval of up to several minutes depending on the density of cells in the suspension, as well as the amplitude of the applied acoustic wave. The particles or cells dragged by the radiation force toward these positions collect in short times of less than five minutes in the device of the invention. Thus, an aggregation process begins that remains stable after ultrasonic actuation. The size of the cell aggregate increases over time with the arrival of more cells from different positions within the receptacle, creating a cluster nucleation where the cells aggregate.Cellular aggregates can acquire irregular or spherical geometries, depending on the shape of the established acoustic pressure node. DESCRIPTION OF THE DRAWINGS In order to complement the description being made and in order to aid in a better understanding of the characteristics of the invention, in accordance with a preferred example of practical embodiment thereof, a set of drawings is attached as an integral part of said description, in which the following is represented for illustrative and non-limiting purposes: Figures 1 and 2.- Show a top view and a side section of a diagram of the device with the structure of nine receptacles arranged in a 3x3 array. Figure 3.- Shows a graph of the relationship between the frequency of the input signal (f) and the measured conductance (G) of the ultrasonic actuator. Figure 4.- Shows a photograph of the formation of various spheroids of PANC-1 pancreatic cancer cells, ultrasonically induced in a time of approximately 4 minutes PREFERRED EMBODIMENT OF THE INVENTION A preferred embodiment of the ultrasonic device and associated method for generating three-dimensional cellular spheroids in short times, a few minutes, from liquid suspensions, is described below with the help of Figures 1-4, also being valid for particles or other microelements. As shown in Figures 1 and 2, the device comprises an ultrasonic actuator (1), particularly a piezoelectric ceramic, mechanically coupled to a polymeric structure (2) with millifluidic receptacles (3) in which a three-dimensional ultrasonic wave is established that acts on each particle or cell in suspension, driving them towards positions of acoustic equilibrium where they collect to form aggregates.The receptacles (3) have a square geometry as does the ultrasonic actuator (1), which has dimensions of 30x30mm. 2on the surface and a thickness of 1.5mm, and is designed to resonate in thickness mode, preferably at a frequency of 491kHz. The ultrasonic actuator (1) and the polymeric structure (2) are concentrically coupled. The device also comprises, as shown in Figures 1 and 2, a signal generator (4), connected to the ultrasonic actuator (1), to supply it with sinusoidal electrical waves. Figure 3 shows the relationship between the frequency of the input signal (f) generated by the ultrasonic actuator (1) and its conductance (G). The graph shows three peaks where the conductance is maximum, at f1 = 469kHz, f2 = 491kHz and f3 = 508kHz, which is where the device is in resonance and shows its maximum acoustic vibration capacity. Of the three resonance frequencies of the device, the one that has been found to be ideal for rapid formation of cellular spheroids has been chosen: f2 = 491kHz. Low levels of P pressure have been required. max= 250 kPa to induce cell aggregates and short ultrasonic irradiation times, below the threshold of cell damage. Furthermore, the polymeric structure (2) can have different configurations: with a single receptacle (3), with four receptacles (3) arranged in a two-dimensional 2x2 array or with nine receptacles (3) arranged in a two-dimensional 3x3 array, all of them centered respectively on the ultrasonic actuator (1). Specifically, in the embodiment with nine receptacles (3), the polymeric structure (2) has dimensions of 127.8x85.5x15.0 ± 0.2 mm 3 and the receptacles are geometrically distributed in 3x3 receptacles (3). In the embodiment with four receptacles (3), the polymeric structure (2) has dimensions of 11x11x0.2 ± 0.2mm 3and the four receptacles (3) are geometrically distributed in 2x2 receptacles (3). Finally, in the embodiment with a single receptacle (1), the polymeric structure (2) has dimensions of 5.5x5.5x0.2 ± 0.2mm 3 . The dimensions of each receptacle (3) are 3.4x3.4x6.4mm 3, with a volume of 74µL. Liquid samples of 50 µL are introduced into each of the receptacles (3). The distance between adjacent receptacles (3) is 4.5mm. Finally, the polymeric structure (2) and the receptacles (3) are preferably made of polyethylene with a density ρ = 1.01g / cm . Furthermore, in one aspect of the invention, a transparent polymer is chosen in order to observe the formation of the spheroids in the receptacles (3). Figure 4 shows a photograph of the formation of various spheroids of PANC-1 pancreatic cancer cells, induced ultrasonically in a time of approximately 4 minutes, which demonstrate the correct operation of the device of the invention.

Claims

CLAIMS 1.- An ultrasonic device for generating three-dimensional cellular spheroids from microelements suspended in liquids, characterized in that it comprises: - a polymeric structure (2) comprising a set of receptacles (3), in which each receptacle (3) comprises a base and sides and is intended to contain a liquid, the difference between the acoustic impedance of the polymeric structure (2) and the liquid being less than 60%, - an ultrasonic actuator (1), concentrically coupled to the receptacles (3) at their base, to transfer mechanical vibrations to them, and - a signal generator (4), connected to the ultrasonic actuator (1), configured to supply sinusoidal electrical waves at a frequency between 450kHz and 520kHz that maximizes the conductance of the ultrasonic actuator (1). 2.- The device of claim 1, wherein the ultrasonic actuator (1) comprises a piezoelectric ceramic, resonant in thickness mode.

3. The device of claim 1, wherein the frequency of the sinusoidal electrical waves generated by the signal generator (4) is selected from 469kHz, 491kHz and 508kHz.

4. The device of claim 1, wherein each receptacle (3) of the polymeric structure (2) comprises a square section base, with sides between 2 and 4 mm in length, and four sides that are flat walls between 8 and 12 mm in height.

5. The device of claim 1, wherein the polymeric structure (2) comprises a single receptacle (3).

6. The device of claim 1, wherein the polymeric structure (2) comprises four receptacles (3) arranged in a 2x2 matrix.

7. The device of claim 1, wherein the polymeric structure (2) comprises nine receptacles (3) arranged in a 3x3 matrix.

8. The device of claim 1, wherein the polymeric structure (2) is made of polyethylene with a density ρ = 1.01 g / cm. 9.- Method for generating three-dimensional cellular spheroids from microelements suspended in liquids, which makes use of the device of any of the preceding claims, and comprising the steps of: - applying the sinusoidal electrical waves to the receptacles (3) of the polymeric structure (2) with the ultrasonic actuator (1) and generated in the signal generator with a variable frequency between 450kHz and 520kHz, - obtaining a measurement of the conductance (G) of the ultrasonic actuator (1) for each of the applied frequencies, - determining at least one working frequency for which the conductance is maximum, and - applying the sinusoidal electrical wave at the working frequency to the receptacles (3) to obtain the spheroids.

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